Linear and Branched Glyco-Lipopeptide Vaccines Follow
Distinct Cross-Presentation Pathways and Generate
Different Magnitudes of Antitumor Immunity
Olivier Renaudet1,2, Gargi Dasgupta1, Ilham Bettahi1, Alda Shi1, Anthony B. Nesburn1, Pascal Dumy2,
1Laboratory of Cellular and Molecular Immunology, The Gavin Herbert Eye Institute, School of Medicine, University of California Irvine, Irvine, California, United States of
America, 2De ´partement de Chimie Mole ´culaire, UMR-CNRS 5250 and ICMG FR 2607, Universite ´ Joseph Fourier, Grenoble, France, 3Institute for Immunology, University of
California Irvine Medical Center, Irvine, California, United States of America, 4Chao Family Comprehensive Cancer Center, University of California Irvine Medical Center,
Irvine, California, United States of America
Background: Glyco-lipopeptides, a form of lipid-tailed glyco-peptide, are currently under intense investigation as B- and T-
cell based vaccine immunotherapy for many cancers. However, the cellular and molecular mechanisms of glyco-
lipopeptides (GLPs) immunogenicity and the position of the lipid moiety on immunogenicity and protective efficacy of GLPs
remain to be determined.
Methods/Principal Findings: We have constructed two structural analogues of HER-2 glyco-lipopeptide (HER-GLP) by
synthesizing a chimeric peptide made of one universal CD4+epitope (PADRE) and one HER-2 CD8+T-cell epitope (HER420–429).
The C-terminal end of the resulting CD4–CD8 chimeric peptide was coupled to a tumor carbohydrate B-cell epitope, based on
a regioselectively addressable functionalized templates (RAFT), made of four a-GalNAc molecules. The resulting HER glyco-
peptide (HER-GP) was then linked to a palmitic acid moiety, attached either at the N-terminal end (linear HER-GLP-1) or in the
middle between the CD4+ and CD8+ T cell epitopes (branched HER-GLP-2). We have investigated the uptake, processing and
cross-presentation pathways of the two HER-GLP vaccine constructs, and assessed whether the position of linkage of the lipid
moiety would affect the B- and T-cell immunogenicity and protective efficacy. Immunization of mice revealed that the linear
HER-GLP-1induceda stronger andlonger lasting HER420–429-specific IFN-c producingCD8+T cell response,whilethe branched
HER-GLP-2 induced a stronger tumor-specific IgG response. The linear HER-GLP-1 was taken up easily by dendritic cells (DCs),
molecules appeared to follow two different cross-presentation pathways. While regression of established tumors was induced
by both linear HER-GLP-1 and branched HER-GLP-2, the inhibition of tumor growth was significantly higher in HER-GLP-1
immunized mice (p,0.005).
Significance: These findings have important implications for the development of effective GLP based immunotherapeutic
strategies against cancers.
Citation: Renaudet O, Dasgupta G, Bettahi I, Shi A, Nesburn AB, et al. (2010) Linear and Branched Glyco-Lipopeptide Vaccines Follow Distinct Cross-Presentation
Pathways and Generate Different Magnitudes of Antitumor Immunity. PLoS ONE 5(6): e11216. doi:10.1371/journal.pone.0011216
Editor: Derya Unutmaz, New York University, United States of America
Received March 7, 2010; Accepted May 26, 2010; Published June 21, 2010
Copyright: ? 2010 Renaudet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants EY-014900 and EY-019896 to LBM from the National Institutes of Health (NIH), The Discovery Eye Foundation, the
Universite ´ Joseph Fourier (UJF Grenoble), the Centre National pour la Recherche Scientifique (CNRS), the Nanobio Program and the COST Action D-34. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Lbenmoha@uci.edu
Aberrant glycosylation leads to the expression of abnormal
tumor associated carbohydrate antigens (TACAs) and are con-
sidered as a unique target for tumor-specific IgG/IgM antibodies
[1,2,3,4,5]. The most common TACA derived B-cell epitope is Tn
antigen (a precursor of Thomsen-Friedenreich or TF antigen),
also known as GalNAc [5,6]. Tn-specific IgG/IgM are detected in
up to 90% of human carcinomas, but their level and affinity is
weak . When administered alone, TACAs activate the antibody
secreting B cells weakly [3,7]. Similarly, tumor-specific CD8+T
cells are also detected in cancer patients, but their level and
function are not sufficient enough to control tumor progression
. Therefore, immunotherapeutic vaccines that can boost the
induction of tumor-specific CD8+T cells and boost their function
are required for tumor protection. An ideal immunotherapeutic
cancer vaccine should comprise both TACA derived carbohydrate
B-cell epitope and tumor associated antigen (TAA) -derived CD8+
T-cell epitopes to boost the sub-optimal antitumor B- and T cell
immune responses often detected in cancer patients [8,9,10,11].
Molecularly defined and human compatible self-adjuvanting
vaccines that are capable of inducing tumor-specific antibody and
CD8+T-cell immunity are limited (reviewed in  and ). The
lowmolecular weight lipid molecule (palmiticacid) is derived from a
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immunologically active lipoprotein of Escherichia coli origin [1,9] and
has been widely used, as an adjuvant, to enhance the immunoge-
nicity of both peptide T-cell epitopes [9,13,14,15,16,17,18] and
carbohydrate B-cell epitopes [19,20,21]. Palmitic acid (PAM)
also acts as a biological ligand for toll receptor 2 (TLR-2) that
is expressed on the surface of antigen presenting cells, such as
dendritic cells, [1,18,22,23] and enhances their phenotypic and
fuctional maturation [1,18,22]. Dendritic cells cross-present exog-
enous palmitic acid-tailed peptide epitopes (i.e. lipopeptides),
associate them with their MHC class I molecules, and present
them to prime CD8+T cells [1,9,21,24,25]. Two major routes for
cross-presentation of lipid-tailed molecules have been described:
(i) processing in the cytosol as they exit from the endocytic
compartment [26,27,28]; and (ii) processing in the endosome and
transfer of the peptides to recycling MHC class I molecules either in
the endocytic pathway or, after regurgitation, at the cell surface
[26,27,28]. We have recently reported that immunization of mice
with an ovalbumin GLP vaccine construct (OVA-GLP) induced B-
and T-cell dependent protective immunity, in both therapeutic and
prophylactic settings [1,21,24]. However, the GLP vaccine strategy
has never been extended to relevant TAA-derived epitopes. In
addition, whether the position of the lipid moiety into GLP
molecules affects the processing and presentation of T cell and B cell
epitopes as well as their in vivo immunogenicity has never been
In this study, we have constructed a prototype HER-2 glyco-
lipopeptide (HER-GLP) cancer vaccine by incorporating all the
necessary components, including TACA as B cell epitopes, CD4+
and CD8+T cell peptide epitopes and an internal immuno-
adjuvant (palmitic acid) in one construct in order to boost potent
and specific antitumor B and T cell immunity. We studied how the
position of the lipid moiety (i.e. either at the N-terminal end or in
the middle of the GLP molecule) affects the uptake of HER-GLP
by DCs, and the processing and cross-priming pathways that lead
its functional presentation to CD8+ T cells. Our results show that
the position of lipid moiety not only affected the uptake and
cross-presentation pathways of GLP in DCs but, interestingly,
modulated the magnitude of antitumor antibody and CD8+ T-cell
protective immunity. These findings have considerable implica-
tions for GLP vaccine development.
Design and assembly of prototype multivalent B, CD4+
and CD8+ epitopes HER-2 glyco-lipopeptide molecules
We designed two prototype HER-2 glyco-lipopeptide mole-
cules: (i) a linear HER-GLP-1 molecule and (ii) a branched HER-
GLP-2 molecule produced using the chemoselective strategy
based on a combined oxime/disulfide bond formation. Both
GLP vaccine molecules were regioselectively assembled and
contained: (i) one CD8+ T-cell epitope peptide (DSLRDSVF)
from HER-2; (ii) one universal CD4+ T-helper epitope (AKX-
VAAWTLKAAA), known as PADRE; (iii) a B-cell epitope made
of Regioselectively Addressable Functionalized Templates (known
as RAFT molecules), which represented a cluster of Tn (a-
GalNAc)4) carbohydrate antigen analogues; and (iv) one palmitic
acid moiety, which plays the role of internal immuno-adjuvant.
The resulting PAM-HER420–429-PADRE-RAFT is designated as
‘‘HER-GLP’’. The corresponding non-lipidated structural analog
HER420–429-PADRE-RAFT is designated as ‘‘HER-GP’’.
Linear HER-GLP-1, branched HER-GLP-2 and non-lipidated
HER-GP were assembled from compound 1 using convergent
ligation chemistry based on oxime and disulfide bond formation
described earlier (Fig. 1A) [1,21,24]. The cyclopeptide RAFT
scaffold 1 displays the cluster of Tn antigen to ensure efficient
antigen delivery and contains a cystein-Npys moiety on the other
addressable domain. This activated cysteine residue permits the
conjugation of peptide or lipopeptide-containing cysteine at the C-
terminal end. The final disulfide coupling reaction between
compound 1 (Fig. 1A) and the peptide HER420–429-PADRE 2 or
(PAM)-PADRE 4 was performed under argon gas in a mixture
of isopropanol and sodium acetate buffer. The purified HER-GP
glyco-peptide, the linear HER-GLP-1; and the branched HER-
GLP-2 glyco-lipopeptides were homogeneous in solution and their
expected primary molecular weights were derived by electrospray
mass spectrometry (Fig. 1B). Multiple freeze-thaw cycles, over a
period of one year, did not disrupt the physicochemical properties
of the HER-GLP vaccine in solution . Finally, the linear HER-
GLP-1, the branched HER-GLP-2 and their parent non-lipidated
HER-GP were labeled unequivocally by fluorescence probe Alexa
Fluor 488 to study their entry into immature dendritic cells by
Branched HER-GLP-2 induced stronger RAFT-specific IgGs
than linear HER-GLP-1
To compare the B-cell immunogenicity of linear HER-GLP-1
and branched HER-GLP-2 molecules (Fig 2A), B10.D1 mice (10
mice/group) were immunized subcutaneously with equimolar
amount of either linear HER-GLP-1 (Group 1) or branched HER-
GLP-2 (Group 2) vaccine constructs in adjuvant-free saline. A
third group of ten B10.D1 mice was immunized with the non-
lipidated HER-GP analog in adjuvant-free saline (Group 3) and
used as control. A fourth group of ten B10.D1 mice was injected
subcutaneously with saline alone (Group 4, Mock). No adverse
reaction such as local inflammation at the sites of injection or
weight loss was observed in any immunized mice rendering the
safety of these adjuvant-free vaccine formulations. Ten days after
the 2ndimmunization, the serum IgG levels specific to carbohy-
drate RAFT were determined in each group by ELISA.
Significant levels of RAFT-specific IgG were induced in both
linear HER-GLP-1 and branched HER-GLP-2 immunized mice
((Fig. 2B) p,0.05 and p,0.01, respectively). However, the
branched HER-GLP-2 appeared as a better immunogen than
the linear HER-GLP-1, suggesting that the position of the lipid
moiety affects the magnitude of antibody responses generated by
the GLP vaccines. Interestingly, immunization of mice with non-
lipidated HER-GP did not induce any significant level of RAFT-
specific IgG responses (p.0.05), revealing the requirement for the
built-in palmitic acid in generating carbohydrate specific IgG Abs.
Constructs missing any of the four components did not produce
Abs response suggesting that it is crucial to have all the
components linked within one molecule (not shown).
The IgGs induced by both linear HER-GLP-1 and branched
HER-GLP-2 construct bind human breast tumor cell line MCF7
expressing Tn molecules (Fig. 2C and 2D). However, higher (350
fold) binding was observed for branched HER-GLP-2 induced
IgGs than linear HER-GLP-1 induced IgGs (150 fold) when
compared to non-immune control IgGs (p,0.01). Under identical
experimental conditions, IgGs induced by both HER-GLP-1 and
HER-GLP-2 immunogen did not show any binding to T2 and RS
cell lines that do not express Tn molecules (not shown).
Taken together, these results indicate that while both linear
HER-GLP-1 and branched HER-GLP-2 molecules induced
RAFT-specific IgGs that bind to human tumor cell lines
expressing the native Tn antigen, the branched HER-GLP-2
appeared to be a stronger B-cell immunogen than the linear HER-
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Linear HER-GLP-1 induced stronger and long lasting
HER420–429-specific IFN-c-producing CD8+ T cell
responses than branched HER-GLP-2
Groups of B10.D1 mice (10/group) were immunized subcuta-
neously three times at fourteen-day intervals with equimolar
amount of linear HER-GLP-1 or branched HER-GLP-2 (Fig 3A).
As a control a third group of ten B10.D1 mice was immunized
with the non-lipidated HER-GP analog. A fourth group of ten
B10.D1 mice was injected subcutaneously with saline alone (Mock).
Ten days after the third immunization, HER420–429-specific CD8+
T cell responses were evaluated in the spleen.
Spleen-derived cells were re-stimulated in vitro with HER420–429
peptide for four days and HER420–429-specific IFN-c producing
CD8+ T cell responseswere measured by ELISpot assays. As shown
in Fig 3B, both linear HER-GLP-1 and branched HER-GLP-2
immunized mice developed significant number of HER420–429-
specific IFN-c producing CD8+ T cells when compared with mock-
immunized control mice (p,0.05). However, the linear HER-GLP-
1 showed higher number of HER420–429-specific IFN-c producing
CD8+ T cells than the branched HER-GLP-2, suggesting that the
position of the lipid moiety in the GLP construct does affect the
magnitude of CD8+ T cell responses. As expected, the HER-GP
immunized mice showed a non-significant number of HER420–429-
specific CD8+ T cells when compared to mock-immunized control
mice (p.0.05), further underlining the requirement for the built-in
palmitic acid immuno-adjuvant for the induction of the T-cell
responses. Constructs missing one of the four components or non-
covalent mixtures of the parts did not induce a T cell response
suggesting that it is crucial to have all the components linked within
one molecule (not shown).
We next performed a kinetic measurement of HER420–429-
specific IFN-c-producing CD8+ T cells in mice immunized with
both linear HER-GLP-1 and branched HER-GLP-2 for up to 60
days post immunization. We observed that the percentage of
HER420–429-specific IFN-c-producing CD8+ T cells was gradually
increased and reached the peak 4.5% for HER-GLP-1 and 3.5%
for HER-GLP-2 on day 28 (Fig. 3C). Thereafter, the percentage of
HER420–429-specific IFN-c-producing CD8+ T cells were gradu-
ally decreased but instead of completely declining, a certain
percentage (2.5% for HER-GLP-1 and 1.8% for HER-GLP-2) of
HER420–429-specific IFN-c-producing CD8+ T cells were main-
tained up to day 60. Taken together, these results indicate that
while both linear HER-GLP-1 and branched HER-GLP-2
molecules are capable of inducing a long-lasting HER420–429-
specific CD8+ T-cell response, the linear HER-GLP-1 appeared
to be a stronger T-cell immunogen than the branched HER-GLP-
2. These results illustrate the importance of the position of palmitic
acid moiety on HER-GLP vaccine construct in terms of
maintaining the long lasting IFN-c-producing CD8 T cells. Unlike
the CD8+ T cell responses, PADRE-specific CD4+ T cell
proliferative responses were not affected by the position of the
lipid moiety (not shown).
Regression of established tumors following
immunotherapeutic immunization with linear HER-GLP-1
and branched HER-GLP-2 vaccines
The immunotherapeutic efficacyof self-adjuvantinglinear HER-
GLP-1 and branched HER-GLP-2 vaccine molecules were
compared by assessing tumor growth and mice survival rate. To
develop tumor, female B10.D1 mice (10 per group) were implanted
subcutaneously in the mammary fat pad with 16105NT2 cells.
Eight to ten days later, when tumor diameter reached 3–4 mm,
mice were immunized subcutaneously four times at seven day
intervals with the linear HER-GLP-1 (group 1), the branched
HER-GLP-2 (group 2), both HER-GLP-1 and HER-GLP-2 (group
3) or injected with PBS alone (group 4, mock). As shown in Fig. 4A,
the tumor diameter (mm), which reflects the tumor progression,
was significantly delayed in mice vaccinated with the linear HER-
(p,0.005). However, therapeutic immunization with HER-GLP-
2 did not lead to a significant reduction the tumor progression
(p.0.005). The inhibition of tumor growth was significantly higher
in linear HER-GLP-1 compared to branch HER-GLP-2 immu-
nized mice (p,0.005). Interestingly, the protective efficacy detected
in mice immunized with both HER-GLP-1 and HER-GLP-2
(group 3) was slightly higher than mice immunized with individual
HER-GLP-1 and HER-GLP-2 constructs. The strong immuno-
therapeutic effect of the linear HER-GLP-1 was also evident from
the survival graph (Fig. 4B). Of 10 mice vaccinated with the linear
HER-GLP-1 and the branched HER-GLP-2 molecules, 8 of 10
and 6 of 10 were still alive over 8-week after tumor inoculation,
respectively. Therapeutic immunization with both HER-GLP-1
and HER-GLP-2 protected 10 of 10 mice from death as compared
to 0 of 10 mock-immunized mice (p,0.005).
Cytoplasmic uptake of linear HER-GLP-1 and branched
HER-GLP-2 constructs by dendritic cells
In an effort to elucidate the mechanisms underlying the
immunogenicity of linear HER-GLP-1 and branched HER-
GLP-2 molecules, we determined the kinetics of their uptake by
immature dendritic cells (DCs). Mouse bone marrow derived
immature DCs were incubated with equimolar amount of Alexa
Fluor 488 labeled HER-GLP-1 or HER-GLP-2 or non-lipidated
HER-GP constructs. The uptake of each vaccine construct on
DCs surface was analyzed by FACS and their cytoplasmic
accumulation was visualized by confocal microscopy. Both linear
HER-GLP-1 and branched HER-GLP-2 were efficiently taken up
by DCs at a concentration as low as 1 uM (Fig. 5A left panel).
Cytoplasmic accumulation of both linear HER-GLP-1 and
branched HER-GLP-2, but not HER-GP, was visualized within
10 min of incubation (Fig. 5A, right panel). The Alexa Fluor 488-
labeled HER-GP was unable to cross DC membrane even after
several trials at higher concentration. This suggests that the
attachment of a palmitic acid moiety play an important role in the
entry of HER-GLP constructs into the cytoplasm of DCs.
Figure 1. Assembly, structures and mass spectrum analyses of prototype multivalent B, CD4+ + and CD8+ + epitopes HER-2 glyco-
lipopeptide molecules. (A) The RAFT moiety 1 was assembled from an orthogonally protected linear decapeptide following the standard Fmoc/
tBu strategy, as we previously described . The amino acid sequences of CD4+and CD8+epitopes and cyclic template are given using one letter
code. Unusual amino acids are designated as dA (L-alanine), Cha (cyclohexyl alanine) and Ahx (L-2-aminohexanoic acid). Each compound displays
clustered Tn analog on the upper domain of the cyclodecapeptide RAFT template. HER-GP and HER-GLP contain respectively either HER420–429-PADRE
chimeric peptide or PAM- HER420–429-PADRE chimeric lipopeptide on the lower domain. (B) Mass spectrum (MS) analysis was obtained by electron
spray ionization (ESI-MS) in the positive mode. The multi-charged ions observed for HER-GP (m/z: 841.7 [M+6H]6+, 1009.7 [M+5H]5+, 1261.7 [M+4H]4+,
1682.2 [M+3H]3+), HER-GLP-1 (m/z: 882.5 [M+6H]6+, 1057.5 [M+5H]5+, 1321.4 [M+4H]4+, 1761.6 [M+3H]3+) and HER-GLP-2 (m/z: 881.3 [M+6H]6+, 1057.6
[M+5H]5+, 1321.4 [M+4H]4+, 1761.7 [M+3H]3+) correspond to the expected deconvoluated masses calculated for [M+H]+(5044.3 for HER-GP, 5282.8 for
HER-GLP-1 and 5282.9 for HER-GLP-2).
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Endocytosis assay uptake showed a total active uptake of HER-
GP and HER-GLPs was measured by FACS and expressed by the
difference in geometric mean of delta fluorescence intensity
(D MFI) that resulted from subtracting the values obtained at
4Cu from the values obtained at 37Cu. Kinetic studies showed that
the linear HER-GLP-1 was quickly taken up at 37uC by DCs
within 10 minutes, whereas the uptake of branched HER-GLP-2
was relatively slower (Fig. 5B). Upon longer incubation at 37uC,
the mean DMFI of the linear HER-GLP-1 construct on DCs was
significantly higher than the branched HER-GLP-2 (p,0.05).
After 30 min incubation, up to 85% of DCs were associated with
the linear HER-GLP-1 and about 75% of DCs were associated
with the branched HER-GLP-2 analog (Fig. 5A). A gradual
increase in the D MFI of both constructs associated with DC was
observed over time, reaching a plateau by 90 min (Fig. 5B).
However, no further changes in the D MFI were observed between
90 to 120 min of incubation. The cytoplasmic entry of both linear
HER-GLP-1 and branched HER-GLP-2 occurred at 37uC but
Figure 2. Immunization with branched HER-GLP-2 induces stronger RAFT-specific IgGs than linear HER-GLP-1. Three groups of B10.D1
mice (n=10) were immunized subcutaneously two times with an interval of 14 days, with linear HER-GLP-1 (50 mM/mouse) or branched HER-GLP-2
(50 mM/mouse) or non-lipidated HER-GP (50 mM/mouse) as shown in (panel A) or injected with PBS alone. Ten days after the 2ndimmunization,
serum was collected from each mouse and the level of RAFT-specific IgG (panel B) was measured by ELISA. MCF7 cells (46105cells) were incubated
for 30 min with 10 ml of mice sera from HER-GLP-1, HER-GLP-2, HER-GP immunized or PBS-injected control mice (Mock) at 1:250 dilutions and
analyzed by flow cytometry using FITC labeled goat anti mouse IgG antibody. The binding efficiency was calculated in terms of mean fluorescent
intensity (MFI) after subtracting the background of serum binding from mock-immunized mice (panel C). A representative data showing the binding
of serum from HER-GLP-1 and HER-GLP-2 immunized mice (solid lines) and serum from mock-immunized mice (broken lines) with MCF7 cells (panel
D). The results are representative of three experiments.
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was inhibited at 4uC (not shown), indicating an active intracellular
delivery mechanism. Together, these results show that both the
linear HER-GLP-1 and branched HER-GLP-2, but not the non-
lipidated HER-GP analog, were taken up quickly by the DCs
and accumulated into DCs cytoplasm. The uptake of linear
HER-GLP-1 appeared to be relatively faster compared to the
branched HER-GLP-2 analog, suggesting that the position of
the lipid moiety might affect the uptake of GLP constructs by
The linear HER-GLP-1 construct induced stronger
dendritic cell maturation and followed by toll-like
receptor 2-dependent T-cell activation
Next we sought to determine whether HER-GP and HER-GLP
constructs are capable of inducing DC maturation and whether
the position of the lipid moiety affects such maturation. Immature
DCs were derived from mouse bone marrow and left untreated
(none) or incubated in vitro for 48 hrs with an equimolar amount
of either linear HER-GLP-1 or branched HER-GLP-2, or non-
lipidated HER-GP and monitored the expression of cell surface
major histocompatibility complex (MHC) class II, and B7 (CD80
and CD86) co-stimulatory molecules which are the well known
phenotypic markers for DC maturation. Incubation of immature
DCs with either linear HER-GLP-1 or branched HER-GLP-2
constructs induced significant up-regulation of MHC class II,
CD80 and CD86 co-stimulatory molecules compared with non-
lipidated HER-GP construct (Fig. 6A). In addition, the incubation
of immature DCs with either linear HER-GLP-1 or branched
HER-GLP-2 was associated with an increase in the production
of IL-12p35 and TNF-a cytokines (Fig. 6B; P,0.005) in a
concentration dependent manner. Under similar conditions,
Figure 3. Immunization with linear HER-GLP-1 induces stronger and long-lasting HER420–429-specific IFN-c producing CD8+ + T cell
responses than branched HER-GLP-2. Three groups of B10.D1 mice (n=10) were immunized subcutaneously three times with HER-GLP-1
(50 mM/mouse), HER-GLP-2 (50 mM/mouse) or HER-GP (50 mM/mouse) as shown in panel A) or injected with PBS alone (Mock) with an interval of 14
days. Ten days after the 3rdimmunization, spleen cells were isolated and stimulated in vitro for 4 days with HER420–429peptide and assayed for IFN-c
producing CD8+ T cells by ELISpot. Mean values (6 SD) of IFN-c spot-forming CD8+ T cells were plotted against each group of mice and are shown in
(B). Kinetics of HER420–429-specific IFN-c producing CD8+ T cells were measured in mice immunized with HER-GLP-1, HER-GLP-2 and HER-GP from 0 to
60 days of post immunization and is shown in (C). The results are representative of three experiments.
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immature DCs incubated with the non-lipidated HER-GP
construct failed to up-regulate the DC markers of maturation
and did not produce the pro-inflammatory cytokines. These data
suggest that the covalent linkage of the lipid moiety not only
facilitated the uptake of the vaccine constructs but also supported
phenotypic maturation of DCs.
In a separate ELISpot assay, we measured the number of
HER420-429-specific IFN-c producing CD8+
presence of bone marrow derived autologous immature DCs
pre-pulsed with equimolar amount of linear HER-GLP-1 or
branched HER-GLP-2 or non-lipidated HER-GP. Higher num-
bers of IFN-c-secreting HER420–429-specific CD8+T-cells were
detected after stimulation with linear HER-GLP-1-pulsed DCs
compared to stimulation with the branched HER-GLP-2-pulsed
DCs (p,0.05) (Fig. 6C). None of the HER420–429-specific CD8+ T-
cells were activated following incubation with immature DCs
alone (none) or with DCs pulsed with a control irrelevant HSV-1
gB495–505peptide (not shown).
To determine whether toll receptors (TLR-2 and TLR-4) on
DCs is playing a potential role in stimulating HER420–429-specific
CD8+ T-cells by linear HER-GLP-1 and branched HER-GLP-2
constructs, blocking experiments were performed. We incubated
autologous immature DCs with either anti-TLR-2 or anti-TLR-4
mAbs, for 30 min before pulsing with the equimolar amounts of
either linear HER-GLP-1, branched HER-GLP-2 or non-
lipidated HER-GP. The anti-TLR-2 mAbs, but not the anti-
TLR-4 mAbs, significantly abrogated the production of the IFN-c
by HER420–429-specific CD8+ T-cells (p=0.002) indicating that
both linear HER-GLP-1 and branched HER-GLP-2 activate DCs
via a TLR-2-dependent pathway (Fig. 6C). Collectively, these
results show that the phenotypic maturation of DCs induced by
the linear HER-GLP-1 and branched HER-GLP-2 occurred
through the TLR-2 signaling pathway.
T-cells in the
The position of the lipid moiety profoundly affects the
cross-presentation pathway of glyco-lipopeptides
To determine the cross-presentation pathways of HER-GLP-
loaded DCs, we used specific antigen-processing inhibitors:
brefeldin A, epoxomycin and monensin. Brefeldin A inhibits
passage from the endoplasmic reticulum to the Golgi, the exocytic
pathway  or inhibits the level of MHC class I molecule
recycling . Epoxomycin acts as a specific proteasome inhibitor
 and inhibits the chymotrypsin-like activity and to a lesser
extent the trypsin-like and peptidyl-glutamyl peptide-hydrolyzing
activities of the proteasome. Epoxomycin is very specific for the
proteasome and does not inhibit non-proteasomal proteases such
as trypsin, chymotrypsin, papain, cathepsin B, calpain, or
tripeptidyl peptidase II . The internalization of exogenous
antigen by endocytosis and subsequent processing by DCs may
occur through the endosomal pathway . Monensin inhibits
endosomal acidification, enzymatic degradation in the lysosomal
compartments and as such might disturb endocytosis [27,34].
To assess the cross-presentation pathway, dendritic cells were
first treated with brefeldin A, Epoxomycin or Monensin, as
described in Materials and Methods, followed by the addition of
linear HER-GLP-1, branched HER-GLP-2 or parent non-
lipidated HER-GP construct. After overnight incubation, DCs
were washed and added to the HER420–429-specific CD8+T cells
for additional 5 hrs. As a positive control, DCs were left untreated
with antigen-processing inhibitors but were pulsed with linear
HER-GLP-1, branched HER-GLP-2 or parent non-lipidated
HER-GP constructs. HER420–429-specific CD8+T cell responses
were detected by ELISpot, as above. As shown in Fig. 7A,
brefeldin A significantly inhibited the magnitude of IFN-c-
producing HER420–429-specific CD8+T-cells induced by both
the linear HER-GLP-1 and branched HER-GLP-2 constructs.
This indicates that the antigen processing of both the linear HER-
Figure 4. Immunotherapeutic efficacy of linear HER-GLP-1 and branched HER-GLP-2 vaccine constructs. NT2 cells (16105/mouse) were
injected s.c. in the mammary fat pad of 40 female B10.D1 mice (5 wk old). Eight days later, when tumor diameter reached 3–4 mm, mice were divided
into 4 groups of 10 mice each. Group 1 was immunized s.c. four times at seven day intervals with the self-adjuvanting linear HER-GLP-1 (GLP-1), group
2 was immunized with the branched HER-GLP-2 (GLP-2), group 3 was immunized with both HER-GLP-1 and HER-GLP-2 (both), and group 4 was
injected with PBS alone as control (Mock). (A) Tumor progression. Local tumor dimensions were measured with calipers as described in Materials
and Methods. The average of tumor diameters (in millimeters) in the course of 50 days is presented. (B) Survival. Mice from the same experiment
were monitored daily for 90 days and were sacrificed when moribund, which corresponded to a tumor diameter of 18 mm. The results are presented
as mean+SEM. Both A and B present p values calculated to compare the two groups of HER-GLP-1 and HER-GLP-2 immunized mice (i.e. group 1 and
group 2). Data are representative of two independent experiments.
PLoS ONE | www.plosone.org7 June 2010 | Volume 5 | Issue 6 | e11216
GLP-1 and branched HER-GLP-2 is governed by the passage
from the endoplasmic reticulum to the Golgi. As shown in Fig. 7B,
epoxomycin almost completely blocked the presentation of the
linear HER-GLP-1 to HER420–429-specific CD8+ T cells. This
indicated that the HER-GLP-1 is processed by the proteasome,
which led to effective presentation of the HER420–429epitope and
stimulation of a stronger IFN-c-producing HER420–429-specific
CD8+T-cells. In contrast to linear HER-GLP-1, the branched
HER-GLP-2 was still processed and presented in the presence of
epoxomycin (Fig. 7B). This result shows that processing of
branched HER-GLP-2 and presentation of HER420–429epitope
to CD8+ T cells is independent on the proteasome pathway. An
opposite result was observed when monensin was used as the
inhibitor (Fig. 7C). Monensin significantly inhibited the presenta-
tion of branched HER-GLP-2 but not the linear HER-GLP-1
indicating that at least partial processing of the branched HER-
GLP-2 in the endosomal compartment and/or recycling through
monensin-sensitive endocytic vesicles. Conversely, presentation of
the linear HER-GLP-1 was not inhibited, suggesting that neither a
monensin-sensitive endogenous pathway nor endosomal acidifica-
tion were used during the processing of linear HER-GLP-1
Glyco-lipopeptides, a form of lipid-tailed glyco-peptides, are
currently under intense investigation as B- and T-cell based
vaccine immunotherapy for many cancers [35,36,37,38,39]. In the
present report, we describe the assembly, immunogenicity and
antitumor efficacy of four-component HER-GLP vaccine con-
struct (one CD4+T-cell epitope, one OVA CD8+T-cell epitope,
one carbohydrate B-cell epitope, and a built-in palmitic acid
adjuvant). The additional incorporation of a palmitic acid moiety
in two different positions results in linear and branched constructs
termed HER-GLP-1 and HER-GLP-2, respectively. We showed
that both constructs are immunogenic. While the linear HER-
GLP-1 induces more potent HER420–429-specific IFN-c-producing
CD8+T cell responses, the branched HER-GLP-2 promotes
stronger tumor-specific IgG responses. Furthermore, although
both constructs enter dendritic cells (DCs) via TLR2 and induced
DCs maturation each construct appeared to be processed and
presented to T cells differently. Accordingly, therapeutic immu-
nization of mice with linear HER-GLP-1 versus branched HER-
GLP-2 induced different levels of antitumor protective immunity.
Thus, the position of the lipid moiety within synthetic GLP
vaccine constructs greatly influence: (i) the magnitude of induced
IgG and CD8+ T cell responses; (ii) the phenotypic and functional
maturation of DCs; (iii) the cross-presentation pathway of the GLP
constructs by DCs; and (iv) the level of therapeutic efficacy against
This work demonstrate that the linear HER-GLP-1 construct
induced higher magnitude of epitope-specific IFN-c-producing
CD8+T-cell response while the branched HER-GLP-2 analogue
preferentially induced stronger IgG antibody response. Besides
Figure 5. Relative uptake of linear HER-GLP-1; branched HER-GLP-2 and non-lipidated HER-GP molecules by bone marrow derived
immature dendritic cells. (A) Primary cultures of bone marrow derived DC populations were incubated for 30 min at 37uC with Alexa Fluor 488-
labeled HER-GLP-1, HER-GLP-2 or HER-GP at an equimolar concentration of 1 uM each. Left panel shows the dot plot representation of loaded HER-
GLP and HER-GP constructs on CD11b/c+cells and right panel shows the subsequent cytoplasmic localization of HER-GLP and HER-GP constructs by
confocal microscopy. (B) Shows the uptake kinetics of HER-GLP and HER-GP constructs by CD11b/c+cells following incubation at different time
intervals as measured by the endocytosis assay uptake. Endocytosis assay uptake of Alexa Fluor 488-labeld HER-GP and HER-GLPs by DCs was
determined for 120 min both at 4Cu and 37Cu. Subsequently, DCs were washed three times with phosphate-buffered saline/bovine serum albumin
1% and total uptake of Alexa Fluor 488-labeld was measured by FACS analyses and expressed by the difference in geometric mean that resulted from
subtracting the values obtained at 4Cu from the values obtained at 37Cu. This formula determines the amount of HER-GP and HER-GLPs that is
actively internalized. The results are representative of five experiments.
PLoS ONE | www.plosone.org8 June 2010 | Volume 5 | Issue 6 | e11216
promoting a stronger CD8+ T cell response, the self-adjuvanting
linear HER-GLP-1 also induced higher-level phenotypic and
functional maturation of dendritic cells. Finally, we established
that the uptake of linear HER-GLP-1 construct did not follow the
classical cross-presentation pathway as evidenced by the endo-
some-to-cytosol inhibition study, but rather appeared to take the
proteasomal pathway. The molecular mechanisms that led to the
exit of the epitopes from endosome to cytosol remain to be
determined. To our surprise, the processing pathway of branched
HER-GLP-2 was different from the linear HER-GLP-1 construct.
The processing of branched HER-GLP-2 was not sensitive to
epoxomycin suggesting that the branched HER-GLP-2 construct
is processed through a proteasome-independent pathway. The
branched HER-GLP-2 processing is, however, sensitive to both
brefeldin A and monensin suggesting the processing is dependent
on lysomal acidification as well as on MHC class I recycling and
transport from endosome to cell surface. To our knowledge, this is
the first report that the position of the lipid moiety within a GLP
vaccine construct greatly affects its cross-presentation pathway
resulting in modulation of the magnitude of induced B and T-cell
The processing of linear HER-GLP-1 is not sensitive to
monensin suggesting that the pathway is independent of lysosomal
acidification. As the processing of linear HER-GLP-1 is sensitive
to both brefeldin A and epoxomycin it suggests that after uptake
by DCs the construct was processed through proteasome mediated
digestion followed by loading of the HER420–429epitope on MHC
class I molecules. In the endosomal system, the linear HER-GLP-1
may be protected from the predominant cleavage by the
proteasome that would destroy the HER420–429 epitope in the
cytosol and may find a suitable enzyme for optimal generation and
presentation of this epitope. However, the identity of this enzyme
remains to be determined. Association with MHC class I
molecules is possible in late endosomes, which were found to
contain MHC class I molecules . Alternatively, the linear
HER-GLP-1 might also egress to the cytosol from a different,
monensin-sensitive compartment than the branched HER-GLP-2
analogue and be digested by an enzymatic activity that is
insensitive to epoxomycin. Finally, we do not exclude that the
affinity of the chemical bond between the palmitic acid moiety and
the peptide backbone might be slightly different during the
synthesis of the linear HER-GLP-1 versus the branched HER-
Figure 6. Phenotypic and functional maturation of dendritic cells induced by linear HER-GLP-1, branched HER-GLP-2 and HER-GP
molecules. (A) Immature DCs were derived from mouse bone marrow and either left untreated (none) or incubated in vitro for 48 hrs with
equimolar amount of linear HER-GLP-1, branched HER-GLP-2, or non-lipidated HER-GP analog. Phenotypic markers for DC maturation (major
histocompatibility complex (MHC) class II, CD80, CD86) were analyzed by FACS and plotted in terms of calculated MFI. (B) IL-12p35 and TNF-a
released by linear HER-GLP-1, branched HER-GLP-2 and HER-GP induced matured DCs were measured by cytokine assay, as described in Materials and
Method. Panel (C) shows the Inhibition of HER420–429-Specific IFN-c spot-forming CD8+ T cells by anti TLR2 antibody as described in Materials and
Method. The results are representative of three experiments.
PLoS ONE | www.plosone.org9 June 2010 | Volume 5 | Issue 6 | e11216
GLP-2. This might play a role in determining how the GLP is
taken-up and processed in DCs, as recently demonstrated using
the palmitoylated encephalitogenic peptides of myelin proteolipid
Our results demonstrated that both linear HER-GLP-1 and
branched HER-GLP-2 constructs are taken up easily by dendritic
cells (Fig. 5), and both constructs induced DCs maturation, albeit
at different levels (Fig. 6A and 6B). Processing of both GLP
constructs by DCs, which leads to different magnitudes of T cell
stimulations (Fig. 3 and Fig. 7), appeared to involve binding/
internalization via TLR-2 molecules. This is supported by our
antibody blocking experiment, where blocking TLR-2, but not
TLR-4, abrogated the presentation of CD8+ T cell epitope to
HER420–429-specific IFN-c-producing T cells (Fig. 6C). The
position of the TLR-2 ligand palmitic acid appeared to influence
the cross-presentation pathway of GLP vaccine constructs within
DCs, and this might be a consequence of a difference in binding/
internalization process via TLR-2. Recent study reported that
peptides linked to TLR-2 ligand Pam(3)Cys of R-configuration
(Pam(R)) lead to better activation of DCs compared to those with
S-configuration (Pam(S)) [42,43]. Although both Pam(R) and
Pam(S) epimers were internalized equally, the study concluded
that the enhanced DC maturation is due to enhanced TLR-2
binding by the Pam(R)-conjugate in contrast to its Pam(S)-
conjugate. Similarly, in case of linear and branched HER-GLP
constructs, one cannot exclude the possibility of two different
affinities of palmitic acid with TLR2, when placed in two different
chemical conformations, cause differential uptake/processing in
DCs . Our results certainly show that the position of TLR-2
ligand palmitic acid, (i.e. linear or branched) greatly affects the
uptake and the cross-presentation pathway of associated epitopes.
In addition, the involvement of other TLRs and non-TLR
receptors are not ruled out in the binding/internalization of linear
HER-GLP-1 and branched HER-GLP-2 . Another TLR-2
ligand lipoteichoic acid was reported be internalized independent-
ly from TLR2 . Investigating the relative role of TLRs and
other non-TLR receptors in: (i) binding/internalization; (ii)
processing; and (iii) enhancing the immunogenicity and protective
efficacy of GLPs is an important goal for future studies. Because
each of the GLP constructs employed in the present study is
molecularly defined, they can be labeled precisely on a single
residue for future mechanistic evaluation.
Production of CD8+T cell epitopes in APCs has been mostly
documented as processing by the proteasome in the cytosol
followed by TAP-mediated transport into the endoplasmic
reticulum and association with nascent MHC class I molecules
[40,47]. Among APCs, only DCs can prime naive CD8+T cells,
and therefore they are required for primary immunization
[40,48,49,50]. Dendritic cells have apparently evolved specific
cross-presentation mechanisms allowing them to prime CD8+T
cells for exogenous Ags that are first internalized by macro-
[31,51,52,53]. Dendritic cells process these antigens and associate
them with their MHC class I molecules . Two major routes for
cross-presentation have been described [31,53,54]: exit from the
endocytic compartment and processing in the cytosol [55,56] and
processing in the endosomal system and transfer of the peptides to
recycling MHC class I molecules either in the endocytic pathway
[57,58] or, after regurgitation, at the cell surface [59,60]. These
mechanisms are essential for the cells to become sensitive to and
develop tolerance to Ags that are not endogenously synthesized in
Cancer cells undergo significant changes in carbohydrate
expression (aberrant glycosylation), and these alterations can be
used as therapeutic targets (reviewed in ). Aberrant glycosyla-
tion of glycoproteins and glycolipids on tumor cells leads to
expression of abnormal tumor associated carbohydrate antigens
(TACAs) [61,62]. The most common TACA is Tn antigen (a
precursor of Thomsen-Friedenreich or TF antigen), also known as
GalNAc, which is a-linked to a serine or threonine residue (a-
GalNAC-O-Ser/Thr) . Tn is detected in up to 90% of human
breast, ovary, and colon carcinomas . However, the induction
of IgG antibodies (Abs) against TACAs is much more difficult than
eliciting similar Abs against pathogen associated carbohydrates
antigens (reviewed in ). This is not surprising, because some
TACAs are self-antigens and are consequently tolerated by the
Figure 7. Cross-presentation pathways of linear HER-GLP-1 and branched HER-GLP-2 vaccine constructs in dendritic cells. (A)
Dendritic cells were pre-incubated with brefeldin A for 1 h, followed by the addition of linear HER-GLP-1, branched HER-GLP-2 or non-lipidated HER-
GP construct (open bars). As a positive control, DCs were left untreated with antigen-processing inhibitors but were pulsed with linear HER-GLP-1,
branched HER-GLP-2 or parent non-lipidated HER-GP (open bars). After overnight incubation, DCs were then washed and added to the HER420–429-
specific CD8+T cells for additional 5 hrs. IFN-c produced by HER420–429-specific CD8+T cells was tested by ELISpot assay. Panel (B) and (C) represents
identical experiments conducted in the presence of epoxomycin and monensin inhibitors, as described in Materials and Methods. Results are
representative of three independent experiments.
PLoS ONE | www.plosone.org10 June 2010 | Volume 5 | Issue 6 | e11216
immune system [3,65,66,67,68]. The shedding of TACAs by
growing tumors exacerbates this tolerance [3,69,70]. However,
under appropriate conditions, Tn can induce tumor-specific IgG
in both mice and non-human primates . Overall the rates of
Tn expression showed statistically significant differences between
healthy and tumorous or transitional tissues [71,72]. Accordingly,
several Tn-based vaccines and immunotherapies that passed
clinical trials showed no major side effects . This raises the
hope that aberrantly glycosylated TACAs can be used as a specific
target for humoral-mediated cancer vaccines.
The studies reported here show that four-component HER-
GLP vaccine molecules elicit robust IgG antibody response. The
position of the lipid moiety significantly affects the level of IgG
production and the optimal binding to breast cancer cell lines
expressing carbohydrate antigens on their surface . Previous
studies demonstrated that lipid-tailed peptides promote a T cell-
independent activation and maturation of B-cells via TLR-2 and
increased the frequency of IgG secreting B-cells . Although B
cells expressing TLR-2 are potential targets, very little is known
about the effect of GLP on B-cells and especially on their potential
for inducing carbohydrate-specific Ab response. Our findings
demonstrate that the branched HER-GLP-2 promoted strong Tn
carbohydrate-specific IgG response. The same Abs bind to intact
Tn expressing breast tumor cells, suggesting that biologically
relevant specificities were produced. Although we found that
PADRE-specific CD4+ T cell proliferative responses were not
affected by the position of the lipid moiety (not shown), this does not
ruled out the alteration of balance between Th1/Th2 helper
cytokines. Why the branched HER-GLP-2 skewed towards
stronger RAFT-specific IgG production than linear HER-GLP-1
remains to be determined. Nevertheless, our findings are
important, particularly in view of the problems associated with
large carrier proteins, such as tetanus toxoid or diphtheria toxoid,
often used to deliver weakly antigenic molecules such as
carbohydrates . In addition, ‘‘booster’’ injections are often
required for conversion of the initial, transient IgM response to a
strong, durable IgG response. However, Ab responses directed
against the vaccine carrier have been shown, in some cases, to
negatively affect the booster response to the vaccine Ag . The
use of a totally synthetic GLP carrier molecule capable of inducing
vigorous helper T cells, but potentially less readily recognized by
Abs might be, in this respect, of significant interest. Together these
data support our belief that GLP should be considered as an
alternative to more complex carriers for use in prophylaxis and
therapeutic cancer vaccines.
The cellular and molecular mechanisms underlying the
immunogenicity of HER-GLP remain to be fully elucidated.
Our data indicate that the lipid moiety is a crucial factor since the
immunogenicity of HER-GLP is superior to its non-lipidated
HER-GP analog. Non-lipidated HER-GP failed to cross cell
membranes, failed to induce maturation of DCs, and failed to
present the HER420–429 peptide epitope to CD8+T cells. In
addition, vaccine constructs missing any individual component out
of the four did not produce any immune responses suggesting that
it is crucial to have all the components linked together in one
molecule. In contrast, lipid-tailed HER-GLP vaccine molecules
easily attach and cross-the cell membrane of DCs and drive their
phenotypic and function maturation, and induced a long-lasting
presentation of the B- and T-cell epitopes in vivo [13,25,77]. This
point is of crucial importance, because the time elapsing between
binding of synthetic peptides and engagement with T cell
precursors in secondary lymphoid organs may well exceed the
peptide life span at the MHC binding groove, especially for low- to
medium-affinity peptides . The use of lipid-tailed HER-GLP
appears to partially overcome this shortcoming by endowing the
antigenic peptide with longer MHC half-life [23,77,79,80,81,82].
It has become increasingly clear that induction and modula-
tion of T cell immunity against tumors require immunogenic
formulations that allow efficient targeting and maturation of
dendritic cells (DCs) [83,84]. Dendritic cells are the professional
Ag-capturing and Ag-presenting cells with a unique ability to
prime naı ¨ve T-cells . Dendritic cells have the unique ability to
present on MHC class I not only peptides from their own
endogenous Ags, but also TAA from their external environment
through a process called cross-presentation . After acquiring
these TAA, DCs carry this information to secondary lymphoid
tissues and present derived epitopes to naive CD8+T cells in ways
that initiate immune responses [10,87]. Critical to this function is a
program of maturation that enhances DCs Ag-presenting and
costimulatory capacity . Immature DCs were left untreated or
incubated in vitro with either the lipid-tailed HER-GLPs, the non-
lipidated HER-GP or the lipid moiety alone and then monitored
for the expression of cell surface MHC class II, CD80 and CD86
co-stimulatory markers indicative of DC maturation. Untreated
immature DCs and lipid moiety treated DCs were used as negative
controls. Under similar conditions, there was no up-regulation of
MHC class II and costimulatory molecules or production of IL-12
or TNF-a following incubation of immature DCs with either the
parental non-lipidated HER-GP alone or the lipid moiety alone
(Fig. 6A and 6B, p,0.05). Non-lipidated HER-GP failed to cross
cell membranes and failed to induce phenotypic and function
maturation of DCs. In contrast, the lipid-tailed HER-GLPs easily
attached and crossed the cell membrane of DCs and drive their
maturation. The linear HER-GLP-1 construct was taken up more
efficiently by DCs than the branched HER-GLP-2 analog. The
present report thus extends previous findings, by showing that in
vitro incubation of immature DCs with HER-GLP molecules
interacts with TLR-2  and increased cell surface expression of
MHC class II and CD80/CD86 co-stimulatory molecules
resulting in mature DCs producing high levels of IL-12p35 and
TNF-a cytokines [1,21,24]. Together, these results indicated that
covalent linkage to the lipid moiety is required for DCs
maturation. This suggests that, as a mechanism behind the
immunogenicity of GLPs, the lipid moiety likely exerts its adjuvant
effect by interacting and stimulating DCs.
Synthetic cancer vaccines, offer safety, reliability and cost
advantages over traditional methods (e.g. live vectors, tumor cells-
APC fusions, genetic immunization), but formidable challenges
still confront their development [21,88,89]. Among these is the
requirement for external immuno-adjuvant, which is critical for
the immunogenicity and protective efficacy of synthetic vaccines
[90,91]. An ideal adjuvant should rescue and increase the immune
response against tumors, with acceptable toxicity and safety even
for those immuno-compromised cancer patients [21,90]. While
several different adjuvants are effective in pre-clinical studies, the
aluminum-based salt (Alum) is currently the only licensed adjuvant
[92,93] for clinical application. Although ‘‘alum’’ is able to induce
a strong antibody (Th2type) response, it has little capacity to
stimulate cellular (Th1 type) immune responses, which are
important for protection against many cancers [8,94]. Therefore,
safe and effective self-adjuvanting molecules are highly desired
[3,21]. These self-adjuvanting cancer vaccine molecules should be
more potent but less toxic than external adjuvants [95,96]. Owing
to the limited success of many vaccines in the clinic, attempts are
being made to improve the safety and efficacy of vaccine
formulations, and to define new adjuvants and antigen delivery
systems. However, the development of new cancer adjuvants as
well as the improvement of efficacy and safety of existing adjuvants
PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11216
still lags far behind [92,97]. We have previously shown that the
‘‘new generation’’ four-component, self-adjuvanting GLP vaccine
molecule, might offer a compromise between highly toxic
adjuvants and no chemical adjuvants at all, while inducing a
strong protective immunity [1,21,24]. In this study we showed the
protective efficacy of a GLP vaccine molecule and demonstrated
that the therapeutic efficacy of HER-GLP is affected by the
position of the lipid moiety.
Bearing in mind the particular constraints for a prospective
human vaccine, the present study designed prototype self-
adjuvanting HER-GLPs vaccines and demonstrated their safety,
immunogenicity and protective efficacy in a mouse tumor model.
Molecularly defined epitope-based vaccines capable of inducing
anti-tumor protective immunity, in a manner compatible with
human delivery, are limited. Few molecules achieve this target
without being delivered with potentially toxic external immuno-
adjuvants. It is important to note that even moderate levels of IFN-
c-producing CD8 T cell responses were induced by HER-GLP
vaccine constructs (Fig. 3), they were sufficient to control tumor
progression (Fig. 4A) and to protect against death (Fig. 4B). This
suggests that the quality, rather than the quantity, of the CD8 T
cell responses was more crucial in protecting against cancer in
this mouse model. We have previously shown that immunization
with MHC I-restricted CTL peptides+helper peptides in IFA
induced higher magnitude of CTL responses compared to when
the same MHC I-restricted CTL peptides+helper peptides are
attached to a lipid moiety (i.e. lipopeptides), delivered without
external immuno-adjuvant [9,13]. However, unlike external toxic
Freund’s adjuvants , the TLR-2 ligand palmitic acid, has been
used as a built-in immuno-adjuvant, and was safe and immuno-
genic in both animals and humans [13,14,16,17,85,99,100].
Because several lipid-tailed peptide vaccine candidates have been
recently employed in clinical trials [101,102,103], we expect that,
once a potent lipid-tailed GLP cancer vaccine is validated in pre-
clinical animal studies, the move to a clinical trial should be
A synthetic liposomal ErbB2/HER2 peptide-based vaccine
construct with the combination of CD8+and CD4+epitopes has
been recently reported to induce prophylactic and therapeutic
protection in mice  but concerns remain about its potentially
toxic adjuvants. Those studies prompted us to construct and test
self-adjuvanting HER-GLP vaccines. Kieber-Emmons and co-
workers showed Pam3CSS moiety serves as a built-in adjuvant and
enhances tumor-specific Abs . Later a three-component GLP
vaccine by Boons and coworkers (a three palmitic acid Pam3-
CysSK4moiety, a CD4+ and a B-Cell epitope) showed induction
of strong tumor-specific IgG responses . To our knowledge, we
are the first to report self-adjuvanting four-component HER-GLP
vaccine molecules. The previously reported complex Pam3-
CysSK4and Pam3CSS molecules have a spontaneous tendency
to form stable aggregates, making the synthesis, purification and
solubility of Pam3CSS-tailed HER-GP extremely difficult. In
contrast, our HER-GLP vaccine constructs were synthesized using
the mono-palmitoyl strategy which is relatively simple to produce
and easy to purify under GMP conditions.
In summary, we have demonstrated that fully synthetic self-
adjuvanting linear and branched HER-GLP vaccine molecules
follow different cross-presentation pathways in DCs and generate
different magnitudes of B- and CD8+T-cell responses. The
position the lipid moiety in the HER-GLP construct profoundly
affects phenotypic and functional maturation of DCs, the
processing of GLP molecules and its cross-presentation in DCs;
as well as the magnitude of IgG and IFN-c producing CD8+T cell
responses. Finally, the position of the lipid moiety also affected the
strength of immunotherapeutic efficacy induced by the GLP
vaccine constructs. The advantage and relative ease of making
these self-adjuvanting GLP cancer vaccines will greatly facilitate
the production of GLP cancer vaccines for large-scale clinical
trials. Their clinical success will depend in great measure on
selection of the appropriate human TAAs and TACAs epitopes.
Materials and Methods
Peptide, glyco-peptide and glyco-lipopeptides synthesis
Protected amino acids and Fmoc-Gly-Sasrin resin
were obtained from Advanced ChemTech Europe (Brussels,
Belgium), Bachem Biochimie SARL (Voisins-Les-Bretonneux,
France) and France Biochem S.A. (Meudon, France). PyBOP
was purchased from France Biochem and other reagents were
obtained from either Aldrich (Saint Quentin Fallavier, France) or
Acros (Noisy-Le-Grand, France). Reverse phase HPLC analyses
were performed on Waters equipment. The analytical (Nucleosil
120 A˚3 mm C18particles, 3064.6 mm2) was operated at 1.3 mL/
min and the preparative (Delta-Pak 300 A˚15 mm C18particles,
200625 mm2for glyco-peptides and DiscoveryH Bio Wide Pore
C5, 25cm610 mm) at 22 mL/min with UV monitoring at 214 nm
and 250 nm using a linear A-B gradient (buffer A: 0.09%
CF3CO2H in water; buffer B: 0.09% CF3CO2H in 90%
acetonitrile). Mass spectra were obtained by electron spray
ionization (ES-MS) on a VG Platform II in the positive mode.
Compound 1 [21,24] (14 mg, 5.89 mmol)
was dissolved in a degazed mixture of sodium acetate buffer 25 mM
pH 5 and isopropanol (6 mL, 1/1). The peptide HER420–429-
PADRE-Cys 2 (16.6 mg, 5.89 mmol) was added and the solution
was stirred until completeness of the reaction (checked by analytical
RP-HPLC). After 2 h, the crude yellow reaction mixture was
purified by semi-preparative RP-HPLC (C18 column, linear
gradient: 95:5 to 0:100 A:B in 30 min, Rt=13.8 min) to obtain
a lyophilized powder corresponding the pure glyco-peptide
HER-GP (8 mg, 27% yield). Analytical data: Rt=8.2 min (C18
analytical column, linear gradient: 95:5 to 0:100 A:B in 15 min);
ES-MS (positive mode) calcd. for C221H361N58O72S25043.6, found
Linear glyco-lipopeptide (HER-GLP-1).
dure was followed from 1 (15 mg, 6.31 mmol) and the lipopeptide
PAM-HER420–429-PADRE-Cys 3 (19.3 mg, 6.31 mmol) for the
synthesis of the glyco-lipopeptide HER-GLP-1. After purification
by semi-preparative RP-HPLC (C5column, linear gradient: 95:5
to 0:100 A:B in 30 min, Rt=22.5 min) the glyco-lipopeptide
HER-GLP-1 was obtained as pure lyophilized powder (6 mg, 18%
yield). Analytical data: Rt=11.8 min (C18 analytical column,
linear gradient: 95:5 to 0:100 A:B in 15 min), ES-MS (positive
mode) calcd. for C237H391N58O73S25281.8, found 5282.8.
procedure was followed from 1 (15 mg, 6.31 mmol) and the
lipopeptide PAM-HER420–429-PADRE-Cys 4 (19.3 mg, 6.31 mmol)
for the synthesis of the glyco-lipopeptide HER-GLP-2. After
purification by semi-preparative RP-HPLC (C5 column, linear
gradient: 95:5 to 0:100 A:B in 30 min, Rt=22.5 min) the glyco-
lipopeptide HER-GLP-1 was obtained as pure lyophilized powder
(5 mg, 15% yield). Analytical data: Rt=11.8 min (C18analytical
column, linear gradient: 95:5 to 0:100 A:B in 15 min), ES-MS
(positive mode) for C237H391N58O73S25281.8, found 5282.9.
A similar proce-
Immunization and serum preparation
Female B10.D1 mice 4 to 5 weeks old were purchased from
Jackson Laboratory (Bar Harbor, ME) and immunized subcuta-
neously at the base of the tail either with (i) linear HER- GLP-1
PLoS ONE | www.plosone.org 12June 2010 | Volume 5 | Issue 6 | e11216
(50 mM/mouse in 100ml of PBS) or with (ii) branched HER-GLP-2
(50 mM/mouse in 100ml of PBS) or with (iii) HER- (50 mM/mouse
in 100ml of PBS) or (iv) PBS alone, three times at 14 day intervals.
Ten days after the last immunization, mice were bled from the
ocular venus plexus. Serum were collected by centrifugation for
5 min at 1250 RPM and stored at 280uC.
Streptavidin coated 96-well ELISA plates were purchased from
NUNC. MultiScreen HTSTMIP plates for ELISpot assays were
purchased from Millipore. Goat anti-mouse IgG-HRP was
purchased from Chemicon. Goat anti-mouse IgG-FITC was
purchased from Sigma. TMB substrate reagent set was purchased
from BD Biosciences.
ELISA and ELISpot assay
Immune and non-immune mouse sera were tested for anti-
RAFT antibodies by using direct ELISA, as we recently described
[21,82]. ELISpot assay was performed by using cytokine ELISpot
pair kit from BD PharMingen, San Diego, CA, as we recently
MCF-7, the human breast cancer cell lines were obtained from
the American Type Culture Collection (Manassas, VA) and grown
to 90% confluence in Modified Dulbecco’s medium, as we recently
reported [5,6,21,105]. The NT2 neu-expressing tumor line
derived from spontaneously arising mammary tumors excised
from neu-N mice was used in experiment of tumor growth
inhibition . NT2 cells express stable neu and MHC class I, as
previously described .
Generation of DCs
Bone marrow-derived DCs were generated using a modified
version of our previously described protocol . Briefly, a total of
26106bone marrow cells were cultured in tissue dishes containing
10 ml of RPMI 1640 supplemented with 2 mM glutamine, 1%
nonessential amino acids (Gibco-BRL), 10% FCS, 50 ng/ml of
GM-CSF and 50 ng/ml IL-4 (PeproTech Inc). Cells were feed
with fresh medium supplemented with 25 ng/ml of GM-CSF and
25 ng/ml of IL-4 every 72 h. After 7 days of culture, this protocol
yielded 506106–606106cells with 70–90% of the non-adherent
cells displaying the typical morphology of DC. This was routinely
confirmed by FACS analysis of CD11c and DEC-205 surface
markers of DC.
Dendritic cell surface and cytosolic uptake assay
One million DC were suspended in at 37uC pre-warmed RPMI
1640 medium containing 5% FCS and incubated for 2 h with 0.1,
1, 10 and 100 ng/ml of linear HER- GLP-1 or branched HER-
GLP-2 constructs labeled with Alexa FluorH488. Cells were
harvested every 10 min, washed in cold FACSH buffer and stained
with 1 ug/ml of FITC-labeled anti-CD11c, anti-Mac-1 (CD11b)
(PharMingen, San Diego, CA). Cells were then acquired using a
FACSCaliburH with two excitation laser sources and analyzed
with CellQuestH software (Becton Dickinson, San Jose, CA), as we
previously described . Confocal microscopy was used to
visualize the cell surface labeling and to follow the intracellular
delivery of linear HER- GLP-1 or branched HER- GLP-2
constructs, as we previously described . The PE or FITC
conjugated secondary antibodies were used to assess the surface
expression of CD80 (clone 10–10A1, IgG), CD86 (clone GL1,
IgG2a) and FITC-MHC class II (clone M5/114.15.2, IgG2b, k)
(PharMingen), respectively. IgG isotype-matched irrelevant mAbs
were used as controls. After staining, 20,000 events were acquired
on a Becton Dickinson (Mountain View, CA) FACSCaliburH and
the expressions of the markers of maturation were analyzed on
CD11c-gated cells using CellQuest software.
Endocytosis assay showing the uptake of Alexa Fluor 488-labeld
HER-GP and HER-GLPs by DCs was measured for 120 min at
both 4Cu and 37Cu respectively. Cells were washed three times
with phosphate-buffered saline containing 1% bovine serum
albumin and total uptake of Alexa Fluor 488-labeld was measured
by FACS. The data are expressed by the difference in geometric
mean after subtracting the values obtained at 4Cu from the values
obtained at 37Cu. This formula determines the amount of HER-
GP and HER-GLPs that is actively internalized.
200 ml of 46105cancer cells were incubated with 10ul of (1:250
dilution) immunized or non-immune serum for 30 min. at 4uC in
FACS staining buffer (PBS containing 5% fetal calf serum and
0.1% sodium azide). Cells were washed two times with buffer
followed by centrifugation for 5 min at 1250 RPM and 5uC after
each wash. Cell pellets were suspended in 200 ml of FACS staining
buffer and incubated with 10ml (undiluted) of secondary antibody
(anti-mouse IgG-FITC) for 30 min. at 4uC. Cells were washed two
times as above. The final cell pellets were suspended in 250 ml of
FACS staining buffer and data acquired immediately on a
FACScan flow cytometer (Becton Dickinson, Mountain View,
CA, USA) and analyzed with CellQuest software (Becton
Blocking TLR-2 receptors on DCs
Blocking of TLR-2 receptors on DCs were performed on day 4
after the initiation of the culture from bone marrow derived cells,
as previously described . Anti-TLR-2 mAb (eBioscience) or an
IgG1 isotype control mAb (10 mg/ml) was added to DCs 30 min
before the addition of the peptide or lipopeptide. Cells were
harvested 48 h later and counted directly in ELISpot Assay.
Monensin antigen-processing inhibitors
Dendritic cells were first treated with by brefeldin A,
Epoxomycin or Monensin, followed by the addition of either
linear HER-GLP-1, branched HER-GLP-2 or parent non-
lipidated HER-GP construct and incubated overnight with
HER420–429-specific CD8+T cells. When used, brefeldin A
(Sigma) or epoxomycin (Alexis Biochemicals) was added 1 h
before the addition of linear HER-GLP-1, branched HER-GLP-2
or parent non-lipidated HER-GP constructs at concentrations of
10 mg/ml and 10 mM, respectively, and then diluted at 2 mg/ml
and 2 mM for the overnight incubation [26,27]. Monensin (Sigma)
was added only 10 min after linear HER-GLP-1, branched HER-
GLP-2 or parent non-lipidated HER-GP addition (in an attempt
to avoid preventing their endocytosis) at a concentration of
50 mM. In all cases, after overnight incubation, DCs were then
washed and added to the HER420–429-specific CD8+T cells (ratio,
1:1) in the presence of brefeldin A (10 mg/ml) for 5 h at 37uC.
CD8+T cells incubated in the presence of non-treated but pulsed
DCs were used as positive controls. After inhibition CD8+T cells
were assessed in ELISpot for IFN-c production as indicated above.
Ten mice in each experimental group were inoculated s.c. in the
upper back with 16105NT2 cells/mouse. Local tumor diameter
was measured with calipers. Starting 8–10 days later, when the
PLoS ONE | www.plosone.org13June 2010 | Volume 5 | Issue 6 | e11216
tumor reached 3–4 mm in diameter, mice were immunized sc.
four times at 7-day intervals with GLP-1 and/orGLP-2 or control
PBS (Mock) on days 0, 7 and 14 and 21, as described above.
Tumor diameter and survival were recorded. The length, width
and height of each tumor were measured using a digital slide
caliper. Tumor volume was calculated by the formula: p/6 6
length 6width 6height.
Statistical differences in tumor sizes between groups of mice
were determined by one-way ANHER. Significance of survival
plots was done with Kaplan-Meier survival platform. For both
analyses, we used the JMP statistics software (SAS Institute).
Conceived and designed the experiments: OR GD LB. Performed the
experiments: OR GD IB AS LB. Analyzed the data: OR GD IB LB.
Contributed reagents/materials/analysis tools: OR GD ABN PD LB.
Wrote the paper: OR GD ABN LB.
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PLoS ONE | www.plosone.org15 June 2010 | Volume 5 | Issue 6 | e11216