Hindawi Publishing Corporation

Clinical and Developmental Immunology

Volume 2011, Article ID 894704, 12 pages
doi:10.1155/2011/894704

Review Article
The Immune Response to Tumors as
a Tool toward Immunotherapy

F. Pandolfi, R. Cianci, D. Pagliari, F. Casciano, C. Bagalà, A. Astone, R. Landolfi, and C. Barone

Department of Internal Medicine, Catholic University of the Sacred Heart, Rome, Italy

Correspondence should be addressed to F. Pandolfi, pandolfi@rm.unicatt.it

Received 11 July 2011; Revised 8 September 2011; Accepted 20 September 2011

Academic Editor: Nima Rezaei

Copyright © 2011 F. Pandolfi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Until recently cancer medical therapy was limited to chemotherapy that could not differentiate cancer cells from normal cells. More
recently with the remarkable mushroom of immunology, newer tools became available, resulting in the novel possibility to attack
cancer with the specificity of the immune system. Herein we will review some of the recent achievement of immunotherapy in such
aggressive cancers as melanoma, prostatic cancer, colorectal carcinoma, and hematologic malignancies. Immunotherapy of tumors
has developed several techniques: immune cell transfer, vaccines, immunobiological molecules such as monoclonal antibodies
that improve the immune responses to tumors. This can be achieved by blocking pathways limiting the immune response, such
as CTLA-4 or Tregs. Immunotherapy may also use cytokines especially proinflammatory cytokines to enhance the activity of
cytotoxic T cells (CTLs) derived from tumor infiltrating lymphocytes (TILs). The role of newly discovered cytokines remains to be
investigated. Alternatively, an other mechanism consists in enhancing the expression of TAAs on tumor cells. Finally, monoclonal
antibodies may be used to target oncogenes.

1. Different Antigenicity of Tumors cancer, hepatocellular carcinoma, pancreatic carcinoma, pro-

state carcinoma, lymphomas and leukaemias, and others [3,
An important role of the immune system is to identify and 4].
eliminate tumors. Transformed cells of tumors express anti- The tumor-associated antigens (TAAs) expressed by tu-
gens that are not found on normal cells; these antigens are mors have several sources.
called tumor-associated antigens (TAAs). The immune sys-
tem recognized these antigens as not self and mounts an (a) Some are derived from oncogenic viruses like human
immune response against tumor cells. However, tumors de- papillomavirus, which causes cervical cancer [5]. The
velop several mechanisms to escape immune recognition. HPV oncoproteins E6 and E7 have crucial roles in
For instance, when T cells interact with tumors, they may various steps of carcinogenesis, inducing degradation
deliver several potential inhibitory signals, including lack of of p53 and destabilization of pRb. Several clinical
proper costimulatory activity by tumor cells and induction trials show that recombinant HPV vaccines are safe
of immunosuppressive Tregs [1, 2]. and effective in preventing persistent infection of
In the recent years, specific antigenic characterization has HPV and associated anogenital lesions. Thus, pro-
permitted us to study an increasing number of tumors, in phylactic HPV vaccination may be an ideal preventive
particular regarding their ability to escape from immune re- method for other HPV-associated cancers. Therefore,
sponse and to downmodulate TAA expression and secreting vaccine against papillomavirus may be considered a
inhibitory molecules. This has resulted in the identification very effective antitumor agent [6–8].
of tumors that elicit different immune responses: (1) strong (b) Other TAAs are cellular proteins usually present in
immunogenic tumors, such as melanoma and renal cell car- the human body that are overexpressed or aberrantly
cinoma, (2) the majority of tumors, however, are poorly expressed in tumor cells; furthermore, others TAAs
immunogenic tumors: these include, for instance, colorectal are also products of mutated genes.
2 Clinical and Developmental Immunology

(c) In addition, TAAs may also be the products of onco- confirmed Virchow’s suggestion of a direct connection be-
genes or mutated oncosoppressors. tween inflammation and cancer. The past 30 years have ac-
cumulated considerable evidence that many tumors elicit a
The most useful response of the immune system against
significant immune response, and a more favourable prog-
tumors is to kill the abnormal cells using CTLs, which
nosis is correlated with the levels of TILs [17]. Nevertheless,
abound among TILs [9, 10]. TAAs are presented on MHC
although tumor microenvironment TILs include tumor-
class I molecules. This allows CTLs to recognize the tumor
reactive T cells, melanoma can escape the immune system
cell as abnormal. NK cells also kill tumor cells by cytotoxicity,
and continue to grow and metastasize [1]. Studying these
especially if the tumor cells have fewer MHC class I mol-
mechanisms of immune escape of the tumor will improve the
ecules on their surface than normal; this being a common
strategies to overcome obstacles to successful immunother-
phenomenon in tumors.
apy of tumors.
Upon activation, CTLs express on their surface the death
Melanoma is characterized by the expression of several
activator designated Fas ligand (FasL) and the engagement of
TAAs, which can be recognized by T cells, resulting in a
Fas/FasL pathway lead to mediated apoptosis of cancer cells
strong immunological response to the tumor. These TAAs
[11, 12].
include gp100, Melan-A/Mart-1, tyrosinase, MAGE-A1, and
Despite the activity of the immune system, clearly, tu-
NY-ESO [1, 18].
mors may evade the immune system and become clinically
Recent data have demonstrated that combined thera-
evident. Tumor cells often have a reduced number of MHC
peutical approach with chemotherapy and cancer vaccines
class I molecules on their surface, thus avoiding detection by
may have positive effects in the treatment of advanced and
killer T cells.
metastatic tumors. Chemotherapy, alone or with the associ-
An important challenge in cancer immunotherapy is the ation of cancer vaccine, can improve the expression of TAAs
identification of effective strategies for enhancing its clinical and induce enhancement of the cancer-reactive CD8+ cyto-
efficacy. One approach is based on adjuvants, capable of toxic T cells (CTLs) [16]. Specific topoisomerase inhibitors
breaking tolerance against TAAs. Interferons-alpha (IFN- can augment melanoma antigens production, suggesting that
alpha) are pleiotropic cytokines belonging to type I IFNs, a combination of chemotherapy and immunotherapy may be
extensively used in the treatment of patients with some of potential value in the treatment of otherwise insensitive
types of cancer and viral disease. IFN-alpha can increase the cancers [19].
expression of surface antigens enhancing the immune re- Moreover, recent data have demonstrated the implication
sponse, acting as an effective adjuvant in cancer immun- of Tregs in the pathogenesis and in the progression of tumors.
otherapy [13, 14]. In melanoma it has been demonstrated Tregs mediate their immunosuppressive action also by the
that IFN-alpha increases the accumulation of gp100-specific, expression of the negative costimulatory receptor CTLA-4.
IFN-gamma-secreting CD8+ T cells in the tumor, demon- Furthermore, in the last years, the identification of,
strating its efficacy as an adjuvant for peptide vaccination and somatic mutations in the gene encoding the serine-threonine
giving insight into its mechanism of action. This provides a protein kinase B-RAF (BRAF) in the majority of melanomas
rationale for clinical trials in which vaccination is combined has resulted in an opportunity to test oncogene-targeted
with IFN-alpha therapy for melanoma [15]. In addition, therapy for this disease. Patients with advanced metastatic
IFN-alpha can promote the differentiation and activity of melanoma have been treated with PLX4032 (Plexxikon;
host immune cells. Notably, a special interest is currently RG7204, Roche Pharmaceuticals), a potent inhibitor of
focused on the use of dendritic cells (DCs) generated in the BRAF with the V600E mutation; this treatment resulted
presence of IFN-alpha (IFN-DC) for the preparation of an- in complete or partial tumor regression in the majority of
ticancer vaccines. An additional approach for enhancing the patients [20, 21].
response to immunotherapy relies on its combination with Melanomas share initiating genetic alterations such as
chemotherapy [16]. oncogenic mutations in BRAF and NRAS and often show
Here we will briefly discuss the immunobiology of tu- recurrent patterns of chromosomal aberrations. Alteration of
mors. Because the topic is too vast for this paper, we will cell cycle proteins (e.g., cyclin D1, pRb, and p16) has a role
discuss two tumors: melanoma as an example of strong im- in transformation and progression in melanocytic tumors.
munogenic tumor and colorectal cancer as an example for Higher expression of PAR-1 (protease-activated receptor-1)
poorly immunogenic tumors. is seen in melanoma cell lines and tissue specimens [22].
Upregulation of PAR-1 mediates high levels of Cx-43
1.1. A Strong Immunogenic Tumor: Melanoma. Malignant (gapjunctional intracellular communication molecule con-
melanoma is one of the most aggressive malignancies in hu- nexin) expression. This molecule is involved in tumor cell
man and is responsible for almost 60% of lethal skin tumors. diapedesis and attachment to endothelial cells [23]. Protein
Therapy with IFN-alpha 2b, the only agent approved in the kinase C (PKC) mediates signals for cell growth and is
USA for adjuvant use in high-risk melanoma patients, has a target of tumor-promoting phorbol esters in malignant
not shown consistent overall survival benefit in randomized transformation [24]. Downregulation of E-cadherin and
trials and is associated with considerable toxicity. Melanoma upregulation of N-cadherin may be seen in melanoma cells.
is one of the first tumors that have been associated to the Such shift of cadherin profile may have a role in uncon-
presence of local cellular inflammation. The description of trolled proliferation, invasion, and migration. Other studies
a lymphocytic infiltration of primary cutaneous melanoma demonstrated the association of vascular endothelial growth
Clinical and Developmental Immunology 3

factor (VEGF) and VEGF-receptor family with progression and in combination with standard-of-care therapies. MVA-
and melanoma metastasis [22]. 5T4 induced potent and sustained immune responses in
Effective cancer immunotherapy is dependent on the approximately 95% of tested patients. With its minimal side
presence of large number of antitumor lymphocytes with effects and ability to produce immune responses, MVA-5T4
appropriate homing and effector functions that enable them is a promising addition to cancer therapy [36]. Moreover,
to seek out and destroy cancer cells in vivo. Adoptive cell preliminary results showed significant associations between
therapy (ACT) refers to an immunotherapy approach in 5T4-antibody responses and overall survival in patients with
which antitumor lymphocytes are identified and grown ex CRC. The 5T4-specific antibodies were present at higher
vivo and then infused into the cancer patients, often along levels in cancer patients compared with healthy donors and
with vaccines or growth factors that can augment the in increased significantly after treatment with MVA-5T4 [37].
vivo impact of the transferred cells. ACT with autologous Furthermore, CRC can express oncogenes; in particular,
tumor infiltrating may mediate durable complete responses KRAS mutations occur in almost 40% of CRC patients.
in patients with metastatic melanoma [25]. KRAS is a cellular signalling effector downstream from the
EGF/EGFR pathway. KRAS mutations are common in color-
1.2. A Poorly Immunogenic Tumor: Colorectal Cancer (CRC). ectal, ovarian, and lung adenocarcinomas. There have been
Colorectal cancer (CRC) is one of the leading causes of death recent attempts to quantify KRAS mutation and predict
in the Western world. Immunotherapy could play a crucial responses to treatment using an EGFR inhibitors (cetuxi-
role in patients with advanced disease at presentation permit- mab) [38].
ting tumor regression or possibly clearance. Despite advances In addition, studying on TILs has permitted us to
in research and treatment modalities, CRC still accounts for differentiate between the immune cellular populations: Tregs
around half a million deaths yearly worldwide. Traditional and Th17 cells are involved in the pathogenesis and the
and even newer pharmaceutical therapeutic regimens are progression and proliferation of CRC malignant cells. In par-
limited in terms of tolerance, efficacy, and cross-resistance. ticular Tregs and Th17 cells are correlated with a poor prog-
Additional non-cross-resistant therapies with nonoverlap- nosis of CRC and with advanced tumors [39]. According to
ping toxicities are needed to improve the outcome for pa- their immune inhibitory function, Tregs depletion results in
tients with CRC. Cancer vaccines, designed to activate stronger TAA-specific immune response [27].
immune effectors (T cells and antibodies) to prevent recur-
rence or treat advanced cancers, have now demonstrated
clinical benefit [26]. 2. Biology of the Immune Response to Tumors
Bonertz et al. have found that in CRC Tregs T-cells re-
sponse is addressed against a limited repertoire of TAAs, 2.1. Immune Pathways That Can Potentially Limit Tumor Ex-
which include p53, carcinoembryonic antigen (CEA), Her2/ pansion
neu, and heparanase pp1 [27].
Colorectal tumor cells frequently express CEA which 2.1.1. Mechanisms of Action in Tumor Vaccines: DCs, CTLs,
correlates with the state of the tumor, augmenting its ex- and Humoral Response. The discovery of high number of
pression in advanced phases. CEA is considered a clinical tumor-infiltrating lymphocytes (TILs) with skewed tumor-
marker of this tumor, with utility in the diagnosis, prognosis specific TCR expression has promoted the development of
and followup of the disease [28, 29]. Some authors have both adoptive immunotherapy with transfer of TAA-primed
used anti-CEA antibodies tagged with radioactive Yttrium- TILs into patients and vaccine-based antitumor therapy
90 [30] against CEA-expressing metastatic malignances or [1, 40]. CTLs mediate tumor destruction by the release of
combined with antivascular antigens, like combretastatin perforin [41] and granzymes or by the activation of the Fas-
and bevacizumab (anti-VEGF), or with gemcitabine [31–33]. /FasL- mediated apoptosis [42].
Moreover, in recent years it has been shown that CRC can Therapeutic tumor vaccines have two main objectives:
express other antigens, such as extracellular surface marker priming Ag-specific T cells and reprogramming memory T-
CD55 [34] and the oncofetal antigen 5T4. This latter is a cells (i.e., a transformation from one type of immunity to
surface glycoprotein expressed on a variety of human adeno- another, e.g., regulatory to cytotoxic). Dendritic cells are es-
carcinomas, including CRC, and plays an important role in sential in the generation of immune responses, and as such
tumor progression and metastasis. The expression patterns represent targets and vectors for vaccination [43]. The main
and functional role in the metastatic process suggest that 5T4 goal of tumor vaccine therapy is the production of mature
is a good target for vaccine development. A modified vaccine dendritic cells (DCs, the most specialized APCs) able to
virus Ankara (MVA) encoding human 5T4 (designated stimulate an antigen-specific T-cells response in vivo [44]. In
TroVax) demonstrated therapeutic effects in murine tumor classical protocol DCs are activated and loaded with TAAs or
models and human T cells recognized 5T4 epitopes in transfected with RNA-encoding tumoral epitopes and then
an HLA-restricted manner. TroVax vaccine has been eval- transferred to tumor-bearing hosts [45, 46]. Notably, most
uated in clinical trials targeting patients with colorectal antigens expressed on tumor cells are self antigens and may
cancer of advanced stage (IV stage), renal cell carcinoma, result in poor antigenicity due to negative selection of high-
and hormone refractory prostate cancer [35]. Results from avidity autoreactive T-cell subsets; moreover antigens expres-
clinical trials on metastatic colorectal cancer demonstrate sion depend on the proteolytic processing by immunopro-
that MVA-5T4 is safe and immunogenic as a monotherapy teasomes and differential binding to allelic MHC variants
4 Clinical and Developmental Immunology

leading to the hiding of “cryptic” specific epitopes [47]. (T-conv). Tregs arise from different populations with unique
Therefore the antigen presentation may be different in dif- TCR repertoires. Enrichment of Tregs within TILs most
ferent cells, and a selection of proper antigenic peptides may likely, therefore, reflects differences in the way that Treg and
be useful to mediate efficient killing of cancer cells [47]. T-conv cells are influenced by the tumor microenvironment.
Elucidating the nature of these influences may indicate how
2.2. Immunological Pathways That Can Limit the Immune Re- the balance between tumor-infiltrating Treg and T-conv cells
sponses can be manipulated for therapeutic purposes [61].

2.2.1. The Role of Tregs. The heterogeneity of CD4 cells has 2.2.2. The Role of CTLA-4 and PD-1. T-cell activation and
been described in the past [48], but only recently a CD4 inhibition are regulated by signalling of several molecules in-
T-cell subpopulation with regulatory function (Tregs) has cluding CD28 that provides costimulation, CTLA-4 (CD152)
been characterized functionally. Regulatory T cells (Tregs) that binds to the same ligands as CD28, but has more affinity
represent about 5–10% of peripheral CD4+ T cells; they and delivers an inhibitory signal, and programmed death-
are characterized by the ability to suppress T-cell responses. 1 (PD-1) that may be involved in tumor evasion. All these
If this function is impaired, the host will be exposed to molecules have a potential role in immunotherapy [39].
dysfunctions in self-tolerance. Several diseases have been Remarkably, CTLA-4 has more affinity than CD28 for
linked to defective Treg activity including type I diabetes, its ligands and can trigger T-cell anergy. CTLA-4 delivers
allergy, and other autoimmune diseases [39, 49–51]. inhibitory signals to T cells blocking their effector functions
In tumors, several studies suggested a direct correlation through different mechanisms including diminishing of TCR
between adverse prognosis and presence of Tregs in periph- signalling, blocking cell cycle progression, and reducing IL-2
eral blood as well in TILs and in draining lymph nodes of production [39].
different tumors [52]. Also PD-1 seems to be involved in immune evasion, and
Tregs express a number of chemokine receptors such as its expression is reported in melanoma TILs contributing to
CCR2, CCR4, CCR5, CCR7, CCR8, and CXCR4 and are able their impaired antitumor responses [62].
to migrate in response to a variety of chemokines such as
CCL22, CCL17, CCL1, and CCL4 [53–55]. Tregs may be re-
cruits to the tumor site by the chemokine CCL22 produced 2.2.3. The Role of Cytokines in Regulation of Tumor Antigens.
by the tumor cells and tumor-associated macrophages Tumors can mediate their ability to escape immune recog-
(TAMs). Tregs accumulated via CCL2-CCR4 recognize tu- nition also secreting immunosuppressive cytokines, such as
mor-associated immunogenic self-antigens (self-Ags) and IL-10 and TGF-beta [63]. Furthermore Tregs can downmod-
proliferate [52]. Moreover, a recent study on breast cancer ulate immune response by cytokine secretion; these include
showed that the hypoxia environment drives the Tregs re- IL-10, TGF-beta, and the discovered novel IL-35 [64–68]. IL-
cruitment through both CXCL12 production by tumor cells 35 has been shown to be constitutively expressed by regula-
and hypoxia-induced CXCR4 expression in Tregs [56]. tory T cells and contributes to their suppressive activity. IL-
Moreover, Tregs selectively recruited within the tumor 35 is an important mediator inducing CD4+CD25+ T-cell
site will be activated by mature DCs likely through TAA; proliferation and IL-10 production [69].
therefore, Tregs induce T-cell suppressions in an antigen- Recent data have demonstrated also a relation between
selective manner [52]. Indeed it is also clear that vaccination cytokines and vitamins. In particular, vitamins A, D, and E
with some of these epitopes, administered with or without an modulate Treg function and IL-10 and TGF-beta production,
adjuvant or presented by ex vivo cultured antigen-presenting involving the immune response mechanisms [70].
cells (APCs), can induce humoral and CTL antitumor re- Moreover, in addition to the immune cells, also tumors
sponses in some cases [57]. can directly secrete immunosuppressive cytokines, further
It has been reported that immunosuppressive factors permitting them to evade the immune response. For example
produced in the growing tumor environment, such as TGF- melanoma secretes oncostatin M (OSM), which transmits
beta, IL-6, and IL-10, created an immunosuppressive envi- its signal via the gp130 cell surface receptor, resulting in
ronment. Therefore, both the tumor cells (by their expres- the selective downmodulation of the melanocyte lineage
sion of tumor antigens and production of these factors) and/ antigens: Melan-A/MART-1, gp100, tyrosinase, tyrosinase-
or the TAMs may act via promoting the antigen-activated related proteins 1 and 2, and the M isoform of microphthal-
T cells to differentiate and proliferate into Treg cells [58]. mia transcription factor [71]. On the other side it is impor-
Tregs suppression may therefore impair cancer immuno- tant to underline that TAAs expression can be modulated
therapies [52]. Therefore a clear understanding of the mech- in both directions. IFN-beta is an additional stimulus to
anisms of action by Tregs in tumor immunity is needed to TAAs expression in melanoma, including Melan-A/MART-
establish a useful tumor vaccine or immunotherapy [59, 60]. 1, gp100, and MAGE-A1, permitting an improve of immune
Tregs are highly specific for antigens, suggesting that they response to melanoma cells [1, 18].
exert T-cell suppression in an antigen-selective manner [27].
Recent data, confirming the high presence of Tregs within 2.2.4. The Role of Heat Shock Protein 90 (HSP90). In recent
TILs in the tumor site and in the tumor-draining lymph years some data have revealed that the molecular chaperone
nodes, however, has demonstrated that regulatory T cells in Heat Shock protein 90 (HSP90) is involved in several
TILs donot originate by conversion of T-conventional cells condition, including cancer. Hsp90 regulates the trafficking
Clinical and Developmental Immunology 5

of proteins in the cell, under stressful conditions, stabilizes arm versus 21.7 in the placebo group) showing a relative
its client proteins, and provides protection to the cell against reduction of 22% in the risk of death as compared with
cellular stressors such as in cancer cells. Through its role placebo arm; also the rate of 3-year survival was increased
in regulating the conformation, stability, and function of for patients receiving Sipuleucel-T (31.7%) as compared with
several key, oncogenic client proteins, HSP90 contributes in those receiving placebo (23%). In particular, patient in the
maintaining malignant transformation and in increasing the Sipuleucel-T group who had an antibody titer of more than
survival, growth, and invasive potential of cancer cells. 400 against PA2024 or prostatic acid phosphatase at any
HSP90-inhibitors, such as geldanamycin and its ana- time after baseline lived longer than did those who had an
logue 17-allylamino-17-demethoxygeldanamycin (17-AAG, antibody titer of 400 or less (P < 0.001 and P = 0.08, resp.).
tanespimycin), determine suppression of MAPK pathway Adverse events that were more frequently reported in the
in malignant cells and may become new anticancer agents Sipuleucel-T group included chills, fever, and headache [79].
[72, 73]. Moreover, Banerji et al. have shown a correlation Several vaccines against melanoma antigens were tested
between oncogenic BRAF and NRAS mutations, frequently in early clinical trials demonstrating a clinical benefit, but
associated with malignant melanoma, and the HSP90. In fact when tested in prospective randomized trials for advance
NRAS mutations are stabilized by the molecular chaperone melanoma, they failed to improve progression-free or overall
HSP90 and they are depleted by the HSP90 inhibitor 17-AAG survival compared with chemotherapy. The first evidence of
[74]. In addition inhibitors may also upregulate TAAs [75]. clinical benefit of vaccination for patients with metastatic
melanoma came from a prospective randomized phase III
3. Clinical Approach to trial, conducted with stage IV or locally advanced stage III
Immunotherapy of Cancer cutaneous melanoma, HLA A0201+ patients, without brain
metastases who received high-dose IL-2 (720.000 IU/kg/
The increased understanding of the mechanisms of immun- dose) as the control group and a gp100 peptide containing a
oregulation has suggested new strategies to design more ef- modified 209-217 (210M) epitope + montanide ISA followed
fective cancer immunotherapies. by high-dose IL-2 as the experimental arm [80]. The mod-
ified g209–217 peptide (referred to as g209-2M) presents
3.1. Vaccines: Prostate Cancer and Melanoma. Cancer vacci- a methionine replacing the natural threonine at position
nation is a kind of immunotherapy that relies on specific 2; it bounds to the HLA-A2 molecule with greater affinity
priming of the immune system in order to stimulate prin- than the unmodified peptide, and it was shown to have
cipally adaptive immunity against vaccine component, in an increased ability to generate melanoma-reactive CTLs.
contrast to nonspecific immunotherapy where the adminis- Response rate was significantly improved in the experimental
tered agent tries to enhance the innate immunity (e.g., Bacille arm as compared with control group (22.1% versus 9.7%
Calmette Guérin). Early attempts to develop effective cancer (P = 0.0223), and also progression-free survival favoured the
vaccines had limited success due to the failure to identificate gp100-immunized patients compared to those treated with
suitable target antigens, to mitigate the immunosuppressive IL2 alone (2.9 months versus 1.6 months, P = 0.01). Over-
environment and generate an effective immune response all survival was longer in the experimental group, but the
[76]. However, an improvement in our understanding of the difference was not significant (17.6 versus 12.8 months, P =
immune system and tumor immunity, in particular, has facil- 0.0964).
itated the development of more promising vaccine strategies Other vaccines containing multiple tumor-associated
[77, 78]. antigens including MAGE proteins, MART-1/MelanA, and
Different vaccination strategies have been investigated gp100 were tested in a phase I/II trial in patients with
including the use of whole-tumor cells or lysates, dendritic advanced melanoma with evidence of clinical activity and
cells, peptide-base approach, recombinant proteins, and viral durable responses [81]. Also vaccines containing dendritic
and DNA delivery vectors. Since antigens are poorly immu- cells pulsed with melanoma-associated antigens or autolo-
nogenic by themselves, vaccines generally require the inclu- gous lysates [82], or electroporated with mRNA encoding
sion of potent immunoadjuvants to induce antitumor re- CD40 ligand, constitutively active toll-like receptor 4, and
sponses and a delivery system to effectively present the an- CD70, are under investigations in metastatic melanoma pa-
tigen to the immune system [77]. tients [83].
Sipuleucel-T represents the first cancer vaccine approved
by the US Food and Drug administration for the treatment of A vaccine containing a tumor-associated antigen such as
metastatic hormone-refractory prostate cancer. Sipuleucel- MAGE-A3 was also tested in a phase II study for patients
T consists of autologous peripheral-blood mononuclear with non-small-cell lung cancer after complete resection with
cells including antigen-presenting cells (APCs) that have improvement in disease-free and overall survival; on the
been activated ex vivo with a recombinant fusion protein basis of these results, a phase III study with this vaccine was
(PA2024) which contains prostatic acid phosphatase fused initiated in 2007 and is currently ongoing [84].
to granulocyte-macrophage colony-stimulating factor [79]. Other vaccines produced promising phase III data such
In a double-blind, placebo-controlled, multicenter phase III as vitespen, an autologous adjuvant vaccine for patients at
trial, patients with metastatic castration-resistant prostate high risk of recurrence after nephrectomy for renal cell car-
cancer who received Sipuleucel-T had a prolonged overall cinoma [85] and Biovaxid, an idiotype vaccine for patients
survival (median survival 25.8 months in the sipuleucel-T with follicular lymphoma in first complete remissions [86].
6 Clinical and Developmental Immunology

Human telomerase reverse transcriptase (hTERT), the an upregulation of MHC class II proteins. The ultimate result
rate-limiting subunit of the telomerase complex, is another is caspase 3 activation, causing cell apoptosis.
attractive target for cancer vaccination since telomerase is Results of studies with rituximab alone as first-line treat-
highly expressed in almost all cancer forms, while the expres- ment of low-grade non-Hodgkin’s lymphoma have been en-
sion in normal tissues is restricted. Phase I/II trials in ad- couraging [92, 93], as well as inhibition of p38 kinase [94].
vanced pancreatic and pulmonary cancer patients after vac- Rituximab has been also combined with conventional CHOP
cination with GV1001, a 16-aminoacids-peptide of hTERT chemotherapy (cyclophosphamide, doxorubicin, vincristine,
sequence, have demonstrated some specific and durable T- and prednisone) for patients with intermediate grade or
cell responses, associated to a prolonged survival, without diffuse large-cell non-Hodgkin’s lymphoma [95, 96].
clinically important toxicity [87, 88]. Alemtuzumab is a mAb targeted at the CD52 antigen,
found on the surface of most chronic lymphocytic leukemia
3.2. Biological Drugs and Their Combination with Cancer (CLL) cells. It is particularly efficient in chemotherapy-re-
Chemotherapy. In contrast to conventional chemotherapy, sistant B-CLL. Binding of alemtuzumab to CD52 on target
immunotherapy of tumors has raised the hope of a more cells may cause cell death by 3 different mechanisms: comple-
specific therapeutical approach in oncology. In fact, immun- ment activation, antibody-dependent cellular cytotoxicity,
otherapy, targeting TAAs, has permitted to use novel more and apoptosis [97, 98].
specific molecules in cancer therapy. In addition, mAbs can be used to deliver a toxin, such as
As discussed above, biological therapies can also stimu- the RFB4(dsFv)-PE38 (BL22), a recombinant immunotoxin
late the immune response against cancer. In addition, as we containing an anti-CD22 variable domain (Fv) fused to trun-
will see, biological therapy can interfere with tumor blood cated pseudomonas exotoxin [99]. CD22 antigen is found
vessel formation therefore blocking its ability to develop. on the surface of hairy cell leukemia (HCL) cells. To target
In some conditions, biological agents may also be admin- relapsed/refractory HCL, immunotherapy has been devel-
istered together with chemotherapy in order to prevent can- oped using anti-CD25 and anti-CD22 recombinant immun-
cer cells from repairing the DNA damage induced by otoxins, or rituximab alone or combined with purine ana-
chemotherapy itself. Biological agents can be grouped in two logs. BL22 is now in phase I and II testing of relapsed/refrac-
main classes; both these classes have an increasing number tory HCL, achieving 47–61% complete remissions, several of
of drugs of potential interest and a complete review of them them ongoing after 9-10 years [100].
will require a volume and it is beyond the scope of this paper. Hematological malignancies show a wide variety of sur-
We therefore will mention those that appear more promising, face markers as potential target for mAbs targeting [91, 101–
having in mind that, by the time our paper will appear, sev- 103]. In comparison to hematological malignancies, solid
eral new products will be introduced in the clinical practice. tumors have fewer specific targets for mAbs that are not
cross-reactive with antigens on normal tissues.
3.3. Monoclonal Antibodies (mAbs). Ideal drugs would be In 2006 the FDA approved trastuzumab, the first mono-
antibodies against specific antigens on cancer cells that are clonal antibody for the treatment of a solid tumour, in HER2
not cross-reactive with those on normal tissues. mAbs achi- overexpressing breast cancer [104]. Trastuzumab works in
eve their therapeutic effect through various mechanisms. several ways: downregulation of HER2 receptor expression;
They can have direct effects in inducing apoptosis or pro- inhibition of proliferation of human tumour cells that
grammed cell death; they can block growth factor receptors, over-express HER2 protein; enhancing immune recruitment
effectively arresting proliferation of tumor cells; they can and antibody-dependent cell-mediated cytotoxicity (ADCC)
bring about antiidiotype antibody formation enhancing the against tumour cells that overexpress HER2 protein, and
patient’s immune response [89, 90]. downregulation of angiogenesis factors. Trastuzumab also
mAbs can be associated to other substance such as a increases the effect of chemotherapy on breast cancer cells
chemotherapy drug, radioactive particle, or a toxin in order (on the average the response rate rises from 50% up to 85%),
to selectively deliver them to a specific cancer cell. and it is currently used in combination with different chem-
The first monoclonal antibody to receive FDA approval otherapy regimens in metastatic disease, in adjuvant and
was rituximab, an antibody directed to the CD20 antigen [89, neoadjuvant setting [105, 106].
90]. CD20 is a transmembrane protein whose intracellular It is important to point out that there are several eviden-
portion contains phosphorylation sequences for protein ces suggesting that blockade of signal transduction may not
kinase C, calmodulin, and casein kinase 2. Rituximab is be the only mechanism of action of mAbs since a potential
active against B-cell lymphoproliferative diseases [89] that role of immunologic mechanisms in the therapeutic efficacy
are the large majority of lymphoproliferative diseases [91]. of ErbB-targeted mAbs (as opposed to TKI) has been report-
When rituximab cross-links CD20 antigen, an increase in ed. Among the variables known to play a role in the anti-
intracellular calcium is observed. This increase appears to tumor activity of TA-targeted mAbs, there is their ability to
activate the SER family of tyrosine kinases, resulting in fur- mediate lysis of tumor cells in vitro by NK cells, monocytes,
ther phosphorylation of the CD20 inner cytoplasmic chain and granulocytes in an ADCC way. The extent of lysis is in
and also phospholipase C-gamma. At the same time there turn influenced by several variables, and they, or at least some
is an upregulation of C-myc and myb messenger ribonucle- of them, may contribute to the differential clinical response
ic acid, an increase in adhesion molecule expression, and of patients treated with mAbs-based immunotherapy [107].
Clinical and Developmental Immunology 7

mAbs also bind complement, leading to direct cell toxicity, in patients with previously treated advanced melanoma with
known as complement-dependent cytotoxicity (CDC). ipilimumab significantly prolonging median overall survival
Cetuximab is mAb effective for treatment of advanced both as a single agent (10.1 months; P < 0.003) and com-
colon cancer in combination with 5-fluorouracil, oxaliplatin, bined with a gp100 vaccine (10.0 months; P < 0.001) com-
or irinotecan chemotherapy. It is also useful in locally ad- pared with vaccine control (6.4 months). Even more note-
vanced head and neck squamous-cell carcinoma when com- worthy was the improvement in long-term survival at 24
bined with radiotherapy or in recurrent head and neck can- months from 13.7% (gp100 alone) to 21.6% and 23.5% for
cer, combined with platinum-based chemotherapy [108]. the combination and single ipilimumab, respectively [115].
Several papers have reported that the activity of cetux- In addition some patients who progressed after an initial
imab, as well as of panitumumab, is related to their link to response (consisting of stable disease for more than six
the epidermal growth factor (EGFR) which prevents cancer months, partial or complete response) were rechallenged
cells from growing. In particular, it has been shown that within 28 days of documented progression with ipilimumab,
these mAbs are effective only in patients whose cancer has no showing a 50% response rate [114]. This pattern of delayed
mutation of K-RAS gene, the so-called wild-type sequence. response is peculiar of anti-CTLA4 antibodies and is the
The mutation can be detected in about 40% of patients. The reason why novel immune-related response criteria were de-
K-RAS mutations keep the EGFR always active so that its veloped, according to which progressive disease is defined as
pathway can no longer be stopped by simply blocking the an increase ≥25% in the sum of tumor diameters confirmed
receptor [109]. by two scans at least 4 weeks apart. However, anti-CTLA4
Other mAbs have the function to enhance T-cell activa- agents also exhibit a severe profile of adverse events including
tion by blocking CTLA-4, a major negative regulator of T- severe rash, grade 3-4 enterocolitis, hypophysitis, hepatitis,
cell-mediated responses. As we discussed previously, CTLA-4 and more rarely, uveitis, pancreatitis, neuropathy, severe leu-
is a homolog of CD28 that functions as an inhibitory receptor copenia, and red cell aplasia which are generally manageable
for B7 costimulatory molecules expressed on mature APCs. and reversible if recognized early and treated promptly with
Anti-CTLA-4 mAbs with a much greater affinity for CTLA-4 corticosteroids [114]. Also ipilimumab produced encourag-
than B7 may provide a survival advantage compared to vac- ing results in phase I trails for patients with hormone-refrac-
cines or chemotherapy alone [90]. On the basis of these pre- tory prostate cancer, ovarian cancer, and non-Hodgkin’s
clinical data, clinical trials have been initiated with two fully lymphoma and in phase II study of patients with metastatic
human anti-CTLA-4 mAbs, with different pharmacokinetic clear cell renal carcinoma [114, 116]. It also significantly
and pharmacodynamic profiles. increased progression-free survival after conventional chem-
Anti-CTLA4 blocking antibodies [110] are effective in otherapy with carboplatin and paclitaxel in patients with
the treatment of malignant melanoma and may increase untreated lung cancer [117]. On the basis of these data,
Th17 cells in peripheral blood of patients with metastatic anti-CTLA4 monoclonal antibodies represent one promising
melanoma. However, anti-CTLA-4 antibody therapy is asso- strategy to support and enhance the patient’s natural antitu-
ciated with autoimmune toxicity, due to the augmented cel- mor response.
lular proinflammatory activity, as consequence of the in-
crease of Th17 cells and of the inhibition of Tregs function 3.4. Antiangiogenic mAbs. Antiangiogenic drugs are biologi-
[17]. cal therapies that stop tumors from creating their own blood
Tremelimumab is a human IgG2 anti-CTLA-4 mAb with vessels. There are different types of drugs that block blood
a serum half-life of approximately 22 days, the same reported vessel growth, including drugs that prevent growth factors
for endogenous human IgG2, which is currently under from reaching cancer cells, drugs that block the growth factor
evaluation at escalating doses (from 3 to 15 mg/kg every three inside the cell, and drugs that affect signals between cells.
months) in several phase I studies in patients with metastatic Vascular endothelial growth factor (VEGF) is one of the main
tumors such as pancreatic, breast [111] and renal cell car- proteins involved in angiogenesis [118, 119].
cinoma in combination with conventional therapies. As a Bevacizumab, by blocking VEGF, can stop the receptors
single agent, tremelimumab did not demonstrate a clinically from sending signals necessary for blood-vessel growing.
significant activity in a phase II study of patients with re- Once a receptor on a cell surface has been triggered and the
fractory metastatic CRC [112]; it generated durable tumor pathway inside the cell activated, only tyrosine kinase inhib-
responses in a phase I/II trial of patients with treated meta- itors (TKIs), such as Sunitinib, can block signals that trigger
static melanoma [113], but it failed to produce a survival the growth of new blood vessels.
advantage in a randomized phase III study compared to con- Thalidomide is another antiangiogenic drug; even if its
ventional chemotherapy with dacarbazine or temozolomide mechanism of action is still not well known, it seems to in-
[20]. terfere with growth signals among cells. It is helpful for re-
Ipilimumab is a fully human monoclonal antibody IgG1 fractory multiple myeloma.
with a shorter half-life, which was tested at a dose of
3 mg/kg with or without dacarbazine and at 10 mg/kg as 3.5. Conjugated mAbs. As discussed above, mAbs may carry
monotherapy every 3 weeks in several phase II studies in other drugs or radiation directly to cancer cells. Mono-
metastatic melanoma patients showing a significant activity clonal antibodies can be conjugated with anticancer drugs,
with durable remissions [114]. These results have been con- radioisotopes, other biologic response modifiers, or other
firmed recently in the first randomized phase 3 trial [115] toxins. When the antibodies bind with antigen-bearing cells,
8 Clinical and Developmental Immunology

Table 1: Summary of some of the most promising drugs currently under investigation, with their target molecule and more promising
diseases of application.

Malignancies showing promising

Class of products Drug name Target
results
Melanoma∗ , Non-Hodgkin’s
Ipilimumab
CTLA-4 lymphoma, prostate cancer, renal
Tremelimumab
cell cancer
B-cell lymphoproliferative
Monoclonal antibodies (mAbs) Rituximab CD20
malignances
Alemtuzumab CD52 B-CLL
Trastuzumab HER2/neu Breast cancer
Cetuximab CRC, head and neck cancer, and
EGFR
Panitumumab others
CRC, metastatic breast cancer,
Bevacizumab VEGF NSCLC, advanced/metastatic
renal cell carcinoma
Tositumomab B-cell lymphoproliferative
Conjugated mAbs CD20
Ibritumomab malignances
Oncogene inhibitors Plexxikon BRAF Melanoma
Sipuleucel-T APC presenting prostatic antigens Prostate cancer
Vaccines
Advanced CRC, renal cell
TroVax APC presenting 5T4 epitope
carcinoma, prostate cancer
17-AAG
HSP90 inhibitors HSP90 Various cancer
geldanamycin
Abbreviations used in the table:
APC: antigen presenting Cell,
B-CLL: B-cell chronic lymphocytic leukemia,
CRC: coloRectal carcinoma,
EGFR: epidermal growth factor Receptor,
HCC: hepatocellular carcinoma,
HSP90: heat shock protein 90,
NSCLC: non-small-cell lung carcinoma,
VEGF: vascular epidermal growth factor,
∗ This agent as most of the others may also be used in combination with TAA-based vaccines, cytokines, and chemotherapy.

they deliver their load of drug directly to the tumour. Tos- Acknowledgments
itumomab and Ibritumomab are two new promising mon-
oclonal antibodies, conjugated with radioisotopes, targeting This work was supported in part by a grant from the Catholic
CD20, that are still under investigation. University of the Sacred Hearth, Rome (Linea D1). The au-
Antibody directed enzyme prodrug therapy (ADEPT) is thors thank Mr. MK for helping in editing the manuscript.
a selective way for carrying an anticancer drug directly to
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