Marco Ruella Adoptive immunotherapy for

Michael Kalos
cancer

Authors’ address Summary: Recent clinical success has underscored the potential for
Marco Ruella1, Michael Kalos1 immunotherapy based on the adoptive cell transfer (ACT) of engi-
1
Department of Pathology and Laboratory Medicine, neered T lymphocytes to mediate dramatic, potent, and durable clinical
University of Pennsylvania, Philadelphia, PA, USA. responses. This success has led to the broader evaluation of engineered
T-lymphocyte-based adoptive cell therapy to treat a broad range of
Correspondence to: malignancies. In this review, we summarize concepts, successes, and
Michael Kalos challenges for the broader development of this promising field, focus-
Lilly Research Laboratories ing principally on lessons gleaned from immunological principles and
Eli Lilly and Company clinical thought. We present ACT in the context of integrating T-cell
450 East, 29th Street and tumor biology and the broader systemic immune response.
New York, NY 10016, USA
Tel.: +1 646-638-5095 Keywords: adoptive cell therapy, immunotherapy, chimeric antigen receptor, cancer,
e-mail: kalos_michael_d@lilly.com immune modulation, tumor microenvironment

Acknowledgements
We are grateful to the multitude of scientists who have laid
the foundations for the field, and to the patients who have
allowed, through their bravery and generosity, the clinical
development of this promising treatment modality. MR is
supported by funds from the “Societa Italiana di Ematologia
Sperimentale” (SIES), from “Associazione Italiana Pazienti Emopatici”
(AIPE) and AFCRI fund number 990-9936-4-900278-
5043-2433-1410 from the University of Pennsylvania. The
authors have read the journal’s policy on disclosure of
potential conflict of interest. MR has nothing to declare. MK
is named on patents related to CART19 technology with the
potential for royalties and milestone payments from the
development and commercialization of CART19 technology.
This article is part of a series of reviews
covering Adoptive Immunotherapy for
Cancer appearing in Volume 257 of
Immunological Reviews.
Video podcast available
Go to
www.immunologicalreviews.com to
watch an interview with Guest Editor
Dr Carl June.
Immunological Reviews 2014
Vol. 257: 14–38
Printed in Singapore. All rights reserved
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews
0105-2896
14
Introduction
The principal biological role of the immune system is to
maintain the integrity of ‘self’. This integrity is maintained
principally by eliminating and destroying diseased and
infected cells, while ensuring that healthy cells and tissues
are not targeted. A vastly complex network of events that
bring together both innate and adaptive immune arm
through both cell mediated and soluble interactions is
responsible for maintaining the integrity of self. The net-
work of cells includes, among others, T and B lymphocytes,
natural killer (NK) cells, antigen-presenting cells (APCs)
such as dendritic cells (DCs) and macrophages, stromal,
endothelial, and epithelial cells. This network of cells is
coordinated by a myriad of soluble factors to sense both
health and damage and in turn orchestrate the immune sys-
tem to clear the body of damaged cells and protect the body
from infections. Data from patients with inherited immune
deficiencies have revealed the critical role for an intact
immune system for organismal health, with autoimmune
disease and increased risks of developing infections and
tumors a consequence of disruptions in immune network
functions (1, 2).
Broadly speaking, immune cells can be defined as having
either effector or regulatory functions in the immune system.
The main known effector cells of the immune system are
understood to be T and NK lymphocytes, mediating,
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014

respectively, adaptive and innate immunity. The variety of
cells with immune regulatory activities is considerably
broader, and includes cells of lymphoid origin such as
CD4+ helper and regulatory cells and B cells, and myeloid
origin such monocytes, macrophage, myeloid derived DCs,
and myeloid derived suppressor cells. Perhaps not surpris-
ingly given the breadth of the immune system, cells not
classically considered to be involved in the immune systems
such endothelial, stromal, and epithelial cells have over the
past few years been shown to have immune regulatory
functions (3–6). The relevance of these cell types in the reg-
ulation of a potent immune response is revealed by the fact
that tumors often evolve under selective pressure by the
immune system to modulate the biology of these cells and
blunt anti-tumor immunity (7).
Abundant evidence exists to document that the immune
system plays an essential role in controlling the growth of
tumors. This evidence has been accumulated from the
study of animal models with defined immune defects (8),
from the increased frequency of cancers in patients with
naturally occurring or acquired immune deficiencies (9),
and also from the observation that tumors often evolve to
become less visible to the immune system. Early efforts to
target cancer through manipulating the immune system in
fact predated the ‘discovery’ of the immune system, most
famously by William Coley who applied heat killed strepto-
cocci (‘Coley’s toxin’) at tumor sites to induce inflamma-
tion with consequent tumor regressions (10). More
recently, the tuberculosis vaccine of Calmette-Guerin (BCG)
has been used with success to treat localized bladder neo-
plasm (11), while adjuvants based on bacterial components
that trigger immunity via engagement of Toll-like-receptors
(TLRs) on immune cells are essential components of FDA-
approved vaccine preparations. Direct evidence of the
potency of effector T cells to target and eradicate tumor
cells was demonstrated through the clinical application of
donor lymphocyte infusion (DLI) to treat leukemia after
allogeneic hematopoietic stem cell transplantation (HSCT),
with the Graft-versus-leukemia (GVL) effect mediated by
alloreactive donor T cells leading to strong anti-leukemic
responses in a significant portion of relapsing patients (12,
13).
As the field of immunotherapy has unfolded over the
past two decades, a number of strategies have been evalu-
ated to target cancer. Such immunotherapeutic strategies
have included a plethora of vaccine-based strategies to
trigger anti-tumor cellular and humoral immunity, anti-
body-based strategies to mediate complement and
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014
Ruella & Kalos
Adoptive immunotherapy for cancer
NK-dependent anti-tumor cytolytic activity, block inhibi-
tory receptors, or modulate the tumor microenvironment
(TMA) (14–19). Unfortunately, each of these approaches
has been compromised by the impact of central and
peripheral immune tolerance, which presents a fundamen-
tal impediment for the effective targeting of tumor cells
by immune effector cells. Specifically, central tolerance, by
deleting the T-cell repertoire to self-antigens often overex-
pressed or aberrantly expressed by tumors, effectively
results in the blunting of the potent anti-tumor T cell rep-
ertoire to such antigens. On the other hand, peripheral
tolerance, commonly exploited by tumors, blunts the abil-
ity of existing effector lymphocytes (T cells, NK cells) to
target tumor cells by establishing an immune-suppressive
state.
The relevance of peripheral tolerance in blunting the anti-
tumor immune response has been highlighted over the past
few years in clinical trials to evaluate antibodies which block
immune-modulating molecules. In these trials, blocking the
immune modulatory activity of cytotoxic T-lymphocyte anti-
gen-4 (CTLA4) (CD152) and programmed death-1 (PD1)
(CD279) has resulted in potent anti-tumor activity, with
document long lasting complete responses in a subset of
patients (20–22); these results have led to the first-in-class
approval of an anti-CTLA4 antibody (ipilumimab/Yervoy),
and the likely approval of PD1-modulating agents in the
near future.
Overcoming central tolerance poses a unique challenge,
since the impact of central tolerance is the lack of a potent
anti-tumor T-cell repertoire to self-antigens. One approach
to overcome central tolerance is the transfer into patients of
potent effector cells, as exemplified by the efficacy of allore-
active T cells in the context of allogeneic cell transplantation.
Practical limitations in terms of availability of suitable
donors as well as GVH-related toxicity issues have limited
the broader use of allogeneic cell transplantation. One alter-
native to allogeneic cell transplantation is adoptive cell trans-
fer (ACT), which involves the infusion into patients of
autologous lymphocytes following ex vivo expansion. Initial
efforts to apply ACT in tumor immunotherapy involved
transfer of bulk T-cell populations into patients, under the
premise that ex vivo manipulation of cells had the potential to
lower activation thresholds of potential tumor-reactive cells
as well as to expand tumor-specific T cells. The promise of
this approach was revealed in clinical trials which showed
that this strategy had the potential to rapidly reconstitute
host immunity (23–25). Recent advances in the ability to
effectively engineer T lymphocytes have provided an
15
Ruella & Kalos
Adoptive immunotherapy for cancer
unprecedented opportunity to be able to expand on the
potential of T-cell transfer and have ushered in what prom-
ises to be a golden age for T-cell-based immunotherapy
based on principles of synthetic biology (26) (Fig. 1).
Indeed, clinical results from recent trials employing engi-
neered T cells are demonstrating the dramatic potency of
this approach to target cancer (27–31).
This review focuses on principles of ACT in the context
of recent conceptual and technical advances that are driving
the field in this emerging era of synthetic biology. We
describe the state of the art for the field with regard to cell
types, engineering approaches, and clinical experience.
Importantly, we focus on viewing ACT in the context of the
systemic immune response, the biological context within
which infused T cells are operative.
A brief history of ACT
The potential to apply ACT as a therapeutic approach was
first evaluated in rodent models over 50 years ago (32).
The first clinical experience in humans was reported from
the Seattle group in 1991 in the context of cytomegalovi-
rus (CMV) prophylaxis during immune reconstitution post
transplantation, where CMV-specific T-cell clones were
expanded ex vivo and infused into patients at high risk
from CMV infection, resulting in the establishment of an
anti-CMV protective immune response (33). This seminal
report demonstrated that the fundamental requirements for
successful ACT, namely ex vivo manipulation, expansion,
and reinfusion into patients were technically feasible, and
precipitated concerted efforts to apply ACT to target malig-
nancy. Early efforts to target malignancy focused on the
use of tumor-infiltrating lymphocytes (TILs) pioneered by
the Rosenberg group at NCI (34), the use of bulk popula-
tions of expanded lymphocytes (23), as well as the use of
tumor antigen-specific clones, isolated following consider-
able tour-de-force efforts (35, 36). These approaches revealed
a number of significant points with regard to the clinical
implementation of ACT: (i) T-cell- based immunothera-
peutic approaches had the potential to effect potent anti-
tumor activity; (ii) tumor targeting strategies would need
to incorporate and address biological considerations for
both the T cell (e.g. the need for cosimulation) and the
tumor (e.g. immunosuppression); and (iii) robust and
practical implementation of ACT to target tumors would
require significant improvements in the ability to rapidly
generate large and potent populations of tumor-specific
lymphocytes.
16
Accordingly, the principal focus in the field of ACT
shifted toward a better understanding of the properties of
the effector cells that were important to mediate and drive
the observed anti-tumor activity and the development of
improved approaches to isolate, manipulate, and expand
such potent effector T cells.
Immune effector cells in ACT
Two effector cell types, T lymphocytes and NK cells have
been principally evaluated for ACT. The essential properties
of these cells that have supported their evaluation have been
the fact that these cells are capable of cytotoxic targeting,
and that these cells can be readily isolated and manipulated
ex vivo. Table 1 summarizes the advantages and disadvantages
for each of the effector cell types that have been evaluated
to date in ACT-based approaches against cancer.
Classical NK cells (CD3 CD56+) represent about 20–
30% of circulating lymphocytes. As part of the innate
immune system, NK cells characterized by rapid and potent
cytolytic activation against virus-infected or transformed
cells, a potential advantage for ACT. A potential advantage
of NK cells is that their principle targets are cells that lack
class I, often a phenotype selected for in tumor cells; on
the other hand the presence of class I on tumors poses
interesting challenges for broad implementation of NK cell-
based therapies. In the context of ACT, classical NK cells,
both as primary cells and cell lines, have been evaluated in
a number of different settings and using a variety of ex vivo
expansion protocols. Classical NK cells have been tested in
ACT non-modified in the context of haplo-identical HSCT
(37), or more recently following genetic modification to
express immune-modulatory cytokines (38). Unfortunately,
despite numerous attempts, these approaches led to at best
modest clinical activity (39, 40). A new and intriguing
approach involves gene engineering of NK cells to express
chimeric antigen receptors (CARs), and these exciting stud-
ies are now starting to be evaluated in early phase clinical
trials (41).
Smaller lymphocytes subsets that represent intermediate
phenotypes between classical NK cells and T cells have
also been evaluated in the context of ACT. NK T cells
(NKT) (CD3+CD56+), iNKT cells (CD3+CD56+Va24-Ja18),
and cytokine-induced killer cells (CIK) (CD3+CD56+) dis-
tinguished by Lu and Negrin (42), are each ‘niche’ effec-
tor lymphocyte subpopulations with potential advantages,
summarized in Table 1, in the context of ACT (43). NKT
cells have been shown to modulate immune responses
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014
 Ruella & Kalos
Adoptive immunotherapy for cancer

NK Treg A Anti-VEGF-R
T Anti-αvβ3 integrin B
B
CART CART homing and
trafficking
C haTCRT
A
NKT
R
T
C D
CART
Tumor cell F

Treg MDSC B
Tumor cell
CART
Lymphodepletion MDSC H Checkpoint inhibitors
Chemothererapy haTCRT Autocrine cytokine
Conditioning Anti-FAP
augmentation
Radiation
TIL
E
G Tumor cell
B haTCRT Treg CART
Treg
TAM B
Treg
Tumor cell
Treg
TIL Tumor cell depletion Tumor cell
MDSC
I B NKT
Treg
B
CART MDSC

NKT
Anti-NKG2D MDSC TAM NK

Molecules Effector cells Regulatory cells Other cells

: Myeloid-Derived Tumor : Tumor Cell

: Surface target epitope CART : CAR-engineered T cell MDSC cell
Suppressor Cell
: MHC:peptide complex
: Endothelial cell
: Endogenously processed haTCRT : Affinity-enhanced TCR- TAM : Tumor Associated
epitope engineered T cell
Macrophage
NK : Natural Killer cell
: CAR
: Tumor infiltrating
: Tumor Associated
: Endogenous TCR complex lymphocyte NKT : Natural Killer T cell
Fibroblast
: Affinity-enhanced TCR Treg : Regulatory T cell
complex B : B cell

Fig. 1. Adoptive cell transfer (ACT) approaches to effect anti-tumor immunity. (A) Engineering T cells to target tumor endothelium facilitates
the entrance of immune cells on tumor site and inhibit angiogenesis. (B) Expression of chemokines (CXCR2, CCR2B) on the surface of T cells
favors homing to tumor site. (C) chemotherapy or radiotherapy before ACT enhance T cells cytotoxicity and persistence by both target tumor and
tumor associated immunosuppressive cells [myeloid-derived suppressor cells (MDSC), Treg, tumor-associated macrophages (TAM)]. (D) Chimeric
antigen receptor engineered T cells (CART) can target TAA and kill tumor cells without the need of HLA presentation. (E) TAA that are processed
and presented in HLA on the surface of tumor cells are recognized by tumor-infiltrating lymphocytes (TIL) and haTCRT. (F) Targeting tumor
associated FAP+ fibroblasts that may suppress T-cell homing and anti-tumor activity in the tumor environment. (G) Ex vivo depletion of Tregs
reduces the number of these inhibitory cells in the infusion product. (H) Checkpoint inhibitors (e.g. anti-CTLA-4 and anti-PD-1) and the
increased expression of activating cytokines (e.g. IL-12) enhance T-cell cytotoxic function. (I) Targeting NKG2D on immune cells re-modulates
tumor environment, reducing the immunosuppressive milieu.

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Immunological Reviews 257/2014 17
Ruella & Kalos
Adoptive immunotherapy for cancer

Table 1. General properties of among tumor-infiltrating lymphocytes (TIL), TCR engineered T cells (TCRT), high affinity TCR engineered
T cells (haTCRT), and chimeric antigen receptor engineered T cells (CART)
Parameter TIL TCRT haTCRT CART
Tissue of origin Tumor Peripheral blood Peripheral blood Peripheral blood
Ease of generation Low Moderate* Moderate* Moderate
Efficiency of generation Moderate High High High
Ex vivo manipulation time 17–30 days 8–12 days 8–12 days 8–12 days
Genetic modification Not necessary Required Required Required
Antigens targeted All All All Surface only
Antigenic specificity Undefined Defined Defined Defined
Need for antigen processing and HLA presentation Yes Yes Yes No
Effector functionality Undefined Low High Very high
Proven clinical efficacy Melanoma Not clear Solid tumors B-cell neoplasms
Potential for Off tissue toxicity Low Low High High
Potential for Off target toxicity Low Low High Low
Feasibility for commercialization Low Low Moderate High
*
Once original TCR has been identified.

against cancer and stimulate effector cell functionality
(44), and they have additionally been reported to be capa-
ble of overcoming peripheral tolerance (45). A potentially
important advantage of iNKT cells is the fact that they rec-
ognize a unique glycol-molecule (agal-Cer) in the context
of the CD1d molecule, which can be used to both specifi-
cally expand these cells ex vivo and also to stimulate their
expansion in patients (46). Additional subsets of NKT cells
have been defined, but to date have not been evaluated in
ACT (47).
cd T cells (CD3+ TCR a b , TCR c+d+), are a popula-
tion of T cells very rare in peripheral blood (4–10%) but
substantially enriched in areas of mucosal immunity such
as gut (48). cd T cells recognize a wide variety of targets
including stress-induced antigens in a classical MHC-unre-
stricted manner and manifest lytic activity and pro-inflam-
matory cytokine secretion. A major population of cd T
cells expresses an invariant Vc9Vd2 T-cell receptor, and has
been shown respond to isopentenyl pyrophosphate (IPP), a
product of the mevalonate pathway which is dysregulated
in tumor cells. These lymphocyte subsets are currently
tested for ACT, both unmodified following bulk expansion
ex vivo (49) and also following gene engineering using
CARs (50).
abT cells (CD3+TCR a+b+) are the most abundant T-cell
population in the blood, the most extensively studied in
general, and the most robustly evaluated for ACT. Both
effector (CD8+) and helper (CD4+) T lymphocytes have
been evaluated in ACT in every conceivable combination,
obtained from the peripheral blood or from tumor sites,
either as bulk populations or antigen-specific clones, or fol-
lowing genetic modification to express tumor re-targeting
receptors.
18
Genetically unmodified T cells
One of the first clinical applications of ACT was the infu-
sion of cells termed lymphokine-induced killer cells (LIK),
which were generated from patient peripheral blood sam-
ples by exposure to high doses of interleukin-2 (IL-2).
Although positive clinical responses were reported in these
studies, these could not be dissociated from the effects of
the co-infusion of high doses of IL-2 (51, 52). Neverthe-
less, the data generated from LIK-based strategies facilitated
the development of next generation approaches, specifically
the evaluation of lymphocytes isolated from tumor biopsies
(TILs) and bulk T lymphocytes activated ex vivo prior to
infusion.
The premise behind TIL-based approaches is that T lym-
phocytes in biopsy specimens are enriched for anti-tumor
reactivity but have become functionally anergized, and that
ex vivo culture of these cells results in their re-activation. TIL-
based strategies were developed principally in the context of
melanoma, thanks to the pioneering efforts of the NCI
group. TILs cultured are selected for anti-tumor activity
following ex vivo culture and recognize tumors in an MHC-
restricted manner via their TCR. Initial studies showed the
feasibility of this approach with expansion of unselected
TILs from subcutaneous and lymph node melanomas with
subsequent re-infusion together with IL-2; although the
response rate for these initial studies was about 30%, TIL
persistence was poor (53, 54). To improve long-term per-
sistence of infused cells, follow-up clinical studies evaluated
the use of non-myeloablative lymphodepleting chemother-
apy (cyclophosphamide and fludarabine) prior to TIL infu-
sion followed by high doses of IL-2, under the premise that
competition for niches and homeostatic cytokines were
impediments to long-term persistence of infused cells.
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Immunological Reviews 257/2014

Incorporation of the lymphodepleting regimen improved
clinical responses to 50%, with enhanced TIL in vivo expan-
sion and persistence (55, 56). In a recent update, 13% of
the patients treated with this regimen experienced complete
regressions ongoing beyond 5 years (57). To evaluate the
effect of enhanced lymphodepletion, two pilot trials were
conducted with total body irradiation (TBI). These treat-
ments resulted in clinical response rate of 49–72% (58).
A major impediment with early TIL trials was that the ex
vivo generation of TIL cultures was extremely labor intensive,
with the generation from each patient of multiple TIL sub-
lines, screening for tumor specific activity against autolo-
gous or HLA-matched tumor cell lines, and long expansion
cycles. As a result, more than a half of patients were
excluded from this treatment. The development of shorter
expansion protocols and more streamlined screening
approaches has considerably improved the broader imple-
mentation of this approach (59). An ongoing phase 2 study
is comparing CD8+ enriched short-term cultured TILs plus
high dose bolus IL-2 after non-myeloablative chemotherapy
against high dose IL-2 alone in metastatic melanoma.
The use of TIL therapy in a broader sense is currently
limited by a number of factors: (i) tumor specimens are not
readily available in many cancers, especially in the metastatic
setting where biopsies are not often obtainable; (ii) TILs are
reproducibly detectable only in a minority of cancers
(mostly melanoma and renal cancer); (iii) expansion proto-
cols remain relatively labor-intensive, expensive, time
consuming, and difficult to standardize; (iv) current condi-
tioning regimens are not feasible in all patients; and (v) TIL
functionality may revert to the immune-suppressed state
when infused TIL re-enter TMA.
Parallel effort to TIL-based approaches involved the ex vivo
expansion of bulk T lymphocytes from the peripheral blood
of cancer patients followed by subsequent re-infusion with
the support of IL-2. This approach was aimed to expand a
population of activated cells with a lowered triggering
threshold and thus with enhanced potential to be triggered
by tumor cells, as well as potentially trigger and expand
tumor-reactive lymphocytes rendered anergic as a result of
peripheral tolerance. Clinical activity was observed in this
approach, with rapid functional immune reconstitution
observed in patients, and the establishment of optimized
timelines for T-cell infusion post lymphodepletion (23). In
a follow-up study, transfer of bulk activated T cells was
associated to the development of autologous GVHD with
rash and colitis in about 25% of treated patients, demon-
strating the potency of this approach (24).
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014
Ruella & Kalos
Adoptive immunotherapy for cancer
Genetically retargeted T cells
The fundamental premise behind genetic retargeting of T
cells is the fact that the endogenous potent tumor-specific
T-cell repertoire has been compromised as a consequence of
central tolerance. Indeed, comparative analyses have demon-
strated that TCRs against tumor have substantially lower
antigen affinity (approximately 0.5 logs) compared with
TCRs directed against virus-derived antigens (60, 61),
providing at least partial explanation for the lack of clinical
efficacy of approaches directed to triggering the self-anti-
gen-reactive T-cell repertoire. Two related approaches have
been developed to genetically redirect T cells: (i) the trans-
fer of affinity-enhanced tumor-specific TCRs and (ii) the
transfer of synthetic CARs. Recent clinical experience has
suggested that both of these approaches can mediate potent
anti-tumor activity.
The ‘canonical’ TCR is composed of a complex of at least
six polypeptide chains (a, b, c, d, e, and f) which assemble
on the surface of T cells. The a and b chains form the pri-
mary binding domain of the TCR, which recognizes intra-
cellularly processed peptides presented on the surface of
target cells by proteins of the MHC. Accessory costimulatory
and adhesion molecules augment the strength and quality of
the TCR-MHC peptide interaction leading to productive
T-cell engagement. Initial efforts to evaluate TCR transfer
involved transfer of self-antigen specific non-enhanced TCR
the genetic transfer of TCR ab chains specific for self-
antigens (62, 63). These pioneering efforts demonstrated
the proof of principle that TCR-based T-cell engineering was
feasible, but also highlighted the need to generate more
potent TCR to overcome the consequences of central toler-
ance on TCR affinity. Approaches to generate more potent
TCRs have included the vaccination of human HLA trans-
genic mice with target tumor antigens to generate high
affinity human antigen-specific TCRs, and this approach has
been used to isolate high affinity TCRs against MDM-2 (64),
p53 (65), CEA (66), gp100 (67), and MAGE-A3 (68). More
recently animals transgenic for human HLA and TCR have
been used to generate high-affinity human ab TCRs (69).
Other approaches to generate high affinity TCRs have
involved taking advantage of gender-restricted expression of
target antigens (such as prostate, ovary) and the lack of tol-
erance to these antigens in the opposite gender (70),
selection of specific T-cell clones from a polyclonal pool of
graft-versus-tumor reactive T cells (71). Early clinical results
using affinity enhanced TCRs have highlighted the promise
of this approach with dramatic anti-tumor activity observed
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Ruella & Kalos
Adoptive immunotherapy for cancer
in a number of cases (72–74). However, as discussed fur-
ther below, the introduction of TCRs with non-physiologi-
cally enhanced affinity to self-antigens perhaps predictably
has led to increased issues related to off target toxicity in
normal tissues (66, 67, 75), an issue of considerable con-
cern that has to be addressed for full implementation of this
approach.
Beyond TCR affinity enhancement approaches, other
efforts to generate higher affinity tumor-specific TCRs have
involved efforts to increase the number of TCRs on the sur-
face of T cells (72, 73, 76), increasing introduced TCR
expression levels (77–80), and a number of strategies to
minimize a and b chains mispairing with endogenous
chains (81–89). Although TCR-based ACT approaches hold
considerable promise as exemplified by early clinical trials,
they do possess certain inherent limitations. More specifi-
cally, the HLA-restricted specificity of engineered TCR limits
the fraction of potential patients to those expressing the rel-
evant HLA allele recognized by the T cells, and the fact that
tumors often downmodulate HLA alleles and/or alter prote-
asomal processing pathways provides for the real possibility
of antigen escape variants and lack of complete responses.
CARs are synthetic receptors that combine the extracellu-
lar single-chain variable fragment (scFv) of an antibody with
a transmembrane (TM) domain and intracellular signaling
domains derived from molecules involved in T-cell signal-
ing. Essentially all current CARs utilize the signaling domain
from the CD3f chain and additionally can contain signaling
domains from molecules involved in T-cell activation or
costimulation. Since the scFv domain binds directly to target
cell surface epitopes CAR-based strategies bypass the need
for MHC-restricted antigen presentation and are thus insen-
sitive to tumor escape mechanism related to HLA down-
modulation and altered processing escape-mechanisms (90).
CARs bypass most of the T-cell triggering limitations related
to epitope density, since the scFv of the monoclonal anti-
body is characterized by high-affinity for the target and
additionally each surface target molecule presents a trigger-
ing epitope. CAR-based redirection represents a near univer-
sal ‘off-the-shelf method to generate large numbers of
antigen-specific helper and cytotoxic T cells. As described in
more detail below, a key to this successful implementation
of CAR-based approaches is the identification of antigens
with surface expression on tumor cells and absent expres-
sion on normal healthy tissues.
The concept of CARs was introduced by Eshhar and col-
leagues in 1989 (91). Since then, many groups have evalu-
ated the possibility to redirect T cells using antibody-based
20
scFv of an antibody fused to the CD3f or less commonly, Fc
receptor c (FcRc) signaling domains. The scFv are usually
derived from murine antibodies and thus have the potential
to trigger immune rejection responses by the host; current
strategies involved the use of humanized or fully human
scFv to mitigate this possibility (92).
Engineering the optimal signaling domain combination
for maximal biological activity has been the focus of exten-
sive preclinical and more recently clinical activity. The most
intensively studied signaling domains have been those asso-
ciated with robust effector or costimulatory activity. Initial
(i.e. first generation) CARs contained only the CD3f chain
signaling domain. Although such CARs were capable of
re-directing T cells to target antigens in vitro demonstrating
the proof of concept that T cells could be engineered using
CAR-based approaches, clinical trials based on such CARs
demonstrated limited efficacy in a variety of cancers, such as
neuroblastoma (93), non-Hodgkin’s lymphoma (94), renal
cancer (95), and ovarian cancer (96). A recurring theme in
these studies was limited in vivo expansion, modest anti-
tumor activity, and lack of in vivo persistence.
To overcome the limitations with first generation CARs,
second generation CARs were developed and evaluated. Sec-
ond generation CARs incorporate additional signaling
domains from costimulatory and accessory functional T-cell
molecules, in attempts to enhance in vivo functionality,
potency, expansion capacity, and persistence. The first
costimulatory molecule to be included in the CAR construct
was CD28, and this led to a dramatic increase of the IL-2
production and killing capacity of the T cells (97). Position-
ing the CD28 domain 5′ to the CD3f domain was shown to
me most effective (97, 98). Moreover, inclusion of the
CD28 domain was shown to enhance resistance to suppres-
sion by regulatory T cells (99, 100). Many other signaling
domains have subsequently been evaluated preclinically,
including 4-1BB (CD137) (101, 102), OX-40 (CD134)
(103, 104), CD244 (105), CD27 (106), and inducible
costimulator (ICOS) (98, 107). In preclinical models, sec-
ond generation CAR T cells have been shown to mediate
enhanced functionalities including expansion and anti-tumor
activity (102). CARs containing the 4-1BB domain have
been shown to result in improved in vivo persistence, anti-
tumor activity and tumor localization compared with first
generation CAR or CAR containing the CD28 domain (100,
101). Inclusion of a CD27 signaling domain to CAR may
also enhance in vivo persistence and improve cytotoxic effect
against xenogeneic tumor (106), while inclusion of the
ICOS signaling domain has been shown to drive human
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T cells to a Th17 phenotype (108). Second generation CARs,
where costimulatory molecules are included in trans, have
also been developed, enabling the ability to more precisely
potentially modulate CAR functionality (109). Most current
clinical trials evaluate second generation CAR designs (110).
Third generation CARs are characterized by the addition
of a second costimulatory domain to further improve T-cell
expansion, cytotoxicity, and in vivo persistence. Third gener-
ation CARs combining CD28 with 4-1BB, and CD28 with
OX-40 have been evaluated (111–113). Conceptually, with
this approach the threshold of T-cell activation is further
reduced in 3rd generation CARs leading to further enhanced
potency but also increased potential for low avidity off target
activation.
In terms of CAR design, both the spacer/hinge and the
TM domains have the potential to influence CAR function.
In fact the hinge region mediates flexibility and it is impor-
tant for the positioning of the binding scFv domain (114,
115). A number of studies have shown that the TM domain
is critical for effective CAR expression on the surface of the
cell, and accordingly many different TM domains have been
evaluated including CD28 (116), OX40 (116), CD3f (117),
FceRIc (118), CD7 (119), CD4 (102, 103), H2-Kb (120),
and CD8 (101, 121). Inclusion of the CD28 TM domain
was found to result in a higher expression of CAR in com-
parison with OX40 and CD3z TM domains (116). Other
reports suggested that intracellular rather than TM domains
modulate CAR surface expression (122). With regard to the
hinge region, IgG1, IgG4, IgD, and CD8 domains have been
mostly evaluated (123, 124). An additional important con-
sideration for CAR design is the length of the hinge region,
which has been shown to influence the quality of the inter-
action between the T cell and target, depending on the loca-
tion of the target epitope on the target antigen (125, 126).
Until more specific rules to guide this process are defined,
this important issue will likely require empiric testing of
multiple CARs against each epitope being targeted. The rela-
tive position of the epitope in the target protein also appears
to be relevant in terms of CAR engineered T cells (CART)
potency. For example, CD22 target epitopes that are proxi-
mal to the cell membrane trigger more potent lytic activity
compared with distal epitopes (126, 127), while other stud-
ies showed longer spacer domains increased the potency of
5T4- and NCAM-specific CAR that recognized membrane
proximal epitopes, potentially by providing increased flexi-
bility to the CAR (125). Indeed, the distance between the T
cell and the tumor cell is influenced by the combination
between the position of the epitope and the length of the
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Ruella & Kalos
Adoptive immunotherapy for cancer
CAR molecule that is mainly related to spacer regions, and
this is able to influence the entity of the activation and kill-
ing activity.
CAR-engineered T cells have been generated against many
different TAA for both solid and hematologic malignancies:
CD19 (128–131), CD20 (94, 132), CD33 (133, 134),
B-cell maturation antigen (135), CD22 (127), CD23 (136),
CD30 (137, 138), CD38 (139), CD44v6 (140), ROR-1
(141), j-light chain (142), Lewis Y antigen (143, 144),
NKp30 (145), TAG-72 (146), CD70 (147), Carboxy-anhy-
drase-IX (CA-IX) (95, 148), human epidermal growth factor
receptor 2 (ErbB-2/Her-2/Neu) (149–153), disialoganglio-
side 2 (GD2) (154–157), GD3 (158), L1CAM (CD171)
(93), VEGF-R2 (159), EGFR (107, 160, 161), MUC-1
(124), MUC-16 (162), prostate-specific membrane antigen
(PSMA) (97, 163, 164), prostate stem cell antigen (PSCA)
(165, 166), 5T4 oncofetal antigen (h5T4) (167), NCAM
(125), mesothelin (111, 168), fibroblast activating protein
(FAP), folate receptor-a (96, 169), NKG2D (170–172), IL-
11 receptor a-chain (173), CEA (115, 174, 175), IL-13Ra2
(117, 176), and erythropoietin-producing hepatocellular
carcinoma A2 (EphA2) (177). As described in more detail
below, a small number of CARs are currently being tested
in early phase clinical trials, with in some cases dramatic
and exciting results that highlight the promise of this
approach against cancer.
A number of groups have initiated efforts to overcome
the need for patient-specific products for ACT. Attempts to
generate ‘universal donor T cells’ have involved the use of
zinc-fingers nucleases to eliminate the expression of the
endogenous TCRs (178). Although this area of research is
still nascent and many issues will need to be identified and
resolved, it has the potential, combined with efforts to
reduce the allo-recognition of engineered T cells for exam-
ple by targeted knockdown of class I and class II loci to
bypass the need to generate patient-specific T cells by gener-
ating universal allogeneic TAA-specific T cells from one
donor that might be administered to multiple recipients.
A novel approach to simultaneously target multiple tar-
gets on tumors is the development of bi-specific CAR. Such
moieties, coined as ‘TanCAR’ simultaneously recognize two
tumor restricted antigens with synergistic enhancement of
effector functionality when both antigens are present on
target cells; T cells engineered with TanCAR retained cyto-
lytic ability with loss of one of the target molecules and
were superior to single CAR engineered T cells in the con-
trol of established experimental tumors in animal models
(179). TanCAR or related approaches have the potential to
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Adoptive immunotherapy for cancer
counteract the existence of antigen-loss variants that often
exist in tumors; however, such approaches may have
enhanced potential for off-tissue toxicity, discussed more
fully below.
An interesting strategy to mitigate off-tissue toxicity is to
develop combinatorial CAR constructs that transduce an
activating signal only when encountering a specific combi-
nation of antigens. Kloss et al. (180) recently described a
novel approach by which T cells are co-transduced with a
CAR that provides suboptimal activation upon binding of
one antigen and a chimeric co-stimulatory receptor (CCR)
that recognizes a second antigen. Using the prostate tumor
antigens PSMA and PSCA as model antigens, this group
demonstrated that combinatorial CAR-engineered T cells
eradicated tumor cells that expressed both but not individ-
ual antigens (180).
A novel strategy to expand the recognition specificity
potential of CAR T cells utilizes a biotin-binding immune
receptor (BBIR), which consists of an extracellular modified
avidin domain linked to an intracellular T-cell signaling
domain; target-specific antibodies can then be loaded onto
BBIR T cells through streptavidin cross-linking (181). Devel-
opment of BBIR T cells may allow the sequential or simulta-
neous targeting of a broad combination of distinct antigens.
A recent report has described the development of CARs
that target the intracellular antigen WT-1 using a scFv that
is able to recognize MHC class I + peptide complexes
(182). Although still extremely preliminary, this approach
has the potential to overcome the limitation of CAR-based
strategies to surface antigens and greatly extend the universe
of target antigens available for CAR-based approaches. On
the other hand, advantages of CAR-based strategies described
above and related to target epitope density and indepen-
dence from HLA expression and processing defects will
likely prove to be impediments for the success of this
approach.
Beyond the introduction of antigen-re-targeting receptors,
gene-engineering efforts have also involved attempts to
selectively target T cells to sites of tumor using chemokine
and homing receptors such as CXCR2 or CCR2B, which
when expressed on engineered T cells have been shown
facilitate the homing of T cells to tumor (137, 183–185).
Engineering of T cells to express immune modulatory cyto-
kines, such as for example IL-12, has been shown to
improve the efficacy of ACT in animal models, at least in
part by increasing the expression of Fas within tumor-infil-
trating macrophages, DCs, and myeloid-derived suppressor
cells (MDSCs) (186). Expression of single chain IL-12 by
22
engineered T cells has been shown to enhance the efficacy
of low doses of MHC-restricted TILs, at least in part by
re-programming tumor associated myeloid cells to cross-
present tumor-associated antigens (187); interestingly, this
approach eliminated the need for lymphodepletion before
CAR therapy (188). Other approaches that involve efforts to
modulation expression of cytokines, such as IL-12 by T
cells, are also currently being evaluated (189). Clearly, the
ability to at-will engineer T cells to express desired
immune-modulating soluble factors and receptors provides
an incredibly exciting and diverse menu of opportunities to
develop more potent, and disease targeted options for ACT,
while also enabling the modulation of SAE associated with
ACT-collateral immune activation.
T-cell subsets for ACT
Over the past few years it has become evident that T-cell
function may be influenced by many other parameters
beyond antigen specificity. The nature of the T cell that is
used for adoptive immunotherapy is highly correlated with
clinical results (190). Initially, it was assumed that the ideal
T cells for adoptive immunotherapy would be effector T
cells (Teff), due to their ability to respond robustly to anti-
gen stimulation as measured by cytotoxicity. However, a
number of early clinical studies showed that highly differ-
entiated tumor specific Teff CD8+ T cells engrafted poorly
and are less capable of tumor killing compared with heter-
ogeneous population of TILs (191, 192). Retrospective
analyses from clinical trials revealed that patients infused
with less differentiated cell products had better clinical
outcome (193), and follow-up studies in animal models
demonstrated that the use of a less differentiated T-cell
population for ACT led to better anti-tumor activity, in vivo
expansion and persistence (194). These results led to
efforts to identify sub-populations of T cells with enhanced
in vivo functionality as well as develop in vitro culture
approaches that generated less differentiated T-cell products
(see below). More recent studies have shown that the fre-
quency of central memory T cells (Tcm) in infusion prod-
ucts correlates with positive clinical response (156).
Furthermore, naive T cells (Tn) have shown higher anti-
tumor effect and longer in vivo persistence than Tcm (195)
and antigen experienced CD8+ Tcm cells persist longer than
effector memory T (Tem) cells (196). More recently a spe-
cific T-cell subset, the stem cell memory T cells (Tscm),
has been described; Tscm have been described to be capa-
ble of self-renewal and to generate potent effectors, this
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Immunological Reviews 257/2014

population may be important for adoptive immunotherapy
(197). Recent exciting work has also revealed the potential
for plasticity in the Tscm repertoire, with data from animal
models suggesting that a very small number of the ‘right’
population of engineered T cells may be sufficient to medi-
ate potent anti-tumor activity accompanied by in vivo expan-
sion and differentiation into T-cell subsets (198).
Tn, Tcm, and Tscm cells express CD62L and CCR7 and
therefore have the potential to home to secondary lymphoid
tissues where they can interact with APCs explaining at least
in part their better clinical activity. On the other hand,
CCR7 cells engineered to express CARs with CD28, OX40,
and TCRf costimulatory domains were recently shown to
be rescued from apoptosis and result in more efficient anti-
tumor efficacy compared with CCR7+ CAR-engineered T
cells (199).
A fundamental property of T cells, which mediate potent
anti-tumor activity appears to be the ability for in vivo expan-
sion, differentiation, and persistence (28). A number of
studies have shown that Teff cells possess a relatively limited
proliferative and engraftment potential compared to Tn,
Tcm, and Tscm, in part due to limited lifespan associated
with shorter telomere length (200–203). Studies using TILs
have demonstrated that a short duration in culture, a rapid
doubling time and longer T-cell telomere length were corre-
lated with better clinical outcome (53, 57, 204–206).
Recent studies indicated that the effector-like CD27 CD8+
cells mediate potent protective immunity and lead to long-
lived memory cells (207). Finally, recent reports suggest
that adoptively transferred Th17 cells demonstrated superior
proliferation, persistence, and anti-tumor activity compared
with Th1 cells (208, 209).
Although data obtained in a number of studies suggest
that less differentiated T cells with longer telomeres, such as
Tn, Tcm, and Tscm, may be the preferred cells for ACT,
there is potential danger of applying reductionist reasoning
to an inherently complex biological by selecting a particular
T-cell subtype for ACT. There is evidence that CD4+ cells
are required for CD8+ memory cell formation that is import
for long-term control of the tumor (210, 211). Indeed, the
most potent and durable clinical responses to date have been
obtained using bulk populations of CD8+ and CD4+ T cells
(27–29).
Considerable effort has been spent principally by the Bay-
lor group to evaluate the ability to isolate and engineer virus
(EBV, CMV)-specific T cells to express CARs, under the pre-
mise that these cells are inherently enriched for memory
cells and additionally can be maintained in vivo by stimulation
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Adoptive immunotherapy for cancer
through their endogenous TCR (212). Clearly this approach
is restricted to patients that have been exposed to the
viruses. EBV-specific T cells had been transduced with anti-
CD30 CARs or anti-GD2 CARs and had been preclinically
tested (138, 155). EBV-specific T cells engineered to express
a chimeric receptor had also been used to treat nasopharyn-
geal carcinoma or AML (133, 213).
T-cell engineering strategies
The development of approaches to at-will engineer T cells
prior to ACT has been a fundamental building block for the
recent success in the field. Although most preclinical and
clinical studies have focused on introducing TCR or CAR
re-targeting receptors into T cells (110), more recent stud-
ies have begun to evaluate the possibility to introduce T
and other immune cell function-modulating molecules in T
cells, such as IL-12 (188), IL-15 (214, 215), CCR2 (216),
IL-2 (217). Initial successful efforts for engineering T cells
utilized pseudotyped c-retroviruses that were capable of
infecting primary human T cells and integrating their DNA
in the host genome; this approach lead to high efficiency
stable transduction of primary T cells that were employed
in multiple clinical trials (218, 219). Concerns about retro-
virus integration site bias coupled with concerns about
long-term retroviral promoter silencing led to the more
recent development of lentivirus-based vectors for gene
transfer. In addition to the fact that lentiviruses vectors
appear to be less susceptible to silencing by host restriction
factors and can deliver larger DNA sequences than retrovi-
ruses (220), lentivirus vectors have been reported to be
able to infect non-cycling cells, particularly when coupled
with cytokine ‘pre-activation’ (221, 222). From an onco-
genic perspective, significant early and justified concern
about the oncogenic potential of retrovirus-based gene ther-
apy as a result of the unfortunate report showing oncogenic
transformation of stem cells due to retrovirus integration
(223, 224) was mitigated initial in animal models (223,
225) and subsequently in long-term longitudinal retrovirus-
based gene-therapy studies now extending beyond 10 years,
which confirmed that retrovirus-based gene transfer into
human T cells appears to be safe (226, 227). Although len-
tivirus-based gene transfer has not been implemented as
long, the fact that in the natural history of HIV no T-cell
oncogenic events have been reported, coupled with the
absence of any reports from to-date lentivirus-based clinical
trials provide reasonable assurances that this approach will
be safe from an oncogenic perspective. Novel integrating
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Adoptive immunotherapy for cancer
virus-based delivery systems such as foamy viruses are
under development (228) but beyond the scope of this
review. Adenovirus-based vectors such as Ad-35 vectors,
which result in long-term episomal transgene expression,
have also been evaluated in human T cells with high
expression efficiencies (229).
The relatively high cost for manufacture and release of
virus-based vectors has precipitated the development of a
variety of non-virus based gene transfer systems. Retro-
transposon systems, such as PiggyBac or Sleeping Beauty
have shown preclinical promise (230–232). Novel and
exciting approaches based on Zinc-finger nuclease, TALEN,
and CRISP/Cas9 based-technologies permit to targeted inser-
tion of transgenes or the targeted editing of the T-cell tran-
scriptome and open up the potential to be able to modulate
transgene expression and T-cell function at will (229, 231,
233, 234).
For some applications, permanent genomic integration of
new constructs may not be necessary for therapeutic efficacy
or may be useful to mitigate potential toxicity. A novel
approach that is currently being investigated to this end is
RNA electroporation; this approach allows the rapid transfer
of the specific mRNA in T cells allowing a high expression
of the specific receptor (122, 235). Since the introduced
mRNA is relatively rapidly degraded, this approach results in
‘biodegradable’ engineered T cells. Although RNA-
engineered T cells have been shown to mediate potent and
antigen specific effector functions, multiple infusions are
required to compensate for the short in vivo half-life of these
cells (236). RNA engineering has been used to deliver tran-
scripts for TCRs, CAR chemokine receptors, or cytokines
(215, 237, 238) and is currently being evaluated in clinical
trials. The biodegradable nature of this approach provides a
considerable safety advantage, in particular when evaluating
T cells engineered to express constructs with potential for
toxicity. RNA engineering also allows the efficient transfer
of multiple transcripts allowing for the ability to engineer T
cells to co-express targeting, homing, and immune modulat-
ing functionalities. An efficient low cost method for gene
transfer to T lymphocytes that includes the use of electropo-
ration and a transposon-based system has recently been
described (239).
T-cell expansion strategies
A fundamental requisite for current ACT strategies is the
ability to engineer and expand T cells prior to infusion into
patients. As described above, the ‘quality’ of T cells
24
employed for ACT appears to be critical for in vivo efficacy.
An early approach, based on the pioneering work of Riddell
and Greenberg (240), employed antibodies specific to CD3e
and cross-linked using allogeneic peripheral blood mononu-
clear cells, with immortalized B-LCL cells added as feeder
cells, and exogenous IL-2. Repetitive stimulation using this
approach generated large numbers of antigen-specific T cells
that were employed in early trials (192, 241–243), unfor-
tunately without significant success, in retrospect due to the
fact that this approach strongly skewed products toward
Teff cells (192, 244). A parallel approach, developed by
Levine, June and colleagues, employed the use of magnetic
beads coated with anti-CD3e and anti-CD28 antibodies to
provide costimulation. This approach generates large num-
bers of T cells with a less differentiated phenotype (245,
246), has been robustly developed (247), and is the
approach that has been recently employed to manufacture
products with extensive in vivo proliferative potential, potent
anti-tumor activity, and long-term functional persistence
currently exceeding 3 years in the initial treated patients
(27–29, see below). Although early studies suggested that
the absolute number of the transferred T cells is almost
always correlated with better clinical responses (205), this
paradigm is now in question given the dramatic potency
achieved with small numbers of cells in a recent trial (27),
and the discovery of T cells with stem cell properties (197,
248, 249). Magnetic bead-based approaches have been used
for preselection of specific subsets of T cells, such as CD8+
cells for TIL immunotherapy (59), CD4+ T cells for transfer
HIV patients (250), CD25+ cells to prevent GVHD after
allogeneic HSCT (251), or to select against particular sub-
sets of cells that may be deleterious for in vivo efficacy, such
as regulatory T cells (Tregs) (252). Based on preclinical
data showing the enhanced persistence and anti-tumor
functionality of Tcm cells (196), strategies to isolate CD8+
Tcm cells under GMP conditions have been developed
(253). T cells may be also isolated based on for antigen
specificity, as discussed above in the context of redirecting
virus specific T cells to target tumors (212), and also in the
context of utilizing virus-specific T cells to treat viral infec-
tions such as CMV (254, 255).
The use of artificial antigen-presenting cells (AAPCs), also
pioneered by the June group (256), to stimulate T cells
under more physiologic conditions provides an interesting
alternative approach to expand T cells for ACT. This
approach, which has not yet been evaluated clinically, has
the potential to provide more physiologic stimulation of
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Immunological Reviews 257/2014

T cells preserving desired differentiation states, and has been
adopted by other groups (257–260). Novel strategies
involving ex vivo expansion of T cells with cytokines are also
currently being investigated. Expansion of CD8+ naive T
cells with IL-15 provides the expansion of T cells with the
characteristics of Tcm cells, with preserved telomere length
(261); compared with T cells expanded with IL-2, the use
of IL-15 has been shown to support superior proliferative
and anti-tumor activity (262). Expression of IL-15RA on
CD8+ T cells has been shown to autonomously enhance the
viability and proliferation of primary CD8+ T cells and cyto-
toxic potential of antigen-specific CD8+ T cells (215). The
combined use of IL-15 and IL-7 in the context of anti-CD3/
anti-CD28 bead-based stimulation has been shown to
facilitate the ex vivo differentiation and expansion of gene
modified CD8+ Tscm cells under GMP compliant conditions
(263). IL-21, when used for T cells expansion, leads to the
production of minimally differentiated T cells with high
proliferative and anti-tumor capacity (264, 265), while
reprogramming of CD19-specific T cells with IL-21 signal-
ing has been shown to improve adoptive immunotherapy of
B-lineage malignancies (266). IL-4 may also have a role in
T-cell expansion, as human T-cells engineered to co-express
IL-4 receptor together with a CAR specific for MUC1 exhib-
ited robust capacity to expand in vitro and effect destruction
of MUC1-expressing tumors (267). Modulation of the
metabolic state of T cells has been shown to influence their
phenotype and function; for example, modulation of PI3K-
AKT-mTOR and Wnt-b-catenin pathways had been shown
to control the differentiation status of T cells; co-culture of
T cells with mTor inhibitors promotes the formation of
memory subsets, while the promotion of the Wnt pathway
leads to the formation of Tscm cells with potent anti-tumor
activity (268).
Modulation of substrate rigidity is another potentially
promising approach to control the quality of ex vivo
expanded T cells. Early in vitro studies in this area have
revealed that modulating substrate rigidity has the potential
to impact T-cell activation and proliferation (269).
Finally, strategies that involve overexpression of genes
involved in survival such as telomerase (270), anti-apoptotic
genes (271), as well as the downregulation of pro apoptotic
molecules such as Fas (272), or dominant-negative receptors
for inhibitory molecules such as dominant negative recep-
tors for TGFb (273) have been evaluated preclinically.
Despite the interesting scientific approach of these strategies,
concerns regarding safety have been raised and should be
excluded before moving to the clinic.
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Adoptive immunotherapy for cancer
T-cell ablation strategies
A concern with ACT has long been the need to ablate trans-
ferred cells, either to counter off target toxicity. To this
end, ‘suicide gene strategies’ have been developed. Initial
suicide system involved use of the herpes simplex virus
(HSV) thymidine kinase (TK), which phosphorylates ganci-
clovir and acyclovir leading to the generation of toxic tri-
phosphate products and cell death (274). Perhaps
predictably, HSV-TK transduction in T cells leads to immu-
nogenic reaction with clearance of TK positive T cells (243,
275). More recently, an elegant system based on the design
and use of a caspase-9-driven apoptosis activity was devel-
oped; in this approach caspase-9 intracellular signaling
domains were fused with a human FK506-binding protein
domains; administration of FK506 (tacrolimus) leads to
dimerization of caspase-9, activation of apoptotic pathways
and potent cell ablation (276, 277). Even more recently an
equally elegant system was developed that involved engi-
neering of cells to express a truncated EGFR polypeptide
devoid of extracellular ligand-binding and intracellular sig-
naling domains but that retained binding to the anti-EGFR
monoclonal antibody Erbitux, enabling both in vitro selection
and in vivo ablation of engineered cells (278). Other
approaches to specifically eliminate engineered T cells
include the co-transduction of CD20 and the use of ritux-
imab to ablate engineered cells by ADC (279).
Target antigens for ACT
The modern age of cancer immunotherapy arguably began
in 1991 with the discovery by Thierry Boon and colleagues
of MART-1, the first human cancer antigen (280). Since
then, a major area of focus in immunotherapy has been the
identification and evaluation, both preclinically and clini-
cally, of candidate target antigens overexpressed or aber-
rantly expressed by tumors (281). Perhaps predictably,
initial efforts in ACT also focused on generating and utiliz-
ing T cells directed to such antigens. A plethora of clinical
data have now led to the conclusion that targeting self-anti-
gens using ACT likely requires the engineering of enhanced
targeting specificity in T cells to overcome the fundamental
impact of central tolerance on the endogenous T-cell reper-
toire (110). Indeed, an unbiased comparison of T cells spe-
cific for self-antigens, including cancer testis (CT) antigens,
overexpressed by tumors and T cells specific for viral anti-
gens has revealed that the latter have affinities for target
antigens approximately one log higher compared with the
former (61). As discussed above, engineering efforts have
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Adoptive immunotherapy for cancer
focused on affinity enhancement of TCR (282) or the use of
high affinity CAR-based strategies.
The definition of ideal target antigens is fundamentally
different for TCR- and CAR- based strategies. For TCR-based
strategies, ideal targets will likely be tumor-specific neo-
antigens, preferably shared across a significant fraction of
tumors to enable systematic tumor targeting, or gender-
restricted antigens that can be used to generate high affinity
TCR in the non-expressing gender (70). Both of these target
classes result in T cells with TCRs that have been shaped by
negative selection in the thymus and thus have minimal
potential for off target recognition of other tissues. In the
case of CARs, because of the potency of this approach, ideal
antigens will either have a tumor-specific expression profile,
or will be uniquely expressed in non-essential tissues such
as prostate, breast, and ovary, which can be removed prior
to ACT to enable tumor-specific targeting. Indeed the
potency of CAR-based approaches and the need for a rela-
tively high threshold of tumor specificity has been high-
lighted by reported which have demonstrated significant
serious adverse events (SAEs) when targeting antigens with
low-level expression in normal tissues such as CAIX and
ERBB2 (see below). If necessary, improvements can also be
performed on CAR–based designs to enhance affinity and
improve potency (141).
Taking into account these considerations, precious few
antigens have been identified that would qualify as ideal. In
terms of neo-antigen targets for TCR, brute force efforts
have in fact identified T cells which recognize tumor-
specific neo-antigens; however, these neo-antigens appear
to in general result from individual tumor-specific genetic
events which result in non-self and thus non-tolerized
proteins (283–285), with T cells and TCRs which are not
amenable to platform development for ACT. In terms of
CAR targets, the profound potency of CAR-engineered cells,
related at least in part to the fact that each surface molecule
for the target protein is a triggering epitope, requires a
lower threshold of expression that is considerably lower
than that required for therapies; this is best exemplified by
the above mentioned SAE with CARs that utilized ScFv
derived from clinically approved antibodies. Thus, ideal
antigens from a CAR-perspective will have no expression in
any non-targeted tissue. Practically speaking, in the emerg-
ing field of T lymphocyte engineering to manifest potent
and redirected tumor specificity, where the physiologic and
evolutionarily selected balance of recognition of self versus
non-self is manipulated, the potential for off-target toxicity
is a risk which ultimately will need to be addressed using
26
comprehensive and innovative preclinical testing followed by
evaluation in clinical trials. A conceptually attractive approach
involves the development of combinatorial activation strate-
gies, with T-cell activation requiring the simultaneous bind-
ing and triggering of multiple target unique CARs (286) and
the use of CART ‘cocktails’ that also may help address issues
related to the escape of antigen loss variants (287).
Animal models, extensively used to evaluate toxicity for
more traditional therapeutic agents, are considerably less
useful in ACT because of lack of epitope conservation across
species and also because they fail to capture the complexity
of human T cells interacting with the broader immune sys-
tem. The clearest demonstration of this is the recent report
of SAE using high-affinity TCR redirected T cells against
MAGE-3; despite extensive preclinical testing of this affinity
enhanced receptor, ACT using cells engineered to express
this receptor resulted in two deaths due to cardivascular
toxicity as a consequence of off-target recognition of an epi-
tope from titin, a protein expressed in differentiated cardio-
myocytes (75, 288).
Beyond targeting antigens expressed by tumors, ACT has
also been applied to target T cells to molecules expressed
by tissues associated with the biology proliferation and sur-
vival of tumors. This effort has principally been carried out
as part of efforts to target solid tumors, and had brought
into juxtaposition the biology of T cells and tumors. Post
ACT T cells initially transit through the heart following the
natural venous drainage and very quickly are transported to
the lungs (289). Localization to lung is a critical initial site
for T-cell trafficking due to potential low level expression
of target antigens by lung epithelium; indeed, off-target
recognition of lung epithelial tissue has been observed in a
trial with Her2-specific CART, due to low-level expression
of Her2 in lung epithelial cells (153). Rapid liver toxicity
has also been recorded in anti-CA IX CART trial due to the
expression of this marker in epithelium of the biliary tree
(95). Different routes on ACT administration, such as intra-
tumor or intraperitoneum, may reduce the potential toxic-
ity (242, 290), although it apparent that, as expected
biologically, T cells subsequently redistribute to secondary
organs; indeed, analysis of tissues from post SAE biopsy of
the titin-related SAE described above revealed broad low-
level distribution of infused T cells in the majority of
organs evaluated (75). Concern has been raised that T cells
may be unable to actively migrate and extravasate into
tumor parenchyma, limiting their effectiveness in vivo. Tar-
geting the tumor vasculature using engineered T cells is
one possible mechanism to facilitate efflux of T cells into
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014

the tumor. Systemic administration of T cells engineered
with CAR targeting avb3 integrin, expressed in tumor
endothelial cells, led to extensive bleeding in tumor tissues
with no evidence of damage to blood vessels in normal tis-
sues; when this approach was coupled with co-delivery of
nanoparticles increased deposition of nanoparticles was
observed in the tumor (291). Combining ACT with agents
that interfere with tumor vasculature such as avastin holds
promise in this regard.
At the tumor site, T cells face a dynamic and intricate
tumor micro-environment (TME) composed of a complex
mixture of tumor cells, endothelial cells, stromal cells, fibro-
blasts, myeloid cells, NKT cells, T cells, and regulatory T
cells, and shaped by the selective pressure to support the
tumor mass both by providing trophic signals and also by
creating a potently anti-inflammatory and immunosuppres-
sive milieu (292–294); these heterogeneous cell types may
occupy up to 90% of the tumor mass (295), where they
interact via soluble and cell-cell dependent mechanisms with
each other, the tumor as well as the effector immune sys-
tem. Tumor cells can modulate their growth environment
through the activity of cytokines and chemokines leading to
chemotactic effects on leukocytes, including monocytes and
macrophages, suppression of the activity of the immune
system, as well as regulation of neovascularization processes
(296, 297); accordingly, targeting cells in the micro-
environment has the potential to fundamentally disrupt the
homeostatic milieu established at tumors thus increasing the
effectiveness of cancer-targeted therapies (298).
Immune cells, both innate and adaptive, are intimately
involved in carcinogenesis by promoting tumor transforma-
tion as seen in chronic inflammatory diseases or tumor asso-
ciated inflammation (299, 300). Tumor cells modify and
affect the behavior of tumor infiltrating cells to promote the
formation of a pro-neoplastic microenvironment (301).
The relevance of the tumor-associated microenvironment
for the efficacy of immunotherapeutic approaches has been
documented in a number of clinical settings. To enumerate
a few examples, the presence of TILs in tumor specimens is
correlated with better clinical outcome in cancer patients
(302, 303). Patients with cancer have elevated numbers of
Tregs in the peripheral blood and within the tumor micro-
environment (304), and tumor infiltration by Tregs is asso-
ciated with poor outcome in many cancer subtypes (305,
306). In colon cancer a T-helper 1 (Th1) immune signature
is predictive of better clinical outcome compared with a
Th17 signature (305). High frequencies of tumor-associated
macrophages (TAMs) are associated with poor prognosis
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014
Ruella & Kalos
Adoptive immunotherapy for cancer
(307, 308). The length of survival among patients with fol-
licular lymphoma correlates with the molecular features of
non-malignant immune cells present in the tumor at diag-
nosis (309). The interplay of engineered T cells with these
cell subsets and others is likely to play an important role for
the development and establishment of potent and long anti-
tumor immunity, as well as in mediating the immune-based
adverse events associated with strong anti-tumor responses
observed in recent CAR-based trials (27–29).
Immune-suppressive mechanisms at play in the tumor
microenvironment involve the suppressive action of regula-
tory T cells, MDSCs, TAMs and other myeloid-lineage
derived suppressive cells, stromal fibroblasts and almost cer-
tainly other cell types not yet defined. These cells inhibit T-
cell function through the production of immunosuppressive
cytokines and other soluble factors, as well as through the
expression of surface molecules that bind to inhibitory
receptors such as CTLA-4, PD-1, TIM-3, BTLA, and LAG-3,
and no doubt others, which are expressed on T cells often
post activation and serve to modulate the T-cell response
(15, 310). These suppressive cells are conditioned by the
tumor microenvironment to become immunosuppressive
and are also stimulated to migrate in the tumor site and
proliferate (311, 312). MDSCs are directly correlated with
chronic inflammation and with the production of IL-1,IL-6,
reactive oxygen species (ROS), and nitric oxide (NO), while
they also promote hypoxia, lactic acid production, and
adenosine accumulation which inhibit APC maturation
(313, 314). The presence of TAMs is generally associated
with worse prognosis, with type 2 TAMs, which produce
immunosuppressive cytokines for Th2 responses, showing
poor antigen-presenting capacity and fail to activate T-cell-
mediated adaptive immunity (315, 316).
Recent clinical experience using antibodies that block the
function of inhibitory receptors have demonstrated the
potency of this mechanism (22, 317, 318); this exciting
line of research has led to the approval of the first in class
‘checkpoint inhibitor’ agent, YervoyTM, which blocks the
functional activity of CTLA4, and the likely approval in the
near future of agents that block the post-activation inhibi-
tory function of PD1 on T cells and PDL1/PDL2 on tumors.
An emerging body of clinical evidence is accumulating that
correlate the presence of inhibitory receptors such as PD-1
in TILs correlate with distant metastasis and poorer outcome
in renal cancer (319) and breast cancers (320). Tregs within
the tumor microenvironment have been shown to markedly
hinder the anti-tumor efficacy of adoptively transferred
tumor-targeted effector T cells (321), and Treg-depleting
27
Ruella & Kalos
Adoptive immunotherapy for cancer
strategies combined with systemic lymphodepletion reduce
the number of Tregs in the tumor microenvironment and
result in higher anti-tumor activity of T cells (322, 323).
Tregs have been shown to also inhibit CART cells in animal
models, with prior treatment with cyclophosphamide effec-
tively reversing the suppression (324).
Manipulating the microenvironment before T-cell infusion
through non-myeloablative chemotherapy and lymphodeple-
tion has been shown to lead to dramatic increases in the per-
sistence of the transferred cells and robust anti-tumor activity
(55, 56). The rationale of prior lymphodepletion was based
on studies in tumor models in mice that showed improved
persistence and anti-tumor activity of transferred T cells fol-
lowing conditioning chemotherapy (325); in these studies,
improved engraftment and expansion was correlated with the
ablation of resident lymphocytes that presumably competed
for cytokines (IL-7 and IL-15) and T-cell growth factors. In
particular, IL-15, which can provide a homeostatic growth
stimulus for adoptively transferred T cells and can lead to the
induction of a memory phenotype with enhanced effector
functionality (56, 323, 326), is not normally detectable, but
it was detected postlymphodepletion. Lymphodepletion also
results in a reduction in the number of regulatory or inhibi-
tory lymphocyte populations present in the tumor (327).
Conditioning chemotherapy may also be useful to eliminate
or reduce myeloid suppressor cells in the tumor microenvi-
ronment, while APCs may be activated by lymphodepletion
with the increase of their susceptibility to Toll-like receptor
signaling (328). The importance of the lymphodepletion has
been confirmed in the clinical setting where most of the
patients not receiving lymphodepletion prior to infusion of
CART19 did not show any objective response of their malig-
nancy (100, 329). The timing of lymphodepletion and con-
ditioning relative to T-cell infusion and the particular
conditioning regimen that are superior is unclear. Early infu-
sion post conditioning seems to be optimal, with infusion on
day +2 apparently superior to later time points in term of
immune reconstitution after high dose chemotherapy and
stem cell infusion (24, 330). A number of regimens have
been employed for lymphodepletion, including TBI, fludara-
bine, and cyclophosphamide, high dose chemotherapy or
bendamustine; however, no direct comparison of these pro-
tocols is available. Finally, it is possible that the optimal che-
motherapeutic regimen will be unique to individual tumor
types with disease specific to most effectively target both the
microenvironment and the disease.
Vasculature is another important mechanistic and structural
component of the tumor milieu; accordingly, the targeting of
28
tumor angiogenesis has been studied preclinically in the con-
text of ACT, to evaluate the potential for synergistic effects
between the two treatment modalities. Adoptive transfer of
CAR T lymphocytes against VEGFR-1 delayed tumor growth
and formation and inhibited pulmonary metastasis in
xenograft models, an effect that was further enhanced by
co-transfer of T lymphocytes that expressed IL-15 (331). In a
separate study, simultaneous targeting of tumor antigens
gp100 (PMEL), TRP-1 (TYRP1), or TRP-2 (DCT) and the
tumor vasculature (VEGFR-2) using ACT revealed synergistic
effects in terms of regression of established tumors in mice
(332).
Tumor stromal fibroblasts (TSFs) are one of the most
prominent cell types in the tumor microenvironment of
many human cancers such as pancreatic, gastrointestinal,
and breast cancers. TSFs appear to play an active role in can-
cer progression by secreting factors that enhance tumor sur-
vival, growth, angiogenesis, and metastasis, in addition to
recruiting other tumor-promoting cell types (333, 334).
The conversion of TSFs to a tumor-promoting state is char-
acterized by expression of surface proteins such as fibroblast
activation protein (FAP), an endopeptidase with expression
largely restricted to TSFs. Accordingly, FAP has been evalu-
ated as a target for ACT-based approaches. Unfortunately, as
recently reported, although highly reactive anti-FAP CARs
had little impact on tumor progression in a variety of synge-
neic mouse tumor implantation models, high doses of FAP-
reactive T cells were observed to induce severe cachexia and
dose-limiting bone toxicity (335), underscoring the previ-
ously made point about the potency and need to critically
evaluate the potential for off-target toxicity of CAR-based
approaches.
ACT-based strategies that utilize engineered T cells can
also be applied to indirectly modulate the host immune sys-
tem and the tumor microenvironment. For example, T cells
engineered to express an NK2GD-targeting CAR were shown
to recruit and activate host lymphocytes in a chemokine-
dependent manner, leading to the development of broader
host immune responses (336).
Clinical overview of ACT
As discussed above, immunotherapy using TILs has a long
clinical history, and this approach has provided important
mechanistic and clinical insights about the potency of ACT.
However, technical issues have prevented the broader imple-
mentation of this approach to target cancers. The ability to
at-will genetically engineer and ex vivo manipulate and
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014

expand potent T cells has brought about what promises to
be the beginning of a golden age for ACT, with multiple
ongoing clinical efforts, unprecedented positive clinical
results, and a real possibility for late stage development and
regulatory approval of ACT in the next few years. Here, we
present a summary of the most salient clinical results
obtained over the past few years that provide insights into
the promise and pitfalls this exciting treatment modality.
TCR-redirected T cells
The first successful clinical experience with TCR redirected T
cells was in the setting of melanoma using a low affinity
HLA-A2-restricted anti-MART1 (clone DMF4). Two of 17
patients showed an objective response with partial tumor
regression with no significant toxicity, with the infused T
cells persisting at very low levels for more than 1 year
(337). A subsequent trial utilizing T cells that expressed a
high affinity anti-MART-1 (clone DMF5) or an anti-gp100
TCR resulted in objective responses in about 25% of the
patients. This trial also demonstrated the first example of on
target toxicity with 29/36 patients experiencing hearing
loss, erythematous skin rash, vitiligo, and/or anterior uveitis
due to the destruction of normal tissues that expressed the
target antigens (67).
A number of clinical trials have been conducted over the
past few years to target members of the CT family of anti-
gens. CT antigens are expressed in germ cell tissues but not
in adult tissues, excluding the immune privileged testes, and
are aberrantly expressed in the cytoplasm of various solid
tumor cells, thus representing in principle ideal targets for
ACT (338). NY-ESO-1 (New York-esophageal squamous cell
carcinoma-1) is one such antigen, expressed in about 80%
of synovial carcinoma, 30–40% of breast, urothelial, esoph-
ageal, thyroid, prostate, melanoma, hepatic, gastric cancers,
and neuroblastomas (34). In a clinical trial at NCI, 4/6
synovial sarcoma patients and 5/11 melanoma patients
showed objective clinical response following treatment with
affinity-enhanced anti-NY-ESO-1 TCR-redirected T cells;
importantly, and perhaps exceptionally - see below, no sig-
nificant toxicities were recorded (31). A number of addi-
tional ACT-based clinical trials to target NY-ESO-1 in
multiple solid tumors are currently ongoing.
The MAGE (melanoma-associated antigen) group of CT
antigens has also been targeted in multiple ACT-based trials.
The MAGE family encompasses a large number of sequence-
related members which are expressed at various frequencies
in a broad range multiple solid tumor types (339, 340).
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014
Ruella & Kalos
Adoptive immunotherapy for cancer
Three recent clinical trials targeting members of the
MAGE family have served to highlight the previously
theoretical safety concerns associated with the potency of
TCR-engineered T-cell therapy. In one trial, T cells were
engineered to express a TCR generated in HLA-A*0201 trans-
genic mice (i.e. not sculpted by the human immune system)
and that recognized an epitope shared between MAGE-A3,
MAGE-A9, and MAGE–A12. Of nine patients treated, five
demonstrated objective clinical responses, but three patients
demonstrated SAE associated with neural toxicity, including
two deaths. Postmortem analysis revealed rare and previously
unrecognized expression of MAGE-A12 in brain tissue (341).
Two trials which evaluated the use of affinity enhanced HLA-
A*01-restricted and MAGE-A3-specific TCR to target mela-
noma and myeloma respectively. The first treated patient in
each of these trials experienced SAE associated with cardio-
toxicity and each patient died within 7 days of T-cell infu-
sion (75). Extensive and detailed retrospective analysis
demonstrated that the affinity enhancement of the TCR
resulted in the off target recognition of a related epitope from
the protein titin expressed in cardiomyocytes (75).
TCRs to target carcino-embryonic antigen (CEA) have
been also recently evaluated in ACT. CEA is a glycoprotein
involved in cell adhesion and is expressed mainly in gastro-
intestinal tissue during fetal development. CEA is overex-
pressed in different cancers, in particular in colorectal
cancer. An anti-CEA high affinity TCR was recently
employed in a clinical trial to treat metastatic colon cancer.
One of three patients treated showed objective response but
all patients developed severe on target toxicity with inflam-
matory colitis. Transient clinical responses as manifested by
reductions in circulating levels of CEA were observed in
each of these patients (66).
Other TCR-based target currently being preclinically eval-
uated include the human onco-protein MDM-2 (64), the
p53 suppressor gene (65, 342), the tyrosinase melanocyte
differentiation antigen, the prostate and breast cancer anti-
gen TARP, the telomerase (343) and the Wilms’ tumor 1
antigen (WT-1) (344, 345). A recent clinical trial evaluated
the infusion of autologous T cells genetically modified for
the expression of a HA1-specific TCR to leukemia patients
relapsing after HSCT (346).
CAR-redirected T cells
The clinical evaluation of CAR-redirected T-cell-based ACT
has exploded over the past few years, with more than 25 clin-
ical trials of CART that are actively recruiting, as registered in
29
Ruella & Kalos
Adoptive immunotherapy for cancer
clinicaltrials.gov (as of 15 July 2013). Among these, half are
evaluating CART-19 in B neoplasms, three CART against Her2
in GBM and sarcoma, and the remaining different other tar-
gets (GD2, j light chain, CD30, EGFR, CD33, CD138, FAP,
CEA, mesothelin).
Initial clinical studies with 1st generation CART targeting
neuroblastoma, lymphoma, renal, and ovarian cancers were
disappointing, with limited clinical activity, lack of in vivo
expansion and long-term persistence in vivo (93–96).
Improvements of CAR engineering led to more positive clin-
ical outcomes and also revealed the potential for SAE. Two
studies in particular, targeting carbonic anhydrase -9 (CA-
IX) in renal carcinoma patients using 2nd generation CARs
and ERBB2 in colon cancer using 3rd generation CARs dem-
onstrated considerable on-target off-tissue toxicity, with
liver toxicity due to targeting of CA-IX-positive biliary epi-
thelium (148) and death from respiratory distress syndrome
due to the activation of T cells presumably in response to
low expression of ERBB2 by lung epithelium (153). Other
notable studies include the targeting of the glycolipid anti-
gen GD2 on neuroblastoma with Epstein-Barr virus (EBV)-
specific T cells engineered to express 2nd generation CARs,
which resulted in tumor regression or necrosis in 4/8
patients, with one CR and a PR, and persistence of infused
cells (155–157, 184).
Most of the dramatic recent success of CART-based ACT
has been achieved in hematological tumors, in particular tar-
geting the CD19 antigen which is expressed during B-cell
development from the early B-cell progenitor (pro-B cell) to
the mature B cells and possibly follicular DCs and by almost
all B-cell malignancies. The absence of expression of this tar-
get in healthy tissues other than B cells has made it an ideal
antigen to target for adoptive immunotherapy.
Infusion of CD19-specific CART cells targeting CD19
showed to be effective in acute lymphoblastic leukemia
(ALL), chronic lymphocytic leukemia (CLL), and non-
Hodgkin lymphomas (NHL) (27, 29, 30, 347). The first
patients treated with second generation CART19 were at the
NCI. These patients received anti-CD19 CARs containing
CD28 and CD3f costimulatory domains; this treatment
resulted in significant regression of tumor and a partial
remission the results of the first eight patients subsequently
enrolled in this trial were recently reported, with six of
eight patients achieving an objective clinical response,
accompanied by transient B-cell ablation (130, 347). A
recently published trial (30) from the MSKCC group in ALL
applied CART therapy as a bridge toward allogeneic trans-
plant; this study showed activity of infused CART19-28f in
30
all five treated patients, including two patients with signifi-
cant disease burden. The CART treatment was followed by
allogeneic cell transplant in 4/5 patients, precluding analy-
sis of long-term efficacy for the CART cells (30). At Baylor
College of Medicine, six patients with relapsed or refractory
NHL were treated with a combination of first- and second-
generation CART19 in the absence of lymphodepletion; sec-
ond generation CARs showed enhanced persistence in this
study. Despite transient stabilization of lymphoma in two
patients, none showed evidence of sustained tumor regres-
sion at the cell doses used (100). Perhaps the most dra-
matic results to date have been reported from our group at
the University of Pennsylvania in the setting of adult CLL
(27, 28) and pediatric ALL (29). These studies employ sec-
ond generation CARs with 4-1BB and CD3f costimulatory
domains, and lentivirus-engineered T cells, expanded using
anti-CD3 and -CD28 coated beads, and infused 2 days after
lymphodepletion. In these studies, CART cells were able to
eradicate very large tumor burdens and mediate complete
and ongoing complete remissions, with ongoing functional
persistence of CART cells now beyond 3 years in 2/3 initial
CLL patients and 1/2 ALL patients, as revealed by ongoing
B-cell aplasia. SAE of varying severity related to delayed
tumor lysis syndrome, cytokine release syndrome, macro-
phage activation syndrome, and hemophagocytic lymphoh-
istocytosis, all of varying severity are common features for
responding patients in these trials. Elevated levels of a num-
ber of cytokines and chemokines, including IFNc, IL-6,
MIP1a, MIp1b, MCP-1 point to the broad and systemic
immune response unleashed by this potent therapy. Toc-
iluzimab, a humanized monoclonal antagonistic antibody
against the IL-6 receptor, has been identified and imple-
mented as a key interventive treatment to mitigate the
impact of the cytokine cascade (348).
Concluding thoughts
More than 18 years from the first clinical demonstration of
the feasibility of T-cell adoptive transfer (349), the field is
poised to capitalize on the multitude of lessons learned as a
result of combining the commitment, insights, and innova-
tions of a multitude of scientists combined with the
profound bravery and generosity of patients. ACT using
engineered T cells is demonstrating dramatic potency in
clinical trials, leading to complete and durable responses in
patients with late stage and treatment refractory disease, and
commercialization of this emerging technology is likely to
occur in the next few years (26).
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunological Reviews 257/2014
 Ruella & Kalos
Adoptive immunotherapy for cancer

In this review, we have highlighted and expanded on a
number of topics of direct relevance for the application of
engineered T cells to ACT, within space and scope con-
straints. In this regard, a number of topics relevant to the
specific implementation of this approach were not fully
developed but merit description. Ultimate effective imple-
mentation of T-cell-based ACT is likely to require innova-
tion and co-development of at least some of these areas, as
summarized here: (i) the issue of targeting aggressive ver-
sus indolent disease, exemplified by T-cell inhibitory effects
mediated by CLL cells (350), and the effect of indolent
diseases on creating an immunosuppressive microenviron-
ment, as illustrated by the differential effect of immune
modulating drugs, such as lenalidomide and bortezomib)
in the indolent setting; (ii) the issue of targeting solid
tumors in general as well as the differential requirements
for targeting individual tumor types such as for example
pancreatic cancer which has been described as resistant to
T-cell infiltration (351). Although the discussion above
with regard to tumor microenvironment is related to this
topic, tumor type-specific and perhaps even patient-specific
issues related to individual tumor biology, architecture,
heterogeneity, and metabolism may well impact potency
and effectiveness of ACT; accessing the relevance and
resolving such issue is likely to require the application of
broad, integrated, and quality supported assay platforms to
identify relevant correlations (352). (iii) Tumor burden
must be considered. With ACT representing a unique para-
digm that involves the in vivo interaction of two viable cell
populations, with the potential for T-cell potency, expan-
sion, and persistence paradoxically dependent on higher
tumor burdens as reflected by the more profound cytokine
elevations observed in the context of higher disease bur-
dens (28–30). (iv) Strategies that combine ACT with the
use of agents that impact tumor biology such as demethy-
lating agents (353), tumor signaling, metabolic pathway
and cell cycle inhibitors (354–356) will need to be further
investigated. The development of such combination strate-
gies, also including checkpoint inhibitor combinations as
discussed above, has the potential to unleash the full-
breadth of the immune response against tumors with
potentially profound anti-tumor activity. (v) Finally, issues
of scale-up, automation, commercialization, and intellectual
property (26, 247), addressing at large scale regulatory
hurdles unique to cell therapy (357), and identifying and
implementing reimbursement models will increasingly need
to be addressed as this field further matures and is applied
to treat patients at large scale.

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Immunological Reviews 257/2014