Eur. J. Immunol. 2006.

36: 1267–1274 Molecular immunology 1267

Induction of IgG antibodies against the GD2 carbohydrate

tumor antigen by vaccination with peptide mimotopes
Angelika B. Riemer1,2, Elisabeth F
rster-Waldl3, Kira H. Brmswig1,3,
Arnold Pollak3, Christoph C. Zielinski4, Hubert Pehamberger2,
Holger N. Lode5, Otto Scheiner1 and Erika Jensen-Jarolim1
1
Department of Pathophysiology, Medical University of Vienna, Vienna, Austria
2
Department of Dermatology, Medical University of Vienna, Vienna, Austria
3
Department of Pediatrics and Juvenile Medicine, Medical University of Vienna,
Vienna, Austria
4
Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria
5
Charit
-Universittsmedizin Berlin, Pediatrics, Campus Virchow, Berlin, Germany

The disialoganglioside GD2, a carbohydrate antigen, is expressed on all tumors of Received 19/7/05
Revised 25/1/06
neuroectodermal origin, including melanoma, neuroblastoma, sarcoma and small cell
Accepted 27/2/06
lung cancer. Due to its specific expression on tumor surfaces, GD2 is an attractive target
for immunotherapies. The mouse/human chimeric anti-GD2 mAb ch14.18 is already [DOI 10.1002/eji.200535279]
applied in melanoma and neuroblastoma trials as a passive immunotherapy. To
establish an active immunotherapy alternative, we aimed to replace the poorly
immunogenic ganglioside with immunogenic peptides. Previously, we used the ch14.18
antibody to select GD2 peptide mimics from a phage display library. In the present
study, two mimics of the ch14.18 epitope were coupled to keyhole limpet hemocyanin
and used for immunizing BALB/c mice. Induction of a specific humoral immune
response towards the original antigen GD2, both purified and expressed on
neuroblastoma and melanoma cells, could be demonstrated in ELISA, Western blot, Key words:
and immunofluorohistochemistry. As the elicited antibodies were of the IgG isotype, the Cancer vaccine
mimotope conjugates were capable of recruiting T cell help and inducing memory
ch14.18
phenomena. In conclusion, we show that an epitope of the carbohydrate antigen GD2
Disialoganglioside
can successfully be translated into immunogenic peptide mimotopes. Our immunization GD2
Epitope mimic
experiments indicate that GD2 mimotopes are suitable for active immunotherapy of
Tumor-associated
GD2-expressing tumors. carbohydrate antigen

Introduction metastasis and progression rates [1]. Well-documented

Tumors expressing high levels of certain tumor-asso-
ciated carbohydrate antigens (TACA) exhibit greater
Correspondence: Erika Jensen-Jarolim, Department of Patho-
physiology, Center of Physiology and Pathophysiology, Medical
University of Vienna, W
hringer Grtel 18–20, 1090 Vienna,
Austria
Fax: +43-1-40400-5130
e-mail: erika.jensen-jarolim@meduniwien.ac.at
Abbreviations: DGG: synthetic C-DGGWLSKGSW-C

GRL: synthetic C-GRLKMVPDLE-C
HAMA: human anti-mouse
antibodies
KLH: keyhole limpet hemocyanin
TACA: tumor-
associated carbohydrate antigen
examples of such TACA are GM2, GD2, and GD3
gangliosides in neuroectodermal tumors [2, 3]. Their
surface localization makes these gangliosides effective
targets for active and passive antibody therapies. The
disialoganglioside GD2 is considered especially inter-
esting, as it is expressed on all tumors of neuroecto-
dermal origin, including melanoma, neuroblastoma,
sarcoma and small cell lung cancer [4, 5], but is absent
in normal tissues and only minimally expressed in brain
[3] and peripheral nerves [5, 6]. Due to its localization
in focal adhesion plaques at the interface of tumor cells
and their substratum, it is thought to play a role in cell
attachment and metastasis [7].

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1268 Angelika B. Riemer et al.
Several mAb against GD2 have been raised, with
14.18 [8] and 3F8 [9] being the most prominent
examples. mAb 3F8 has been given to neuroblastoma
patients in its murine form [10], and from these studies
it was seen that the antigen GD2 is rarely lost following
mAb treatment [11]. mAb 14.18 has been further
developed into a human/mouse chimeric derivative,
ch14.18. Both 14.18 and ch14.18 trigger tumor cell lysis
via antibody-dependent cellular cytotoxicity and com-
plement-dependent cytotoxicity [12, 13]. In preclinical
models, the 14.18 antibodies have been shown to
prevent the outgrowth of experimental melanoma
[14] and neuroblastoma [15] tumors. Consequently,
ch14.18 was applied in several clinical trials against
melanoma [16], neuroblastoma [17, 18] and also
osteosarcoma [18] with an acceptable safety profile.
The effects of these passive antibody therapies were
thought to be enhanced by a combination with systemic
cytokines, GM-CSF [19, 20] and IL-2 [21]. In the next
step, ch14.18 was expressed as a fusion protein with
IL-2, to target the cytokine directly to the tumor
site [22]. This compound is already being tested in
clinical trials [23].
As an alternative to these passive antibody applica-
tions, active immunizations against GD2 have been
investigated. However, the antigen itself cannot be used
as an effective immunogen due to its glycolipid nature.
Carbohydrates are mostly T cell-independent antigens,
and as no T cell help is involved, relatively low antibody
titers and no memory responses are induced. Therefore,
GD2 has to be antigenically enhanced for immunization
purposes. One possibility is coupling of the ganglioside
to an immunogenic carrier [24]; another is the
translation of the carbohydrate antigen into a mimicking
peptide [25] or protein [26–28] structure. Our group
has generated circular decapeptide mimics of the 14.18
epitope on the GD2 antigen using the phage display
technique described in a previous study [25]. They were
proven to be true epitope mimics (so called mimotopes)
by mimicry tests in the ELISA format and by 3-D
computer modeling. The aim of the present study was to
evaluate the mimotopes for their capability of inducing a
GD2-specific humoral immune response.
Results
The vaccine constructs
Out of the 13 mimotopes generated for the ch14.18
epitope, the two cyclic peptides showing the best
mimicry results [25], C-GRLKMVPDLE-C and C-
DGGWLSKGSW-C, were selected for immunization
studies. For this, they were produced synthetically.
Before proceeding to immunizations, the synthetic
f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2006. 36: 1267–1274
peptides were again checked for specific recognition
by the selecting antibody. Indeed, synthetic C-
GRLKMVPDLE-C and C-DGGWLSKGSW-C (subse-
quently referred to as “GRL” and “DGG”), circularized
by disulfide bridging between the flanking cysteines,
were recognized by ch14.18, but not by the isotype
control antibody (Fig. 1, left panel). To ensure
immunogenicity and availability of sufficient T helper
epitopes, the mimotopes were coupled to the immuno-
genic carrier, keyhole limpet hemocyanin (KLH).
Specific mimicry of the ch14.18 epitope through
mimotope conformation was also preserved in the
conjugated form. KLH itself was only nonspecifically
recognized by ch14.18 (Fig. 1, right panel).
Selected GD2 mimotopes elicit cross-reactive
antibodies
Three groups of mice were immunized four times with
either GRL-KLH, DGG-KLH, or KLH alone. Subsequently,
blood was drawn and IgG serum levels were determined.
To enable comparison between the groups, in a first step,
antibodies directed against the carrier molecule, KLH,
were measured in the ELISA format. All three groups
showed comparable anti-KLH values (OD 450–570 nm
0.388  0.004, 0.396  0.005, and 0.418  0.012 for
mice immunized with GRL-KLH, DGG-KLH, and KLH,
respectively), indicating successful immunization in all
three groups. We then proceeded to determine the anti-
mimotope titers. Uncoupled mimotope peptides, a
control peptide, and the carrier protein were dotted
onto a nitrocellulose membrane, and incubated with
serial dilutions of sera from mimotope-KLH-immunized
mice. As seen in the ELISA assay, anti-KLH titers were
comparable for both groups (Fig. 2, KLH panels).
Mimotope-induced antibodies were found to be cross-
reactive for both peptides, i.e., GRL-KLH-immunized
mice recognized GRL as well as DGG, and DGG-KLH-
Figure 1. Synthetic peptide mimics of the ch14.18 epitope on
disialogangliside GD2 are specifically recognized by ch14.18.
Left panel: Cyclic peptides GRL and DGG, dotted in triplicates,
are recognized by ch14.18, but not by the isotype control
antibody cetuximab, or detecting anti-human IgG antibody
(buffer control). Right panel: After conjugation to the im-
munogenic carrier molecule KLH, cyclic GRL and DGG
mimotope conformation is preserved. ch14.18 recognized the
mimotope conjugates (GRL-KLH and DGG-KLH), but only
nonspecifically interacts with the carrier KLH. Controls are
again clear.
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Eur. J. Immunol. 2006. 36: 1267–1274 Molecular immunology 1269

Figure 2. Sera of mice immunized with mimotope conjugates

are cross-reactive with both peptides mimicking the ch14.18
epitope on GD2. Mice were immunized with GRL-KLH or DGG-
KLH. Total Ig titer determinations by DotBlot against the
peptides GRL and DGG, against the carrier KLH, and against a
control peptide are shown. Serum dilutions are given in the
figure.
immunized mice recognized DGG as well as GRL (Fig. 2,
GRL and DGG panels). As both peptides, although
different in sequence, were characterized to be 3-D
mimics of the same epitope, this finding is not
surprising, but supports their structural equivalence.
Interestingly, both GRL-KLH- and DGG-KLH-immunized
mice reacted more strongly with the GRL peptide.
Neither group recognized a cyclic decamer control
peptide (Fig. 2, bottom panels), so the induced
antibodies are demonstrated to be specific for the
GD2 mimotopes.
Mimotope-induced antibodies recognize the
original antigen GD2
Figure 3. Mimotope-induced antibodies recognize the original
antigen, disialoganglioside GD2. Mice immunized with GRL-
KLH or DGG-KLH developed IgG antibodies specifically reacting
with GD2, but not with the control ganglioside GM1, in ELISA.
KLH-immunized mice show no reactivity with either ganglio-
side. Box plots show median, 25th and 75th percentiles of IgG
levels of respective groups. Both differences of mimotope
groups in GD2/GM1 recognition, as well as differences between
mimotope and control groups in GD2 recognition, are
statistically significant (p<0.05) by two-tailed Student's t-test.
carrier groups again was found to be significant
(p<0.001 for GRL-KLH, and p<0.01 for DGG-KLH).
We thus demonstrated that immunization with the
selected GD2 mimotopes indeed induces a specific anti-
GD2 immune response. Ig subclasses and exact amounts
of anti-GD2 antibodies (shown in Table 1) elicited by
mimotope vaccination were also determined by gang-
lioside ELISA. In serum dilution series, IgM could be
detected up to dilutions of 1:3000 in both mimotope

Table 1. Anti-GD2 antibody quantities (lg/mL) in mouse sera

To assess whether the induced antibodies not only react
after mimotope immunization
with the actual immunogen, but also with the original
antigen, GD2, a ganglioside ELISA was performed. Both Mimotope used for immunization
mimotope-KLH conjugates induced antibodies that Antibody subclass
C-GRLKMVPDLE-C C-DGGWLSKGSW-C
reacted specifically with GD2, but not with the control
IgM 210.00 50.00
ganglioside GM1 (Fig. 3). This difference in recognition
was found to be statistically significant (p<0.001 for IgG1 2.30 2.20
GRL-KLH-immunized mice, and p<0.05 for DGG-KLH- IgG2a 0.55 1.78
immunized mice). Mice immunized with the carrier KLH IgG2b 0.47 0.20
alone did not recognize either ganglioside, and the
IgG3 – –
difference in GD2 recognition between mimotope and

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1270 Angelika B. Riemer et al.
Figure 4. Immunodetection of GD2 by anti-mimotope sera in a
Western blot of a SK-N-AS cell lysate. Blotted GD2 is detected by
ch14.18 as a band at 55 kDa. Sera from mice immunized with
the GRL-KLH and DGG-KLH conjugates also recognize GD2
(serum dilutions given in figure, total Ig detected), whereas sera
from mice immunized with the carrier KLH alone show no
reactivity with the antigen.
groups, IgG1 up to 1:300 in the GRL-KLH and up to
1:1000 in the GDD-KLH group, IgG2a up to 1:1000 in
both groups, and IgG2b up to 1:300 and 1:100,
respectively. No IgG3 or IgA could be detected in the
sera.
Mimotope-induced antibodies recognize GD2 in
melanoma and neuroblastoma cell extracts and
on the cell surface
To further confirm the relevance of the mimotope-
induced antibodies, we sought to show that they also
recognize GD2 on tumor cells. Cell extracts of GD2-
expressing M21 melanoma cells and SK-NA-S neuro-
blastoma cells were prepared and separated electro-
phoretically. In both extracts, ch14.18, which was
applied as a positive control, and sera from mimo-
Figure 5. Immunofluorescence staining of GD2 on M21 melanoma cells. Anti-GD2 IgG antibodies were detected by FITC-
conjugated secondary antibodies. Nuclei were stained with Hoechst dye. Cells were viewed with a Zeiss Axioplan 2 fluorescence
microscope. (A) Anti-GD2 mAb ch14.18 (positive control). (B) Sera from mice immunized with GRL-KLH. (C) Sera from mice
immunized with DGG-KLH. (D) Sera from mice immunized with KLH alone do not specifically recognize GD2.
f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2006. 36: 1267–1274
tope-immunized mice recognized GD2 at a band at
55 kDa in Western blots. Binding intensity was found to
be dose dependent. Serum from mice immunized with
the carrier KLH alone did not recognize the antigen
(Fig. 4, results for the SK-NA-S cell extract). After
deglycosylation of the blots, the 55-kDa band was no
longer detectable with ch14.18 or the immune sera,
indicating that the antibodies indeed recognize the
carbohydrate moiety of GD2. These data show that the
mimotope-induced antibodies recognize GD2 not only
in the purified preparation, which had also been used in
characterizing the mimotopes [25], but also in its native
form.
To demonstrate whether anti-mimotope antibodies
also react with GD2 on cell surfaces, we performed
immunofluorescence staining of M21 and SK-NA-S cells.
mAb ch14.18 was used as a positive control (Fig. 5A,
results of the M21 staining). Sera of mice immunized
with GRL-KLH (Fig. 5B) or DGG-KLH (Fig. 5C) showed
marked membranous staining of GD2-expressing cells.
As seen in the GD2 ELISA, anti-GRL-KLH antibodies
reacted slightly more strongly with the target cells. Only
background staining was observed when serum anti-
bodies from mice immunized with KLH alone were
tested (Fig. 5D). SW480 control cells did not stain with
any antibody (data not shown).
Discussion
In the present study, we show that an epitope of the
carbohydrate antigen GD2 can be successfully trans-
lated into immunogenic peptide epitope mimics. Dis-
ialoganglioside GD2 belongs to the class of TACA, and is
expressed on malignancies of neuroectodermal origin.
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Eur. J. Immunol. 2006. 36: 1267–1274
For these tumors, such as melanoma and neuroblasto-
ma, therapeutic options in advanced disease are limited.
Therefore, novel treatment modalities are being sought.
GD2 has so far been exploited as a passive immunother-
apy target with the chimeric mAb ch14.18 in neuro-
blastoma and melanoma trials. However, passive anti-
body applications have several drawbacks. First, these
antibodies need to be engineered to be human/mouse
chimeric or preferably fully humanized, to prevent
formation of human anti-mouse antibodies (HAMA) in
the patient. Also with ch14.18, which is a human/mouse
chimeric antibody, the development of human anti-
chimeric antibodies has been seen [20]. Second,
'artificial' antibodies have to be repeatedly administered
to achieve continuous serum levels that are needed for
anti-tumor efficacy. This dependency on weekly infu-
sions is extremely demanding for cancer patients. Third,
the production costs of the amounts of chimeric or
humanized antibodies needed for passive immunother-
apy are enormous. For all these reasons, an active
immunotherapy that would induce anti-GD2 antibodies
would be an attractive solution, both circumventing
multiple infusions, as well as the danger of inducing an
immune response against the non-human parts of the
artificial antibodies. The continuous availability of
'natural', polyclonal antibodies induced by active
immunization is expected to show all the beneficial
properties of passive immunotherapy, with the addi-
tional advantage that the induced antibodies are not of
a single given antibody subclass. By being both
polyclonal and of different isotypes, these antibodies
are expected to be capable of triggering the whole array
of antibody-mediated immune functions against the
tumor.
Indeed, active immunizations against GD2 have been
pursued for a long time. In initial studies, gangliosides
were used directly as immunogens [29, 30], a strategy
that encountered problems as carbohydrates are in-
trinsically T cell-independent antigens, which only elicit
weak immune responses and mostly antibodies of the
IgM subclass. To overcome these limitations, the weakly
immunogenic gangliosides were coupled to the im-
munogenic carrier molecule KLH [24]. Currently, a
GD2-KLH conjugate vaccine is being tested in patients
with malignant melanoma [31].
Yet other approaches seek to replace the carbohy-
drate moiety with its immunological drawbacks alto-
gether. The first possibility to mimic carbohydrates with
a protein structure was the development of anti-
idiotypic antibodies (anti-Id). Indeed, several anti-Id
mimicking GD2 have been generated [26–28], and one
of them has already been used as a vaccine in advanced
melanoma patients [32]. However, anti-Id are still mAb,
and, when applied as antigens, can give rise to HAMA.
So either they need to be humanized, or produced as
f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Molecular immunology 1271
recombinant Fab without the Fc portion. This limits their
practicability for vaccine formulation.
Another possibility to generate mimics of any
structure recognized by an antibody is the phage display
technique [33]. It is a technique to define peptides
mimicking natural epitopes, including carbohydrate
structures [34–36]. Phage display peptide libraries
consist of filamentous phage particles displaying
random peptides of defined length on their surface.
By biopanning [37] such phage display libraries with an
antibody of interest, epitope mimics, so-called mimo-
topes, can be selected. These peptides mimic the original
antigen by virtue of their 3-D structure, in which the
amino acids of the peptide adopt the same shape as the
epitope recognized by the selecting antibody [38, 39].
Thus, the poorly immunogenic carbohydrate antigens
can be converted into peptide epitope mimics, which
have a much greater potential of both antigenicity and
memory induction. Another strength of vaccinations
with these epitope mimics is that by virtue of their not
being identical to the original antigen, immunological
tolerance, which exists for self antigens, can be
circumvented. Additionally, in the vaccine formulation,
a highly immunogenic carrier molecule is included,
providing epitopes for eliciting T cell help. Moreover, the
vaccine is administered with an immunological
adjuvant. Taking these three strategies together,
immunological tolerance to GD2 can be overcome.
The mimotope strategy has already been successfully
pursued for a number of carbohydrate antigens, including
several TACA [35, 40–42], with the resulting mimotopes
being used for immunizations either coupled to KLH [43],
or encoded as minigenes in a DNA vaccine approach [44].
In a previous study, our group defined such mimotopes of
the epitope recognized by mAb ch14.18 on GD2 [25].
These were, to the best of our knowledge, the first peptide
mimics generated for this ganglioside antigen. Early last
year, another group described the definition of a GD2
mimotope [45], which they encoded in a minigene for a
DNA vaccination approach. In the present study, we
aimed to assess whether 'our' cyclic mimotopes were
immunogenic and whether the induced antibodies
recognize the original antigen GD2. To ensure correct
cyclization and thus peptide conformation, we chose to
have them produced synthetically, and coupled to KLH.
We were able to show that two selected GD2 epitope
mimics elicited a humoral immune response. The induced
antibodies were cross-reactive for the two peptides, a
finding that strengthens the mimotope hypothesis.
Obviously, both peptides, although different in sequence,
share the same 3-D characteristics, which mimic the
ch14.18 epitope. Importantly, the anti-mimotope anti-
bodies confirmed this mimicry by specifically recognizing
the original antigen, GD2 in ELISA, Western Blot, and on
the cell surface. We thus demonstrated that the GD2
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1272 Angelika B. Riemer et al.
ganglioside antigen can be translated into peptide
mimotopes for vaccination purposes.
Peptide vaccines that can be synthesized custom-
made are cost-effective, simple to produce, and can
easily be quality-controlled during the manufacturing
process. They are chemically stable and contain no
oncogenic, toxic, or infectious material. As active
immunogens, synthetic mimotopes can continuously
induce available tumor-targeting antibodies. These
characteristics answer the points raised as disadvantages
of passive antibody therapies, i.e., high costs and
multiple necessary infusions. Additionally, the induction
of undesired antibodies (e.g., HAMA) is ruled out, as not
only a pure antigen, but a specific epitope is applied.
Moreover, the resulting 'natural' humoral and cellular
immune responses against GD2, compared to passive
anti-GD2 immunotherapy, may even mediate enhanced
anti-tumor effects.
In conclusion, we have shown that an epitope of a
carbohydrate antigen can successfully be translated into
immunogenic peptide mimotopes. Furthermore, immu-
nizations with these epitope mimics induced antibodies
again recognizing the original carbohydrate, in our case
the disialoganglioside GD2. As the elicited antibodies
were of the IgG isotype, we could show that with
repeated vaccinations the mimotope conjugates are
capable of recruiting T cell help and inducing memory
phenomena. We thus provide evidence that GD2
mimotopes are suitable candidates for active immu-
notherapy of GD2-expressing tumors, such as melanoma
and neuroblastoma.
Materials and methods
Cell lines and cell lysates
Two GD2-positive cell lines were used in this study, the human
melanoma cell line M21 and the human neuroblastoma cell
line SK-NA-S. The human colon adenocarcinoma cell line
SW480, which is GD2 negative, was employed as a negative
control. All cells were grown in RPMI medium (Gibco BRL,
Inchinnan, UK) supplemented with 10% fetal calf serum, 1%
glutamine, and 1% penicillin/streptomycin.
Total cell lysates were prepared as described previously
[38], with a lysis buffer containing 20 mM Tris, 150 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and a protease
inhibitor cocktail (Complete, Roche, Basel, Switzerland), at
pH 7.5. The protein concentration was determined photome-
trically, using bicinchoninic acid (BCA Protein Assay Kit,
Pierce, Rockford, IL). Extracts were aliquoted and stored
at –80 C.
Monoclonal antibodies
ch14.18, a mouse/human IgG1 mAb, directed against
disialogangliside GD2, was kindly provided by Dr. S. D. Gillies
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Eur. J. Immunol. 2006. 36: 1267–1274
(EMD-Lexigen, Lexington, MA). Cetuximab, also a mouse/
human chimeric IgG1 mAb, directed against EGFR, was used as
an isotype control.
Synthesis of vaccine constructs and DotBlot assay
The 1,12-cyclic peptides C-GRLKMVPDLE-C (GRL) and C-
DGGWLSKGSW-C (DCC) were manufactured synthetically
(piChem, Graz, Austria). Conformational accuracy was
verified with ch14.18 in a DotBlot assay. In brief, the peptides
were solubilized in PBS/20% dimethylformamide, and dotted
onto a nitrocellulose membrane at 1 mg/mL. Blot strips were
incubated with ch14.18 or cetuximab, respectively, and bound
antibody was detected by peroxidase-conjugated anti-human
IgG (Jackson ImmunoResearch, West grove, PA) using the
ECL+ chemiluminescence detection protocol (Amersham
Pharmacia Biotech, Little Chalfont, UK). Subsequently, the
peptides were coupled via a linker (GPGPG) and S-acetyl-thio-
acetate on their C terminus to a succinimidyl-4-(N-maleini-
midomethyl)cyclohexan-1-carboxylate-activated immuno-
genic carrier, KLH. The conjugate was again checked for
ch14.18 binding capability in a DotBlot assay (as above).
Immunization of BALB/c mice
Three groups (n=8) of BALB/c mice (Charles River Labora-
tories, Sulzfeld, Germany) were immunized i.p. with 10 lg of
the mimotope conjugates, GRL-KLH and DGG-KLH, or the
carrier protein KLH alone, respectively, on days 1, 15, 36 and
57. Aluminum hydroxide was used as an adjuvant in all groups.
Blood was taken from the tail vein on days 0 (preimmune
serum), 22, 43 and 64. Mice were treated according to
European Union Rules of Animal Care, with permission no.
66.009/35-BrGT/P2004 from the Austrian Federal Ministry of
Education, Science and Culture.
Titer determination
All titers were determined using the third immune sera
(collected on day 64). Anti-carrier titers were assessed by
ELISA. For anti-carrier titers, ELISA plates (Nunc, Roskilde,
Denmark) were coated with KLH, 1 lg/mL in bicarbonate
buffer, pH 9.6, by overnight incubation at 4 C. Plates were
then washed with PBS/0.05% Tween 20, and nonspecific
binding was blocked by incubation with PBS/1% dry milk
powder. Sera were added at a dilution of 1:100 in PBS/0.01%
dry milk. Bound antibodies were detected with a peroxidase-
conjugated rat-anti-mouse IgG antibody (Jackson Immuno
Research Laboratories, Inc., West Grove, PA). The reaction was
developed with TMB substrate (BD Biosciences, San Diego,
CA). OD was measured in an ELISA reader (Dynatech,
Denkendorf, Germany) at 450–630 nm.
Anti-peptide titers were determined by incubation of serial
dilutions of pooled sera (1:5000; 1:10 000; 1:50 000;
1:100 000; 1:1 000 000) with dotted mimotope peptides, a
control peptide, and KLH. Bound antibodies were detected
with a peroxidase-conjugated sheep-anti-mouse Ig antibody
(Amersham) as in the DotBlot assay described above.
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Eur. J. Immunol. 2006. 36: 1267–1274
Ganglioside ELISA
Purified gangliosides GD2 and GM1 (Sigma, St. Louis, MO)
were coated at a concentration of 1 lg/mL to ELISA plates
(Nunc) by dilution in ethanol and subsequent evaporation of
the diluent. Plates were washed and blocked as described
above. Third immune sera were added in a dilution series of
1:100–1:3000 in PBS/0.01% dry milk, and bound antibodies
were detected with a peroxidase-conjugated rat-anti-mouse
IgG antibody (Jackson). For subclass determinations, mouse Ig
standard dilution series were coated onto the same ELISA
plates. Third immune sera were added to the ganglioside-
coated wells as described above, while standards were
incubated with PBS/0.01% dry milk. Rat anti-mouse Ig
subclass antibodies were then added to all wells at a dilution
of 1:500 in PBS/0.01% dry milk, and bound antibodies
detected with a peroxidase-conjugated mouse anti-rat IgG
antibody (Jackson). The peroxidase/substratum reactions
were developed and measured as described above.
Cell lysate Western blots
M21 and SK-N-AS cell lysates (prepared as described above)
were separated by SDS-PAGE, and blots incubated with
ch14.18 or serum pool dilutions as detecting antibodies.
Bound ch14.18 was detected by a peroxidase-conjugated goat
anti-human IgG antibody (Jackson), and bound mouse
antibodies by a peroxidase-conjugated sheep anti-mouse Ig
antibody (Amersham), using the ECL+ chemiluminescence
detection protocol (Amersham) and Biomax-MS films (Kodak).
Immunofluorohistochemistry
M21 and SK-NA-S cells were plated at 5  104 cells/mL on
eight-well Lab-Tek tissue culture chamber slides (Miles
Laboratories Inc., Naperville, IL). SW480 cells were used as
negative controls. Cells were grown overnight until half-
confluent. Chamber slides were then cooled to 4 C, and
washed with ice-cold PBS. Cells were fixed with 4%
paraformaldehyde in PBS for 30 min, and chamber slides
were incubated with 50 mM NH4Cl in PBS to quench fixation,
and blocked with 1% BSA in PBS. Subsequently, cells were
incubated with ch14.18 (positive control), or with pooled anti-
GRL-KLH and anti-DGG-KLH third immune sera, and bound
antibodies were detected by FITC-conjugated goat anti-mouse
IgG (Caltag Laboratories, Burlingame, CA). Pooled third
immune sera from the KLH-immunized mice were used as
control. Nuclei were stained with 0.1 lg/mL Hoechst dye
(Sigma) in PBS for 10 min. Cells were mounted in Mowiol
mounting medium and viewed with a Zeiss Axioplan 2 (Carl
Zeiss, Jena, Germany).
Acknowledgements: The work was supported by
BioLife Science GmbH, Vienna, Austria; by project grant
no. 10965 of the Austrian National Bank Science Fund;
and by the Center of Excellence in Clinical and
Experimental Oncology (CLEXO), Austrian Federal Min-
istry of Education, Science and Culture (GZ 200.062/2-VI/
1/2002). A.B. Riemer and K.H. Br
mswig are recipients of
f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Molecular immunology 1273
Hans & Blanca Moser Fund scholarships. The work was
also supported by DFG (Lo 635–2) and Frdergesellschaft
Kinderkrebs-Neuroblastomforschung to H.N. Lode. We
thank Harald Kurz and Silke Gruber for excellent
technical assistance.
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