Characterization of the Antibody Response against NeuGcGM3
Ganglioside Elicited in Non-Small Cell Lung Cancer Patients
Immunized with an Anti-Idiotype Antibody1
Ana María Herna ´ndez,2* Darie ´n Toledo,* Darel Martínez,* Tania Grin ˜a ´n,* Victor Brito,*
Amparo Macías,†Sailyn Alfonso,‡Teresa Rondo ´n,* Eduardo Sua ´rez,* Ana María Va ´zquez,*
and Rolando Pe ´rez*
1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides,
sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing
gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses
elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the
hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3
ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing
X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of
the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id?Ag?Abs could reflect the
activation of an autologous idiotypic cascade into the patients. Both Id?Ag?and Id?Ag?fractions were separated by affinity
chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in
the Id?Ag?fraction. Both Id?Ag?and Id?Ag?Abs were able to specifically recognize and induce cell death in NeuGcGM3-
expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median
survival times. The Journal of Immunology, 2008, 181: 6625–6634.
lung cancer (NSCLC),3which constitutes ?80% of the total num-
ber of new cases (1, 2). Surgery is currently the only curative
treatment for NSCLC, but because the majority of the patients are
diagnosed with advanced disease this is seldom an effective course
of action. The majority of NSCLC patients will require systemic
chemotherapy, but unfortunately the median survival time even
after the best available combination of systemic active drugs is
limited to ?8–9 mo, and a 1-year survival rate is roughly 30–36%
(3–7). These facts underscore the urgent need to develop new ther-
apeutic approaches for NSCLC.
NeuGc-containing gangliosides are attractive targets for cancer
immunotherapy as these glycolipids are not normally expressed in
humans and are therefore foreign Ags, but they have been detected
ung cancer is the leading cause of cancer-related mortal-
ity, with 1.2 million new cases worldwide diagnosed each
year. The most frequent histological type is non-small cell
in a range of human tumors by Abs and chemical analysis (8–12).
Additionally, recent experimental data suggest that NeuGcGM3 is
relevant for tumor progression (13). One strategy to generate im-
mune responses to these glycolipids is the use of anti-Id Abs
(Ab2). This approach arose from Jerne’s idiotypic network theory
(14), which postulates that, due to the large potential diversity of
Ig variable regions, the Id repertoire can mimic the universe of self
and foreign epitopes. Thus, properly selected anti-idiotypic Abs
could act as tumor-associated ganglioside surrogates. In addition to
our own experience, two other anti-idiotypic mAbs mimicking
gangliosides have been used in clinical trials with cancer patients,
but with limited results (15, 16).
We previously reported a vaccine preparation featuring a mu-
rine anti-Id mAb related to the NeuGc-containing ganglioside
Ag model. This Ab2, named 1E10 (17), was generated from the
immunization of BALB/c mice with P3, an idiotypic Ab (Ab1)
that recognizes NeuGc-containing gangliosides, sulfated glyco-
lipids, and Ags present in different human tumors including
those from the lung, and which contains a regulatory Id accord-
ing to Bona’s concept (18–24). Preparations containing 1E10
mAb were able to induce antitumor effects against lung metas-
tases in murine models, and phase I clinical trials have proven
the safety and immunogenicity of 1E10 Id vaccination in mel-
anoma and breast cancer patients (20, 25, 26). From these latter
studies, high titer Ab responses to NeuGc-containing ganglio-
sides were measured in the sera of cancer patients. A fraction of
non-suppressible anti-NeuGc-containing ganglioside Abs was
demonstrated through adsorption of these sera with 1E10 mAb,
suggesting that 1E10 Id vaccination might enhance antitumor
natural immune response (20, 26). Furthermore, NeuGcGM3-
specific IFN-?-secreting cells were measured by ELISPOT
from PBMC of 1E10-vaccinated breast cancer patients (27).
*Department of Antibody Engineering and†Department of Clinical Trials, Center of
Molecular Immunology, Havana, Cuba; and‡Oncology Unit, Celestino Herna ´ndez
Robau Hospital, Villa Clara, Cuba
Received for publication January 8, 2008. Accepted for publication August 20, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Recom-Bio S.L. and by the Cuban Government.
2Address correspondence and reprint requests to Dr. Ana María Herna ´ndez
Va ´zquez, Department of Antibody Engineering, Center of Molecular Immunol-
ogy, 216 Street and 15 Avenue, Atabey, Playa, Havana 11600, Cuba. E-mail
3Abbreviations used in this paper: NSCLC, non-small cell lung cancer; Ab2, anti-Id
Ab; Ab1, idiotypic Ab; HPTLC, high performance TLC; PI, propidium iodide; CI,
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
More recently we performed a clinical study in NSCLC patients that
showed encouraging clinical benefits (28). We now report the im-
mune response elicited in 20 advanced NSCLC patients treated with
1E10 mAb, and demonstrate the induction of Abs that are Id?Ag?,
but also Id?Ag?. Both Ab fractions recognized and induced the
death of myeloma cells expressing NeuGcGM3 by a complement-
independent mechanism. Those patients who developed IgG
and/or IgMAbs againstNeuGcGM3
We hypothesize 1E10 Id vaccination could induce an idio-
typic cascade, which would amplify the Ag-specific immune
response to a tumor-associated neo-self ganglioside Ag. This
therapeutic concept goes well beyond the classical concept of
Materials and Methods
Gangliosides and cells
Gangliosides NeuAcGM3 and NeuGcGM3 purified from dog and horse
erythrocytes, respectively, as described earlier (29), were provided by Dr.
L. E. Ferna ´ndez (Vaccine Department, Center of Molecular Immunology,
Havana, Cuba). P3-X63-Ag8.653 (X63) murine myeloma cell line (ATCC
NCRL 1580), expressing NeuGcGM3 in their membrane (30), and H82
human lung carcinoma cell line (ATCC HTB-175), negative for the ex-
pression of the ganglioside, were grown in DMEM (Invitrogen) supple-
mented with 10% heat-inactivated FCS (HyClone), 2 mM L-glutamine, 25
mM HEPES, 100 U/ml penicillin, 100 ?g/ml streptomycin, and maintained
at 37°C with 5% CO2.
Anti-Id Ab (1E10)
Ab2 1E10 mAb (IgG1, ?) was generated by immunizing BALB/c mice
with P3 mAb (IgM, ?; Refs. 17, 18). 1E10 mAb was purified from ascites,
and the aluminum hydroxide-precipitated mAb vaccine was produced in
accordance with the Good Manufacturing Practice guidelines and certified
by the Quality Control Department of the Center of Molecular Immunol-
ogy, as previously reported (20).
Twenty patients with histo- or cytological confirmed advanced NSCLC
were eligible for enrollment in a compassionate use study, after pro-
viding written, informed consent. All the patients have previously re-
ceived the oncospecific treatment established in the Oncological Ther-
apeutic Standards, according to their stage at the moment of the
diagnosis by the National Comprehensive Cancer Network guidelines
(version 2.0, 2006). First line chemotherapy, consisting mainly on cis-
platin/vinblastin, had to be completed 4 wk before the patients entered
the study. Other eligibility criteria included World Health Organization
performance status ?2, age ?18 years, normal hematopoietic, hepatic,
and renal functions, and life expectancy higher than 3 mo. The most
important exclusion criteria included the presence of brain metastases,
pregnancy or lactation, serious chronic diseases, and active infections.
The study was approved by the Institutional Review Board of the hos-
pital where the study was developed.
Patients were injected intradermally with 15 doses of 1 mg of aluminum
hydroxide-precipitated 1E10 mAb, as base treatment. The first five
doses were administered every 14 days, and the remaining 10 doses
were administered every 28 days. After 15 doses, reinmunizations were
administered at 28-day intervals, if the patients maintained a favorable
clinical status. Serum was obtained before and during treatment. Pa-
tients who received one or more vaccine doses were evaluable for tox-
icity and clinical results, and those who received at least four doses of
the Ab response induced in vaccinated NSCLC patients.
Hyperimmune sera from patients immunized with alu-
minum hydroxide-precipitated 1E10 mAb were prein-
cubated with the isotype-matched ior C5 mAb, and later
the reactivity against 1E10 and ior C5 mAbs was as-
sessed by ELISA. ?, p ? 0.01, Mann-Whitney U test,
Immunodominance of 1E10 mAb Id in
Table I. Number of doses received, maximal titer, and isotype analyses
against 1E10 mAb and NeuGc-GM3, and survival times in vaccinated
(mo)IgM IgG IgMIgG
6626 ANTI-NeuGcGM3 Abs ELICIT BY AN ANTI-Id mAb
aluminum hydroxide-precipitated 1E10 mAb were considered immuno-
Measurement of Ab response
To measure Ab3 reactivity against 1E10 mAb and purified gangliosides
in sera from NSCLC patients, solid-phase ELISAs were performed as
previously described (20). The highest serum dilution giving OD values
?0.25 and being at least three times the value corresponding to the
preimmune serum at the same dilution was considered as titer. Assays
were performed in triplicate for each sample and the coefficient of vari-
ation was ?15%. The OD of the blanks was ?0.1. The presence of Abs
specific to gangliosides was detected by immunostaining on high per-
formance TLC (HPTLC) plates, and by ELISA, as previously described
Ab-binding inhibition assays
To define the extent of the Id-specific response against 1E10 mAb, pa-
tients’ hyperimmune sera were incubated overnight at 4°C with isotype-
matched irrelevant mAb ior C5, specific for a glycoprotein expressed on
human colorectal cells (31) at a final concentration of 0.5 ?g/ml, to adsorb
the human Abs against the isotypic determinants of 1E10 mAb. Then,
samples were added onto 1E10 mAb-coated plates and remnant reactivity
was assessed by the ELISA procedures previously described (20). Ior C5
mAb-coated plates were used to measure the isotypic response in non-
adsorbed sera and as controls of the absorption efficiency.
Evaluation of the anti-ganglioside reactivity after preabsorption of
patients’ sera with 1E10 mAb was performed by ELISA. Serum samples
were preincubated with 1E10 mAb at a final concentration of 0.5 ?g/ml,
and added onto plates coated with NeuGcGM3.Serum reactivity was
assessed by the ELISA procedures previously described (20). ELISA
plates coated with 1E10 mAb were used as a control of the adsorption
Isolation of Id positive and negative Ab fractions
Hyperimmune sera from NSCLC patients and their preimmune sera,
used as negative control, were diluted 1/2 with PBS to a final volume
of 200 ?l and incubated with 100 ?l of Sepharose 4B-coupled 1E10
mAb matrix, overnight, at 4°C with shaking. The supernatant was re-
covered and Abs bound to the column were eluted with 200 ?l of
glycine-HCl (pH 2.8) and neutralized with 2 M Tris. Reactivity of the
eluted and unbound fractions against 1E10 mAb and gangliosides was
tested by ELISA as previously described (20).
Patient serum samples or Ab fractions were incubated with 5 ? 105X63
cells for 30 min on ice. After washing, the cells were incubated with FITC-
conjugated goat anti-human immunoglobulins (Jackson ImmunoResearch
Laboratories) for 30 min on ice. H82 lung carcinoma cells were used as
negative control. To determine whether the reactivity of the isolated Id?
and Id?Abs against the cells was not due to the presence of remnant Abs
reacting with 1E10 mAb, both fractions were preincubated with 1E10 mAb
at a final concentration of 0.5 ?g/ml overnight at 4°C. Percentage of pos-
itive stained cells were determined in a FACScan instrument (BD Bio-
sciences). The WinMDI 2.8 444 program was used to analyze a total of 104
cells acquired on every FACS assay.
Induction of cell death
Patients’ sera or Ab-purified fractions were incubated with 2 ? 105X63
cells in 100 ?l of RPMI 1640 culture medium supplemented with 1% FCS
in 5% CO2atmosphere at 37°C for 4 h. The cell death induction was
detected by the addition of propidium iodide (PI; Sigma-Aldrich) at a final
concentration of 10 ?g/ml and analyzed by flow cytometry. Similar ex-
periments were performed with patients’ samples previously heated 30 min
at 56°C for complement inactivation.
The mean value and the SD of the values obtained in the triplicates of each
sample were calculated. Each experiment was repeated at least twice. The
mean values and the SDs were plotted using the Microcal Origin program.
Mann-Whitney U test was used as a non-parametric test for pair-wise com-
parisons. Survival times were estimated using the Kaplan-Meier method
via the SPSS Program, version 10, and the differences in the median sur-
vival times between responders and non-responders were compared using
the log-rank test.
1E10 mAb induced a specific Ab response against its Id (Id?)
in NSCLC patients
NSCLC patients were considered as immunologically evaluable
when they had received at least five doses of the aluminum
hydroxide-precipitated 1E10 mAb. Ab responses induced by
immunization with 1E10 mAb were tested in sera obtained from
patients before and during the treatment. Of the 20 patients, 18
bound to microtiter plates coated with NeuGc and NeuAcGM3 (200 ng/well), and the reaction was developed with biotinylated goat anti-human IgG?IgM,
followed by the addition of alkaline phosphatase-streptavidin complex. B, Gangliosides (1 ?g) were chromatographed with chloroform:methanol:0.2%
CaCl2in 2.5 M NH3 (5:4:1; v/v/v) and visualized with orcinol. For the immunostaining, the plates were incubated with the patients preimmune (pI) and
hyperimmune (hI) sera, diluted 1/200, and the reaction was developed with alkaline phosphatase conjugated goat anti-human IgG?IgM.
Kinetics and specificity of Ab3 response against NeuGcGM3 in the sera of NSCLC. A, Sera from vaccinated patients diluted 1/400 were
6627The Journal of Immunology
developed Abs against 1E10 mAb. This Ab response was of the
IgG isotype with titers ranging from 1/3,200 to 1/25,600 (Table
I); no IgM Abs were detected at the lowest serum dilution tested
To confirm that a specific response against 1E10 mAb Id was
generated by the immunization, patients’ hyperimmune sera were
preabsorbed with the isotype-matched control mAb, and the re-
maining reactivity against 1E10 mAb was measured by ELISA. As
shown in Fig. 1, strong reactivity against 1E10 mAb was detected
in patient sera after preabsorption with the control mAb. The level
of Ab response against 1E10 mAb Id was higher than the reactivity
of the non-preadsorbed sera against the isotype in all patients (p ?
0.05, Mann-Whitney U test, one tail).
1E10 mAb immunization induced a specific Ab response
against NeuGcGM3 in the patients (Ag?)
Pre- and postimmunization sera samples were tested by ELISA
for the recognition of NeuGcGM3 and NeuAcGM3 to deter-
mine whether the treatment of NSCLC patients with 1E10 mAb
induced Abs with the same specificity of P3 mAb (Ab1). Six-
teen patients developed Abs of IgM and/or IgG isotype that
NeuAcGM3was detected (Fig. 2A). Titers of up to 1/25,600 and
1/12,800 of IgM and IgG responses were obtained, respectively
(Table I). Analysis of the isotype of the anti-NeuGcGM3 re-
sponse indicated that 14 patients generated both IgG and IgM
Abs, one patient only showed IgG, and in another patient only
IgM Abs specific to NeuGcGM3 were detected. The Ab re-
sponse against NeuGcGM3 ganglioside was increased with the
course of vaccination, reaching a peak after patients received
the fourth or fifth doses of the anti-idiotypic mAb. The speci-
ficity of the anti-ganglioside Ab response was confirmed by
HPTLC immunostaining where an evident specific binding of
hyperimmune patients’ sera with NeuGcGM3 was observed. No
reaction was detected when preimmune patients’ sera were
tested (Fig. 2B).
and the remnant reactivity to 1E10 mAb and NeuGcGM3was assessed by ELISA. B, Id?Ab fraction isolated from patient 7 was incubated with 1E10 mAb
or NeuGcGM3 on ELISA plates. C, Gangliosides (1 ?g) were chromatographed with chloroform:methanol:0.2% CaCl2in 2.5 M NH3 (5:4:1; v/v/v) and
visualized with orcinol. For the immunostaining, the plates were incubated with the Id?Ab fractions isolated from the preimmune (pI) and hyperimmune
(hI) sera of patient 7, diluted 1/200, and the reaction was developed with alkaline phosphatase-conjugated goat anti-human IgG?IgM.
Reactivity against NeuGcGM3 in patient’s sera absorbed with 1E10 mAb. A, Sera from NSCLC patients were preincubated with 1E10 mAb
6628 ANTI-NeuGcGM3 Abs ELICIT BY AN ANTI-Id mAb
1E10 mAb immunization generated Ag?Id?and Ag?Id?Abs
in the immunized patients
As 1E10 mAb immunization generated different isotype pattern
Ab responses against the mAb molecule and the ganglioside,
we studied whether 1E10 mAb induced the activation of
NeuGcGM3-related idiotypic networks through the detection of
Abs characterized to bind to NeuGcGM3 and not to 1E10 mAb
(Ag?Id?) in patients’ sera. To accomplish this, hyperimmune pa-
tient sera were preincubated with saturating amounts of 1E10
mAb, and the remaining reactivity against NeuGcGM3 was mea-
sured by ELISA. Plates coated with 1E10 mAb were used as con-
trols of the adsorption efficiency, showing that there was no bind-
ing with 1E10 mAb by the preadsorbed sera (Id?). Significant
binding activity to NeuGcGM3 was still detected in preabsorbed
sera of all the patients studied, suggesting the presence of a frac-
tion of Abs characterized to be Ag?Id?(Fig. 3A). To further char-
acterize these Abs, the Id?fraction was separated from the Id?
fraction by incubating hyperimmune patient sera with a Sepharose
4B-coupled 1E10 mAb matrix. The bound (Id?) and the unbound
(Id?) Ab fractions were then recovered, and their reactivity against
1E10 mAb and NeuGcGM3 was confirmed by ELISA using frac-
tions isolated from preimmune sera as negative controls. As is
shown in Fig. 3B, no binding with the 1E10 mAb could be detected
in the Id?fraction. However, this fraction contained not only
IgM, but also IgG Abs against NeuGcGM3. The specificity of
these Abs was further corroborated by HPTLC immunostaining,
where Id?Abs reacted with NeuGcGM3 but not with the N-
acetylated variant of the ganglioside chromatographed on the
TLC plate (Fig. 3C).
We next evaluated the capacity of patient sera to recognize
naturally expressed NeuGcGM3 on tumor cells. To do this, pre-
immune and hyperimmune sera of the patients were incubated
with the myeloma cell line X63, which expresses high levels of
NeuGcGM3 (75% of all glycolipids expressed at the cell mem-
branes; Ref. 30), and the binding was measured by flow cytom-
etry. Hyperimmune sera from 10 of the 12 patients tested
showed significant binding to X63 myeloma cells compared
with their preimmune ones. In contrast, both pre- and hyperim-
mune sera from patients showed very low reactivity to the
NeuGcGM3 negative H82 cells. Fig. 4 shows the results
and hyperimmune sera of 1E10 mAb-
treated NSCLC patients to myeloma
cell line P3-X63-Ag8.653. Patients pre-
immune (light gray) and hyperimmune
(black) sera, diluted 1/10, were incu-
bated with the NeuGcGM3 expressing
myeloma cell line P3-X63-Ag8.653 or
the control human cell line H82. The
jugated anti-human IgG?IgM. The
numbers represent the percentage of
hyperimmune sera-reacting cells after
the subtraction of the value of preim-
mune sera-reacting cells.
Binding of preimmune
6629The Journal of Immunology
obtained with the sera from three representative patients. To
determine which fractions (Id?or Id?) were responsible for this
recognition, the binding of each to the X63 myeloma cells was stud-
ied. As shown in Fig. 5, both Ab fractions obtained from hyperim-
mune patients’ sera bound to the myeloma cells, and a very low per-
centage of the cells was recognized by the Ab fractions isolated from
preimmune sera. The reactivity of the Id?Ab fractions obtained from
hyperimmune sera was not due to the presence of remnant Abs re-
acting with 1E10 mAb at the dilution tested, because preincubation of
these fractions with the anti-Id mAb did not inhibit their binding. In
contrast, the adsorption of the Id?Ab fractions with 1E10 mAb ab-
rogates their recognition of the myeloma cells (Fig. 5). These results
together indicate that 1E10 mAb treatment induced in the patients the
generation of Abs specific to NeuGcGM3 due to their recognition of
1E10 Id, and also of Abs specific to the nominal Ag, but not to the
1E10 mAb generated Abs capable of inducing cell death
With the objective to study whether the Abs developed in the
immunized patients were able not only to recognize, but also to
kill X63 myeloma cells, patients’ sera were incubated for 4 h at
37°C with the cells, and the effect on their viability was studied
by flow cytometry using the PI exclusion assay. In 9 of 15
patients studied, an increase in PI incorporation in the cells
incubated with hyperimmune patient sera was observed over
that caused by preimmune sera (Fig. 6). The results shown in
Fig. 6 suggest that this cell death was induced by a mechanism
independent of complement cascade activation, because it was
not inactivated by heating the sera (30 min at 56°C) before use
in the assay. We next studied whether both Id?and Id?Ab
fractions also possess this cytotoxic capacity by incubating
them with the myeloma cells, as previously described. As is
shown in Fig. 7, both Id?and Id?Abs were able to induce the
death of the cells. These results suggest that 1E10 mAb treat-
ment induced in the patients the generation of Abs with the
kill tumorcells expressing
1E10 mAb-immunized patients who developed anti-NeuGcGM3
Abs had longer survival times
Patients enrolled in the study were evaluated for safety. The over-
all toxicity of 1E10 anti-Id vaccine was classified as grade 1 and 2,
according to the National Cancer Institute Common Toxicity Cri-
teria (version 3.0). The most common side effects were local re-
action in the injection site with erythema and induration, occasion-
ally associated with mild pain that disappeared in a few days
(24–72 h). Some patients had fever, pruritus, arthralgias, and mild
headache. All the symptoms were independent of the number of
doses administered, and they lasted between 1 and 3 days and
disappeared spontaneously. No other severe or unexpected adverse
events were observed, and neither biochemical nor hematological
abnormalities were reported.
Abs to the myeloma cell line P3-X63-
Ag8.653. Id?and Id?Abs, diluted
1/10, were preincubated or not with sat-
urating amounts of 1E10 mAb, and the
binding with the myeloma cell line P3-
X63-Ag8.653 was detected by flow cy-
tometry with FITC-conjugated anti-
human IgG?IgM. Numbers represent
the percentage of labeled cells.
Binding of Id?and Id?
6630ANTI-NeuGcGM3 Abs ELICIT BY AN ANTI-Id mAb
Table I shows the individual survival of the NSCLC patients
that participated in this study. The overall median time survival
of the patients treated with 1E10 mAb was 10.6 mo (95% con-
fidence interval (CI), 3.0–17.3 mo). There were statistically sig-
nificant differences between the median survival time of the
NeuGcGM3 (median survival time 14.26 mo; 99% CI, 5.97–
17.3 mo) and the median survival time of the patients that did
not develop Abs against the ganglioside (median survival time
6.35 mo; 95% CI, 4.97–9.67 mo; p ? 0.01, log-rank; Fig. 8).
and/orIgG Abs against
In this study we characterize the immune response against
NeuGcGM3 ganglioside induced in NSCLC patients due to the
immunization with the anti-Id 1E10 mAb. A predominant IgG Ab
response against 1E10 mAb Id was developed in most of the im-
munized patients. The Id immunodominance was operationally de-
fined by incubating patient sera with an irrelevant mAb to adsorb
the reactivity against the murine isotype and then measuring the
remaining immunoreactivity against 1E10 mAb and comparing it
to the anti-isotype response. The magnitude of the remaining anti-
Id response was significantly higher than the response detected in
non-adsorbed sera against the control Ab (anti-isotype response;
Fig. 1). This Id immunodominance was observed not only in this
study, but also in two previous phase I clinical trials conducted in
advanced melanoma and breast cancer patients (20, 26). Moreover,
similar results were obtained when monkeys and chickens were
immunized with 1E10 mAb (32), suggesting that 1E10 mAb Id
immunodominance is not a species-depending property. The 1E10
mAb Id immunodominance is a characteristic specially important
for a murine Ab that is devoted to use for cancer patient treatment,
because it could avoid the induction of a high immune response
against the murine isotype, which in some cases can generate a
diminishment of the treatment efficacy and adverse reactions that
usually worsen with the increase of the dose number (33, 34).
A low frequency of side effects was observed in our study. Thir-
teen patients received more than 10 doses of the anti-Id mAb, and
confirmation of the safety of the treatment with this anti-Id vaccine
preparation was reported in the previous clinical trials (21, 26, 27).
The treatment of NSCLC patients with 1E10 mAb elicited Abs that
shared the capacity of P3 mAb to recognize NeuGcGM3. The pres-
ence of these Ag?-specific Abs was demonstrated by direct bind-
ing to the purified ganglioside assessed by ELISA and TLC im-
munostaining, and by their recognition of the NeuGcGM3-positive
myeloma cell line X63 by flow cytometry. In most of the patients
we detected a relatively high titer of anti-NeuGcGM3 Abs of both
IgM and IgG isotypes. This is a relevant result taking into account
that is difficult to obtain an IgG Ab response against these Ags
(35). Even the use of anti-Id Abs as protein mimicries of ganglio-
sides does not guarantee the induction of this kind of response.
Previously, it was reported that most of the melanoma patients
immunized with 1A7 mAb able to mimic GD2 ganglioside devel-
oped specific IgG Abs against this ganglioside (15). In contrast,
when melanoma and small cell lung cancer patients were treated
with the anti-Id BEC-2 mAb, the percentage of patients that de-
veloped anti-GD3-specific Ab response was low, mainly of IgM
isotype. The presence of these Abs was detected by ELISA, but
could not be confirmed by TLC immunostaining or flow cytometry
cells were incubated for 4 h at 37°C with patients 1, 7, and 8 preimmune (pI) and hyperimmune (hI) sera, diluted 1/10, without any treatment (Active
complement) or previously inactivating the complement cascade by heating (Inactive complement). The percentage of cell death was determined by the
PI incorporation assay.
NSCLC vaccinated patient’s sera induced cell death to the myeloma cell line X63 by a mechanism independent of complement cascade. X63
6631The Journal of Immunology
(36, 37). The differential induction of Ab responses against gan-
gliosides could be dependent on their different expression in nor-
mal tissues. In fact, studies previously reported showed the relation
between the level of ganglioside expression in human and murine
normal tissues and their immunogenicity (38–40).
The precise cellular and molecular mechanisms that mediate
tolerance against gangliosides have not yet been elucidated. Indi-
rect evidence suggests that B2 cell repertoire is regulated, avoiding
the generation of a mature Ab response against these Ags (38).
The strong Ab response against NeuGcGM3 induced in patients
by the 1E10 mAb can be explained because NeuGc-containing
gangliosides are not self-Ags in humans, as the gene for the en-
zyme responsible for NeuGc biosynthesis is inactivated (41–43).
The very small amounts of NeuGc conjugates detected in some
human normal tissues appear to originate from exogenous sources
(44, 45). One explanation for the higher expression of NeuGc-
containing gangliosides in some tumors is the recent demonstra-
tion that tumor hypoxia induces the transcription in the cells of a
sialic acid transporter that facilitates the incorporation of NeuGc
molecule into tumor gangliosides, as NeuGcGM2 (46). It is note-
worthy that our previous preclinical studies proved that 1E10 mAb
can behave as an immunogenic mimic, depending on the presence
or absence of NeuGcGM3 ganglioside in the normal tissues of the
immunized species (32).
The different isotype pattern in the response against 1E10 mAb
molecule, where only IgG Abs were detected, in comparison with
the IgG and IgM Ab response against NeuGcGM3, suggested that
different B cell populations could be activated in the patients to
produce Abs against this ganglioside. An important finding of this
study was the detection of a high level of non-suppressible im-
mune reactivity to NeuGcGM3 ganglioside by the adsorption of
patient sera with 1E10 mAb. A similar finding in preclinical stud-
ies was reported by Lange and Lemke in mice immunized with an
Ab against phenyl-oxazolone (47), and by ourselves in chickens
immunized with 1E10 mAb (32). The presence of these Id?Ag?
Abs due to the immunization of NSCLC patients with 1E10 mAb
also confirm the results obtained in advanced melanoma and breast
cancer patients treated with this anti-Id mAb (20, 26).
Our studies included a fractionation of the patient serum sam-
ples to further characterize the Id?Ag?Abs. We then isolated an
Id?Ag?Ab-enriched fraction from patient immune sera by remov-
ing the Id?Ab fractions with 1E10 mAb-coupled to Sepharose 4B
matrix. Their reactivity against NeuGcGM3 and not against 1E10
mAb was demonstrated by ELISA (Fig. 3B). To our knowledge,
there is no other evidence to date supporting the induction of
Id?Ag?and Id?Ag?Abs related to a ganglioside-specific im-
mune response in cancer patients.
The formation of immune complexes between tumor ganglio-
side Ags and the induced Ab3 (Id?Ag?) could eventually amplify
the specific anti-ganglioside Ab response, either by improving Ag
presentation somehow or by modifying the antigenicity of such
tumor gangliosides in the context of the immune complexes, pro-
viding an alternative explanation to the induction of the Id?Ag?
Abs. However, this explanation is not likely true for the Id?Ag?
Abs detected in our study, because they, like Ab3 Abs, were highly
specific for NeuGcGM3, being able to discriminate between this
ganglioside and the acetylated version (NeuAcGM3). The only
difference between these two gangliosides is a single oxygen atom
at the N-acetyl moiety of the sialic acid, which is involved in the
interaction between NeuGcGM3 and Ab3 Abs. Then we should
expect a different epitope recognition pattern for Abs induced by
the putative immune complexes.
vaccinated patient’s sera induced cell death to the myeloma cell line X63.
Cells were incubated for 4 h at 37°C with the Id?and Id?Ab fractions
isolated from patient 1, 7, and 8 preimmune (bold line) and hyperimmune
(shaded area) sera, diluted 1/10. The percentage of cell death was deter-
mined by the PI incorporation assay.
The Id?Ag?and Id?Ag?fractions isolated from NSCLC-
IgM and/or IgG Abs against NeuGcGM3 (black) and of the patients that
did not develop Abs against the ganglioside (gray; p ? 0.01, log-rank). On
the y-axis, the percentage of surviving is reported; on the x-axis, the time
from entrance the study (months) is reported.
Kaplan-Meier survival curves of patients that developed
6632ANTI-NeuGcGM3 Abs ELICIT BY AN ANTI-Id mAb
Preclinical data already published by our group suggest that P3
and 1E10 mAb could be able to activate idiotypic networks, in-
volving both B and T cells. Pe ´rez et al. (23) showed that lymph
node cells from BALB/c mice immunized with P3 mAb prolifer-
ated in vitro, in a dose-dependent manner, not only in response to
P3 mAb but also to 1E10 mAb, suggesting the existence of a
naturally occurring B-T cell idotypic network. Rodríguez et al.
(48) showed that chickens immunized with P3 mAb (Ab1) or 14F7
mAb (Ab1), another Ab specific for NeuGcGM3, developed an
anti-idiotypic response against both immunizing Abs. Only those
chickens, however, immunized with P3 mAb were able to develop
a strong and specific Ab response against N-glycosylated ganglio-
sides. The detection of Abs with this specificity in animals immu-
nized with an Ab1 suggested that the elicited Ab2 Abs behaved in
vivo as a ganglioside surrogate inducing a specific Ab3 response
against these Ags.
This previous evidence and the immunochemical results pre-
sented in this study suggest that the vaccination of cancer patients
with 1E10 mAb could induce the activation of an idiotypic cas-
cade. Although any idiotope could be able to mimic any Ag, only
those related to Ags that have been fixed by evolution, due to their
relevance for organism homeostasis, will be immunodominant and
capable of inducing natural idiotypic cascades (49). Among the
available Ab3 idiotopes induced by 1E10 mAb immunization in
our model, those related to the NeuGcGM3 binding site could be
recognized by Ab4 natural Abs in a different way than by 1E10
mAb and may possess a different type of mimicry. These kind of
Ab4 Abs might then induce NeuGcGM3-specific Ab5 Abs that do
not recognize 1E10 mAb Id (Id?Ag?). The involvement of T cells
in the activation of idiotypic Ab cascades has been reported pre-
viously (23, 50), and the existence of these anti-idiotypic T cells in
our case could explain the induction of specific IgG Abs against
At present, most of the Id vaccine approaches are based exclu-
sively in the mimetic capacity of the anti-Id Abs, without searching
for their immunoregulatory potential. The use of anti-Id Abs as
immunogens could offer the possibility not only of generating Ab3
Abs against their own idiotopes, but also inducing a cascade of
Id-anti-Id interactions leading to an amplified and long lasting im-
mune response against the nominal Ag. The expansion of natural
Ab repertoire by Id vaccination could even participate in the lysis
of tumor cells. In fact, recent reports showed that natural IgM Abs
that detect specific structures on aberrant cells can remove these
cells by inducing apoptotic stress (51–53).
One question we wanted to address was whether the generation
of NeuGcGM3-specific Abs in patients could have some biological
impact in tumor cells expressing this ganglioside. Our results from
the flow cytometry studies showed that most of the patients’ sera
tested bound to NeuGcGM3-positive myeloma cells and were
killed by a complement-independent mechanism. Furthermore, we
demonstrate that the Id?and the Id?fractions isolated from pa-
tients’ hyperimmune sera are capable of inducing this cytotoxic ef-
fect. We do not know yet which cytotoxic mechanism is activated by
these Abs. One possibility could be that they mediate mechanisms of
programmed cell death, because it has been reported for other Abs
specific to non-protein Ags, like gangliosides (54–57).
Several clinical trials using anti-Id Abs in cancer patients have
referred a correlation between a better clinical response and the
induction of Abs against the nominal Ag (15, 58–60). Thus, we
evaluated whether the Ab response developed in NSCLC patients
due to 1E10 mAb immunization had clinical relevance. The results
of the analysis showed that there was a relation between longer
survival times and the induction in the patients of anti-NeuGcGM3
Abs. A randomized, double blind phase II clinical trial is ongoing
to evaluate the clinical effect of 1E10 mAb vaccine in NSCLC and
to define the value of the Abs induced by the anti-Id treatment as
real predictors of clinical outcome.
We thank Dr. Stephen P. Schoenberger for critically reviewing the
manuscript. We also thank Dr. L. E. Ferna ´ndez for generously provid-
The authors have no financial conflict of interest.
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6634 ANTI-NeuGcGM3 Abs ELICIT BY AN ANTI-Id mAb