108 Recent Patents on Biotechnology 2011, 5, 108-117

Pre-Clinical and Clinical Aspects of Peptide-based Vaccine Against Hu-

man Solid Tumors

Seyed Amir Jalali1,2 and Giorgio Parmiani1*

1
Unit of Immuno-Biotherapy of Melanoma and Solid Tumors, Division of Molecular Oncology, San Raffaele Scientific
Institute, Milan, Italy; 2Nanotechnology and Biotechnology Research Center, Mashhad University of Medical Sciences,
Mashhad, Iran

Received: April 30, 2011 Revised: June 16, 2011 Accepted: June 18, 2011

Abstract: Peptides deriving from tumor-associated antigens and recognized by patient T cells have been firstly defined in
the early 90’s, and then used as vaccine in animal models and in cancer patients. Early trials showed a variable, often even
high frequency of patients developing peptide-specific T-cell mediated immune response usually accompanied by a lower
frequency of clinical response. Modified, long peptides could be synthesized with a higher in vitro binding to the corre-
sponding HLA allele that only seldom translated into a clear improvement in the tumor response. However, we show here
that more recent studies of multipeptide-based vaccines resulted in a higher and more robust T cell response causing also a
more effective clinical response particularly in melanoma and prostate cancer patients. In this article, we also used some
of the recent patents describing different inventions related to pre-clinical and clinical aspects of peptide based vaccines
against human solid tumors.
Keywords: Peptides, tumor antigens, vaccination, clinical response.

1. INTRODUCTION
From the middle of the 1990s, many cancer vaccination
strategies have been proposed, including peptide-based vac-
cines. Such a strategy was made possible by the molecular
characterization of tumor-associated antigens (TAAs) recog-
nized by T cells the first of which was identified in the mela-
noma antigen (MAGE), and found to be presented by HLA-
A*0101 [1].
Additional TAAs were characterized in the following
years by other research groups worldwide [2]. One possible
classification of TAAs recognized by T cells is provided in
Table 1.
It is clear that the majority of these TAAs is represented
by normal, self-proteins that, as such, are weakly immuno-
genic owing to different forms of tolerance to these mole-
cules. However, the simplicity of producing large scale clini-
cal-grade, well defined peptides allowed their use in the
clinic. Thus, several phase I-II , peptide-based clinical vacci-
nation trials were initiated. Despite the ability of such pep-
tide-based vaccines to induce a weak T cell-specific immune
response in up to 50-60% of patients, cancer vaccines failed
to become a standard therapy because evidence of clinical
activity was obtained only in a minority of subjects [3-5] as
exemplified in Table 2 for metastatic melanoma patients.
This is likely due to the weak immunogenicity of self
TAAs used to immunize patients. In addition, many tumor
immune escape mechanisms that could contribute to this
*Address correspondence to this author at the Unit of Immuno-Biotherapy
of Melanoma and Solid Tumors, Division of Molecular Oncology, San
Raffaele Scientific Institute, Milan, Italy; Tel: +39 02 26437440; Fax: +39
02 26435602; E-mail: parmiani.giorgio@hsr.it
effect have been described during the last few years. A list of
such mechanisms is presented in Table 3. Thus, in an effort
to counteract this immune evasion by tumor cells, different
approaches have been developed to by-pass or impair tumor
escape mechanisms, to induce a stronger immune response
by peptide-based vaccination, and achieve a better control of
tumor growth (see Section 5, Current and future develop-
ments).
2. IDENTIFYING TUMOR-SPECIFIC CYTOTOXIC T
LYMPHOCYTES (CTL) AND T-HELPER PEPTIDE
EPITOPES
Targeting of T cell defined epitopes is dependent on both
the antigenic profile of the tumor and the HLA haplotype of
the patients. The CTL epitope identification strategy is based
essentially on three steps. In the first one, a motif analysis
selects epitopes associated with a high probability of binding
to specific HLA receptors [13]. The second step includes in
vitro biochemical assays like the high-throughput receptor-
ligand assays that allow the assessment of the actual binding
capacity to specific HLA receptors of the peptides selected in
the first step [14]. The third step is the in vitro and in vivo
immunogenicity as assessed by the stimulation of autologous
T cells and/or generation of T cell immune response after in
vivo immunization, respectively [15].
2.1. Identification of CTL Epitopes by Different Ap-
proaches
The discovery of CTL epitopes of TAAs has proceeded
along two different experimental lines. The first one involves
a documented T-cell reaction against tumor cells, the charac-
terization of T-cell specificity and the identification of the
nature of the antigen that is recognized. This last step

1872-2083/11 $100.00+.00 © 2011 Bentham Science Publishers

Peptide-based Vaccines in Solid Tumors Recent Patents on Biotechnology 2011, Vol. 5, No. 2 109

Table 1. Examples of Tumor Antigens Recognized by T Cells

Category Antigens Description

Differentiation MART-1, CEA, PSA Shared self differentiation proteins expressed also on normal cells

MUC1, RAS/m, Survivin,

Overexpressed hTERT, HER-2/neu , Shared but over-expressed on tumor cells
TPD52, SART, lck

MAGE,
Cancer Testis Shared self, expressed by different tumors and normal testis or placenta
NY-ESO-1, MCAK

Tumor-specific - CDK4/m,
-actin-m, Caspase-8/m Unique, mutated and expressed only by a single tumor

HPV: L1, E6, E7

Tumor-specific, virus-related Expression in cervical carcinoma, hepatocellular carcinoma.
HBV: HCV
CDK4/m= cycline-dependent kinase/mutated; CEA= carcino-embryonic antigen; HBV= hepatitis B virus; HCV= hepatitis C virus; HER2= human epithelial receptor2; HPV= human
papilloma virus; hTERT= human telomerase reverse transcriptase; MAGE= melanoma antigen; lck= lymphocyte specific kinase; MCAK= mitotic centromere associated kinesin;
MUC1 = mucin 1; PSA= prostate specific antigen; SART= squamous antigen rejecting tumor; TPD52= tumor protein D52.

Table 2. First Generation (1996-2006) of Self Peptide-based Vaccination of Metastatic Melanoma Patients (Phase I/II Studies)*

Clinical Response (CR+PR)

Type of Peptide TAA N. of Patients Immune Response (%)
(Mean%)

Lineage related (e.g. Melan-A/MART1) 159 14 20-65

Cancer/Testis (e.g. MAGE) 92 17 30-50

DC peptides 124 16 56

DC lysates 106 18 46
CR= complete response; PR= partial response; TAA= Tumor-associated antigens.
*Data collected from several publications [6-12].

Table 3. Tumor Escape Mechanisms that Suppress the Anti-Tumor Immune Response

Implicated Molecule Proposed Mechanisms of Tumor Escape

FasLigand Induces apoptosis in TIL by tumor-derived FasL

Apoptosis of TILs by binding of the lymphocytic programmed death 1 molecule (PD1) and
B7-H1
tumor-associated B7-H1
CCL22/CCL17 These two chemokines attract Treg that down-regulate anti-tumor response.
Disability in the accumulation and proliferation of tumor-specific T cells by tumor release of indolamine 2,3-
IDO
dioxygenase (IDO)
Expression of CEA/CAM1 TIL effector functions can be inhibited by CEA/CAM1.
MMPs High affinity IL-2 receptor of lymphocytes may be cleaved by MMPs
Generation of epitope loss variants Spontaneous or T cell-induced loss of TAAs occurs in tumor cells
Loss, down-regulation of HLA class I Ags It may occur due to mutations/deletion of HLA or
2-microglobulin genes
Expression of HLA class Ib molecules HLA-E and HLA-G protect from NK cell destruction by masking the loss of HLA class I molecules
High tumor cell burden Chronic contact to TAAs without co-stimulatory signaling leads to activation-induced cell death.
Release of ROS Tumor cells produce ROS that may contribute to the loss of signaling molecules like CD3- in TILs.
Impairment of peptide processing Down-regulated expression of TAP and immunoproteasome components.
The production of pro-inflammatory cytokines can be inhibited by the constitutive activation by tumor cells
Suppression of pro-inflammatory signals
of some molecules (e.g. STAT3).
CEA/CAM=CEA cell adhesion molecule; HLA: human leukocyte antigens; IDO=indolamine dioxygenase; MMPs= Matrix Metallo proteinase; NK= natural killer; ROS=reactive oxygen
species; ; STAT= signal transducer and activation of transcription; TAA= tumor-associated antigens; TAP=transporter-associated with presentation; TIL= tumor infiltrating lymphocytes
110 Recent Patents on Biotechnology 2011, Vol. 5, No. 2
requires a gene cloning approach [16, 17]. The other one
uses the reverse immunology [18, 19] to predict in silico T-
cell epitopes from the already known sequence of TAAs. For
example, Ramakrishna et al., using liquid chromatography
and mass spectrometry, were able to identify 16 novel HLA-
A2-bound peptides from a very large and complex pool of
MHC class I-bound peptides purified from HLA-A2+ ovar-
ian cancer cell lines [20].
The knowledge of peptide binding motifs for the com-
mon HLA class I molecules has allowed the in silico screen-
ing of TAAs for identification of dominant T-cell epitopes
with predicted binding capacity to be selected for designing
a cancer vaccine. The algorithms utilize different peptide
binding motifs and different arithmetic methods, based on
the contribution to binding of each aminoacid in a peptide
independently on the overall peptide structure [21]. There-
fore, from a practical point of view, the combination of two
or more methods is advisable to reduce the number of non-
selected peptides with binding capacity.
Schiewe and Haworth recently developed an algorithm
called PePSSI for the flexible structural prediction of peptide
binding to MHC molecules, and they applied this algorithm
to identify peptide epitopes from the cancer-testis (CT)
TAAs based on the potential of these peptides to bind to the
human MHC molecule HLA-A2. This algorithm may pro-
vide a new method for identifying more T-cell epitopes for
peptide vaccines [22].
2.2. Experimental Verification of Reverse Immunology
In this approach, the actual binding capacity needs to be
shown because the ranking of the predictions does not per-
fectly correlate with the actual binding measurements and
false positive prediction of binding occurs [23]. Two strate-
gies have been developed to evaluate the predicted peptide
for their biological activity. In many studies, naive T cells
have been stimulated in vitro using, as antigen-presenting
cells (APCs), peripheral blood mononuclear cells (PBMCs)
from healthy volunteers ex-vivo loaded with the predicted
epitope [24]. Furthermore, peripheral blood lymphocytes
(PBL) or tumor-infiltrating lymphocytes (TIL) from patients
whose tumor expressed the relevant TAA and the appropri-
ate HLA restriction element, have been used [25, 26]. An
alternate approach is based on testing in vivo the immuno-
genicity of peptide in HLA transgenic mice [27, 28].
These two assays have their own unique advantages. For
example, the use of in vitro assays allows testing of the per-
formance of different vaccine constructs directly in human
cells, while the HLA transgenic mice allow testing of spe-
cific formulations in vivo in mice that expresses the human
HLA molecule of interest. One set of experiments demon-
strated concordance between results obtained with human
PBLs in vitro and in HLA transgenic mice in vivo [29]).
2.3. Attempts to Optimize the Selection of Candidate Epi-
topes and Minimize the Experimental Efforts
The tumor-specific CTL epitopes that were identified by
reverse immunology during the early studies (until 2001)
were predicted by taking into account only the HLA class I
peptide binding capacity [17, 30]. Different reverse immu-
Jalali and Parmiani
nogenic approaches were attempted to optimize the selection
of candidate epitopes [31] and to avoid the effect of some
variable that may affect the performance of peptide-based
vaccines like cellular processing and transporter associate
with presentation (TAP) efficiency [32, 33].
In the case of CTL epitopes, those following the C-
terminus of the epitope or protein appear to be most impor-
tant because they can modulate the efficiency by which dif-
ferent CTL epitopes are generated by proteasome processing
and transported to the endoplasmic reticulum [34-36]. For
example, positive charges were associated with optimal re-
sponses in about 80% of the cases. These data implied that
suboptimal responses noted in the case of specific epitopes
in cancer vaccines may be due to suboptimal processing.
3. DEVELOPING MORE EFFECTIVE PEPTIDE-
BASED VACCINES
The extensive use of synthetic, mostly HLA class I-
restricted peptides as cancer vaccines in the first 10 years of
studies (1994-2004) resulted in a limited clinical outcome as
exemplified for metastatic melanoma patients (see Table 2).
Among the different reasons of this modest clinical outcome,
some of which have been alluded above (e.g. escape mecha-
nisms), on should consider: a) the nature and tissue distribu-
tion of TAAs (see Table 1), b) the types of immune adju-
vants used, c) the lack of HLA class II epitopes that have
been rarely included in the peptide-based anti-cancer vac-
cines. Therefore, efforts were made in the last few years to
increase the immunogenicity of peptide TAAs by addressing
some of these issues by different approaches.
3.1. The Nature and Tissue Distribution of TAAs
The first TAAs that were molecularly characterized and
made available for clinical use belonged to the groups of
differentiation antigens, i.e. those expressed also on the nor-
mal cells from which the tumor derives (e.g. MART1, PSA)
or to the group of cancer /testis antigens (e.g. MAGE, NY-
ESO-1) expressed in different human tumors and on a tiny
fraction of normal cells of the testis and placenta (Table 1).
Being expressed also by normal cells these TAAs are weakly
immunogenic owing to different forms of peripheral or cen-
tral tolerance developed by our body to such normal tissue
components. This tolerance may have caused a weak im-
mune response against vaccines including such self TAA
that represented the large majority of the TAAs administered
during the early years of clinical studies. The only truly tu-
mor-specific antigens, like those deriving from somatic point
mutations, have yet to be used in the clinic because their
molecular characterization at the single patient level is cum-
bersome and costly. However, the new developments in ge-
nomic technology allow to profile somatic mutations at the
single tumor level and to identify the mutated epitopes rec-
ognizable by the patient T cells [37].
3.2. Using Adjuvants to Improve Immune Reactivity
Administration of immune-activating agents (adjuvants)
is used to augment the effects of a vaccine by recruiting pro-
inflammatory cells at the vaccine site and to prevent the deg-
radation of the peptide/protein vaccine by proteolytic en-
Peptide-based Vaccines in Solid Tumors
zymes thus leading to a higher stimulation of the patients’
immune system. There are many types of immunological
adjuvants, including standard adjuvants of bacterial origin
like bacillus of Calmette-Guérin (BCG), and cytokines. Sur-
prisingly, only few studies are available on the direct com-
parison of different adjuvants used with the same vaccine
[38, 39].
It is known that innate immune responses are induced
through the recognition of microbial products by Toll-like
receptors (TLRs) expressed by different immune cells (DCs,
macrophages, etc.) and that a number of adjuvants bear TLR
ligands thus showing considerable activity in preclinical and
clinical settings. For example, Imiquimod, a TLR-7 agonist,
has been shown to effectively induce immune responses in
patients with low-grade epithelial human tumors [40, 41].
Moreover, several studies have shown an immunostimula-
tory effect for synthetic oligodeoxynucleotides containing
un-methylated CG dinucleotides (CpG ODN). CpG patterns
are recognized by TLR-9, and administration of CpG has
been found to result in antitumor activity in animals and hu-
mans. In fact, studies by Speiser et al., targeting the mela-
noma antigen MART1 with CpG, resulted in strongly in-
creased MART1 specific T-cell frequencies and enhance-
ment of T-cell activation in comparison with standard adju-
vants in patients with advanced melanoma [42]
Granulocyte Macrophage-colony stimulating factor (GM-
CSF) is the cytokine most frequently used as cancer vaccine
adjuvant. However, as we reported previously, GM-CSF
may increase the vaccine-induced immune response when
administered repeatedly at relatively low doses (range 40–80
mcg for 1–5 days) [43] while impairing the immune re-
sponse when given at high dose. This paradoxic effect is
likely to be due to the increased activation of the myeloid-
derived suppressor cells (MDSC) [44]. This conclusion was
recently supported by two different clinical studies [45, 46].
Several aspects of this issue have been recently discussed in
more details by Clive and colleagues [47].
Another adjuvant widely used in the past but currently
used only as an immunotherapeutic agent for intravesical
treatment of superficial bladder cancer is the tuberculosis
vaccine BCG. It has also been shown to be as effective as
established cytokine therapy in renal cell cancer but with less
toxicity and lower cost [48] and to significantly improve the
quality of life of lung cancer patients [49].
3.2. Peptide Pulsed onto Dendritic Cells
DCs are “nature’s adjuvant,” since they can prime naive
T cells and are potent stimulators of cytolytic and helper T
cell immunity. The goal of DC-based vaccination is the in-
duction of an effective anti-tumor immune response, which
is long lasting and generates long-lived memory. One of the
first DC-based vaccination trial was reported for B cell lym-
phoma patients. The DC were loaded with idiotype antigen
plus keyhole limpet hemocyanin (KLH) as non-specific ad-
juvant; all vaccinated patients developed an immune re-
sponse and one patient underwent a complete tumor regres-
sion [50].
With the safety of DC transfers established, the challenge
of the ongoing clinical studies will be, a) to determine effec-
Recent Patents on Biotechnology 2011, Vol. 5, No. 2 111
tive therapeutic doses and obtain evidence for clinical effi-
cacy, b) to optimize culture conditions for DCs, especially if
tailored subsets of polarized DCs are to be produced, c) to
define cytokine/chemokines profiles that characterize differ-
ent DC subsets, d) to establish conditions for DC polariza-
tion or repolarization toward clinically beneficial type 1 T
cell responses, and, e) to find the means for maintaining DC
functions in the hostile tumor microenvironment.
In new clinical studies, different types of human tumors
were treated by DC-based vaccines but, at the moment,
without strong evidence of clinical benefit though crucial
data were collected that allow now the design of novel clini-
cal trials [51]. However,an important exception was the vac-
cine Sipuleucel-T consisting of autologous DC/APCs, pre-
exposed in vitro to the fusion protein between prostate asso-
ciated phosphatase and GM-CSF (PA2024) This phase III
study resulted in a prolonged overall survival among patients
with metastatic castration-resistant prostate cancer. In April
2010, Sipuleucel-T was approved by the United States Food
and Drug Administration as vaccine for prostate cancer ther-
apy [52].
3.3. Modified Epitope Peptide: Fixed Anchor and Het-
eroclitic Analogs
Fixed anchor analogs peptides are created by modifying
the main anchor residues; these peptides can associate with
increased binding to HLA receptors to create a complex that
can interact more efficiently with the cognate TCR. The
MelanA/MART-1 decamer 26-35 2L [53] and the gp100
209/2M [54] are examples of these modified epitopes for
melanoma vaccines but other modified peptides, like CEA,
appears to be less efficient in HLA binding [55]. A patent
filed by Figdor et al. describes the usefulness of Melanoma
associated peptide analogues for the vaccine against Mela-
noma. The invention is concerned with the treatment and
diagnosis of cancer [56].
Some interesting data have been obtained regarding a
newer category of epitope analogs, called heteroclitic ana-
logs. Heteroclitic analogs are generated by analoging resi-
dues other than the main anchors. A number of different re-
ports have shown that heteroclitic analogs are associated
with T-cell hyperstimulation [57, 58], and this more potent
response is mediated by increased binding of the peptide-
HLA complex to the T-cell receptor (TCR) ([59]. Accord-
ingly, heteroclitic analogs have been related with a striking
capacity to break tolerance, as shown in a variety of different
studies [60, 61]. In addition, vaccines based on heteroclitic
peptides are less expensive because lower concentrations are
required to achieve a response.
May et al. [61] embedded a synthetic immunogenic ana-
log CD8(+) inside the longer peptide (aa 122–140), by mu-
tating residue 126, and produced heteroclitic Wilm’s tumor
(WT) 1 peptides as well as WT1 CD4(+) (aa 122–140).
These authors showed that the former two epitopes are able
to stimulate a peptide-specific CD4(+) response that can rec-
ognize WT1(+) tumor cells in multiple HLA-DRB1 settings.
The use of modified peptides, however, has been compli-
cated by the recent findings that CD8+ T cells generated by
immunizing with these TAA epitopes, though effective in
112 Recent Patents on Biotechnology 2011, Vol. 5, No. 2
recognizing the given peptide included in the vaccine [62],
often are unable to recognize and kill tumor cells expressing
the original, natural peptide [63, Rivoltini/Parmiani, unpub-
lished].
3.4. Help for the Cytotoxic T Lymphocytes by Addition
of CD4+ T Cells Peptide Epitopes
Response to the vaccine in our and others’ previous pro-
tocols [64-67] was often transient, peaking after few vaccine
injections and returning to basal levels at the end of the
treatment. The modality of this anti-vaccine immune re-
sponse was attributed at least in part to the lack of activation
of CD4+ T helper lymphocytes by HLA class II-restricted
epitopes in the vaccine [68]. The helper mechanism is based
on several features of CD4+ T cells because they: a) secrete
the lymphocyte growth hormone IL-2, b) activate the APC
via CD40-CD40L interactions that can subsequently license
the CD8+ T cell to kill its target cell [69], c) are required for
the induction of CD8+ T cell memory [70] , d) physical link-
ing of T-helper peptide and CTL peptide-epitope further in-
creased the magnitude of the CTL response because of pres-
entation of both peptide epitopes on a single APC.
The first synthetic peptide vaccine that protected against
a lethal challenge with Lymphocyte choriomeningitis virus
LCMV [71] not only contained a CD8+ T cell epitope, but
also a CD4+ T cell epitope [72]. Deletion of the CD4+ T
cells almost completely abrogated this protective effect,
showing the importance of the simultaneous activation of
CD4+ and CD8+ T cells.
Nonspecific MHC class II-restricted epitopes, such as
pan-DR Th epitope (PADRE), were used in two clinical vac-
cination trials in melanoma and cervical carcinoma subjects
that were vaccinated with MAGE-3 and HPV16 E7 CTL
peptides, respectively with contradictory results since anti-
helper epitope response seldom was accompanied by a paral-
lel increase in the CTl CD8 response [73, 74]. Other clinical
studies [75] demonstrated that HER-2/neu–derived MHC
class II helper peptides containing HLA-A2–binding motifs
were able to induce long-lasting HER-2–specific CD8 T
cells. In addition, epitope spreading was observed in 84% of
patients and significantly correlated with the generation of a
HER-2/neu protein-specific T-cell immunity.
3.5. The Rationale of Multi-Epitope, Long Peptide Vac-
cines in Cancer Immunotherapy
To improve the immunogenicity of peptides it was pro-
posed to use synthetic long peptides (SLP) that can be pro-
duced by chemical linkage of multiple immunogenic epi-
topes including both HLA class I and class II epitopes [76].
In addition to artificial polyepitope vaccines, naturally linked
and usually overlapping CTL and Th epitopes also per-
formed well as vaccines in preclinical mouse models [77].
This strategy presents many advantages. In fact, since
SLP are unable to directly bind to MHC class  or  mole-
cules as exogenous peptides, they can only be taken up,
processed and presented by professional APCs [76] thus
avoiding binding to non-professional APCs, which can in-
duce a temporary CTL response or tolerance [78]. Moreover,
tumor cells may fail to stimulate the T cell response and es-
Jalali and Parmiani
cape immune destruction through the down-regulation of a
specific HLA allele or mutation of a single epitope [79].
Thus vaccines containing multiple epitopes may interact
with more HLA class I and class II alleles and avoid in vivo
selection of less immunogenic tumor cells. Furthermore,
inclusion of multiple epitopes may be associated with in-
creased potency and may overcome T-cell tolerance ([80].
For example, four different TAAs: p53, CEA, MAGE 2/3
and Her-2/neu are expressed in many cases of breast, ovarian
and lung cancer [14] and multiple TAAs such as MART 1,
gp100, tyrosinase, MAGE, TRP1, TRP2 have been found to
be expressed in the case of melanoma with induction of tu-
mor-specific T-cell responses against at least one of them
[81]. In fact, while any given TAA is usually not expressed
in all of tumors, combining different TAA in the vaccine
allows coverage of the 80 to 90% of tumors.
Moreover, according to the epitope spreading phenome-
non, immune responses against immunodominant epitopes in
SLP may increase natural responses to other subdominant
determinants of the same protein and to epitopes of other
protein antigens [82, 83]. Targeting multiple TAAs thus en-
sures that different patients affected by a given tumor type
are covered by this approach [83].
There are also possible disadvantages of multi-epitope
vaccines. For example, there may be competition between
peptides based on MHC affinity. Another potential negative
aspect is the possibility that there would be multiple weaker
responses rather than strong responses to one or two of the
peptides. Furthermore, in the case of multiple T-cell epitopes
vaccine, presence of junctional epitopes (those created by the
juxtaposition of two epitopes) has the potential to influence
processing efficiency. These neo-epitopes can cause inap-
propriate responses that may reduce the overall immuno-
genicity of the vaccine construct [84]. Recently, the Episort
program was developed which allows selection of epitope
arrangements and spacer configurations associated with a
minimal number of potential junctional epitopes.
However, in a mouse study antitumor vaccination using a
SLP which included MHC class I- and II-restricted male (H-
Y) epitopes, an unexpected mortality was observed [85]
Mice with an increased frequency of anti–H-Y CD4 T cells
were primed with SLP+CpG and boosted 10 d later. Within
hours of boost, mice displayed shock-like signs with high
mortality. Serum cytokine levels were high and TNF-
se-
creted by the CD4 T cells was identified as the key effector
molecule [85]. However, a similar finding has not been re-
ported in clinical studies in which multiple or SLP were used
as described below.
4. CLINICAL TRIALS USING MULTI-PEPTIDE-
BASED VACCINES
Several trials using multipeptide vaccines have been per-
formed with a variety of results; a selection of the most in-
teresting ones is presented in Table 4 and commented below.
A multi-epitope melanoma peptide vaccine was designed by
Slingluff et al. [86] as a mixture of gp100(280 –288 and 17-
25), tyrosinase (369–377D and 240-251S), and the tetanus
toxoid as a helper peptide that were either administered with
Montanide ISA51, IL-2 and/or GM-CSF or pulsed onto
autologous immature DCs in a phase II trial of metastatic
Peptide-based Vaccines in Solid Tumors Recent Patents on Biotechnology 2011, Vol. 5, No. 2 113

Table 4. Multipeptide based Therapeutic Vaccines

Immune Clinical
Vaccine peptide(s) Adjuvant N. of Patients Reference
Response Response

Melanoma

Montanide 13 42% 15%

4 peptides: Gp 100 (280-288); Gp 100 (17-25);
+IL2 80% >0S [86]
Tyrosinase (369- 377); Tyrosinase (240-251) +
+GM-CSF 75% -
TT*
DC 13 11% 8%

4 peptides: Gp 100 (280-288);Gp 100 (17-25); Montanide

Tyrosinase(369-377); Tyrosinase (240-251) + +IL2 early 40 37-38%
>0Sº (trend) [87]
TT* +IL2 late 53-83%

6 peptides: MART1, MAGE, Gp100, Tyrosinase

No 39 81% >0S [88]
+class II+TT*

Montanide +GM- 26 100% >0S [89]

12 vs. 4 peptides (see above) + TT*
CSF 25 77% >DFS

Montanide 48 85% >PFS (trend) [90]

Gp100(209-219), Tyrosinase (368-374)
+GM-CSF

Montanide 22 0% DFS not in- Rivoltini et al.,

Gp100(209-219), MART1 (27L), NY-ESO-1 creased submitted
Vs.
(165V), Survivin (97M)+cyclophosphamide
Observation 21 75%

Prostate cancer

28 72% PFS 8.5mos vs. [91]

PPV+Estramustine vs.
Montanide 29 0% 2.8mos
Estramustine
(P=0.0012)

11 peptides: PSA (141-150) (146-154) (154- Imiquimod, GM- 19 NA 21% (PSADT) [92]
163), PSMA(4-12), PSCA(14-22)(105-113), CSF, RNA Pro-
Survivin (5-14)(95-104)(97-111), Prostein (31- teamine
39), TRP (187-195), PSMA (459-473)

PSMA1 (4-12), PSMA2 (711-719), Survivin Montanide 20 90%# 70% (<PSADT) [93]
(96-104)+cyclophosphamide

NSCLC
CEA(24V9) (605D6)(691H5); Her2(689); p53 Montanide 63 85% >OS [94]
(139L233),(49M2); MAGE-3 (1215)

Renal Cell Cancer (38%) [95]

CA9(219-227) (288-296)(323-331) Montanide 23 70% (3PR, 6SD)

Breast Cancer 101 [96]

E75/HER2/neu GM-CSF vs. 75% (DTH) P<0.04 (PFS)
85

Glioblastoma 75% 66% [98]

ITK-1 peptides Montanide 12 (CTL) (1PR, 7SD)

EGFRvIII KLH 18 43% >OS? [99]

(Ab)
CA9= cancer antigen 9; CEA= carcino-embryonioc antigen; CR= complete response; DC= dendritic cells; DFS= disease-fee survival; DTH= Delayed type hypersensitivity; EG-
FRvIII= epidermal growth factor receptor variant III; HER= human epidermal receptor ; Gp100= glycoprotein 100; KLH= keyhole limpet hemocyanin; MAGE-3= melanoma anti-
gen-3; NSCLC= non-small cell lung cancer; OS= overall survival; PFS= progession free survival; PR= partial response; PSA= prostate specific antigen; PSADT= prostate specific
doubling time ¸ PSMA= prostate specific membrane antigen; PPV= personalized peptide vaccine; PSCA= prostate specific cell surface antigen; SD= stable disease; TRP= transmem-
brane protein; TT=Tetanus Toxoid.
114 Recent Patents on Biotechnology 2011, Vol. 5, No. 2
melanoma . Patients receiving the four peptides together
with GM-CSF developed a better immune response and
clinical response as compared with patients who received
only DC-pulsed peptides. However, the rate of immunologi-
cal response remained higher than that of clinical response,
perhaps reflecting the immunogenicity of these TAAs strong
enough to elicit a detectable immune response but not a
clinical response [86]. In another study by the same group,
40 advanced melanoma patients were vaccinated with the
four above mentioned gp100-and tyrosinase-derived peptides
admixed in Montanide ISA 51, together with GM-CSF and
low dose IL-2 given in two different schedules, up-front and
delayed. The immunological evaluation showed that IL-2
given upfront elicited a weaker T cell response and had a
shorter 5-year survival as compared with delayed IL-2 [87].
In a phase I/II trial, a vaccine comprised of 6 melanoma-
associated peptides as antigenic targets for melanoma-
reactive T helper cells was tested for safety and immuno-
genicity. Vaccination with the helper peptide vaccine was
well tolerated and the induction of CD4 T-cell against mela-
noma class II peptides was found in 81% of patients [88]
(Table 4).
In another trial conducted by the same group, the 12 pep-
tide regimen was compared with that including 4 peptides; a
significant increase in both the frequency and potency of the
T cell response was noted in the arm receiving 12 peptides
and this response was also accompanied by a better clinical
outcome [89].
An additional trial of vaccination was conducted by the
group of Slingluff [45] in which 12 different melanoma pep-
tides (deriving from MAGE, MART1, Tyrosinase, NY-
ESO1) admixed with Montanide were used to vaccinate
melanoma patients with or without the addition of GM-CSF
(Table 4). Surprisingly GM-CSF impaired the frequency of
the immune response as compared with the patients that did
not receive this cytokine, confirming previous observations
of our and other groups [43, 46, 47]. The three years OS and
DFS estimates were 76% and 52% respectively, but the
events were too few to find meaningful differences. This is
at variance with the study of Weber and colleagues [90] who
found that, in a similar clinical context, GM-CSF increased,
though modestly, the immune response of vaccinated pa-
tients (see Table 4).
A multipeptide-based phase I-II, randomized vaccination
trials was performed by our group in stage II-III melanoma
patients (Table 4). Before lymph node surgery, 43 patients
received (N=21) or not (N=22) 3 different peptides and a low
dose of cyclophosphamide, the latter in an attempt to down-
modulate Tregs frequency and/or function . T cell reactions
were found in 75% of vaccinated subjects but not in the ob-
servation arm though no difference in Tregs frequency and
progression-free survival (PFS) was noted in the interim,
early assessment (Rivoltini L et al., submitted).
As for the prostate cancer, of great interest is the study
by the group of Itoh [91] that selected the peptides to be used
as vaccine in the trial in each subject according to the pre-
clinical in vitro immunogenicity of each peptide, a cumber-
some but rationale approach to the construction of personal-
ized vaccines. When given with estramustine, the vaccine
conferred a statistically significant advantage in PFS to the
Jalali and Parmiani
patients (Table 4). Vaccination in hormone sensitive prostate
cancer patients with biochemical relapse was performed by
Feyerabend and colleagues [92] who injected 11 different
HLA class I and II prostate peptides with or without different
adjuvants and obtained a 21% clinical response rate as as-
sessed by PSA doubling time.
We have also conducted a multipeptide vaccination study
in locally advanced hormone-resistant prostate cancer using
peptides from PSMA-1 and -2, PAP and survivin along with
low dose of cyclophosphamide [93]. As shown in Table 4,
90% of patients responded to at least one peptide, most fre-
quently to survivin and 70% of them showed a statistically
significant decline of the PSA during vaccination but the
PSA-doubling time (DT) returned to pre-vaccine values after
vaccine discontinuation.
A polyepitope vaccine composed of 5 TAAs (ie, CEA,
p53, HER-2/neu, and MAGE-2 and MAGE-3) and 10 pep-
tides derived from them, has been evaluated in 63 patients
with non-small cell lung cancer (NSCLC). A strong CTL
response was observed in the majority of patients without
significant adverse effects and a trend toward survival bene-
fit was evident [94].
A vaccination trial with CA9 peptides was conducted by
the group of Itoh, with an approach similar to that used for
prostate cancer, in renal cell carcinoma and with interesting
immunological and clinical findings [95] (see Table 4). Ad-
vanced breast cancer patients were also treated with the
E75/HER2/neu peptides and GM-CSF in a phase II random-
ized trial of patients with or without lymph node involve-
ment. The study showed a statistically significant increase in
PFS in vaccinated subjects compared to patients not receiv-
ing the vaccine [96]
A milestone paper is that reporting immunological and
clinical results obtained by Kenter and colleagues [97] in a
therapeutic setting in which patients with HPV16-(E6, E7)
positive vulvar intraepithelial neoplasia vaccinated with a
mixture of long HPV peptides showed a high rate (79%) of
clinical response including complete rejection of the tumor.
5. CURRENT AND FUTURE DEVELOPMENTS
Peptide-based vaccines are easy to construct and to de-
liver to cancer patients though they need to be administered
along with immunological adjuvants . However, the first
generation of clinical trials failed to provide relevant clinical
outcomes though allowing collecting important new infor-
mation. Such information ,together with that obtained in pre-
clinical mechanistic investigations in vitro and in animal
models, has promoted the design of novel protocols. Most of
them are aimed at improving the immune response to peptide
vaccines, by counteracting mechanism of immune escape
like a) MHC down-regulation of tumor cells (100), b) Treg
and/or MDSC activity (101), c) tumor-release suppressive
factor (e.g.IDO)(102); d) use of multiple synthetic long pep-
tides, (SLP) and e) targeting of immune co-stimulatory
molecules (e.g. CTLA4, PD-1). The use of such combined
strategies is generating efficacious clinical responses in this
new wave of clinical trials that will impact on the clinical
outcome of future cancer therapy [67, 103, 104].
Peptide-based Vaccines in Solid Tumors
ACKNOWLEDGMENTS
The work of Dr. G. Parmiani has been supported by
Grants of the EU FP6 (Brussels), the Italian Association of
Cancer Research (AIRC, Milan, Italy), the Alliance against
Cancer (Rome) and by Antigenics, Inc., New York.
Seyed Amir Jalali was supported by a Fellowship of the
Ministry of Science, Research and Technology of Iran.
CONFLICT OF INTERESTS
The authors declare no conflict of interests.
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