Critical Reviews in Clinical Laboratory Sciences, 2009; 46(4): 167–189

REVIEW ARTICLE

Cancer immunotherapy
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13

Constantin N. Baxevanis, Sonia A. Perez, and Michael Papamichail

Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, Athens, Greece
Referee: Professor Guido Forni, Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of
Torino, Torino, Italy

Abstract
Recent scientific advances have expanded our understanding of the immune system and its response to
malignant cells. The clinical goal of tumour immunotherapy is to provide either passive or active immu-
nity against malignancies by harnessing the immune system to target tumours. Monoclonal antibodies,
cytokines, cellular immunotherapy, and vaccines have increasingly become successful therapeutic agents
for the treatment of solid and haematological cancers in preclinical models, clinical trials, and practice. In
this article, we review recent advances in the immunotherapy of cancer, focusing on new strategies and
future perspectives as well as on clinical trials attempting to enhance the efficacy of immunotherapeutic
modalities and translate this knowledge into effective cancer therapies.
Keywords:  Adoptive transfer; cancer immunotherapy; cancer vaccines; chemotherapy; cytokines;
immunomodulation; monoclonal antibodies; NK cells; NKT cells; regulatory cells
For personal use only.

Abbreviations:  AICD, activation-induced cell death; AML, acute myeloid leukemia; BiTEs, bispecific
T-cell engager molecules; CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; CTL,
cytotoxic T lymphocyte; CTLA, cytotoxic T lymphocyte antigen; DC, dendritic cells; DTH, delayed-type
hypersensitivity; EpCAM, epithelial cell adhesion molecule; FLIP, flice inhibitory protein; GC, glucocorticoid;
GvHD, graft versus host disease; HC, hydrocortisone; HLA, human leucocyte antigen; IDO, indoleamine-
2,3-dioxygenase; IFN, interferon; IL, interleukin; KIR, killer cell immunoglobulin-like receptors; mAb,
monoclonal antibody; MHC, major histocompatibility complex; MUC1, mucin-1; NK, natural killer cell; NKT,
natural killer T cell; NSCLC, n
­ on-small-cell lung cancer; PADRE, pan-DR epitope; PD, programmed death;
TAA, tumour associated antigen; TCR, T cell receptor; TGF, transforming growth factor; Th, T helper cell; TLR,
toll-like receptor; TRAIL, tumour necrosis factor-inducing ligand; Tregs, T regulatory cells; VEGF, vascular
endothelial growth factor; PBMC, peripheral blood mononuclear cells; PSA, prostate specific antigen; rV,
recombinant vaccinia virus; scFv, single-chain antibody fragments.

Introduction renal cell carcinoma.1 Monoclonal antibodies have

Recent scientific advances have expanded our
­understanding of the immune system and its response
to malignant cells. These breakthroughs provided for
rapid progress in the field of cancer immunology and
immunotherapy. The clinical goal of tumour immu-
notherapy is to provide either passive or active immu-
nity against malignancies by harnessing the immune
system to target tumours.
Cytokine therapy, applied in the form of exoge-
nously administered recombinant interferon-gamma
(IFN) or interleukin-2 (IL-2), was the first type of
immunotherapy to be applied in patients with vari-
ous types of cancer, mostly including melanoma and
become increasingly used therapeutic agents for the
treatment of various types of malignancies. Many are
now being tested as components of adjuvant or first-
line therapies to assess their efficacy in improving or
prolonging survival.2 Genetic constructs displaying
antibody-like specificity represent promising tools
for therapeutic interventions.3,4 Vaccine therapy rep-
resent attempts to activate the immune system to
recognize cancer cells and eliminate them from the
body. Technological advances have allowed the con-
struction of various types of vaccines such as DNA
or RNA vaccines, recombinant viruses expressing
tumour genes, genetically engineered tumour cells
or dendritic cells (DC), and synthetic long-peptides

Address for correspondence:  Professor M. Papamichail, Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, 171 Alexandras
Avenue, Athens 11522, Greece. E-mail: papamichail@ciic.gr
(Received 29 December 2008; revised 12 February 2009; accepted 26 March 2009)
ISSN 1040-8363 print/ISSN 1549-781X online © 2009 Informa UK Ltd
DOI: 10.1080/10408360902937809 http://www.informahealthcare.com/lab
 168   C. N. Baxevanis et al.
representing a plethora of tumour immunogenic
epitopes.5 All these innovative approaches have
considerably improved the field of cancer vaccines.
Vaccines will certainly have an impact on the inci-
dence of certain cancers, and research is ongoing
into therapeutic vaccines for various cancers, thereby
opening up the possibility of administering vacci-
nations to patients during disease-free intervals to
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
prevent recurrences. Combining immunotherapy
with conventional therapies may prove to be the most
effective strategy for patient benefit.6 Moreover, vacci-
nation against cancer-specific antigens can sensitize
the tumour to subsequent chemotherapeutic treat-
ment.7 Adoptive-cell (T, NK, NKT) transfer therapy
has developed into an effective treatment, mainly for
patients with metastatic melanoma.8 Current applica-
tion of this therapy relies on the ex vivo generation of
highly active, highly avid tumour-reactive lymphocyte
cultures from endogenous tumour-infiltrating lym-
phocytes9 or on the genetic engineering of cells using
antigen receptor genes to express de novo tumour
antigen recognition.3,10
In this article, we review recent advances and per-
spectives in the immunotherapy of cancer, focusing
For personal use only.
on new strategies to enhance the efficacy of immu-
notherapeutic modalities as well as on clinical trials
attempting to translate this knowledge into effective
cancer therapies.
FDA approved cytokines for cancer
immunotherapy
IFN, was the first cytokine to demonstrate anti-tumour
activity in patients with advanced melanoma. IFN-2b,
a type I IFN, is a highly pleiotropic cytokine with immu-
noregulatory, antiproliferative, differentiation-induc-
ing, apoptotic, and antiangiogenic properties in mul-
tiple malignancies,1 and objective tumour response
rates of approximately 20% were observed in phase
I/II trials for metastatic disease.11,12 In 1995, recom-
binant IFN-2b became the first immunotherapy
approved for adjuvant treatment of stage IIB/III
melanoma. The value of adjuvant high-dose IFN-2b
for the treatment of high-risk melanoma has been eval-
uated in three US cooperative group studies.13–15 These
studies showed that high-dose IFN-2b significantly
reduced the risk of recurrence; two of three studies
also demonstrated a significant improvement in over-
all survival rates compared with observation or the
GM2 ganglioside conjugated to keyhole limpet hemo-
cyanin mixed with QS-21 adjuvant vaccine.16 IFN-2b
has been also approved for the treatment of renal and
kidney carcinoma, follicular lymphoma, hairy cell
leukemia, and chronic myelogenous leukemia.
IL-2 was a second exogenous cytokine to dem-
onstrate anti-tumour activity against melanoma
and was approved for the treatment of adults with
advanced metastatic melanoma. IL-2 plays a central
role in immune regulation and T-cell proliferation.17
High-dose bolus intravenous recombinant IL-2 was
shown to have anti-tumour effects when administered
with or without lymphokine-activated killer cells in
phase II clinical trials involving advanced metastatic
melanoma patients.17,18 In these studies, an objective
response rate of 16% (median response duration,
8.9 months; range, 4 to 106+) with a durable response
rate of 4% was demonstrated.18–21 IL-2 has been also
approved for the treatment of renal and kidney carci-
noma as well as haematological malignancies.
Cytokines with high potential in treating
cancer
IL-7 is a 25 kDa T-cell growth factor that costimu-
lates T cell receptor (TCR) signalling.22–24 IL-7 is
required for T-cell development and T-cell survival
in the ­periphery.22 It signals via the IL-7 receptor (R),
comprising c and IL-7R.24,25 Expression of IL-7R
marks cells destined to become memory cells during
evolution of the immune response.26–28 In preclinical
studies, IL-7 was demonstrated to act as a vaccine
adjuvant exerting profound effects on subdominant
responses.29 It has also been shown to enhance CD4+
and CD8+ effector and CD8+ memory populations
and to support tumour protection following vaccina-
tion with DC.28 IL-7 is essential for the augmented
anti-tumour effects during lymphopenia.27,28 Two
phase I/II trials utilizing recombinant IL-7 have been
completed in patients with cancer.30,31 There was no
toxicity while dramatic increases in total body CD4+
and CD8+ T cells were observed with no selective
increase in Tregs. There appeared to be a preferential
expansion of naïve T cells with an increased T-cell
repertoire diversity. IL-7 may be considered a vaccine
adjuvant to enhance the efficacy of tumour vaccines
targeting self/tumour antigens for which an estab-
lished self-tolerance exists.
IL-15 is a cytokine and T-cell growth factor that is
produced by macrophages and DC and acts on CD8+
T cells, CD4+ T cells, and NK cells.32 It signals through
IL-15R, which presents IL-15 in trans to IL-2/15 
and the  chain receptor.33,34 IL-15 has various func-
tions, of which the most prominent are (i) inhibition of
antigen-induced cell death (AICD) of T cells (in con-
trast to IL-2, which promotes AICD),34–36 (ii) reversal of
T-cell anergy,34–36 (iii) promotion of induction of long-
lived CD8+ T cells with potent anti-tumour efficacy,37
(iv) substitution of CD4+ T-cell help in cytotoxic T

lymphocyte (CTL) induction,35 (v) activation of NK
cells,38 and (vi) induction of tumour regression in
mice.39–41 IL-15 may potentially be applicable as a
vaccine adjuvant to induce longer-lived, more effica-
cious CD8+ T cells, as a single agent to overcome T-cell
anergy, as a T-cell growth factor in conjunction with
adoptive T-cell immunotherapy, and as a systemic or
intratumoural cytokine therapy for cancer.
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
IL-12 is a 70 kDa glycoprotein that binds IL-12R on
NK, T cells, DC, and macrophages.42 It promotes IFN
release by IL-12R-expressing T, NK, and NKT cells
and induces T helper 1 (Th1) polarization as well as
proliferation of IFN expressing T cells.43,44 IL-12 also
displays anti-angiogenic activity and plays an impor-
tant role in anti-cancer development and immunity
in animal models.45–47 Phase I and II clinical data48–53
have demonstrated low efficacy when recombinant
IL-12 was administered as monotherapy in patients
with melanoma and renal cell carcinoma. Because of
its potency to induce robust IFN production by TCR-
activated T cells, IL-12 may induce more impressive
effects when co-administered with a vaccine. Its use as
an adjuvant may both polarize Th1 responses and aug-
ment CD8 responses in any antigen-specific strategy.
For personal use only.
IL-21 belongs to the -chain family of cytokines
(together with IL-2, IL-4, IL-7, IL-9, and IL-15) and it
is primarily produced by CD4+ T cells.54,55 It induces
differentiation of pro-inflammatory CD4+ Th17 cells,
stimulates memory and naïve CD8+ T-cell expansion
synergistically with IL-7 or IL-15, and improves degree
of expansion and affinity of antigen-specific CTL clones
generated in vitro.54–57 Its effects on NK cells include
augmentation/differentiation and anti-tumour
activity.56–58 IL-21 also inhibits maturation, activation,
and cytokine production of immature DC.59,60 In pre-
clinical models in vivo, IL-21 has been demonstrated
to induce tumour rejection, prevention of metastases,
and immune memory.59,60 Because of its effects on
CD8+ T cells, IL-21 may be essential for in vitro gen-
eration and expansion of tumour antigen-specific CTL
lines/clones to be used in adoptive immunotherapy.
Moreover, it can be used as a systemic immunomodu-
lator combined with monoclonal antibody therapy for
potentiating NK cell-mediated ADCC.
Monoclonal antibodies for cancer treatment
The development of hybridoma technology by Kohler
and Milstein61 led to the production of monoclonal
antibodies (mAbs), which have considerably modified
the field of clinical oncology. The poor performance of
mAbs in early clinical settings was attributed to short
antibody half-life and the immunogenicity of the
mouse protein in humans (resulting in the production
Cancer immunotherapy   169
of neutralizing human anti-mouse antibodies.62 These
limitations were overcome by generating chimeric or
humanized mAbs.63 Moreover, growing knowledge of
key cellular pathways in tumour induction and pro-
gression provided the platform for appreciating the
role of humoral immunity in cancer defence, thus
paving the way for an increasing proportion of new
drugs entering clinical trials.
Some molecules, such as trastuzumab, rituximab,
alemtuzumab, cetuximab, are now widely used in
clinical practice.2,64,65 These antibodies are now being
tested in different indications alone or in combina-
tion with standard chemotherapy. They are also being
developed for the treatment of inflammatory diseases
(rituximab). Numerous other antibodies are currently
in pre-clinical and clinical development phases for
several malignancies, including renal carcinoma,
melanoma, lymphomas, leukaemia, and breast, ovar-
ian, and colorectal cancer.2,61 An alternative approach
is to conjugate the mAb to a toxin,66 a cytotoxic agent,
or a radioisotope (Table 1).67 In other cases, these anti-
bodies aim to modify the tumour microenvironment
through inhibition of angiogenesis or by enhancing
host immune responses against cancer.
If the molecule targeted by the antibodies is clearly
identified, most often the precise mechanism of
action of these immunoglobulins is understood. They
can have direct effects in inducing apoptosis or pro-
grammed cell death,15 but they can also block growth
factor receptors, efficiently arresting proliferation of
tumour cells.64,68 Indirect effects include (i) recruit-
ing cells that exert cytotoxicity, such as monocytes,
macrophages, and, most important, T and NK lym-
phocytes (an immune pathway known as antibody-
dependent cell-mediated cytotoxicity)62,68 and (ii)
binding complement, which leads to toxicity known
as complement-dependent cytotoxicity.68 mAbs may
also act as regulators of immune responses in vivo.
We have recently demonstrated that breast cancer
patients on trastuzumab exhibited decreased levels
of serum HER-2/neu, which correlated with a drop in
the numbers of peripheral T regulatory cells (Tregs)
and objective clinical responses.69 This novel func-
tion of mAbs may have important implications for the
down-regulation of tumour-associated peripheral
and local immune suppression mechanisms.
Immunomodulatory mAbs for the treatment of
cancer
Immunostimulatory mAbs directed to immune recep-
tors have emerged as a new and promising strategy
to fight cancer. In general, mAbs can be designed
to bind molecules on the surface of lymphocytes or
 170   C. N. Baxevanis et al.
antigen-presenting cells to provide activating signals
(e.g. CD28, CD137, CD40, and OX40).70,71 On the other
hand, mAbs can also be used to block the action
of surface receptors that normally downregulate
immune responses [cytotoxic ­T-lymphocyte–associated
antigen 4 (CTLA-4) and PD-1/B7-H1]. In com-
bined regimes of immunotherapy, these mAbs are
expected to improve therapeutic immunizations against
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
tumours as observed in preclinical studies.
Anti-4-1BB (agonistic anti-CD137) mAb has been
successfully tested as an anti-cancer molecule in pre-
clinical studies.70 4-1BB is a member of the tumour
necrosis factor/nerve growth factor family of receptors
and has a natural ligand (4-1BBL) that is expressed on
activated T lymphocytes as well as on NK cells and
DC.72 This mAb, which acts against CD137, has the
ability to stimulate potent anti-tumour responses73
and, paradoxically, ameliorates autoimmune mani-
festations in mice.73
Therapy with mAbs against CTLA-4, which block
the inhibitory action of CTLA-4 on T cells, is capable
of inducing anti-tumour responses in mice as well
as in humans, but is accompanied by adverse events
in the form of autoimmune reactions.74 Anti-CTLA-4
For personal use only.
mAb was developed to bind to the CTLA-4 on T cells
and thereby prevent the inhibitory cascade triggered
by CTLA-4 binding to B7.75–77 In a murine model, anti-
CTLA-4 mAb has demonstrated anti-tumour activity
in moderately antigenic and highly immunogenic
tumours.75 Anti-CTLA-4 mAb alone is not sufficient
to alter the growth of poorly immunogenic tumours,
such as MC38.78,79 Preclinical investigations have been
performed using anti-CTLA-4 mAb in combination
with cancer vaccines to increase the magnitude of
the T-cell response and the avidity of antigen-specific
T cells.77 Based on the strength of these preclinical
data, clinical trials have been initiated with two fully
human anti-CTLA4 mAbs.
Tremelimumab induced durable objective
responses with low-grade toxicities when used as
second-line monotherapy in a phase I study with
melanoma patients treated with single, escalating
doses.80 Subsequently, in a phase II trial, patients
with advanced melanoma randomly were assigned
to receive either  10 mg/kg tremelimumab monthly
or 15 mg/kg tremelimumab every three months.81
Although the response rate was not significantly dif-
ferent between the two arms, the 15 mg/kg every
three months regimen was associated with a lower
incidence of grade 3/4 adverse events. Consequently,
the 15 mg/kg every three months dosing regimen
was selected for further study and is currently being
investigated for single-agent anti-tumour activity in a
larger phase II open-label trial and in a randomized
comparative phase III study against dacarbazine or
temozolomide in patients with advanced relapsed or
refractory melanoma.82
Phase I studies of ipilimumab were performed
in patients with prostate cancer,83 melanoma,84 and
ovarian cancer.85 In these studies, patients after a
single administration of ipilimumab achieved some
clinical efficacy, as manifested by incomplete reduc-
tion of tumour size and extensive tumour necro-
sis with leukocyte infiltration. In phase II studies,
repeated administrations with ipilimumab allowed
more patients to achieve objective responses.86 The
combination of ipilimumab with chemotherapeu-
tics [dacarbazine87 or docetaxel88], with IL-289 or with
melanoma-associated peptide vaccines90 improved
the rate of complete responses in patients compared
with the monotherapy arms, demonstrating that com-
bining anti-CTLA-4 therapy (i) with cytotoxic therapy,
such as chemotherapeutics, may create an enhanced
environment for immunomodulatory treatment
by potentially releasing endogenous antigens from
tumours and/or altering the immune milieu, and
(ii) with treatments that have different mechanisms
of action, it may be possible to produce additive or
synergistic effects.
Genetic constructs specifically targeting
tumour-associated antigens
Chimeric antigen receptors
Chimeric antigen receptors (CARs) represent a tech-
nological improvement in the field of cancer immu-
notherapy for retargeting the functional program
of immune lymphocytes towards specific molecu-
lar targets expressed on the cell surface of tumour
cells. CARs are genetic constructs consisting of an
antigen-recognizing antibody molecule linked to a
T-lymphocyte signalling domain that can be modi-
fied to include co-stimulatory signals that improve the
activation of CAR-expressing cells.3The main advan-
tage of CARs is their ability to redirect T-lymphocyte
specificity and their killing/effector activity towards
a selected target in a non-major histocompatibility
complex (MHC)-restricted fashion, exploiting the
antigen-binding properties of mAbs.3 Ligand binding
by the chimeric receptor triggers phosphorylation of
the immunoglobulin tyrosine activation motifs in the
cytoplasmic region of the molecule and this activates a
signalling cascade that is required for the induction of
cytotoxicity, cytokine secretion, and proliferation.91,92
CAR-expressing cells, by recognizing tumour antigens
in a non-MHC-restricted manner, are not affected by
down-regulation of HLA (human leucocyte antigen)
class I antigens and by defects in the antigen-processing

machinery of tumour cells.3 Moreover, this technol-
ogy enables the generation of large numbers of lym-
phocytes with specificity for their autologous tumours
to be used in adoptive cellular immunotherapy.92–94
CAR technology has been successfully applied to
enhance tumour recognition by primary T cells,95–97
and several studies have demonstrated specific kill-
ing of tumour target cells in vitro and in vivo following
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
redirection of T and NK cell lines98–100 or primary NK
cells101,102 with chimeric receptors.
Several clinical trials using lymphocytes engineered
to express CARs have been implemented; however, so
far, no positive clinical responses have been noted.103,104
One major obstacle to this form of therapy is the pres-
ence of high serum levels of soluble monovalent anti-
gen, which upon binding to grafted cells would render
them anergic. Thus, such modalities of therapy would
be restricted to patients with low levels of soluble
tumour antigen. The antigenic diversity in tumour-cell
populations may also hamper positive clinical results.
The use of lymphocytes grafted with CARs recognizing
more than one antigen may circumvent this problem.
Preclinical studies have demonstrated the capacity of
T cells expressing bispecific CARs to lyse tumour cells
For personal use only.
expressing both carcinoembryonic antigen (CEA) and
TAG-72103 or HIV-infected cells expressing wild-type
and mutant envelope proteins.105
Another limitation of CARs is their potential immu-
nogenicity. Since single-chain antibody fragments
(scFv) are most often derived from a mAb of murine
origin, this might trigger a host immune response and
therefore accelerate T-cell clearance. Humanizing
the mouse portions of CARs or by using previously
humanized scFvs106,107 may offer a solution to this
problem. Another aspect that should be taken into
consideration when utilizing lymphocytes grafted
with CARs in clinical trials is that T cells of cancer
patients may possess abnormalities in their TCR-
mediated signalling.108,109 Thus, the question to be
addressed is whether potential downstream defects
in the signalling cascade will impair signal transduc-
tion initiated by the chimeric receptor(s).
Aptamers
The idea of using nucleic acid molecules as thera-
peutic agents was initially entered into praxis with
the development of antisense strategies. Antisense
compounds are single-stranded nucleic acids
that disrupt the synthesis of a targeted protein by
hybridizing in a sequence-dependent manner to
the mRNAs that encode it.110 The mechanism of
inhibition by single-stranded nucleic acid aptamers
is different in that these directly inhibit a protein’s
Cancer immunotherapy   171
function by folding into a specific three-dimen-
sional structure that dictates high-affinity binding
to the targeted protein.4,111 Because they inhibit the
activity of existing proteins directly, aptamers are
more similar to mAb or small molecule drugs than
to antisense compounds, and this property greatly
increases the number of clinical indications that are
potentially treatable by nucleic acid-based com-
pounds.111 Aptamers exhibit specificity and avidity
comparable to or exceeding that of mAbs and can
be generated against most targets.4 Unlike mAbs,
aptamers can be synthesized in a chemical process
and hence offer significant advantages in terms of
reduced production cost and simpler regulatory
approval processes.4,112
In many instances, aptamers have been shown
to inhibit the function of their targets, presumably
by blocking binding of the cognate ligand.4,113 An
aptamer targeting vascular endothelial growth factor
(VEGF) became the first aptamer approved for human
therapy to treat age-related macular degeneration,
arguably a milestone in the application of aptamer
technology.114 Modulation of immune responses
in vivo by using aptamers was first described by
Santulli-Marotto et  al.,115 who generated aptamers
that bind and inhibit the function of murine CTLA-4,
thereby enhancing tumour immunity. Bivalent and
multivalent configurations of other immunomodulat-
ing aptamers binding to 4-1BB or OX40 costimulated
T-cell activation in vitro and promoted tumour rejec-
tion in vivo.112,116
Aptamers offer important advantages over antibod-
ies to manipulate the immune system for therapeutic
purposes. Antibodies are cell-based products requir-
ing a complex and regulatory approval process that in
many cases provide a serious obstacle to their availa-
bility for clinical application. Because aptamers can be
chemically synthesized, the manufacturing and regu-
latory approval processes should be much simpler and
less costly. The use of ­immune-modulating aptamers
as adjuvants to cancer takes advantage of the inherent
lack of immunogenicity of aptamers.4 Antibodies are
unlikely to be suitable for this application. Thus, for the
field of therapeutic aptamers to reach its full potential,
translational researchers and biotechnology compa-
nies must take advantage of the unique properties of
aptamers to address important, unexplored clinical
issues.
Bispecific scFv antibodies
Bispecific T-cell engager molecules (BiTEs) consti-
tute a class of two flexibly linked scFvs that have the
potential to redirect tumour-resident and circulating
 172   C. N. Baxevanis et al.
T cells to lyse tumour cells.10 One scFv interacts with
epitopes on tumour cells, whereas the other scFv
binds an epitope on the TCR/CD3 complex of T cells.
BiTEs have the potency and efficacy to target tumour
cells at low T-cell numbers without the need for T-cell
co-stimulation. BiTEs have been demonstrated to
induce lytic synapses between human T cells and a
MHC class I-negative, tumour cell line, resulting in
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
potent target-cell lysis.117 This shows that certain T-cell
recognition molecules on target cells are dispensable
for synapse formation and BiTE activity and suggests
that BiTE-activated polyclonal T cells may ignore
major immune evasion mechanisms of tumour cells
in vivo, such as loss of MHC class I expression. The
concepts underlying BiTE technology have been
successfully validated in preclinical studies with two
different target antigens, namely CD19 and epithelial
cell-adhesion molecule (EpCAM).118–121 An EphA2-
specific bispecific single-chain antibody (bscAb),
linked to anti-CD3 bscAb, was recently designed to
target epitopes that are uniquely exposed on malig-
nant cells.122 bscEphA2xCD3 had the specificity and
potency to redirect low numbers of T cells for lysis of
EphA2-expressing tumour cells that possessed few
For personal use only.
available sites for bscEphA2xCD3, both in cell cultures
and animal models. A CD19/CD3-directed bscAb
(referred to as MEDI-538, MT103, or bscCD19xCD3)
is currently being investigated in the clinic as a poten-
tial therapy for B-cell malignancies.
The concept of anti-tumour vaccination
Tumour vaccines have been designed to either
increase immune recognition of tumour cells or to
enhance the anti-tumour immune response through
lymphocyte activation.123 Vaccines have utilized
mixtures of antigens or single antigens specific to
the target. Preclinical studies with vaccines showed
evidence of complete tumour regression and/or
prolonged stabilization of tumour growth.124 In early
clinical trials, vaccines prepared from whole tumour
cells were associated with limited activity, presumably
due to the already biased nature of the host immune
response to specific tumour-associated antigens.125
The discovery and cloning of a number of tumour-
associated antigens spurred interest in active immu-
nization for cancer immunotherapy. Attempts at
immunotherapy of cancer have included therapeutic
vaccination with recombinant viral vectors encoding
tumour-associated antigens,126 recombinant proteins
with appropriate adjuvants,127 antigen-loaded DC,128
DNA encoding tumour-associated antigens,5 and syn-
thetic peptides.129 However, apart from in melanoma,
in which impressive clinical responses have been
observed in a small minority of patients, the overall
results have been disappointing.130 The negative expe-
rience with anti-tumour vaccines may suggest that
development of novel vaccine strategies for cancer are
needed to improve recognition, immune response,
effector functions, and trafficking of T cells induced
by vaccination. These goals may be achieved by con-
current administration of novel immunotherapeutics
with an immunopotentiating profile.
Blocking tumour-induced immune suppression
Mabs capable of neutralizing the biologic activity of
suppressive factors released by the tumour, such as
anti-VEGF antibody bevacizumab, which, in addi-
tion to its effect on tumour vasculature may decrease
VEGF-induced inhibition of DC and T-cell function,70
mAbs to transforming growth factor (TGF)-, and
IL-10 are also potential means to reverse immune
escape induced by tumour-released molecules. In
addition, nonsteroidal anti-inflammatory drugs,
including the specific cyclooxygenase-2 inhibitor
celecoxib, could be a means of inhibiting cyclooxyge-
nase-2 that leads to prostaglandin E2 production.131
Removal or blocking the function of cells with
suppressive activity
The immunosuppressive microenvironment of a
tumour reduces the effect of therapeutic vaccines, both
during the induction of immunity and in the effector
phase of the response. One way to improve the induc-
tion phase is to block the negative regulators of the
activation of effector T cells.132 Antibodies against one
such molecule, CTLA-4, are being evaluated in clini-
cal trials.133 CTLA-4 is expressed on activated T cells,
where it serves as an inhibitor of activation. Blocking
the activity of CTLA-4 allows greater expansion of all
T-cell populations, presumably including those with
anti-tumour reactivity (see above). The depletion of
immunosuppressive cells from the tumour environ-
ment is also mandatory for the development of robust
anti-tumour immunity. Denileukin diftitox (DAB/IL2;
Ontak) is a recombinant DNA-derived cytotoxic pro-
tein composed of diphtheria toxin fragments A and B
and the full-length IL-2 molecule. DAB/IL2 binds to
CD25 (the IL-2R chain) and, following internalization,
inhibits protein synthesis, causing cell death within
hours.134 DAB/IL2 is FDA approved for the treatment
of patients with persistent or recurrent cutaneous T cell
lymphoma whose malignant cells express CD25. When
administered to patients with melanoma, this protein
depletes the blood of regulatory T cells. In most patients

(90%), this treatment has resulted in the production of
melanoma-specific CD8 T cells.135 The function of the
immunosuppressive enzyme indoleamine 2,­3-dioxyge-
nase (IDO) can be inhibited by ­1-methyl-tryptophan,136
and clinical trials testing its effects in patients with can-
cer are planned. This reagent would have the potential
of synergizing with immunotherapy by inhibiting
immune suppression by IDO.
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
Induction of sensitivity to apoptosis
Modulation of tumour necrosis factor-inducing ligand
(TRAIL) receptors, which are preferentially expressed
by cancer cells, may synergize with vaccination ther-
apy and other targeted therapy approaches. Histone
deacetylase inhibitors have been shown to up-regulate
the TRAIL death receptor DR5, leading to sensitization
of cancer cells to TRAIL-induced death,137 and therefore
would be candidate drugs to use in combination with
TRAIL-ligand constructs, TRAIL ­receptor-activating
antibodies, and vaccine-induced immune effector
cells. Furthermore, the demethylating agent decitab-
ine has been shown to sensitize melanoma cell lines
For personal use only.
to the cytotoxic effects of IFNs by inducing the expres-
sion of proapoptotic and growth-inhibitory genes.138
The antiapoptotic members of the Bcl-2 family are
potential targets for immunosensitizing drugs. Bcl-2 is
also up-regulated in many cancers,139 and inactivating
its function may result in a proapoptotic cancer cell
inflammatory milieu facilitating the cytotoxic effect
of vaccine-induced immune cells. ABT-737 is a small-
molecule inhibitor of the anti-apoptotic proteins
Bcl-2, Bcl-XL, and Bcl-w. Mechanistic studies reveal
that ABT-737 does not directly initiate the apoptotic
process but enhances the effects of death signals,
displaying synergistic cytotoxicity with chemothera-
peutics and radiation.140 Apoptotic pathways may
also be targeted by altering transcription of regula-
tors of apoptosis. Inhibition of nuclear factor-B in
melanoma sensitizes cells to TRAIL-induced (but not
Fas or TNF-) apoptosis.141 An additional approach
would be the use of the proteasome inhibitor bort-
ezomib, which is known to inhibit nuclear factor-B
and down-regulate the antiapoptotic molecule flice
inhibitory protein (FLIP).142
Novel approaches for improving clinical
results with cancer vaccines
Preventive vaccination
Compromised immunity in patients with advanced
cancer is largely unavoidable and, currently,
Cancer immunotherapy   173
untreatable. This is broadly true even in instances
in which conventional anticancer treatments have
been used to generate a scenario of minimal resid-
ual disease in which to administer immunotherapy.
Therapeutic vaccination in the adjuvant setting is
rarely capable of eradicating residual tumour cells
that are present after surgery and eliciting a complete
response. This inability probably reflects the fact that
debulking tumours removes only one component of
the systemic immune suppression that they estab-
lish, namely the intratumoural suppressive milieu.
This indicates that the suppressive T cells/DC that
have already escaped the tumour may be active in
the lymph nodes and that the immunosuppressive
cytokine profile present in the peripheral blood of
the patient is capable of limiting vaccine efficacy
even in the absence of the immunosuppressive
tumour microenvironment.
Such considerations have led to the hypothesis
that immunization with a therapeutic cancer vaccine
at the earliest stages of carcinogenesis—before local
and systemic immunosuppressive environments
are established by the tumour—will yield the best
immune responses to vaccination and therefore con-
fer excellent protection against the progression of can-
cer development. An example of a currently available
vaccine that may benefit from early administration
resulting from improved screening methods, includes
a breast cancer vaccine consisting of a HER-2/neu
peptide (E75) mixed with received granulocyte-mac-
rophage colony-stimulating factor (GM-CSF) that has
been successful in preventing disease recurrence in
node-positive patients with minimal disease bur-
den.143 In addition, Holmes et al.144 performed a phase
I clinical trial of the novel Ii-Key/HER-2(776-790)
hybrid preventive vaccine in disease-free, node-neg-
ative breast cancer patients. In this study, the Ii-Key
modified vaccine showed enhanced immunological
responses in the absence of an immunoadjuvant. In a
randomized, double-blind study, patients with stage
II breast cancer, with no evidence of disease and at
low risk for recurrence, received injections with oxi-
dized mannan-MUC1 to immunize against MUC1 and
prevent cancer recurrence/metastases.145 Immunized
patients had no recurrences (0/16) for more than five
years after treatment, whereas the recurrence rate
in patients receiving placebo was 27% (4/15). In the
majority of vaccinated patients, both humoral and
cellular immunity to MUC1 was evident.
Therapeutic vaccines combined with chemotherapy
The combination of active immunization with
standard treatments is provocative because of the
 174   C. N. Baxevanis et al.
immunosuppressive effects of most standard treat-
ments. The in vivo preclinical data by Machiels et al.146
addressing the issue of chemotherapy and cancer
vaccines indicated that taxane therapy (employing
taxol) may in fact increase T-cell precursors rather
than deplete them. Later on, Arlen et al.147 designed
one phase II study of patients with metastatic
androgen-independent prostate cancer randomized
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
to receive vaccine alone or vaccine with low-dose
docetaxel. The vaccination regimen was composed
of (a) recombinant vaccinia virus (rV) that expresses
the prostate-specific antigen gene (rV-PSA) admixed
with (b) rV that expresses the B7.1 costimulatory gene
(rV-B7.1) and (c) sequential booster vaccinations with
recombinant fowlpox virus (rF-) containing the PSA
gene (rF-PSA). Patients received GM-CSF with each
vaccination.
The median increase in T-cell precursors to PSA was
equal in both arms following three months of therapy.
In addition, immune responses to other prostate can-
cer-associated tumour antigens were also detected
post-vaccination. Eleven patients who progressed on
vaccine alone crossed over to receive docetaxel at the
time of progression. Median progression-free survival
For personal use only.
on docetaxel was 6.1 months after receiving vaccine
compared with 3.7 months with the same regimen in
a historical control. This was the first clinical trial to
show that docetaxel can be administered safely with
immunotherapy without inhibiting vaccine specific
T-cell responses. Based on their findings, the authors
hypothesized that patients previously vaccinated
with an anticancer vaccine may respond longer
to docetaxel compared with a historical control of
patients receiving docetaxel alone.
In another phase I study,148 17 patients with different
types of advanced cancer were immunized with anti-
gen cytochrome P450 1B1 (CYP1B1), which is overex-
pressed on almost all human tumours.149 Six patients
developed immunity to CYP1B1, three of whom devel-
oped disease stabilization. All but one of 11 patients
who did not develop immunity to CYP1B1 progressed
and did not respond to salvage therapy. Five patients
who developed immunity to CYP1B1 required salvage
therapy for progressive metastatic disease and showed
marked response to their next treatment regimen,
which for most lasted longer than one year.
Antonia et al.150 vaccinated 29 patients with exten-
sive stage small cell lung cancer with a DC-based p53
vaccine. Objective clinical response to vaccination
as well as subsequent chemotherapy was evaluated.
p53-specific T-cell responses to vaccination were
observed in 57.1% of patients. One patient showed
a clinical response to vaccination, whereas most of
the patients had disease progression. However, they
observed a high rate of objective clinical responses
to the chemotherapy (61.9%) that immediately
followed vaccination. Clinical response to subse-
quent chemotherapy was closely associated with
induction of immunologic response to vaccination.
Furthermore, Wheeler et al.151 analyzed survival and
progression times in 25 vaccinated (13 with and 12
without subsequent chemotherapy) and 13 non-vac-
cinated de novo glioblastoma (GBM) patients receiv-
ing chemotherapy. Vaccinated patients receiving
subsequent chemotherapy exhibited significantly
longer times to tumour recurrence after chemother-
apy relative to their own previous recurrence times
as well as significantly longer post-chemotherapy
recurrence times and survival relative to patients
receiving isolated vaccination or chemotherapy.
These data suggest that vaccination against
cancer-specific antigens can sensitize the tumour
against subsequent chemotherapeutic treatment.
Although the mechanisms that underlie such a
synergistic effect have not been elucidated yet, it is
speculated that the vaccination-induced increase
in the frequency of primed T cells may constitute
a major advantage by the time tumour microen-
vironment is modified by cytotoxic drugs [e.g. dis-
ruption of tumour stroma, decreased suppressive
activity, increased sensitivity of tumour cells to
granzymes and up-regulation of tumour-associated
antigens11].
Adjuvants and multiple peptide vaccines, including
T-helper epitopes
Over the years, various vaccines containing exactly
fitting MHC class I-binding peptides have been
tested for their therapeutic efficacy against both
virally and non-virally induced cancers.123 However,
the observed immunological and clinical responses
were rather poor. Therefore, ways to develop more
powerful cancer vaccines should be thoroughly
explored. Preclinical studies demonstrate that
tumour-specific CD4+ Th cells critically contribute
to the development and efficacy of anti-tumour
responses.152–154 The effectiveness of these Th cells
probably lies in their capacity to deliver essential
activation signals to DC, needed for an optimal
priming of tumour-specific CTLs.153,154 In addition,
antigen-specific CD4+ T cells may provide CTL with
essential growth stimuli during the effector phase.43
In line with this notion, several studies show that
effective CTL priming can be induced by the inclu-
sion of Th epitopes in peptide vaccines.152,155
Another strategy to potentiate vaccines is the use
of molecularly defined strong DC-activating adju-
vants, such as oligodeoxynucleotide (ODN)-CpG,

monophosphoryl lipid A (MPL), anti-CD40 Ab, and
GM-CSF.156–158 An increase in the length of the peptide
used for vaccination strongly affects the magnitude of
the induced CD8+ T-cell response. Vaccination with
long peptides22–45 amino acids in length) containing
either a human papillomavirus (HPV)-derived, an
adenovirus-derived or a modified ovalbumin-derived
CTL epitope resulted in more robust and effective CTL
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
responses than vaccination with the minimal CTL
peptide.152,159 Even under conditions that precluded
T-cell help, a single vaccination with a long-peptide
vaccine induced significantly higher CD8+ T-cell
responses in MHC class II knockout mice and in CD40
knockout mice than a minimal CTL peptide.152,159
A re-evaluation of the early reports on the success-
ful use of peptide vaccines in animal models reveals
that most of the vaccines used were actually longer
than the exact minimal CTL peptide epitope.160–163
Physical linking of Th and CTL epitopes or conjugation
of toll-like receptors (TLR) ligands to CTL peptides
increases the size of the ultimate peptide used and
as such might have improved the CTL response. The
mechanism that explains the difference between long
and minimal CTL peptides lies in the fact that injec-
For personal use only.
tion of short peptides leads to the exogenous load-
ing in vivo of MHC class I molecules on all cells that
have such molecules, including T cells and B cells.164
Moreover, these B and T cells circulate and, therefore,
also arrive in lymph nodes throughout the body in the
absence of immunostimulatory adjuvants. This una-
voidably leads to immunological tolerance because T
and B cells, in contrast to properly activated DC, lack
the co-stimulatory surface molecules required for
appropriate effector CTL generation.
Another advantage of long-peptide vaccines over
minimal CTL-peptide vaccines is the increased dura-
tion of in vivo epitope presentation in the antigen-
draining lymph node,164 previously shown to be impor-
tant for clonal expansion165 and for IFN production
by effector T cells.166 Such improved in vivo presenta-
tion and concomitant induction of T-cell expansion
by an extended CTL peptide epitope is particularly
apparent if the CTL epitope concerned displays
weaker MHC class I binding.164 Another advantage of
long-peptide vaccines is that they include a plethora
of immunogenic epitopes and thus are capable of
eliciting T-cell responses against many different HLA
class I and class II alleles.167 This will certainly prevent
escape of tumours from immunosurveillance through
mutation of T-cell epitopes and down-regulation of
certain HLA alleles. Therefore, injection of long-pep-
tide vaccines will ensure pluralism in the anti-tumour
response in which several tumour-specific CD8+ and
CD4+ T cells will contribute to more efficient tumour
killing.
Cancer immunotherapy   175
Clinical trials using polyepitope
long-peptide vaccines
The necessity of CD4 T-cell help to generate and sustain
MHC class I-restricted CD8 T-cell responses has led
not only to the use of MHC class II-restricted epitopes
derived from the same protein,168,169 but also to uni-
versal, nonspecific MHC class II-restricted epitopes,
such as PADRE, in clinical vaccination trials.170,171 Two
clinical studies172,173 determined whether HER-2/neu–
specific CD8 T-cell immunity could be elicited using
HER-2/neu–derived MHC class II helper peptides,
which contained encompassed HLA-A2–binding
motifs. Nineteen HLA-A2 patients with HER-2/neu–
overexpressing cancers received a vaccine prepara-
tion consisting of putative HER-2/neu helper peptides
p369–384, p688–703, and p971–984. Contained within
these sequences are the HLA-A2–binding motifs p369–
377, p689–697, and p971–979. Following vaccination,
the mean peptide-specific T-cell precursor frequency
to the HLA-A2 peptides increased in the majority of
patients. In addition, the peptide-specific T cells were
able to lyse tumours. The responses were long lived
and detectable for more than one year after the final
vaccination in patients. These results demonstrated
that HER-2/neu MHC class II epitopes containing
encompassed MHC class I epitopes are able to induce
long-lasting HER-2–specific IFN-–producing CD8 T
cells. In addition, the generation of protein-specific
immunity after peptide immunization was associated
with epitope spreading reflecting the initiation of an
endogenous immune response.
A polyepitope peptide vaccine, named IDM-
2101, has been evaluated for safety and induction of
immune responses in phase I trials in colon cancer
and metastatic non-small-cell lung cancer (NSCLC)
patients174 and for clinical efficacy in phase II tri-
als in NSCLC patients.175 The IDM-2101 vaccine was
designed to induce CTLs against five tumour-asso-
ciated antigens (TAAs) frequently overexpressed in
NSCLC (i.e. carcinoembryonic antigen, p53, HER-
2/neu, and melanoma antigens [MAGE] 2 and 3).
IDM-2101 is composed of 10 synthetic peptides from
these TAAs, nine of the peptides representing CTL
epitopes. Each CTL epitope is restricted by HLA-A2.1
and at least one other member of the HLA-A2 super-
family of major histocompatibility complex class I
molecules, providing coverage of approximately 45%
of the general population. The tenth synthetic peptide
is the pan-DR epitope PADRE. In the phase I studies,
no significant adverse effects were observed, and a
strong CTL response in the majority of patients was
noticed.174
In the phase II study, patients achieved initial sta-
ble disease and overall survival in comparison with
 176   C. N. Baxevanis et al.
historical patients who received conventional treat-
ment.175 A novel melanoma vaccine comprising six
melanoma-associated peptides as antigenic targets
for melanoma reactive helper T cells was evaluated
for safety and immunogenicity in a phase I/II trial.176
Vaccination with the helper peptide vaccine was well
tolerated. Proliferation assays revealed induction of
T-cell responses to the melanoma helper peptides
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
in 81% of patients. Among patients with measurable
disease, objective clinical responses were observed in
two patients (12%), with response durations of 1 and
3.9 years. Durable stable disease was observed in two
additional patients for periods of 1.8 and 4.6 years.
Welters et  al.177 initiated a phase I/II clinical vac-
cination study in end-stage cervical cancer patients
with a vaccine consisting of a set of 13 overlapping
peptides, 27–35 amino acids in length, of the complete
sequence of the HPV16 E6 and E7 oncogenic proteins.
The vaccine, in Montanide adjuvant, was well toler-
ated, and one out of 35 patients had a complete and
lasting disease regression. Five additional patients
achieved ­stable disease with salvage chemotherapy
after vaccination lasting up to two years, but, on aver-
age, all other patients died within five months. This
For personal use only.
indicated that therapeutic vaccination in this cate-
gory of patients has only limited therapeutic action by
itself, but might exert much better therapeutic activity
in conjunction with conventional chemotherapy or
radiation therapy.
At the same time, six patients who had been sur-
gically treated for HPV16-positive cervical cancer
were subcutaneously vaccinated with the same set of
overlapping long peptides. Vaccine-induced HPV16
E6-specific T-cell responses were detected in six out
of six patients and HPV16 E7-specific T-cell responses
in five out of six patients. The responses were broad,
comprised both HPV16-specific CD4+ and CD8+
T-cells, and could be detected up to 12 months after
the last vaccination. The vaccine-induced responses
were dominated by effector-type CD4+CD25+FOXP3−
type 1 cytokine IFN-producing T cells (Th cells),
but also included the expansion of T cells with a
CD4+CD25+FOXP3+ phenotype (Tregs). The pres-
ence and vaccine-induced increase of HPV16-specific
Tregs indicate that strategies to eliminate or disarm
these cells should be considered for immunothera-
peutic strategies against HPV-induced cancers.
The same group conducted a phase II vaccina-
tion study178 in patients with HPV16-positive vulvar
intraepithelial neoplasia grade III, a premalignant
condition. Patients were vaccinated four times with
the same vaccine, which consisted of 13 overlapping
long peptides of the E6 and E7 oncoproteins of HPV16.
Of the patients analyzed, 25% achieved complete
regression of all lesions, and a number of patients
showed partial regression (>50% reduction in size)
of the lesions with marked relief of symptoms. These
results are encouraging because further improvement
can be expected from co-formulation of this vaccine
with a potent TLR ligand.
We are currently utilizing peptide HER-2/neu
(776–790), chemically linked to a four amino-acid
long peptide from the regulatory region of the MHC
class II invariant chain (Ii-Key),152 for enhanced
immunogenicity, in a phase I trial with HER-2/neu+
prostate cancer patients. Only grade I toxicity has
been observed in 10% of the patients, whereas 80% of
those (24 of 30) have responded with increased immu-
nological responses to the vaccine in vivo (measured
as delayed-type hypersensitivity (DTH)) and in vitro
(as determined by the IFN-based ELISPOT assay).
We are observing similar results using this Ii-Key
conjugated HER-2/neu peptide in an ongoing phase
II study with HER-2/neu+ node-positive and high risk
node-negative breast cancer patients for prevention of
recurrences. Although our peptide vaccine is injected
admixed with GM-CSF as an adjuvant, strong DTH
responses in some of the patients also can be detected
in the absence of GM-CSF, demonstrating the high
effectiveness of the Ii-Key/peptide formulation.
Adoptive cellular immunotherapy
Adoptive transfer of autologous tumour-specific T
cells, as initially reported in the late 1980s, has been
shown to reproducibly mediate the destruction of
bulky tumours in a respectable percentage of the
patients treated.179 This approach is mainly based on
growing tumour-infiltrating lymphocytes ex vivo from
surgically excised tumour specimens of patients and
then adoptively transferring them into the patient
along with bolus high-dose IL-2.8 This type of immu-
notherapy has the advantage that one can administer
extremely high numbers of non-modified or geneti-
cally engineered lymphocytes with high avidity for
tumour antigens.9 Furthermore, adoptive cellular
immunotherapy (ACT) can include both CD4+ and
CD8+ T cells capable of recognizing tumour antigens
and synergistically acting in the anti-tumour immune
response.180,181 These cells are capable of massive
expansion in both mice and humans. Another advan-
tage of ACT is that the adoptively transferred T cells
are capable of trafficking to virtually every somatic
site.182 However, these promises have not translated
into clinical successes for cancer patients. Even
where tumour regression or complete responses were
achieved, there was usually relapse of the disease.183–
185
Increasing numbers of reports over recent years
have highlighted potential immunosuppressive

mechanisms that act on tumours and systemically
in cancer patients to block effective anti-tumour
immune responses.186 One method to overcome the
immunosuppressive environment induced by the
tumours is to use lymphodepleting preparative regi-
mens to provide an altered environment for trans-
ferred cells.187,188
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
Augmentation of adoptive immunotherapy through
lymphodepletion
Adoptive transfer of ex vivo expanded anti-tumour
T cells after a nonmyeloablative lymphodepleting
preparative regimen with chemotherapeutic rea-
gents is the most effective treatment for patients with
metastatic melanoma. When anti-tumour T cells are
given after lymphodepletion, usually 50% of patients
refractory to other treatments experience objective
clinical responses with this treatment.187,189 Not all
patients’ tumours yield anti-tumour T cells, however,
and only about one-third of the patients experience
durable tumour regressions. Thus, there are signifi-
cant efforts to continue to improve current therapies.
For personal use only.
Preclinical data show that increasing the intensity of
lymphodepletion to a myeloablative preparative regi-
men that requires hematopoietic stem cell transplant
might further improve the effectiveness of adoptive
cell transfer.190,191 Nevertheless, this type of immuno-
therapy, besides melanoma, should also be applied to
other types of cancer.
How does lymphodepletion improve adoptive T-cell
therapy?
While the exact mechanisms by which lymphodeple-
tion regimens lead to improved responses to adoptive
T-cell transfer remain to be defined, three ­non-mutually
exclusive models have been proposed. In one scenario,
prior depletion of lymphocytes may create “space” for
the adoptively transferred cells within the lymphocyte
compartment. In this model, homeostatic mechanisms
result in increased proliferation and enhanced survival
of the transferred T cells, largely through increased
access to endogenous common-chain cytokines, such
as IL-2, IL-7, and IL-15.23,192
Myeloablative lymphodepletion regimens involv-
ing total body irradiation also induce superior anti-tu-
mour responses when combined with adoptive T-cell
transfer, as shown in both human cancer patients
and in animal models.192 Radiation induces damage
to the gut, leading to the translocation of microbes
Cancer immunotherapy   177
from the gut and the systemic liberation of bacterial-
derived lipopolysaccharide. This TLR 4 agonist, then,
acts to activate the innate immune system, leading
to increased levels of proinflammatory cytokines,
activated DC, and enhanced anti-tumour function of
adoptively transferred T cells.
The third mechanism by which lymphodeple-
tion is thought to augment ACT is through the
depletion of immune cells that act to suppress
immune responses in vivo.193 Tregs are a subset
of CD4+ T  cells that are known to inhibit both the
activation and proliferation of cytotoxic and helper
T cells, and they are likely to play a major role in
the induction of functional immune tolerance to
tumours often observed in cancer patients.194 Other
regulatory CD4+ T-cell subsets likely targeted by
lymphodepletion include Tr1 and Th3 cells, which
produce the immunosuppressive cytokines IL-10
and TGF-ß, respectively.195,196 Increased numbers of
Tregs in cancer patients have been correlated with
an unfavourable prognosis, arguing that elimination
of these cells may result in an improved efficacy of
adoptive immunotherapy.197 A novel mechanism
that promotes anti-tumour immunity was recently
suggested by Shvets et al.,198 who found that in irra-
diated mice undergoing homeostatic proliferation,
T cells displayed severely decreased expression of
inhibitory molecules PD-1 and CTLA-A4, which
rendered them insensitive to tumour-induced sup-
pressor mechanisms. In addition, these cells could
not be converted to Tregs since they were refractory
to Foxp3 expression upon treatment with TGF.
Although lymphodepletion is effective at eliminat-
ing regulatory T cells in vivo, recent studies have
shown that these cells repopulate the periphery very
rapidly, highlighting the transient and possibly lim-
iting nature of this approach.199
The improved clinical results from lymphodeple-
tion in the context of ACT, combined with the recent
discovery and characterization of diverse regulatory
immune cell subsets, provided new strategies for
tumour immunotherapy. These are based on the very
important point that immunoregulatory mechanisms
that suppress anti-tumour responses need to be iden-
tified and addressed in a more efficacious manner. It
probably will not matter how many effector T cells we
can generate or how efficiently they can kill tumour
cells in vitro if they are being totally suppressed in vivo.
Thus, it is becoming apparent that by understanding
the regulatory complexicity of the immune system,
one can begin to design truly rational treatment strate-
gies designed to shift the balance in favour of immune
activation.
 178   C. N. Baxevanis et al.
Cancer immunotherapy using adoptive transfer of
in vitro-activated NKT cells
Lipid antigens bind to CD1 molecules with their
alkyl chains buried in hydrophobic pockets and
expose their polar lipid headgroup whose fine struc-
ture is recognized by the TCR of CD1-restricted NKT
cells.200–202 CD1-restricted T cells carry out effector,
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
helper, and adjuvant-like functions and interact
with other cell types, including macrophages, DC,
NK cells, T cells, and B cells, thereby contributing to
both innate and adaptive immune responses.203–205
Insights gained from mice and humans now deline-
ate the extensive range of diseases in which CD1-
restricted T cells play important roles and reveal
differences in the role of CD1a, CD1b, and CD1c
in contrast to CD1d.206 Invariant TCR alpha chains
(V24JQ gene segment in humans and V14J281
in mice), self-lipid reactivity, and rapid effector
responses uncover a subset of CD1d-restricted T
cells (NKT cells) having unique effector functions
without a counterpart among MHC-restricted T
cells.
Changes in V24+Vb11+ NKT cell number and
For personal use only.
function are associated with human autoimmune
diseases and cancer.206–209 Restoration of this cor-
responding NKT cell population in mice or in vivo
activation with alpha-galactosylceramide (-GalCer;
KRN7000) can prevent or reduce tumour growth and
autoimmunity.210–212 Although the therapeutic value
of these NKT cells in man remains to be determined,
large numbers of functional antigen-specific NKT
cells can be expanded in vitro. V24+V11+ human
NKT cells can be expanded continuously by repeated
stimulation with KRN7000, from unfractionated
donor peripheral blood mononuclear cells (PBMC),
in the presence of IL-2 for more than two months
with a potential yield of >1012 cells.213 Expanded NKT
cells retain their CD4+ or CD4- phenotype after res-
timulation and are functional as shown by cytokine
secretion, killing of antigen-pulsed target cells, and
activation of NK cell cytotoxicity.213 n vitro expanded
V24 NKT cells show substantial cytotoxicity against
tumour target cells.213,214
Adoptive transfer of in vitro activated NKT cells
was performed in patients with lung cancer who
were refractory to standard treatment.215 Intravenous
injection of activated V24 NKT cells was car-
ried out to test immunological, safety, and clinical
responses. Transferred V24 NKT cells, co-cultured
with -GalCer and IL-2 for two or three weeks, mostly
expressed either double negative or CD8+ NKT cells,
which have been reported to produce Th1-type
cytokines.216 After two administrations of activated
V24 NKT cells, the circulating V24
NKT cells increased in two out of three cases, and
augmentation of IFN--producing cells in PBMCs was
detected by an ELISPOT assay. Intracellular cytokine
staining showed the majority of IFN- producing cells
detected after activated NKT cell administration to be
CD3- CD56+ NK cells, but not CD3+ cells. Therefore,
-GalCer-activated V24 NKT cells appeared to sub-
sequently stimulate NK cells to produce IFN-.215
Clinical trials aiming at endogenous activation
of NKT cells have also been reported. In a phase
I study, Giaccone et  al.217 administered KRN7000
intravenously in 24 patients with solid tumours.
No dose-limiting toxicity was observed over a wide
range of doses (50–4800 µg/m2). Immunomonitoring
demonstrated that NKT cells (CD3+V24+Vß11+)
typically disappeared from the blood within 24 hours
of KRN7000 injection. Additional biological effects
included increased serum cytokine levels (TNF- and
GM-CSF) in five of 24 patients and a transient decrease
in peripheral blood NK cell numbers and cytotoxic-
ity in seven of 24 patients. Importantly, the observed
biological effects depended on pre-treatment NKT-
cell numbers rather than on the dose of KRN7000.
Pre-treatment NKT-cell numbers were significantly
lower in patients compared with healthy controls
(P = 0.0001). No clinical responses were recorded,
and seven patients experienced stable disease for a
median duration of 123 days.
n preclinical studies, several laboratories have
demonstrated that -GalCer loaded onto DC is more
efficient than soluble -GalCer in stimulating activa-
tion and expansion of endogenous anti-tumour NKT
cells.218,219 In a phase I study,220 -GalCer-pulsed DC
were intravenously administered in 11 lung cancer
patients at escalating doses. No severe adverse events
were observed during this study in any patient. After
the first and second injection of -GalCer-pulsed
DC, a dramatic increase in peripheral blood Va24
NKT cells was observed in one case, and significant
responses were seen in two cases. No patient was
found to meet the criteria for partial or complete
responses, whereas two cases remained unchanged
for more than a year with good quality of life.
Nieda et  al.221 designed a phase I study to treat
patients having metastatic malignancies with
-GalCer-pulsed CD1d-expressing DC. The results
showed that this therapy had substantial, rapid, and
highly reproducible specific effects on V24+V11+
NKT cells and provided the first human in vivo evi-
dence that V24+V11+ NKT cell stimulation leads
to activation of both innate and acquired immu-
nity, resulting in modulation of NK, T-, and B-cell
numbers and increased serum IFN. Uchida et al.222
conducted a phase I study with -GalCer-pulsed
antigen presenting cells (APCs) administered to the

nasal submucosa of 11 patients with head and neck
cancer and evaluated the safety and feasibility of such
a treatment. During the clinical study period, no seri-
ous adverse events were observed. Following the first
and second administration of a-GalCer-pulsed APCs,
an increased number of NKT cells were observed in
four patients, and enhanced natural killer activity was
detected in the peripheral blood of eight patients.
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
Although the objective anti-tumour response
rate remains low at present, continuous refine-
ment to augment the cytolytic activity is needed,
without losing the safety profile that is the marked
advantage of cancer immunotherapy. Considering
the mechanisms of the anti-tumour effect via NKT
cells, the development of a combination therapy
seems quite reasonable, since the NKT cells show an
adjuvant effect and modulate other immune effec-
tor cells. Combinations with mAb-based immuno-
therapy may be interesting additional approaches
to investigate. As previously mentioned, other rea-
gents activating distinct cell populations that pro-
mote adaptive immunity, such as peptide vaccine
to activate tumour antigen-specific CD8+ T cells,
might also be useful for activating tumour-specific
For personal use only.
immunity in combination with NKT cell-activation
immunotherapy.
Clinical application of NK cells
NK cells kill many tumour cell lines and may also
play a critical role in anti-tumour immunity.223,224
For tumours with low-to-undetectable MHC mol-
ecule expression, NK cells may be the predominant
immune effector mechanism of defence.225 NK cells
can also kill tumour cells that express normal levels of
MHC class I through interaction between activation
of natural cytotoxicity receptors and specific MHC-
like ligands on target cells.226,227 In addition to direct
cytotoxic activity against malignant cells, NK cells are
believed to play a crucial role in the early phase of
adaptive immunity engagement against both infec-
tious agents and tumour cells, thus providing a key
link between innate and adaptive immunity.228,229 In
particular, NK cell-mediated tumour killing appears
to elicit an efficient T-cell response, likely by inducing
DC maturation.230,231 IL-2, a powerful stimulator of NK
cell cytotoxic activity, has been successfully combined
with different vaccination regimens in humans,232–234
but the relative contribution to the anti-tumour effect
is difficult to determine and has been rarely investi-
gated.235 Moreover, the toxicity associated with the
systemic administration of IL-2 has hampered the
clinical utilization of this cytokine. The administra-
tion of other NK cell activators (e.g. IL-12, IL-15, IL-18,
Cancer immunotherapy   179
IL-21) as part of vaccination strategies has been suc-
cessfully tested in preclinical cancer models.224,236,237
Human NK cells discriminate between differ-
ent allelic forms of MHC molecules via killer cell
­immunoglobulin-like receptors (KIRs), which are
clonally distributed, and each cell in the repertoire
bears at least one receptor that is specific for self-MHC
class I molecules.238 Consequently, when faced with
mismatched allogeneic targets, NK cells in the reper-
toire will sense the missing expression of self-MHC
class I alleles and will mediate alloreactions.239,240
Donor versus recipient NK cell alloreactivity derives
from a mismatch between donor NK clones (carrying
specific inhibitory receptors for self-MHC class I mol-
ecules) and MHC class I ligands on recipient cells.
When faced with mismatched allogeneic targets, these
donor NK clones sense the missing expression of self-
HLA class I alleles and mediate alloreactions.241,242
Transplantation from NK alloreactive haploidenti-
cal donors controls acute myeloid leukemia (AML)
relapse and improves engraftment without causing
graft versus host disease (GvHD).243 The effectiveness
of NK-cell alloreactivity, as revealed in the haploi-
dentical transplant setting, has led to the establish-
ment of specific criteria for donor selection that have
enhanced survival rates of leukaemia patients,243–245
These results will encourage extending the use of
mismatched transplants to more leukaemia patients
without a matched donor. Other developments
might include designing protocols that will facilitate
exploitation of NK-cell alloreactivity in the setting of
unrelated donor transplantation. Perspectives from
murine models suggest that alloreactive NK cells
might become part of the conditioning regimen, as
they may potentiate the graft versus leukaemia effect
of the transplant,243–246 help engraftment, and protect
against T-cell–mediated GvHD. They might also be
infused post-transplant to help prevent or control
leukaemia relapse.
Studies are currently seeking to apply donor allore-
active NK cells beyond the field of transplantation as
adoptive immunotherapy for cancer. One can even
envisage ways to overcome autologous NK-cell inhi-
bition by self-HLA molecules to direct autologous
NK cells against tumours, thus obviating the need for
immune suppression and/or allogeneic transplanta-
tion. A study of relapsing acute myeloid leukemia
patients has demonstrated that the infusion of NK cells
from haploidentical donors is safe. Cells expanded
in vivo after lymphopenia was induced by high-dose
cyclophosphamide and fludarabine. Interestingly,
remissions were observed when potentially alloreactive
KIR-ligand mismatched donors were used.247 Another
report shows that NK-cell overexpression of activating
receptors overcomes the inhibitory signals delivered
 180   C. N. Baxevanis et al.
through receptors for HLA molecules and enhances
NK killing of NK-resistant leukaemia targets.101
The limiting factor in clinical protocols utilizing
NK cells for adoptive transfer remains the number of
appropriately activated NK that can be obtained.248
Usually the donor undergoes a four-to-five hour leu-
kapheresis, which is a protracted and cumbersome
procedure. We have recently reported249 novel data
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
on the effect of glucocorticoids (GCs), along with
stimulatory signals from IL-15 or IL-2, on NK cell
expansion and function. Although GCs have been
reported to exert an inhibitory effect on NK cells,
we have demonstrated that NK cells activated with
IL-15 (or IL-2) in the presence of hydrocortisone
(HC) not only retain their cytotoxic activity, but are
also protected from apoptosis, and their prolifera-
tive rate is significantly elevated. HC plus IL-15 is
capable of inducing up to a 400-fold expansion of
human NK cells within 20 days of culture compared
to about a 50-fold with IL-15 alone, thus making
feasible their large-scale production from a limited
volume of peripheral blood ( 200 mL of peripheral
blood could give rise to more than 109 NK cells
using this protocol) and their potential application
For personal use only.
in clinical protocols. Furthermore, expanding NK
cells in the presence of HC, thus rendering them
pre-conditioned to the presence of GCs, is advanta-
geous, since their intravenous injection into a recip-
ient exposes them to a microenvironment where
active cortisol is naturally present at doses similar
to those used in vitro.
Autologous NK cells, activated and expanded in
vitro in the presence of IL-15 and HC, have been
found to be effective in vivo in a lung metastasis
mouse model. These NK cells retained their ability
to efficiently infiltrate tumour-bearing lung tissue
and to persist for more than three days after NK cell
infusion. Furthermore, survival of animals with pre-
established lung tumours, treated with NK cells acti-
vated and expanded with IL-15 and HC, was more
than six-fold extended compared to untreated ani-
mals (unpublished data). We have initiated a phase
I clinical study in patients with advanced NSCLC,
who are adoptively transferred with allogeneic NK
cells expanded ex vivo with IL-2 or IL-15 and HC,
in order to take advantage of both, i.e. the novel NK
cell-expansion protocol to obtain large numbers
of appropriate effector cells and the KIR mismatch
between donor and recipient. The selection of lung
cancer is based on the observation that the majority
of intravenously administered NK cells accumulate
in the lung and are retained at the tumour site.250 This
protocol has been applied so far in 15 patients with
no toxic effects.
Conclusion
A plethora of knowledge has been obtained from pre-
clinical studies and clinical trials on the capacity of
the immune system to develop anti-cancer responses
and the multiple pathways through which immuno-
therapeutic modalities can boost a patient’s immune
system to fight its own tumour. Further elucidating
the molecular mechanisms underlying interactions
between tumours and the immune system effectors
will re-enforce our investigational capacity to devise
novel immunotherapeutic modalities, which will
prove to be more efficacious. It is also obvious that
the mechanisms underlying anti-tumour responses
need to be better delineated in pre-clinical models
and clinical trials. There are important conclusions to
be drawn from our current knowledge, and thus the
future of cancer immunotherapy is likely to include
the following strategies.
Increasing bodies of evidence suggest that cyto-
toxic drugs positively influence immune responses
against tumours. This “side” effect on tumour immu-
nology modifies the clinical response to chemother-
apy. An emerging modified modality suggests that
therapeutic vaccination increases clinical responses
to subsequent salvage chemotherapy. Thus, the
combination of vaccination and chemotherapy may
provide substantial clinical benefits for patients with
advanced cancer. Chemotherapy may also improve
immune-mediated destruction of tumours by facili-
tating the induction of an immune response against
tumour stroma.
Targeting tumour stroma as an immunotherapeutic
strategy may be very effective. Radiotherapy can also
be combined with vaccination strategies to improve
their effectiveness so long as attention is paid to dos-
ages and the timing of each treatment.
Therapeutic vaccination in late disease is at
present very ineffective, although we might be able
to see clinical responses in the face of multiepitope
vaccines (synthetic long peptides and naturally
occurring polypeptides) incorporating various CTL
and Th epitopes. Polypeptides will be exlusively
processed by DC, which will then trigger robust
anti-tumour responses. This effect can be further
enhanced by the use of DC-activating molecules
such as TLR ligands.
Targeted immunotherapies with anti-tumour
mAbs either as monotherapy or in combination with
chemotherapy or vaccines have shown great promise
in advanced cancers. mAbs exert their effects either by
inducing tumour cell apoptosis/necrosis or by form-
ing immune complexes with soluble tumour antigens,
which, in this way, will be processed and presented by

DC more quickly (i.e. through Fc receptor phagocyto-
sis). Moreover, mAbs may act to potentiate anti-tumour
immunity by depleting regulatory T cells, triggering
costimulatory molecules, or blocking coinhibition.
As a general option, immunotherapy intervention
requires tumour debulking and, therefore, should
be combined with surgery and chemotherapy.
Immunotherapeutic approaches should be tested in
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
patients whose tumours have been resected and who
are receiving adjuvant chemotherapy, but are at high
risk for recurrence. Tumour debulking will greatly
diminish immunosuppressive mechanisms that may
otherwise be induced by the cancer.
Last, and most important, there is growing evidence
pointing to the value of vaccinating at early stages of
cancer progression. Such preventive vaccines, when
applied in patients with cancer at early stages, when
the tumour load is low enough to establish a local and
systemic immunosuppressive network, may induce
efficient anti-tumour responses and enhanced clini-
cal benefit. Advances in early cancer detection meth-
ods and the development of efficacious cancer vac-
cines will allow vaccination of patients with various
types of cancer at early stages of disease, when they
For personal use only.
still having intact immune systems. This may become
the most promising strategy for preventing tumour
progression. Currently, realization of preventive vac-
cination will be feasible in patients with certain types
of cancer (e.g. breast, prostate, ovary, and colon) who
are free of disease after treatment with conventional
therapy, but at high risk of recurrence. It is prob-
able that an effective preventive peptide-vaccination
modality will require polyepitope vaccines, combined
with strong adjuvants (e.g. TLR agonists) and immu-
nomodulatory mAbs. Phase I/II studies assessing
preventive combined immunotherapy will definitely
provide the platform for designing more efficacious
protocols for cancer therapy.
However, autoimmunity is a possible side effect
of cancer immunotherapy in those instances when
intense immunotherapy is necessary, and thus the
incidence of autoimmunity in early or especially in
advanced cancer should be evaluated in the short or
long term following immunotherapy.
A large body of evidence has been gathered about
the potential of the immune system to control tumour
promotion and the various pathways through which
immunotherapy can induce robust immune responses
for fighting cancer. Such knowledge has stimulated the
invention of novel immunotherapeutic modalities,
including therapeutic antibodies, vaccines, and cell-
based treatments. Moreover, improvements in our
understanding of antiapoptotic and immune escape
mechanisms have resulted in the discovery of biologi-
cally active drugs capable of specifically targeting and
Cancer immunotherapy   181
altering such mechanisms. This progress in cancer
immunology will in the near future introduce elegant
combinatorial treatments involving targeting drugs
with novel immunotherapeutic strategies aiming at
immunostimulation with simultaneous elimination of
tumour-induced suppressive mechanisms. These new
therapies in cancer are expected to induce improved
clinical responses and, eventually, cancer prevention.
Declaration of interest: The authors report no con-
flicts of interest. The authors alone are responsible for
the content of this paper.
References
1. Kirkwood J. Cancer immunotherapy: the interferon-alpha
experience. Semin Oncol 2002; 29: 18–26.
2. Boyiadzis M, Foon KA. Approved monoclonal antibodies for
cancer therapy. Expert Opin Biol Ther 2008; 8: 1151–1158.
3. Baxevanis CN, Papamichail M. Targeting of tumor cells by
lymphocytes engineered to express chimeric receptor genes.
Cancer Immunol Immunother 2004; 53: 893–903.
4. Nimjee SM, Rusconi CP, Sullenger BA. Aptamers: an emerg-
ing class of therapeutics. Annu Rev Med 2005; 56: 555–583.
5. van der Burg SH, Bijker MS, Welters MJ, Offringa R, Melief CJ.
Improved peptide vaccine strategies, creating synthetic arti-
ficial infections to maximize immune efficacy. Adv Drug
Deliv Rev 2006; 58: 916–930.
6. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G.
Immunological aspects of cancer chemotherapy. Nat Rev
Immunol 2008; 8: 59–73.
7. Ramakrishnan R, Antonia S, Gabrilovich DI. Combined
modality immunotherapy and chemotherapy: a new perspec-
tive. Cancer Immunol Immunother 2008; 57: 1523–1529.
8. Rosenberg SA, Packard BS, Aebersold PM, Solomon D,
Topalian SL, Toy ST, Simon P, Lotze MT, Yang JC, Seipp CA
et al. Use of tumor-infiltrating lymphocytes and interleukin-2
in the immunotherapy of patients with metastatic melanoma.
A preliminary report. N Engl J Med 1988; 319: 1676–1680.
9. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS,
Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US,
Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-
Freezer LJ, Mavroukakis SA, Rosenberg SA. Cancer regres-
sion in patients after transfer of genetically engineered
­lymphocytes. Science 2006; 314: 126–129.
10. Wolf E, Hofmeister R, Kufer P, Schlereth B, Baeuerle PA.
BiTEs: bispecific antibody constructs with unique anti-tu-
mor activity. Drug Discov Today 2005; 10: 1237–1244.
11. Kirkwood JM, Ernstoff M. Melanoma: therapeutic options
with recombinant interferons. Semin Oncol 1985; 12: 7–12.
12. Kirkwood JM, Ernstoff MS, Davis CA, Reiss M, Ferraresi R,
Rudnick SA. Comparison of intramuscular and intravenous
recombinant alpha-2 interferon in melanoma and other
cancers. Ann Intern Med 1985; 103: 32–36.
13. Kirkwood JM, Strawderman MH, Ernstoff MS, Smith TJ,
Borden EC, Blum RH. Interferon alfa-2b adjuvant therapy
of high-risk resected cutaneous melanoma: the Eastern
Cooperative Oncology Group Trial EST 1684. J Clin Oncol
1996; 14: 7–17.
14. Kirkwood JM, Ibrahim JG, Sondak VK, Richards J, Flaherty LE,
Ernstoff MS, Smith TJ, Rao U, Steele M, Blum RH. High- and
low-dose interferon alfa-2b in high-risk melanoma: first
 182   C. N. Baxevanis et al.
analysis of intergroup trial E1690/S9111/C9190. J Clin Oncol
2000; 18: 2444–2458.
15. Kirkwood JM, Ibrahim JG, Sosman JA, Sondak VK,
Agarwala  SS, Ernstoff MS, Rao U. High-dose interferon
alfa-2b significantly prolongs relapse-free and overall
survival compared with the GM2-KLH/QS-21 vaccine in
patients with resected stage IIB-III melanoma: results of
intergroup trial E1694/S9512/C509801. J Clin Oncol 2001;
19: 2370–2380.
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
16. Kirkwood JM, Bender C, Agarwala S, Tarhini A, Shipe-
Spotloe J, Smelko B, Donnelly S, Stover L. Mechanisms and
management of toxicities associated with high-dose inter-
feron alfa-2b therapy. J Clin Oncol 2002;. 20: 3703–3718.
17. Smith KA. Interleukin-2: inception, impact, and implica-
tions. Science 1988; 240: 1169–1176.
18. Atkins MB, Kunkel L, Sznol M, Rosenberg SA. High-dose
recombinant interleukin-2 therapy in patients with meta-
static melanoma: long-term survival update. Cancer J Sci
Am 2000; 6(Suppl 1): S11–S14.
19. Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G,
Margolin  K, Abrams J, Sznol M, Parkinson D, Hawkins M,
Paradise C, Kunkel L, Rosenberg SA. High-dose recom-
binant interleukin 2 therapy for patients with metastatic
melanoma: analysis of 270 patients treated between 1985
and 1993. J Clin Oncol 1999; 17: 2105–2116.
20. Dutcher JP, Creekmore S, Weiss GR, Margolin K,
Markowitz AB, Roper M, Parkinson D, Ciobanu N, Fisher RI,
Boldt DH et  al. A phase II study of interleukin-2 and lym-
phokine-activated killer cells in patients with metastatic
For personal use only.
malignant melanoma. J Clin Oncol 1989; 7: 477–485.
21. Parkinson DR, Abrams JS, Wiernik PH, Rayner AA,
Margolin KA, Van Echo DA, Sznol M, Dutcher JP, Aronson FR,
Doroshow JH et  al. Interleukin-2 therapy in patients with
metastatic malignant melanoma: a phase II study. J Clin
Oncol 1990; 8: 1650–1656.
22. Schluns KS, Kieper WC, Jameson SC, Lefrancois L.
Interleukin-7 mediates the homeostasis of naive and mem-
ory CD8 T cells in vivo. Nat Immunol 2000; 1: 426–432.
23. Fry TJ, Mackall CL. Interleukin-7: master regulator of
peripheral T-cell homeostasis? Trends Immunol 2001; 22:
564–571.
24. Geiselhart LA, Humphries CA, Gregorio TA, Mou S,
Subleski  J, Komschlies KL. IL-7 administration alters the
CD4:CD8 ratio, increases T cell numbers, and increases T
cell function in the absence of activation. J Immunol 2001;
166: 3019–3027.
25. Fry TJ, Moniuszko M, Creekmore S, Donohue SJ, Douek DC,
Giardina S, Hecht TT, Hill BJ, Komschlies K, Tomaszewski J,
Franchini G, Mackall CL. IL-7 therapy dramatically alters
peripheral T-cell homeostasis in normal and SIV-infected
nonhuman primates. Blood 2003; 101: 2294–2299.
26. Storek J, Gillespy T, 3rd, Lu H, Joseph A, Dawson MA,
Gough  M, Morris J, Hackman RC, Horn PA, Sale GE,
Andrews RG, Maloney DG, Kiem HP. Interleukin-7 improves
CD4 T-cell reconstitution after autologous CD34 cell trans-
plantation in monkeys. Blood 2003; 101: 4209–4218.
27. Fry TJ, Mackall CL. The many faces of IL-7: from lymphopoi-
esis to peripheral T cell maintenance. J Immunol 2005; 174:
6571–6576.
28. Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA,
Palmer  DC, Spiess PJ, Hwang LN, Yu Z, Wrzesinski C,
Heimann DM, Surh CD, Rosenberg SA, Restifo NP. Removal
of homeostatic cytokine sinks by lymphodepletion enhances
the efficacy of adoptively transferred tumor-specific CD8+ T
cells. J Exp Med 2005 202: 907–912.
29. Melchionda F, Fry TJ, Milliron MJ, McKirdy MA, Tagaya Y,
Mackall CL. Adjuvant IL-7 or IL-15 overcomes immunodo-
minance and improves survival of the CD8+ memory cell
pool. J Clin Invest 2005; 115: 1177–1187.
30. Rosenberg SA, Sportes C, Ahmadzadeh M, Fry TJ, Ngo LT,
Schwarz SL, Stetler-Stevenson M, Morton KE, Mavroukakis
SA, Morre M, Buffet R, Mackall CL, Gress RE. IL-7 admin-
istration to humans leads to expansion of CD8+ and CD4+
cells but a relative decrease of CD4+ T-regulatory cells.
J Immunother 2006; 29: 313–319.
31. Sportes C, Hakim FT, Memon SA, Zhang H, Chua KS,
Brown MR, Fleisher TA, Krumlauf MC, Babb RR, Chow CK,
Fry TJ, Engels J, Buffet R, Morre M, Amato RJ, Venzon DJ,
Korngold R, Pecora A, Gress RE, Mackall CL. Administration
of rhIL-7 in humans increases in vivo TCR repertoire diver-
sity by preferential expansion of naive T cell subsets. J Exp
Med 2008; 205: 1701–1714.
32. Burton JD, Bamford RN, Peters C, Grant AJ, Kurys G,
Goldman CK, Brennan J, Roessler E, Waldmann TA. A lym-
phokine, provisionally designated interleukin T and produced
by a human adult T-cell leukemia line, stimulates T-cell pro-
liferation and the induction of lymphokine-activated killer
cells. Proc Natl Acad Sci USA 1994; 91: 4935–4939.
33. Burkett PR, Koka R, Chien M, Chai S, Boone DL, Ma A.
Coordinate expression and trans presentation of inter-
leukin (IL)-15Ralpha and IL-15 supports natural killer cell
and memory CD8+ T cell homeostasis. J Exp Med 2004; 200:
825–834.
34. Dubois S, Mariner J, Waldmann TA, Tagaya Y. IL-15Ralpha
recycles and presents IL-15 in trans to neighboring cells.
Immunity 2002; 17: 537–547.
35. Tan JT, Ernst B, Kieper WC, LeRoy E, Sprent J, Surh CD.
Interleukin (IL)-15 and IL-7 jointly regulate homeostatic
proliferation of memory phenotype CD8+ cells but are not
required for memory phenotype CD4+ cells. J Exp Med 2002;
195: 1523–1532.
36. Becker TC, Wherry EJ, Boone D, Murali-Krishna K, Antia R,
Ma A, Ahmed R. Interleukin 15 is required for proliferative
renewal of virus-specific memory CD8 T cells. J Exp Med
2002; 195: 1541–1548.
37. Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K,
Cardones AR, Finkelstein SE, Palmer DC, Antony PA,
Hwang ST, Rosenberg SA, Waldmann TA, Restifo NP. Central
memory self/tumor-reactive CD8+ T cells confer superior
antitumor immunity compared with effector memory T
cells. Proc Natl Acad Sci USA 2005; 102: 9571–9576.
38. Sotiriadou NN, Perez SA, Gritzapis AD, Mahaira LG,
Salagianni M, Baxevanis CN, Papamichail M. Beneficial
effect of short-term exposure of human NK cells to IL15/IL12
and IL15/IL18 on cell apoptosis and function. Cell Immunol
2005; 234: 67–75.
39. Kobayashi H, Dubois S, Sato N, Sabzevari H, Sakai Y,
Waldmann TA, Tagaya Y. Role of trans-cellular IL-15 pres-
entation in the activation of NK cell-mediated killing, which
leads to enhanced tumor immunosurveillance. Blood 2005;
105: 721–727.
40. Klebanoff CA, Finkelstein SE, Surman DR, Lichtman MK,
Gattinoni L, Theoret MR, Grewal N, Spiess PJ, Antony  PA,
Palmer DC, Tagaya Y, Rosenberg SA, Waldmann TA,
Restifo NP. IL-15 enhances the in vivo antitumor activity of
tumor-reactive CD8+ T cells. Proc Natl Acad Sci USA 2004;
101: 1969–1974.
41. Teague RM, Sather BD, Sacks JA, Huang MZ, Dossett  ML,
Morimoto J, Tan X, Sutton SE, Cooke MP, Ohlen C,
Greenberg PD. Interleukin-15 rescues tolerant CD8+ T cells

for use in adoptive immunotherapy of established tumors.
Nat Med 2006; 12: 335–341.
42. Yadav D, Sarvetnick N. Cytokines and autoimmunity: redun-
dancy defines their complex nature. Curr Opin Immunol
2003; 15: 697–703.
43. Kennedy R, Celis E. Multiple roles for CD4+ T cells in
anti-tumor immune responses. Immunol Rev 2008; 222:
129–144.
44. Baxevanis CN, Gritzapis AD, Papamichail M. In vivo anti-
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
tumor activity of NKT cells activated by the combination of
IL-12 and IL-18. J Immunol 2003; 171: 2953–2959.
45. Smyth MJ, Thia KY, Street SE, Cretney E, Trapani JA,
Taniguchi M, Kawano T, Pelikan SB, Crowe NY, Godfrey DI.
Differential tumor surveillance by natural killer (NK) and
NKT cells. J Exp Med 2000; 191: 661–668.
46. Noguchi Y, Jungbluth A, Richards EC, Old LJ. Effect of inter-
leukin 12 on tumor induction by 3-methylcholanthrene.
Proc Natl Acad Sci USA 1996; 93: 11798–11801.
47. Nanni P, Nicoletti G, De Giovanni C, Landuzzi L, Di Carlo E,
Cavallo F, Pupa SM, Rossi I, Colombo MP, Ricci C, Astolfi A,
Musiani P, Forni G, Lollini PL. Combined allogeneic tumor
cell vaccination and systemic interleukin 12 prevents mam-
mary carcinogenesis in HER-2/neu transgenic mice. J Exp
Med 2001; 194: 1195–1205.
48. Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor
immunity and immunotherapy. Cytokine Growth Factor Rev
2002; 13: 155–168.
49. Voest EE, Kenyon BM, O’Reilly MS, Truitt G, D’Amato  RJ,
Folkman J. Inhibition of angiogenesis in vivo by
For personal use only.
­interleukin 12. J Natl Cancer Inst 1995; 87: 581–586.
50. Cebon J, Jager E, Shackleton MJ, Gibbs P, Davis ID,
Hopkins  W, Gibbs S, Chen Q, Karbach J, Jackson H,
MacGregor DP, Sturrock S, Vaughan H, Maraskovsky E,
Neumann A, Hoffman E, Sherman ML, Knuth A. Two phase I
studies of low dose recombinant human IL-12 with Melan-A
and influenza peptides in subjects with advanced malignant
melanoma. Cancer Immun 2003; 3: 7.
51. Peterson AC, Harlin H, Gajewski TF. Immunization with
Melan-A peptide-pulsed peripheral blood mononuclear
cells plus recombinant human interleukin-12 induces clini-
cal activity and T-cell responses in advanced melanoma.
J Clin Oncol 2003; 21: 2342–2348.
52. Hamid O, Solomon JC, Scotland R, Garcia M, Sian S, Ye W,
Groshen SL, Weber JS. Alum with interleukin-12 augments
immunity to a melanoma peptide vaccine: correlation with
time to relapse in patients with resected high-risk disease.
Clin Cancer Res 2007; 13: 215–222.
53. Gollob JA, Mier JW, Veenstra K, McDermott DF, Clancy D,
Clancy M, Atkins MB. Phase I trial of twice-weekly intra-
venous interleukin 12 in patients with metastatic renal cell
cancer or malignant melanoma: ability to maintain IFN-
gamma induction is associated with clinical response. Clin
Cancer Res 2000; 6: 1678–1692.
54. Spolski R, Leonard WJ. The Yin and Yang of interleukin-21
in allergy, autoimmunity and cancer. Curr Opin Immunol
2008; 20: 295–301.
55. Leonard WJ, Zeng R, Spolski R. Interleukin 21: a cytokine/
cytokine receptor system that has come of age. J Leukoc Biol
2008; 84: 348–356.
56. Wilk E, Kalippke K, Buyny S, Schmidt RE, Jacobs R. New
aspects of NK cell subset identification and inference of
NK cells’ regulatory capacity by assessing functional and
genomic profiles. Immunobiology 2008; 213: 271–283.
57. Ouyang W, Kolls JK, Zheng Y. The biological functions of T
helper 17 cell effector cytokines in inflammation. Immunity
2008; 28: 454–467.
Cancer immunotherapy   183
58. Perez SA, Mahaira LG, Sotiropoulou PA, Gritzapis AD,
Iliopoulou EG, Niarchos DK, Cacoullos NT, Kavalakis YG,
Antsaklis AI, Sotiriadou NN, Baxevanis CN, Papamichail M.
Effect of IL-21 on NK cells derived from different umbilical
cord blood populations. Int Immunol 2006; 18: 49–58.
59. Chen Z, Laurence A, O’Shea JJ. Signal transduction path-
ways and transcriptional regulation in the control of Th17
differentiation. Semin Immunol 2007; 19: 400–408.
60. Davis ID, Skak K, Smyth MJ, Kristjansen PE, Miller DM,
Sivakumar PV. Interleukin-21 signaling: functions in cancer
and autoimmunity. Clin Cancer Res 2007; 13: 6926–6932.
61. Köhler G, Milstein C. Continuous cultures of fused cells
secreting antibody of predefined specificity. Nature.
1975;256(5517):495–497.
62. Iannello A, Ahmad A. Role of antibody-dependent cell-me-
diated cytotoxicity in the efficacy of therapeutic anti-cancer
monoclonal antibodies. Cancer Metastasis Rev 2005; 24:
487–499.
63. Clynes R. Antitumor antibodies in the treatment of cancer:
Fc receptors link opsonic antibody with cellular immunity.
Hematol Oncol Clin North Am. 2006;20(3):585–612.
64. Strome SE, Sausville EA, Mann D. A mechanistic perspective
of monoclonal antibodies in cancer therapy beyond target-
related effects. Oncologist 2007; 12: 1084–1095.
65. Castillo J, Winer E, Quesenberry P. Newer monoclonal anti-
bodies for hematological malignancies. Exp Hematol 2008;
36: 755–768.
66. Kreitman RJ. Immunotoxins for targeted cancer therapy.
Aaps J 2006; 8: E532–E551.
67. Reff ME, Heard C. A review of modifications to recombinant
antibodies: attempt to increase efficacy in oncology applica-
tions. Crit Rev Oncol Hematol 2001; 40: 25–35.
68. Weiner GJ. Monoclonal antibody mechanisms of action in
cancer. Immunol Res 2007; 39: 271–278.
69. Perez SA, Karamouzis MV, Skarlos DV, Ardavanis A,
Sotiriadou NN, Iliopoulou EG, Salagianni ML, Orphanos G,
Baxevanis CN, Rigatos G, Papamichail M. CD4+CD25+ regu-
latory T-cell frequency in HER-2/neu (HER)-positive and
HER-negative advanced-stage breast cancer patients. Clin
Cancer Res 2007; 13: 2714–2721.
70. Melero I, Hervas-Stubbs S, Glennie M, Pardoll DM, Chen L.
Immunostimulatory monoclonal antibodies for cancer ther-
apy. Nat Rev Cancer 2007; 7: 95–106.
71. Baxevanis CN, Perez SA, Papamichail M. Combinatorial
treatments including vaccines, chemotherapy and mono-
clonal antibodies for cancer therapy. Cancer Immunol
Immunother 2009; 58: 317–324.
72. Goodwin RG, Din WS, Davis-Smith T, Anderson DM,
Gimpel  SD, Sato TA, Maliszewski CR, Brannan CI,
Copeland NG, Jenkins NA et al. Molecular cloning of a ligand
for the inducible T cell gene 4-1BB: a member of an emerg-
ing family of cytokines with homology to tumor necrosis fac-
tor. Eur J Immunol 1993; 23: 2631–2641.
73. Sica G, Chen L. Modulation of the immune response through
4-1BB. Adv Exp Med Biol 2000; 465: 355–362.
74. Teft WA, Kirchhof MG, Madrenas J. A molecular perspective
of CTLA-4 function. Annu Rev Immunol 2006; 24: 65–97.
75. Egen JG, Kuhns MS, Allison JP. CTLA-4: new insights into its
biological function and use in tumor immunotherapy. Nat
Immunol 2002; 3: 611–618.
76. Peggs KS, Quezada SA, Korman AJ, Allison JP. Principles
and use of anti-CTLA4 antibody in human cancer immuno-
therapy. Curr Opin Immunol 2006; 18: 206–213.
77. Hodge JW, Chakraborty M, Kudo-Saito C, Garnett CT,
Schlom J. Multiple costimulatory modalities enhance CTL
avidity. J Immunol 2005; 174: 5994–6004.
 184   C. N. Baxevanis et al.
78. Leach DR, Krummel MF, Allison JP. Enhancement of anti-
tumor immunity by CTLA-4 blockade. Science 1996; 271:
1734–1736.
79. Hodge JW, Schlom J. Comparative studies of a retrovirus ver-
sus a poxvirus vector in whole tumor-cell vaccines. Cancer
Res 1999; 59: 5106–5111.
80. Ribas A, Camacho LH, Lopez-Berestein G, Pavlov D,
Bulanhagui CA, Millham R, Comin-Anduix B, Reuben JM,
Seja E, Parker CA, Sharma A, Glaspy JA, Gomez-Navarro J.
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
Antitumor activity in melanoma and anti-self responses in a
phase I trial with the anti-cytotoxic T lymphocyte-associated
antigen 4 monoclonal antibody CP-675,206. J Clin Oncol 2005;
23: 8968–8977.
81. Ribas A, Hanson DC, Noe DA, Millham R, Guyot DJ,
Bernstein SH, Canniff PC, Sharma A, Gomez-Navarro J.
Tremelimumab (CP-675,206), a cytotoxic T lymphocyte
associated antigen 4 blocking monoclonal antibody in clini-
cal development for patients with cancer. Oncologist 2007;
12: 873–883.
82. Taniguchi T, Matsui H, Fujita T, Takaoka C, Kashima N,
Yoshimoto R, Hamuro J. Structure and expression of a cloned
cDNA for human interleukin-2. Nature 1983; 302: 305–310.
83. Small EJ, Tchekmedyian NS, Rini BI, Fong L, Lowy I,
Allison JP. A pilot trial of CTLA-4 blockade with human anti-
CTLA-4 in patients with hormone-refractory prostate can-
cer. Clin Cancer Res 2007; 13: 1810–1815.
84. Tchekmedyian NS, Glaspy JA, Korman AJ. MDX-010 (human
anti-CTLA4): a phase I trial in malignant melanoma. Proc
Am Soc Clin Oncol 2002; 21(abstr 56): 15a.
For personal use only.
85. Hodi FS, Mihm MC, Soiffer RJ, Haluska FG, Butler M,
Seiden MV, Davis T, Henry-Spires R, MacRae S, Willman A,
Padera R, Jaklitsch MT, Shankar S, Chen TC, Korman  A,
Allison JP, Dranoff G. Biologic activity of cytotoxic T lym-
phocyte-associated antigen 4 antibody blockade in pre-
viously vaccinated metastatic melanoma and ovarian
carcinoma patients. Proc Natl Acad Sci USA 2003; 100:
4712–4717.
86. Maker AV, Yang JC, Sherry RM, Topalian SL, Kammula US,
Royal RE, Hughes M, Yellin MJ, Haworth LR, Levy C, Allen T,
Mavroukakis SA, Attia P, Rosenberg SA. Intrapatient dose
escalation of anti-CTLA-4 antibody in patients with meta-
static melanoma. J Immunother 2006; 29: 455–463.
87. Hersh E, Weber J, Powderley J. Disease control and long term
survival in chemotherapy naive patients with advanced
melanoma treated with ipilimumab (MDX-010) with or with-
out dacarbazine. J Clin Oncol 2008; 26(abstr 9022): 488s.
88. Small EJ, Higano C, Tchekmedyian NS. Randomized phase II
study comparing 4 monthly doses of ipilimumab (MDX-010)
as a single agent or in combination with a single dose of
docetaxel in patients with hormone refractory prostate can-
cer. J Clin Oncol 2006; 24(abstr 4609): 243s.
89. Maker AV, Phan GQ, Attia P, Yang JC, Sherry RM,
Topalian SL, Kammula US, Royal RE, Haworth LR, Levy C,
Kleiner D, Mavroukakis SA, Yellin M, Rosenberg SA.
Tumor regression and autoimmunity in patients treated
with cytotoxic T lymphocyte-associated antigen 4 block-
ade and interleukin 2: a phase I/II study. Ann Surg Oncol
2005; 12: 1005–1016.
90. Attia P, Phan GQ, Maker AV, Robinson MR, Quezado MM,
Yang JC, Sherry RM, Topalian SL, Kammula US, Royal RE,
Restifo NP, Haworth LR, Levy C, Mavroukakis SA, Nichol G,
Yellin MJ, Rosenberg SA. Autoimmunity correlates with
tumor regression in patients with metastatic melanoma
treated with anti-cytotoxic T-lymphocyte antigen-4. J Clin
Oncol 2005; 23: 6043–6053.
91. Hombach A, Wieczarkowiecz A, Marquardt T, Heuser C,
Usai L, Pohl C, Seliger B, Abken H. Tumor-specific T cell
activation by recombinant immunoreceptors: CD3 zeta sig-
naling and CD28 costimulation are simultaneously required
for efficient IL-2 secretion and can be integrated into one
combined CD28/CD3 zeta signaling receptor molecule.
J Immunol 2001; 167: 6123–6131.
92. Gritzapis AD, Mamalaki A, Kretsovali A, Papamatheakis  J,
Belimezi M, Perez SA, Baxevanis CN, Papamichail M.
Redirecting mouse T hybridoma against human breast and
ovarian carcinomas: in vivo activity against HER-2/neu
expressing cancer cells. Br J Cancer 2003; 88: 1292–1300.
93. Mamalaki A, Gritzapis AD, Kretsovali A, Belimezi M,
Papamatheakis J, Perez SA, Papamichail M, Baxevanis CN.
In vitro and in vivo antitumor activity of a mouse CTL hybri-
doma expressing chimeric receptors bearing the single chain
Fv from HER-2/neu- specific antibody and the gamma-chain
from Fc(epsilon) RI. Cancer Immunol Immunother 2003; 52:
513–522.
94. Eshhar Z. The T-body approach: redirecting T cells with anti-
body specificity. Handb Exp Pharmacol 2008; 181: 329–342.
95. Kershaw MH, Jackson JT, Haynes NM, Teng MW, Moeller M,
Hayakawa Y, Street SE, Cameron R, Tanner JE, Trapani JA,
Smyth MJ, Darcy PK. Gene-engineered T cells as a superior
adjuvant therapy for metastatic cancer. J Immunol 2004; 173:
2143–2150.
96. Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar  Z,
Mavroukakis SA, White DE, Wunderlich JR, Canevari  S,
Rogers-Freezer L, Chen CC, Yang JC, Rosenberg SA, Hwu P. A
phase I study on adoptive immunotherapy using gene-mod-
ified T cells for ovarian cancer. Clin Cancer Res 2006; 12:
6106–6115.
97. Pinthus JH, Waks T, Malina V, Kaufman-Francis K,
Harmelin  A, Aizenberg I, Kanety H, Ramon J, Eshhar Z.
Adoptive immunotherapy of prostate cancer bone lesions
using redirected effector lymphocytes. J Clin Invest 2004;
114: 1774–1781.
98. Uherek C, Tonn T, Uherek B, Becker S, Schnierle B,
Klingemann HG, Wels W. Retargeting of natural killer-cell
cytolytic activity to ErbB2-expressing cancer cells results in
efficient and selective tumor cell destruction. Blood 2002;
100: 1265–1273.
99. Schirrmann T, Pecher G. Tumor-specific targeting of a cell
line with natural killer cell activity by asialoglycoprotein
receptor gene transfer. Cancer Immunol Immunother 2001;
50: 549–556.
100. Muller T, Uherek C, Maki G, Chow KU, Schimpf A, Klingemann
HG, Tonn T, Wels WS. Expression of a CD20-specific chimeric
antigen receptor enhances cytotoxic activity of NK cells and
overcomes NK-resistance of lymphoma and leukemia cells.
Cancer Immunol Immunother 2008; 57: 411–423.
101. Imai C, Iwamoto S, Campana D. Genetic modification of
primary natural killer cells overcomes inhibitory signals and
induces specific killing of leukemic cells. Blood 2005; 106:
376–383.
102. Pegram HJ, Jackson JT, Smyth MJ, Kershaw MH, Darcy PK.
Adoptive transfer of gene-modified primary NK cells can
specifically inhibit tumor progression in vivo. J Immunol
2008; 181: 3449–3455.
103. Alvarez-Vallina L. Genetic approaches for antigen-selective
cell therapy. Curr Gene Ther 2001; 1: 385–397.
104. Ren-Heidenreich L, Lum LG. Life or death of T cells with
antigen-specific receptors—using T cells for cancer adop-
tive immunotherapy/gene therapy. Curr Gene Ther 2001; 1:
253–255.

105. Patel SD, Moskalenko M, Smith D, Maske B, Finer MH,
McArthur JG. Impact of chimeric immune receptor extracel-
lular protein domains on T cell function. Gene Ther 1999; 6:
412–419.
106. Hombach A, Schneider C, Sent D, Koch D, Willemsen RA,
Diehl V, Kruis W, Bolhuis RL, Pohl C, Abken H. An entirely
humanized CD3 zeta-chain signaling receptor that directs
peripheral blood t cells to specific lysis of carcinoembry-
onic antigen-positive tumor cells. Int J Cancer 2000; 88:
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
115–120.
107. Hoogenboom HR, Marks JD, Griffiths AD, Winter G. Building
antibodies from their genes. Immunol Rev 1992; 130: 41–68.
108. Levey DL, Srivastava PK. Alterations in T cells of cancer-bear-
ers: whence specificity? Immunol Today 1996; 17: 365–368.
109. Zier K, Gansbacher B, Salvadori S. Preventing abnormali-
ties in signal transduction of T cells in cancer: the promise
of cytokine gene therapy. Immunol Today 1996; 17: 39–45.
110. Melisi D, Troiani T, Damiano V, Tortora G, Ciardiello F.
Therapeutic integration of signal transduction targeting
agents and conventional anti-cancer treatments. Endocr
Relat Cancer 2004; 11: 51–68.
111. Dollins CM, Nair S, Sullenger BA. Aptamers in immuno-
therapy. Hum Gene Ther 2008; 19: 443–450.
112. McNamara JO, Kolonias D, Pastor F, Mittler RS, Chen L,
Giangrande PH, Sullenger B, Gilboa E. Multivalent 4-1BB
binding aptamers costimulate CD8+ T cells and inhibit
tumor growth in mice. J Clin Invest 2008; 118: 376–386.
113. White RR, Sullenger BA, Rusconi CP. Developing aptamers
into therapeutics. J Clin Invest 2000; 106: 929–934.
For personal use only.
114. Ng EW, Shima DT, Calias P, Cunningham ET, Jr., Guyer DR,
Adamis AP. Pegaptanib, a targeted anti-VEGF aptamer
for ocular vascular disease. Nat Rev Drug Discov 2006; 5:
123–132.
115. Santulli-Marotto S, Nair SK, Rusconi C, Sullenger B, Gilboa E.
Multivalent RNA aptamers that inhibit CTLA-4 and enhance
tumor immunity. Cancer Res 2003; 63: 7483–7489.
116. Dollins CM, Nair S, Boczkowski D, Lee J, Layzer JM, Gilboa E,
Sullenger BA. Assembling OX40 aptamers on a molecular
scaffold to create a receptor-activating aptamer. Chem Biol
2008; 15: 675–682.
117. Offner S, Hofmeister R, Romaniuk A, Kufer P, Baeuerle PA.
Induction of regular cytolytic T cell synapses by bispecific
single-chain antibody constructs on MHC class I-negative
tumor cells. Mol Immunol 2006; 43: 763–771.
118. Dreier T, Baeuerle PA, Fichtner I, Grun M, Schlereth B,
Lorenczewski G, Kufer P, Lutterbuse R, Riethmuller G,
Gjorstrup P, Bargou RC. T cell costimulus-independent and
very efficacious inhibition of tumor growth in mice bear-
ing subcutaneous or leukemic human B cell lymphoma
xenografts by a CD19-/CD3- bispecific single-chain anti-
body construct. J Immunol 2003; 170: 4397–4402.
119. Hoffmann P, Hofmeister R, Brischwein K, Brandl C,
Crommer S, Bargou R, Itin C, Prang N, Baeuerle PA. Serial
killing of tumor cells by cytotoxic T cells redirected with a
CD19-/CD3-bispecific single-chain antibody construct. Int J
Cancer 2005; 115: 98–104.
120. Brischwein K, Schlereth B, Guller B, Steiger C, Wolf A,
Lutterbuese R, Offner S, Locher M, Urbig T, Raum T,
Kleindienst P, Wimberger P, Kimmig R, Fichtner I, Kufer P,
Hofmeister R, da Silva AJ, Baeuerle PA. MT110: a novel bis-
pecific single-chain antibody construct with high efficacy
in eradicating established tumors. Mol Immunol 2006; 43:
1129–1143.
121. Schlereth B, Fichtner I, Lorenczewski G, Kleindienst P,
Brischwein K, da Silva A, Kufer P, Lutterbuese R, Junghahn I,
Kasimir-Bauer S, Wimberger P, Kimmig R, Baeuerle PA.
Cancer immunotherapy   185
Eradication of tumors from a human colon cancer cell line
and from ovarian cancer metastases in immunodeficient
mice by a single-chain Ep-CAM-/CD3-bispecific antibody
construct. Cancer Res 2005; 65: 2882–2889.
122. Hammond SA, Lutterbuese R, Roff S, Lutterbuese P,
Schlereth B, Bruckheimer E, Kinch MS, Coats S, Baeuerle PA,
Kufer P, Kiener PA. Selective targeting and potent control of
tumor growth using an EphA2/CD3-Bispecific single-chain
antibody construct. Cancer Res 2007; 67: 3927–3935.
123. Ribas A. Update on immunotherapy for melanoma. J Natl
Compr Canc Netw 2006; 4: 687–694.
124. Bijker MS, Melief CJ, Offringa R, van der Burg SH. Design and
development of synthetic peptide vaccines: past, present
and future. Expert Rev Vaccines 2007; 6: 591–603.
125. Kanodia S, Da Silva DM, Kast WM. Recent advances in strat-
egies for immunotherapy of human papillomavirus-induced
lesions. Int J Cancer 2008; 122: 247–259.
126. Pol S, Michel ML. Therapeutic vaccination in chronic
hepatitis B virus carriers. Expert Rev Vaccines 2006; 5:
707–716.
127. Gilboa E. DC-based cancer vaccines. J Clin Invest 2007; 117:
1195–1203.
128. Cavallo F, Offringa R, van der Burg SH, Forni G, Melief CJ.
Vaccination for treatment and prevention of cancer in ani-
mal models. Adv Immunol 2006; 90: 175–213.
129. Melief CJ, van der Burg SH. Immunotherapy of established
(pre)malignant disease by synthetic long peptide vaccines.
Nat Rev Cancer 2008; 8: 351–360.
130. Kim CJ, Dessureault S, Gabrilovich D, Reintgen DS,
Slingluff  CL Jr. Immunotherapy for melanoma. Cancer
Control 2002; 9: 22–30.
131. Steinman RM, Mellman I. Immunotherapy: bewitched,
bothered, and bewildered no more. Science 2004; 305:
197–200.
132. Sutmuller RP, van Duivenvoorde LM, van Elsas A,
Schumacher TN, Wildenberg ME, Allison JP, Toes RE,
Offringa R, Melief CJ. Synergism of cytotoxic T lymphocyte-
associated antigen 4 blockade and depletion of CD25(+) reg-
ulatory T cells in antitumor therapy reveals alternative path-
ways for suppression of autoreactive cytotoxic T lymphocyte
responses. J Exp Med 2001; 194: 823–832.
133. Kirkwood JM, Tarhini AA, Panelli MC, Moschos SJ, Zarour
HM, Butterfield LH, Gogas HJ. Next generation of immuno-
therapy for melanoma. J Clin Oncol 2008; 26: 3445–3455.
134. Mahnke K, Schonfeld K, Fondel S, Ring S, Karakhanova S,
Wiedemeyer K, Bedke T, Johnson TS, Storn V, Schallenberg S,
Enk AH. Depletion of CD4+CD25+ human regulatory T
cells in vivo: kinetics of Treg depletion and alterations in
immune functions in vivo and in vitro. Int J Cancer 2007;
120: 2723–2733.
135. Rasku MA, Clem AL, Telang S, Taft B, Gettings K, Gragg H,
Cramer D, Lear SC, McMasters KM, Miller DM, Chesney J.
Transient T cell depletion causes regression of melanoma
metastases. J Transl Med 2008; 6: 12.
136. Munn DH, Mellor AL. IDO and tolerance to tumors. Trends
Mol Med 2004; 10: 15–18.
137. Insinga A, Monestiroli S, Ronzoni S, Gelmetti V, Marchesi F,
Viale A, Altucci L, Nervi C, Minucci S, Pelicci PG. Inhibitors
of histone deacetylases induce tumor-selective apoptosis
through activation of the death receptor pathway. Nat Med
2005; 11: 71–76.
138. Gollob JA, Sciambi CJ. Decitabine up-regulates S100A2
expression and synergizes with IFN-gamma to kill uveal
melanoma cells. Clin Cancer Res 2007; 13: 5219–5225.
139. Vlaykova T, Talve L, Hahka-Kemppinen M, Hernberg M,
Muhonen T, Collan Y, Pyrhonen S. Immunohistochemically
 186   C. N. Baxevanis et al.
detectable bcl-2 expression in metastatic melanoma: asso-
ciation with survival and treatment response. Oncology
2002; 62: 259–268.
140. Maiuri MC, Le Toumelin G, Criollo A, Rain JC, Gautier F,
Juin P, Tasdemir E, Pierron G, Troulinaki K, Tavernarakis N,
Hickman JA, Geneste O, Kroemer G. Functional and physi-
cal interaction between Bcl-X(L) and a BH3-like domain in
Beclin-1. Embo J 2007; 26: 2527–2539.
141. Franco AV, Zhang XD, Van Berkel E, Sanders JE, Zhang XY,
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
Thomas WD, Nguyen T, Hersey P. The role of NF-kappa  B
in TNF-related apoptosis-inducing ligand (TRAIL)-
induced apoptosis of melanoma cells. J Immunol 2001; 166:
5337–5345.
142. Sayers TJ, Brooks AD, Koh CY, Ma W, Seki N, Raziuddin A,
Blazar BR, Zhang X, Elliott PJ, Murphy WJ. The protea-
some inhibitor PS-341 sensitizes neoplastic cells to TRAIL-
mediated apoptosis by reducing levels of c-FLIP. Blood 2003;
102: 303–310.
143. Peoples GE, Gurney JM, Hueman MT, Woll MM, Ryan GB,
Storrer CE, Fisher C, Shriver CD, Ioannides CG, Ponniah S.
Clinical trial results of a HER2/neu (E75) vaccine to prevent
recurrence in high-risk breast cancer patients. J Clin Oncol
2005; 23: 7536–7545.
144. Holmes JP, Benavides LC, Gates JD, Carmichael MG,
Hueman  MT, Mittendorf EA, Murray JL, Amin A, Craig  D,
von Hofe E, Ponniah S, Peoples GE. Results of the first
phase I clinical trial of the novel II-key hybrid preventive
HER-2/neu peptide (AE37) vaccine. J Clin Oncol 2008; 26:
3426–3433.
For personal use only.
145. Apostolopoulos V, Pietersz GA, Tsibanis A, Tsikkinis A,
Drakaki H, Loveland BE, Piddlesden SJ, Plebanski M,
Pouniotis DS, Alexis MN, McKenzie IF, Vassilaros S. Pilot
phase III immunotherapy study in early-stage breast cancer
patients using oxidized mannan-MUC1 [ISRCTN71711835].
Breast Cancer Res 2006; 8: R27.
146. Machiels JP, Reilly RT, Emens LA, Ercolini AM, Lei RY,
Weintraub D, Okoye FI, Jaffee EM. Cyclophosphamide,
doxorubicin, and paclitaxel enhance the antitumor immune
response of granulocyte/macrophage-colony stimulating
factor-secreting whole-cell vaccines in HER-2/neu tolerized
mice. Cancer Res 2001; 61: 3689–3697.
147. Arlen PM, Gulley JL, Parker C, Skarupa L, Pazdur M,
Panicali  D, Beetham P, Tsang KY, Grosenbach DW,
Feldman J, Steinberg SM, Jones E, Chen C, Marte J, Schlom J,
Dahut  W. A randomized phase II study of concurrent
docetaxel plus vaccine versus vaccine alone in metastatic
androgen-independent prostate cancer. Clin Cancer Res
2006; 12: 1260–1269.
148. Gribben JG, Ryan DP, Boyajian R, Urban RG, Hedley  ML,
Beach K, Nealon P, Matulonis U, Campos S, Gilligan  TD,
Richardson PG, Marshall B, Neuberg D, Nadler LM.
Unexpected association between induction of immunity to
the universal tumor antigen CYP1B1 and response to next
therapy. Clin Cancer Res 2005; 11: 4430–4436.
149. Maecker B, Sherr DH, Vonderheide RH, von Bergwelt-
Baildon MS, Hirano N, Anderson KS, Xia Z, Butler MO,
Wucherpfennig KW, O’Hara C, Cole G, Kwak SS, Ramstedt U,
Tomlinson AJ, Chicz RM, Nadler LM, Schultze JL. The
shared tumor-associated antigen cytochrome P450 1B1 is
recognized by specific cytotoxic T cells. Blood 2003; 102:
3287–3294.
150. Antonia SJ, Mirza N, Fricke I, Chiappori A, Thompson  P,
Williams N, Bepler G, Simon G, Janssen W, Lee JH, Menander K,
Chada S, Gabrilovich DI. Combination of p53 cancer vaccine
with chemotherapy in patients with extensive stage small cell
lung cancer. Clin Cancer Res 2006; 12: 878–887.
151. Wheeler CJ, Das A, Liu G, Yu JS, Black KL. Clinical respon-
siveness of glioblastoma multiforme to chemotherapy after
vaccination. Clin Cancer Res 2004; 10: 5316–5326.
152. Sotiriadou NN, Kallinteris NL, Gritzapis AD, Voutsas IF,
Papamichail M, von Hofe E, Humphreys RE, Pavlis T,
Perez SA, Baxevanis CN. Ii-Key/HER-2/neu(776-790) hybrid
peptides induce more effective immunological responses
over the native peptide in lymphocyte cultures from patients
with HER-2/neu+ tumors. Cancer Immunol Immunother
2007; 56: 601–613.
153. Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF,
Heath WR. Help for cytotoxic-T-cell responses is mediated
by CD40 signalling. Nature 1998; 393: 478–480.
154. Schoenberger SP, Toes RE, van der Voort EI, Offringa R,
Melief CJ. T-cell help for cytotoxic T lymphocytes is mediated
by CD40-CD40L interactions. Nature 1998; 393: 480–483.
155. Scheibenbogen C, Schadendorf D, Bechrakis NE,
Nagorsen  D, Hofmann U, Servetopoulou F, Letsch A,
Philipp  A, Foerster  MH, Schmittel A, Thiel E, Keilholz U.
Effects of granulocyte-macrophage colony-stimulating fac-
tor and foreign helper protein as immunologic adjuvants on
the T-cell response to vaccination with tyrosinase peptides.
Int J Cancer 2003; 104: 188–194.
156. Salucci V, Mennuni C, Calvaruso F, Cerino R, Neuner P,
Ciliberto G, La Monica N, Scarselli E. CD8+ T-cell tolerance
can be broken by an adenoviral vaccine while CD4+ T-cell
tolerance is broken by additional co-administration of a
Toll-like receptor ligand. Scand J Immunol 2006; 63: 35–41.
157. Ahonen CL, Doxsee CL, McGurran SM, Riter TR, Wade WF,
Barth RJ, Vasilakos JP, Noelle RJ, Kedl RM. Combined TLR
and CD40 triggering induces potent CD8+ T cell expansion
with variable dependence on type I IFN. J Exp Med 2004;
199: 775–784.
158. Schwarz K, Storni T, Manolova V, Didierlaurent A, Sirard JC,
Rothlisberger P, Bachmann MF. Role of Toll-like receptors
in costimulating cytotoxic T cell responses. Eur J Immunol
2003; 33: 1465–1470.
159. Zwaveling S, Ferreira Mota SC, Nouta J, Johnson M,
Lipford  GB, Offringa R, van der Burg SH, Melief CJ.
Established human papillomavirus type 16-expressing
tumors are effectively eradicated following vaccination with
long peptides. J Immunol 2002; 169: 350–358.
160. Kast WM, Brandt RM, Melief CJ. Strict peptide length is
not required for the induction of cytotoxic T lymphocyte-
mediated antiviral protection by peptide vaccination. Eur J
Immunol 1993; 23: 1189–1192.
161. Gao XM, Zheng B, Liew FY, Brett S, Tite J. Priming of influ-
enza virus-specific cytotoxic T lymphocytes vivo by short
synthetic peptides. J Immunol 1991; 147: 3268–3273.
162. Minev BR, McFarland BJ, Spiess PJ, Rosenberg SA, Restifo NP.
Insertion signal sequence fused to minimal peptides elicits
specific CD8+ T-cell responses and prolongs survival of thy-
moma-bearing mice. Cancer Res 1994; 54: 4155–4161.
163. Reinholdsson-Ljunggren G, Ramqvist T, Ahrlund-Richter L,
Dalianis T. Immunization against polyoma tumors with syn-
thetic peptides derived from the sequences of middle- and
large-T antigens. Int J Cancer 1992; 50: 142–146.
164. Bijker MS, van den Eeden SJ, Franken KL, Melief CJ, van der
Burg SH, Offringa R. Superior induction of anti-tumor CTL
immunity by extended peptide vaccines involves prolonged,
DC-focused antigen presentation. Eur J Immunol 2008; 38:
1033–1042.
165. Lefrancois L, Marzo A, Williams K. Sustained response initi-
ation is required for T cell clonal expansion but not for effec-
tor or memory development in vivo. J Immunol 2003; 171:
2832–2839.

166. Obst R, van Santen HM, Melamed R, Kamphorst AO,
Benoist  C, Mathis D. Sustained antigen presentation
can promote an immunogenic T cell response, like den-
dritic cell activation. Proc Natl Acad Sci USA 2007; 104:
15460–15465.
167. Offringa R, van der Burg SH, Ossendorp F, Toes RE, Melief CJ.
Design and evaluation of antigen-specific vaccination strat-
egies against cancer. Curr Opin Immunol 2000; 12: 576–582.
168. Widmann C, Romero P, Maryanski JL, Corradin G,
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
Valmori D. T helper epitopes enhance the cytotoxic response
of mice immunized with MHC class I-restricted malaria
peptides. J Immunol Methods 1992; 155: 95–99.
169. Fayolle C, Deriaud E, Leclerc C. In vivo induction of cyto-
toxic T cell response by a free synthetic peptide requires
CD4+ T cell help. J Immunol 1991; 147: 4069–4073.
170. Weber JS, Hua FL, Spears L, Marty V, Kuniyoshi C, Celis E. A
phase I trial of an HLA-A1 restricted MAGE-3 epitope peptide
with incomplete Freund’s adjuvant in patients with resected
high-risk melanoma. J Immunother 1999; 22: 431–440.
171. Ressing ME, van Driel WJ, Brandt RM, Kenter GG, de Jong JH,
Bauknecht T, Fleuren GJ, Hoogerhout P, Offringa R, Sette A,
Celis E, Grey H, Trimbos BJ, Kast WM, Melief CJ. Detection of
T helper responses, but not of human papillomavirus-spe-
cific cytotoxic T lymphocyte responses, after peptide vac-
cination of patients with cervical carcinoma. J Immunother
2000; 23: 255–266.
172. Disis ML, Gooley TA, Rinn K, Davis D, Piepkorn M,
Cheever MA, Knutson KL, Schiffman K. Generation of T-cell
immunity to the HER-2/neu protein after active immuniza-
For personal use only.
tion with HER-2/neu peptide-based vaccines. J Clin Oncol
2002; 20: 2624–2632.
173. Knutson KL, Schiffman K, Disis ML. Immunization with a
HER-2/neu helper peptide vaccine generates HER-2/neu
CD8 T-cell immunity in cancer patients. J Clin Invest 2001;
107: 477–484.
174. Beebe M, Qin M, Moi M, Wu S, Heiati H, Walker L, Newman M,
Fikes J, Ishioka GY. Formulation and Characterization
of a Ten-peptide Single-vial Vaccine, EP-2101, Designed
to Induce Cytotoxic T-lymphocyte Responses for Cancer
Immunotherapy. Hum Vaccin. 2008; 4(3):37–45.
175. Barve M, Bender J, Senzer N, Cunningham C, Greco FA,
McCune D, Steis R, Khong H, Richards D, Stephenson J,
Ganesa P, Nemunaitis J, Ishioka G, Pappen B, Nemunaitis M,
Morse M, Mills B, Maples PB, Sherman J, Nemunaitis  JJ.
Induction of immune responses and clinical efficacy
in a phase II trial of IDM-2101, a 10-epitope cytotoxic
T-lymphocyte vaccine, in metastatic non-small-cell lung
cancer. J Clin Oncol 2008; 26: 4418–4425.
176. Slingluff CL, Jr., Petroni GR, Olson W, Czarkowski A,
Grosh  WW, Smolkin M, Chianese-Bullock KA, Neese  PY,
Deacon DH, Nail C, Merrill P, Fink R, Patterson JW, Rehm PK.
Helper T-cell responses and clinical activity of a melanoma
vaccine with multiple peptides from MAGE and melanocytic
differentiation antigens. J Clin Oncol 2008; 26: 4973–4980.
177. Welters MJ, Kenter GG, Piersma SJ, Vloon AP, Lowik MJ,
Berends-van der Meer DM, Drijfhout JW, Valentijn AR,
Wafelman AR, Oostendorp J, Fleuren GJ, Offringa R,
Melief  CJ, van der Burg SH. Induction of tumor-specific
CD4+ and CD8+ T-cell immunity in cervical cancer patients
by a human papillomavirus type 16 E6 and E7 long peptides
vaccine. Clin Cancer Res 2008; 14: 178–187.
178. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-
van der Meer DM, Vloon AP, Drijfhout JW, Wafelman AR,
Oostendorp J, Fleuren GJ, Offringa R, van der Burg SH,
Melief CJ. Phase I immunotherapeutic trial with long pep-
tides spanning the E6 and E7 sequences of high-risk human
Cancer immunotherapy   187
papillomavirus 16 in end-stage cervical cancer patients
shows low toxicity and robust immunogenicity. Clin Cancer
Res 2008; 14: 169–177.
179. Rosenberg SA, Dudley ME. Cancer regression in patients
with metastatic melanoma after the transfer of autologous
antitumor lymphocytes. Proc Natl Acad Sci USA 2004; 101:
14639–14645.
180. Surman DR, Dudley ME, Overwijk WW, Restifo NP. Cutting
edge: CD4+ T cell control of CD8+ T cell reactivity to a model
tumor antigen. J Immunol 2000; 164: 562–565.
181. Touloukian CE, Leitner WW, Topalian SL, Li YF, Robbins PF,
Rosenberg SA, Restifo NP. Identification of a MHC class
II-restricted human gp100 epitope using DR4-IE transgenic
mice. J Immunol 2000; 164: 3535–3542.
182. Palmer DC, Balasubramaniam S, Hanada K, Wrzesinski  C,
Yu  Z, Farid S, Theoret MR, Hwang LN, Klebanoff CA,
Gattinoni L, Goldstein AL, Yang JC, Restifo NP. Vaccine-
stimulated, adoptively transferred CD8+ T cells traffic indis-
criminately and ubiquitously while mediating specific tumor
destruction. J Immunol 2004; 173: 7209–7216.
183. Leen AM, Rooney CM, Foster AE. Improving T cell therapy
for cancer. Annu Rev Immunol 2007; 25: 243–265.
184. Greenberg PD, Cheever MA, Fefer A. Eradication of dis-
seminated murine leukemia by chemoimmunotherapy with
cyclophosphamide and adoptively transferred immune
syngeneic Lyt-1 + 2- lymphocytes. J Exp Med 1981; 154:
952–963.
185. Greenberg PD, Cheever MA, Treatment of disseminated
leukemia with cyclophosphamide and immune cells: tumor
immunity reflects long-term persistence of tumor-specific
donor T cells. J Immunol 1984; 133: 3401–3407.
186. Croci DO, Zacarias Fluck MF, Rico MJ, Matar P, Rabinovich GA,
Scharovsky OG. Dynamic cross-talk between tumor and
immune cells in orchestrating the immunosuppressive
network at the tumor microenvironment. Cancer Immunol
Immunother 2007; 56: 1687–1700.
187. Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL,
Restifo NP, Royal RE, Kammula U, White DE, Mavroukakis SA,
Rogers LJ, Gracia GJ, Jones SA, Mangiameli DP, Pelletier MM,
Gea-Banacloche J, Robinson MR, Berman DM, Filie AC,
Abati A, Rosenberg SA. Adoptive cell transfer therapy fol-
lowing non-myeloablative but lymphodepleting chemo-
therapy for the treatment of patients with refractory meta-
static melanoma. J Clin Oncol 2005; 23: 2346–2357.
188. Rapoport AP, Stadtmauer EA, Aqui N, Badros A, Cotte J,
Chrisley L, Veloso E, Zheng Z, Westphal S, Mair R, Chi N,
Ratterree B, Pochran MF, Natt S, Hinkle J, Sickles C, Sohal A,
Ruehle K, Lynch C, Zhang L, Porter DL, Luger S, Guo C,
Fang  HB, Blackwelder W, Hankey K, Mann D, Edelman R,
Frasch C, Levine BL, Cross A, June CH. Restoration of immu-
nity in lymphopenic individuals with cancer by vaccination
and adoptive T-cell transfer. Nat Med 2005; 11: 1230–1237.
189. Gattinoni L, Powell DJ Jr, Rosenberg SA, Restifo NP. Adoptive
immunotherapy for cancer: building on success. Nat Rev
Immunol 2006; 6: 383–393.
190. Wrzesinski C, Paulos CM, Gattinoni L, Palmer DC, Kaiser A,
Yu Z, Rosenberg SA, Restifo NP. Hematopoietic stem cells
promote the expansion and function of adoptively trans-
ferred antitumor CD8 T cells. J Clin Invest 2007; 1172:
492–501.
191. Muranski P, Boni A, Wrzesinski C, Citrin DE, Rosenberg SA,
Childs R, Restifo NP. Increased intensity lymphodepletion
and adoptive immunotherapy—how far can we go? Nat Clin
Pract Oncol 2006; 3: 668–681.
192. Paulos CM, Kaiser A, Wrzesinski C, Hinrichs CS, Cassard L,
Boni A, Muranski P, Sanchez-Perez L, Palmer DC, Yu Z,
 188   C. N. Baxevanis et al.
Antony PA, Gattinoni L, Rosenberg SA, Restifo NP. Toll-like
receptors in tumor immunotherapy. Clin Cancer Res 2007;
13: 5280–5289.
193. Lizee G, Radvanyi LG, Overwijk WW, Hwu P.
Immunosuppression in melanoma immunotherapy: poten-
tial opportunities for intervention. Clin Cancer Res 2006; 12:
2359s–2365s.
194. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+
regulatory T cells in immunological tolerance to self and
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
non-self. Nat Immunol 2005; 6: 345–352.
195. Roncarolo MG, Bacchetta R, Bordignon C, Narula S,
Levings MK. Type 1 T regulatory cells. Immunol Rev 2001;
182: 68–79.
196. Weiner HL. Induction and mechanism of action of trans-
forming growth factor-beta-secreting Th3 regulatory cells.
Immunol Rev 2001; 182: 207–214.
197. Lizee G, Radvanyi LG, Overwijk WW, Hwu P. Improving anti-
tumor immune responses by circumventing immunoregu-
latory cells and mechanisms. Clin Cancer Res 2006; 12:
4794–4803.
198. Shvets A, Chakrabarti R, Gonzalez-Quintial R, Baccala R,
Theofilopoulos AN, Prud’homme GJ. Impaired negative reg-
ulation of homeostatically proliferating T cells. Blood 2008;
133: 622–625.
199. Powell DJ, Jr, de Vries CR, Allen T, Ahmadzadeh M.
Rosenberg  SA. Inability to mediate prolonged reduction
of regulatory T Cells after transfer of autologous CD25-
depleted PBMC and interleukin-2 after lymphodepleting
chemotherapy. J Immunother 2007; 30: 438–447.
For personal use only.
200. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki  K,
Ueno H, Nakagawa R, Sato H, Kondo E, Koseki H,
Taniguchi M. CD1d-restricted and TCR-mediated activation
of valpha14 NKT cells by glycosylceramides. Science 1997;
278: 1626–1629.
201. Brossay L, Chioda M, Burdin N, Koezuka Y, Casorati G,
Dellabona P, Kronenberg M. CD1d-mediated recognition
of an alpha-galactosylceramide by natural killer T cells is
highly conserved through mammalian evolution. J Exp Med
1998; 188: 1521–1528.
202. Spada FM, Koezuka Y, Porcelli SA. CD1d-restricted recogni-
tion of synthetic glycolipid antigens by human natural killer
T cells. J Exp Med 1998; 188: 1529–1534.
203. Fujii S, Shimizu K, Smith C, Bonifaz L, Steinman RM.
Activation of natural killer T cells by alpha-galactosylcera-
mide rapidly induces the full maturation of dendritic cells in
vivo and thereby acts as an adjuvant for combined CD4 and
CD8 T cell immunity to a coadministered protein. J Exp Med
2003; 198: 267–279.
204. Fujii S, Liu K, Smith C, Bonito AJ, Steinman RM. The linkage
of innate to adaptive immunity via maturing dendritic cells
in vivo requires CD40 ligation in addition to antigen pres-
entation and CD80/86 costimulation. J Exp Med 2004; 199:
1607–1618.
205. Hayakawa Y, Takeda K, Yagita H, Kakuta S, Iwakura Y, Van
Kaer L, Saiki I, Okumura K. Critical contribution of IFN-
gamma and NK cells, but not perforin-mediated cytotoxic-
ity, to anti-metastatic effect of alpha-galactosylceramide.
Eur J Immunol 2001; 31: 1720–1727.
206. Brigl M, Brenner MB. CD1: antigen presentation and T cell
function. Annu Rev Immunol 2004; 22: 817–890.
207. Motohashi S, Kobayashi S, Ito T, Magara KK, Mikuni O,
Kamada N, Iizasa T, Nakayama T, Fujisawa T, Taniguchi M.
Preserved IFN-alpha production of circulating Valpha24
NKT cells in primary lung cancer patients. Int J Cancer 2002;
102: 159–165.
208. Tahir SM, Cheng O, Shaulov A, Koezuka Y, Bubley GJ,
Wilson SB, Balk SP, Exley MA. Loss of IFN-gamma produc-
tion by invariant NK T cellsps in advanced cancer. J Immunol
2001; 167: 4046–4050.
209. Taniguchi M, Harada M, Kojo S, Nakayama T, Wakao H.
The regulatory role of Valpha14 NKT cells in innate and
acquired immune response. Annu Rev Immunol 2003; 21:
483–513.
210. Linsen L, Somers V, Stinissen P. Immunoregulation of
autoimmunity by natural killer T cells. Hum Immunol 2005;
66: 1193–1202.
211. Yamamura T, Sakuishi K, Illes Z, Miyake S. Understanding
the behavior of invariant NKT cells in autoimmune diseases.
J Neuroimmunol 2007; 191: 8–15.
212. Molling JW, Moreno M, de Groot J, van der Vliet HJ,
von Blomberg BM, van den Eertwegh AJ, Scheper RJ,
Bontkes HJ. Chronically stimulated mouse invariant NKT
cell lines have a preserved capacity to enhance protection
against experimental tumor metastases. Immunol Lett
2008; 118: 36–43.
213. Rogers PR, Matsumoto A, Naidenko O, Kronenberg M,
Mikayama T, Kato S. Expansion of human Valpha24+ NKT
cells by repeated stimulation with KRN7000. J Immunol
Methods 2004; 285: 197–214.
214. Nishi N, van der Vliet HJ, Koezuka Y, von Blomberg BM,
Scheper RJ, Pinedo HM, Giaccone G. Synergistic effect of
KRN7000 with interleukin-15, -7, and -2 on the expansion of
human V alpha 24+V beta 11+ T cells in vitro. Hum Immunol
2000; 61: 357–365.
215. Motohashi S, Ishikawa A, Ishikawa E, Otsuji M, Iizasa T,
Hanaoka H, Shimizu N, Horiguchi S, Okamoto Y, Fujii S,
Taniguchi M, Fujisawa T, Nakayama T. A phase I study of
in vitro expanded natural killer T cells in patients with
advanced and recurrent non-small cell lung cancer. Clin
Cancer Res 2006; 12: 6079–6086.
216. Lee PT, Benlagha K, Teyton L, Bendelac A. Distinct func-
tional lineages of human V(alpha)24 natural killer T cells.
J Exp Med 2002; 195: 637–641.
217. Giaccone G, Punt CJ, Ando Y, Ruijter R, Nishi N, Peters M,
von Blomberg BM, Scheper RJ, van der Vliet HJ, van den
Eertwegh AJ, Roelvink M, Beijnen J, Zwierzina H, Pinedo HM.
A phase I study of the natural killer T-cell ligand alpha-ga-
lactosylceramide (KRN7000) in patients with solid tumors.
Clin Cancer Res 2002; 8: 3702–3709.
218. Toura I, Kawano T, Akutsu Y, Nakayama T, Ochiai T,
Taniguchi M. Cutting edge: inhibition of experimental tumor
metastasis by dendritic cells pulsed with alpha-galactosyl-
ceramide. J Immunol 1999; 163: 2387–2391.
219. Fujii S, Shimizu K, Kronenberg M, Steinman RM. Prolonged
IFN-gamma-producing NKT response induced with alpha-
galactosylceramide-loaded DCs. Nat Immunol 2002; 3:
867–874.
220. Ishikawa A, Motohashi S, Ishikawa E, Fuchida H,
Higashino K, Otsuji M, Iizasa T, Nakayama T, Taniguchi M,
Fujisawa T. A phase I study of alpha-galactosylceramide
(KRN7000)-pulsed dendritic cells in patients with advanced
and recurrent non-small cell lung cancer. Clin Cancer Res
2005; 11: 1910–1917.
221. Nieda M, Okai M, Tazbirkova A, Lin H, Yamaura A, Ide K,
Abraham R, Juji T, Macfarlane DJ, Nicol AJ. Therapeutic
activation of Valpha24+Vbeta11+ NKT cells in human sub-
jects results in highly coordinated secondary activation of
acquired and innate immunity. Blood 2004; 103: 383–389.
222. Uchida T, Horiguchi S, Tanaka Y, Yamamoto H, Kunii N,
Motohashi S, Taniguchi M, Nakayama T, Okamoto Y. Phase I

study of alpha-galactosylceramide-pulsed antigen present-
ing cells administration to the nasal submucosa in unresect-
able or recurrent head and neck cancer. Cancer Immunol
Immunother 2008; 57: 337–345.
223. Cheng WF, Hung CF, Lin KY, Ling M, Juang J, He L, Lin CT,
Wu TC. CD8+ T cells, NK cells and IFN-gamma are important
for control of tumor with downregulated MHC class I expres-
sion by DNA vaccination. Gene Ther 2003; 10: 1311–1320.
224. Ma HL, Whitters MJ, Konz RF, Senices M, Young DA,
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Notre Dame Australia on 06/14/13
Grusby MJ, Collins M, Dunussi-Joannopoulos K. IL-21 acti-
vates both innate and adaptive immunity to generate potent
antitumor responses that require perforin but are independ-
ent of IFN-gamma. J Immunol 2003; 171: 608–615.
225. Cerwenka A, Baron JL, Lanier LL. Ectopic expression of
retinoic acid early inducible-1 gene (RAE-1) permits natural
killer cell-mediated rejection of a MHC class I-bearing tumor
in vivo. Proc Natl Acad Sci USA 2001; 98: 11521–11526.
226. Moretta A, Bottino C, Vitale M, Pende D, Cantoni C,
Mingari MC, Biassoni R, Moretta L. Activating receptors and
coreceptors involved in human natural killer cell-mediated
cytolysis. Annu Rev Immunol 2001; 19: 197–223.
227. Wu J, Lanier LL. Natural killer cells and cancer. Adv Cancer
Res 2003; 90: 127–156.
228. Mocikat R, Braumuller H, Gumy A, Egeter O, Ziegler H,
Reusch U, Bubeck A, Louis J, Mailhammer R, Riethmuller G,
Koszinowski U, Rocken M. Natural killer cells activated by
MHC class I(low) targets prime dendritic cells to induce pro-
tective CD8 T cell responses. Immunity 2003; 19: 561–569.
229. Diefenbach A, Raulet DH. The innate immune response
For personal use only.
to tumors and its role in the induction of T-cell immunity.
Immunol Rev 2002; 188: 9–21.
230. Kelly JM, Darcy PK, Markby JL, Godfrey DI, Takeda K,
Yagita H, Smyth MJ. Induction of tumor-specific T cell mem-
ory by NK cell-mediated tumor rejection. Nat Immunol
2002; 3: 83–90.
231. Moretta A. Natural killer cells and dendritic cells: rendez-
vous in abused tissues. Nat Rev Immunol 2002; 2: 957–964.
232. Lotem M, Shiloni E, Pappo I, Drize O, Hamburger T,
Weitzen  R, Isacson R, Kaduri L, Merims S, Frankenburg S,
Peretz T. Interleukin-2 improves tumour response to DNP-
modified autologous vaccine for the treatment of metastatic
malignant melanoma. Br J Cancer 2004; 90: 773–780.
233. Osanto S, Schiphorst PP, Weijl NI, Dijkstra N, Van Wees A,
Brouwenstein N, Vaessen N, Van Krieken JH, Hermans J,
Cleton FJ, Schrier PI. Vaccination of melanoma patients with
an allogeneic, genetically modified interleukin 2-producing
melanoma cell line. Hum Gene Ther 2000; 11: 739–750.
234. Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P,
Marincola FM, Topalian SL, Restifo NP, Dudley ME,
Schwarz  SL, Spiess PJ, Wunderlich JR, Parkhurst MR,
Kawakami Y, Seipp CA, Einhorn JH, White DE. Immunologic
and therapeutic evaluation of a synthetic peptide vaccine
for the treatment of patients with metastatic melanoma. Nat
Med 1998; 4: 321–327.
235. Moller P, Moller H, Sun Y, Dorbic T, Henz BM, Wittig B,
Schadendorf D. Increased non-major histocompatibility
complex-restricted lytic activity in melanoma patients vac-
cinated with cytokine gene-transfected autologous tumor
cells. Cancer Gene Ther 2000; 7: 976–984.
Cancer immunotherapy   189
236. Berzofsky JA, Ahlers JD, Belyakov IM. Strategies for designing
and optimizing new generation vaccines. Nat Rev Immunol
2001; 1: 209–219.
237. Lee P, Wang F, Kuniyoshi J, Rubio V, Stuges T, Groshen S,
Gee C, Lau R, Jeffery G, Margolin K, Marty V, Weber J. Effects
of interleukin-12 on the immune response to a multipeptide
vaccine for resected metastatic melanoma. Clin Oncol 2001;
19: 3836–3847.
238. Moretta L, Moretta A, Unravelling natural killer cell func-
tion: triggering and inhibitory human NK receptors. Embo
J 2004; 23: 255–259.
239. Moretta L, Ciccone E, Moretta A, Hoglund P, Ohlen C,
Karre  K. Allorecognition by NK cells: nonself or no self?
Immunol Today 1992; 13: 300–306.
240. Karre K. NK cells, MHC class I molecules and the missing
self. Scand J Immunol 2002; 55: 221–228.
241. Moretta A, Bottino C, Pende D, Tripodi G, Tambussi  G,
Viale  O, Orengo A, Barbaresi M, Merli A, Ciccone E,
Moretta  L. Identification of four subsets of human CD3-
CD16+ natural killer (NK) cells by the expression of clon-
ally distributed functional surface molecules: correlation
between subset assignment of NK clones and ability to
mediate specific alloantigen recognition. J Exp Med 1990;
172: 1589–1598.
242. Colonna M, Brooks EG, Falco M, Ferrara GB, Strominger JL.
Generation of allospecific natural killer cells by stimula-
tion across a polymorphism of HLA-C. Science 1993; 260:
1121–1124.
243. Ruggeri L, Capanni M, Tosti A, Urbani E, Posati S, Aversa F,
Martelli MF, Velardi A. Innate immunity against hematologi-
cal malignancies. Cytotherapy 2002; 4: 343–346.
244. van der Meer A, Schaap NP, Schattenberg AV, van
Cranenbroek B, Tijssen HJ, Joosten I. KIR2DS5 is associated
with leukemia free survival after HLA identical stem cell
transplantation in chronic myeloid leukemia patients. Mol
Immunol 2008; 45: 3631–3638.
245. Verheyden S, Demanet C. NK cell receptors and their ligands
in leukemia. Leukemia 2008; 22: 249–257.
246. Ruggeri L, Capanni M, Martelli MF, Velardi A. Cellular ther-
apy: exploiting NK cell alloreactivity in transplantation. Curr
Opin Hematol 2001; 8: 355–359.
247. Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA,
Yun GH, Fautsch SK, McKenna D, Le C, Defor TE, Burns LJ,
Orchard PJ, Blazar BR, Wagner JE, Slungaard A, Weisdorf DJ,
Okazaki IJ, McGlave PB. Successful adoptive transfer and in
vivo expansion of human haploidentical NK cells in patients
with cancer. Blood 2005; 105: 3051–3057.
248. Arai S, Klingemann HG. Natural killer cells: can they be use-
ful as adoptive immunotherapy for cancer? Expert Opin Biol
Ther 2005; 5: 163–172.
249. Perez SA, Mahaira LG, Demirtzoglou FJ, Sotiropoulou PA,
Ioannidis P, Iliopoulou EG, Gritzapis AD, Sotiriadou NN,
Baxevanis CN, Papamichail M. A potential role for hydrocor-
tisone in the positive regulation of IL-15-activated NK-cell
proliferation and survival. Blood 2005; 106: 158–166.
250. Albertsson PA, Basse PH, Hokland M, Goldfarb RH,
Nagelkerke JF, Nannmark U, Kuppen PJ. NK cells and the
tumour microenvironment: implications for NK-cell function
and anti-tumour activity. Trends Immunol 2003; 24: 603–609.