Download Vaccination of Patients with Small-Cell Lung Cancer with Synthetic

6094 Vol. 10, 6094 – 6100, September 15, 2004
Clinical Cancer Research
Vaccination of Patients with Small-Cell Lung Cancer with Synthetic
Fucosyl GM-1 Conjugated to Keyhole Limpet Hemocyanin
Lee M. Krug,1 Govind Ragupathi,2
Chandra Hood,2 Mark G. Kris,1
Vincent A. Miller,1 Jennifer R. Allen,3
Stacy J. Keding,3 Samuel J. Danishefsky,3
Jorge Gomez,1 Leslie Tyson,1 Barbara Pizzo,1
Valerie Baez,1 and Philip O. Livingston2
Thoracic Oncology Service and 2Laboratory of Tumor Vaccinology,
Department of Medicine, and 3Laboratory of Bioorganic Chemistry,
Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center,
Weill Medical College of Cornell University, New York, New York
against fucosyl GM1 and tumor cells expressing fucosyl
GM1, comparable with the response induced by the bovine
derivative. We plan to combine synthetic fucosyl GM1 vaccine at a dose of 30 ␮g with vaccines against three other
antigens—GM2, Globo H, and polysialic acid—to test in
patients with SCLC after initial chemotherapy.
1
ABSTRACT
Purpose: Immunotherapy directed toward cell surface
antigens may provide a novel approach to the eradication of
chemoresistant micrometastatic disease in patients with
small-cell lung cancer (SCLC). Studies in SCLC cell lines
and human tissues suggest that the ganglioside fucosyl GM1
is an abundant yet specific target. A prior clinical study
demonstrated the potent immunogenicity of fucosyl GM-1
derived from bovine thyroid gland, conjugated to keyhole
limpet hemocyanin (KLH) and administered with QS-21
adjuvant.
Experimental Design: We tested the immunogenicity of
three different doses of a synthetic version of fucosyl-GM1
in patients with SCLC after a major response to initial
therapy. The primary end point was to establish the lowest
effective dose capable of inducing antibody production.
Results: Five of six patients at the 30-␮g dose and three
of five patients at the 10-␮g dose mounted IgM responses of
1:80 or greater. These antibodies were confirmed by flow
cytometry in seven of eight cases. None of the patients at the
3-␮g dose had titers above 1:80. One patient at the 30-␮g
dose had an IgG response with a titer of 1:80. The sera from
six of the eight responders induced potent complementmediated cytotoxicity of tumor cells.
Conclusions: Vaccination with the synthetic fucosyl
GM1-KLH conjugate induces an IgM antibody response
Received 3/9/04; revised 5/21/04; accepted 6/16/04.
Grant support: Supported by NIH grants PO1CA33049 (P. Livingston), GM19578 (J. Allen), and AI16943 and CA28824 (S. Danishefsky); an American Society of Clinical Oncology Career Development
Award (L. Krug); and the Lawrence and Selma Ruben Foundations.
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.
Requests for reprints: Lee M. Krug, Memorial Sloan-Kettering Cancer
Center, 1275 York Avenue, Box 327, New York, NY 10021. Phone:
212-639-8420; Fax: 212-794-4357; E-mail: [email protected].
©2004 American Association for Cancer Research.
INTRODUCTION
The currently accepted treatment for small-cell lung cancer
(SCLC) involves four to six cycles of etoposide plus cisplatin or
carboplatin, with concurrent thoracic radiation added for patients with limited-stage disease. In the majority of cases, patients derive substantial tumor reduction with this treatment, and
for about 20 to 25% of patients with limited-stage disease, this
can represent curative therapy. However, the remaining patients
with limited-stage disease, and essentially all patients with extensive-stage disease, still die from chemoresistant disease that
often returns just months after initial therapy is completed.
Because our currently available treatments do not eradicate
residual disease, different strategies need to be explored. One
approach involves the induction of a host immune response to
attack chemoresistant tumor cells.
Because of its neuroectodermal origin, SCLC has a number
of specific antigens that could serve as immune targets. To
identify the most appropriate carbohydrate antigens to use, we
analyzed a series of tumor samples and normal tissues by
immunohistochemistry (1). With the use of a specific mouse
monoclonal antibody, F12, (2) fucosyl-GM1 (Fuc␣1–2Gal␤1–
3GalNAc␤1– 4[NeuAc␣2–3]-Gal␤1– 4Glc␤1–1Cer) was identified in nearly all cases of SCLC (3, 4). In contrast, fucosyl GM1
was detected in only 2 of 10 squamous cell tumors, 1 of 5
large-cell tumors, 0 of 8 adenocarcinomas, and 0 of 3 carcinoids.
Uptake was also noted in small round cells of the thymus,
spleen, small intestine, and pancreatic islet cells. No fucosyl
GM1 was detected in normal lung or other normal tissues.
Fucosyl GM1 is expressed more frequently and more abundantly on SCLC tumors than are two other gangliosides, GD3
and GM2 (5). The presence of fucosyl GM1 has been demonstrated by high-performance thin-layer chromatography immunostaining in culture media from SCLC cell lines, and in tumor
extracts and serum of nude mouse xenografts (6). Furthermore,
it was detected in the serum of 4 of 20 SCLC patients, all of
whom had extensive-stage disease. Fucosyl GM1 was not detected in the serum of 12 patients with non–SCLC or in 20
healthy volunteers (6).
A prior clinical study demonstrated the potent immunogenicity of bovine fucosyl GM1 (7). Thirteen SCLC patients (4
limited stage, 9 extensive stage) who had achieved a major
response to initial chemotherapy were immunized with fucosylGM1 (30 ␮g) extracted from bovine thyroid gland, conjugated
to keyhole limpet hemocyanin (KLH), and administered with
the adjuvant QS-21. Six vaccinations were planned over 16
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Clinical Cancer Research 6095
weeks. All 10 patients who completed five vaccinations
mounted both IgM and IgG titers of ⱖ1:40 against Fuc-GM1 as
measured by ELISA. Toxicities were mild and transient and
included local skin reactions, flu-like symptoms, diarrhea, and
fatigue. Six patients had sensory peripheral neuropathy worsen
by one grade. Three patients with limited-stage SCLC were
relapse-free at 18, 24, and 30 months at the time this trial was
reported.
Fucosyl-GM1 was subsequently synthesized with a glycal
assembly method similar to that used previously for the synthesis of other carbohydrate antigens (8 –10). The construct was
armed with a terminal pentenyl group in place of ceramide or
allyl glycoside. In the presently reported trial, we tested the
immunogenicity of the synthetic version of fucosyl-GM1 in
patients with SCLC after a major response to initial therapy. As
in prior trials, we conjugated fucosyl GM1 to the carrier protein
KLH, and coadministered it with the immunologic adjuvant
QS-21 to overcome immunologic tolerance (11–14).
PATIENTS AND METHODS
Patient Selection. We enrolled adult patients with
SCLC, limited or extensive stage, who had completed initial
therapy with chemotherapy (and radiation if needed) at least 4,
but not more than 12 weeks previously. Patients needed to have
a Karnofsky performance status of 70% or greater. Required
hematologic and biochemical measurements included a total
white blood count ⱖ3.0 ⫻ 106 cells/␮L, a total lymphocyte
count ⱖ0.5 ⫻ 106 cells/␮L, aspartate aminotransferase level
ⱕ1.5 ⫻ upper limit of normal, serum bilirubin ⱕ1.5 mg/dl, and
serum alkaline phosphatase level ⱕ1.5 ⫻ upper limit of normal.
Patients with immune deficiency or autoimmune disease, prior
splenectomy or splenic radiation, or patients on oral corticosteroids were excluded. Patients could not have peripheral neuropathy greater than grade 1 at baseline. Because of the potential
cross-reaction with islet cells, patients with type II diabetes
mellitus were excluded. Pregnant or lactating women, patients
with New York Heart Association class III or IV heart failure,
or patients with another active malignant disease in the last 5
years were also excluded.
Within 3 weeks of starting treatment, all of the patients
underwent a history and physical examination including neurologic examination, chest X-ray, complete blood count, and biochemical profile, including amylase. Premenopausal women
were required to have a negative pregnancy test. A computed
tomography scan was done after completion of chemotherapy to
document disease status. For patients lost to follow-up, survival
status was determined by using the Social Security Death Index.4
Immunization. To establish the lowest effective dose
capable of inducing antibody production, three dose levels of
fucosyl GM1-KLH conjugate (30, 10, and 3 ␮g) were studied.
The initial dose level of 30 ␮g was chosen based on the prior
study of the fucosyl-GM1 vaccine extracted from bovine thyroid
gland. The dose of QS-21 was 100 ␮g at all dose levels.
Vaccinations were administered intradermally on weeks 1, 2, 3,
4, 8, and 16.
4
www.ssdi.genealogy.rootsweb.com.
Vaccine Preparation. Fucosyl-GM1 was synthesized as
a pentenyl glycoside with the glycal assembly method (8, 10).
The pentenyl group of the fully synthetic fucosyl-GM1 construct was converted to an aldehyde group by ozonolysis and
linked to the –NH2 group of the heterobifunctional cross-linker
4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazine
(MMCCH) as previously described (15). The maleimide group
of MMCCH was then reacted with thiolated KLH. The schema
of synthesis and the structure of the synthetic fucosyl GM1
compared with the previously tested extracted fucosyl GM1 are
demonstrated in Fig. 1. In the previous study, the pentenyl in
fucosyl-GM1 isolated from bovine extracts was converted to an
aldehyde group by ozonolysis and treatment with methyl sulfide, which was followed by direct linkage to NH2 groups on
KLH with sodium cyanoborohydride. KLH was obtained from
Progenics (Tarrytown, NY). The fucosyl-GM1-KLH conjugate
was washed and filtered to confirm sterility, and conjugate
containing 30 ␮g of fucosyl-GM1 was aliquoted into individual
vials containing saline with 100 ␮g of the immunologic adjuvant QS-21 [obtained from Antigenics Pharmaceuticals (New
York, NY)].
Safety Testing. Samples from the vial materials were
tested for sterility and safety, and immunogenicity was confirmed in mice.
Serologic Assays. Techniques for ELISA assays, flow
cytometry, and complement-dependent cytotoxicity assays, detailed previously (7), are briefly described below. All of the data
points were repeated once on a separate day, and three or more
times in some cases.
ELISA. Serial dilutions of patient sera were placed on
Nunc microwell plates coated with purified natural Fuc-GM1
ganglioside. Alkaline-phosphatase-conjugated goat antihuman
IgM or IgG was added. After incubation and washing, plates
were developed with Sigma 104 phosphatase substrate (Sigma
Diagnostics, St. Louis, MO). Absorbance was measured at 414
nm, and the highest dilution with an absorbance of at least 0.100
was defined as the antibody titer.
Fluorescence-Activated Cell Sorting. Fluorescenceactivated cell sorting (FACS) was done on the human SCLC cell
line DMS 79. Patient sera were added to cell pellets and were
incubated with either FITC-labeled goat antihuman IgM or
FITC-labeled goat antihuman IgG. The percentage of positive
cell population and the mean fluorescence intensity of the
stained cells were analyzed by flow cytometry (FACScan, Becton Dickinson, CA). Pre- and postvaccination sera were analyzed together. Prevaccination sera were used to set the FACScan result at 10% as background for comparison with
percentage positive cells with postvaccination sera. The mouse
anti-Fuc-GM1 monoclonal antibody, F12, was used as a positive
control.
Complement-Dependent Cytotoxicity Assays. Complement-dependent Cytotoxicity Assay (CDC) was done by a
2-hour chromium release assay with serum diluted at 1:4, 1:100,
and 1:1000 with DMS 79 cells and human complement.
Immune Thin-Layer Chromatography. Immune staining by thin layer chromatography (ITLC) was done with patient
sera before and after immunization. Purified gangliosides and
tumor extracts were spotted on silica gel glass plates as described previously (16). After plexigum coating, blocking, and
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6096 Vaccination with Synthetic Fucosyl GM1-KLH Conjugate
Fig. 1 Schema for synthesis of
synthetic Fucosyl GM1 and comparison of different forms of Fucosyl GM1
washing, horseradish peroxidase-conjugated antihuman IgM
and IgG antibodies were added for 2 hours, and the plates were
developed with 4-chloro-1-naphthol with H2O2.
Statistical Considerations. The primary end point of
this study was to determine the immunogenicity of synthetic
fucosyl GM1-KLH conjugate plus QS-21 in patients with SCLC
who have had a major response after initial chemotherapy or
chemoradiotherapy. We planned to enroll up to six patients at
each dose level. The lowest dose level at which four of the six
patients developed a significant immune response was to be
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Clinical Cancer Research 6097
selected as the phase II dose. Because of low drug supply, only
five patients were enrolled at the two lower dose levels.
A significant immune response was defined as (a) an
antibody titer of ⱖ1:80 by ELISA against Fucosyl-GM1 confirmed by ITLC and a titer of 1:20 against tumor cells expressing Fucosyl-GM-1 for patients with no detectable baseline titer;
or (b) an antibody titer ⱖ8-fold increase over baseline for
patients with a detectable baseline titer.
RESULTS
Six patients were enrolled at the 30-␮g dose level, and five
patients were enrolled at the 10- and 3-␮g dose levels. Baseline
patient characteristics are specified in Table 1. Three patients at
the 30-␮g dose level and one patient at the 3-␮g dose level
received five of six planned injections, and two patients at the
3-␮g dose level received four injections. All of these patients
were taken off study for disease progression. The remaining 10
patients received all six vaccinations.
No grade 3 or 4 toxicity occurred. The most common
toxicity, injection site reaction, was observed in 87% of patients
(56% grade 1, 31% grade 2). Five patients (31%) experienced
grade 1 myalgias. Grade 1 flu-like symptoms, arthralgias, fever,
or chills were reported by one patient each. Three patients (19%)
experienced peripheral sensory neuropathy, one of which was
grade 2 (from a grade 1 background). Other symptoms reported
by more than one patient included cough (31%) and fatigue
(25%). One patient had a grade 2 elevation of amylase and one
patient had grade 1 hyperglycemia with no clinical sequelae.
The peak responses for each patient are summarized in
Table 2. Five of six patients at the 30-␮g dose and three of five
patients at the 10-␮g dose mounted IgM responses of 1:80 or
greater. The IgM ELISA titers for all six of the patients vaccinated at the 30-␮g dose are shown in Fig. 2. These antibodies
were confirmed against synthetic- and tumor-extracted Fucosyl
GM1 by ITLC (data not shown) and against fucosyl GM1positive tumor cells by flow cytometry in seven of eight cases.
Flow cytometry results against the DMS-79 SCLC cell line for
all six patients treated at the 30-␮g dose level are shown in Fig.
3. Only patient 6, who had low-level ELISA and ITLC reactivity, was negative. However, none of the patients at the 3-␮g
dose had titers above 1:80. This suggests a dose response with
the 10- and 30-␮g doses superior to the 3-␮g dose. One patient
at the 30-␮g dose had an IgG response with a titer of 1:80.
Whereas these IgG ELISA titers are lower than those seen with
the previous bovine fucosyl GM1-KLH conjugate vaccine, the
IgM titers were similar, and sera from six of the eight responders
induced potent complement-mediated cytotoxicity of tumor
cells, a result that was comparable with the CDC observed in the
previous trial.
The clinical outcomes, including time to progression and
survival from the start of vaccination, are shown in Table 3. No
radiologic responses were observed.
DISCUSSION
There are advantages and disadvantages to the use of
synthetic fucosyl GM1 for vaccine production. Whereas the
synthesis itself is technically demanding, it simplifies vaccine
production rather than obtaining it from bovine tissues. Nuclear
magnetic resonance and mass spectrometry analyses provided
assurance that the carbohydrate portion of the synthetic fucosyl
GM1 is identical to that which is found expressed on tumor
cells. However, it is possible that the different functionalities
present at the reducing end of the oligosaccharide (pentenyl
versus ceramide) may alter the architecture or three-dimensional
shape of the entire fucosyl GM1 construct. We demonstrate here
that vaccination with the synthetic fucosyl GM1-KLH conjugate
Table 1 Patient characteristics
Female
Median age (range)
Karnofsky performance status
100%
90%
80%
70%
Initial stage
Limited
Extensive
Chemotherapy
Etoposide ⫹ cisplatin
Etoposide ⫹ carboplatin
Radiation
Thoracic
Prophylactic cranial irradiation
Radiation for brain metastasis
Bone
None
Response
Complete
Partial
30 ␮g
(n ⫽ 6)
10 ␮g
(n ⫽ 5)
3 ␮g
(n ⫽ 5)
Total
(N ⫽ 16)
4
58 (45–71)
4
64 (43–73)
4
69 (57–82)
12 (75%)
61 (43–82)
0
4
2
0
1
2
1
1
0
0
3
2
1
6
6
3
3
3
4
1
3
2
10
6
5
1
4*
1
3
2
12
4
3
3
1
0
2
3
2
0
1
1
3
2
0
0
2
9
7
1
1
5
2
4
5
0
4
1
11
5
* One patient received five cycles of etoposide ⫹ cisplatin and one cycle etoposide ⫹ carboplatin.
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6098 Vaccination with Synthetic Fucosyl GM1-KLH Conjugate
Table 2
Dose and
patient no.
Summary of immunology results for patients vaccinated with fucosyl GM1-KLH ⫹ QS21
Peak reciprocal
IgM ELISA titer
IgM FACS
(% DMS 79 cells pos)
Pre
Post
Peak reciprocal
IgG ELISA titer
CDC
(% DMS 79 lysis)
Pre
Post
ITLC
Pre
Post
0
40
80
20
0
0
10
11
10
10
9
11
11
9
16
11
14
12
⫹⫹⫹
⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫹
13
4
0
0
2
0
83
43
78
51
77
0
29
96
97
23
86
0
40
0
0
20
10
11
10
10
10
10
12
13
13
13
⫺
⫹
⫹
⫺
⫹
14
2
0
0
9
41
89
79
3
66
13
36
12
15
21
0
0
0
40
0
9
10
10
10
11
10
14
10
27
11
NP
NP
NP
NP
NP
30
0
0
11
0
26
0
0
10
0
30 ␮g
1
2
3
4
5
6
2560
20
640
80
2560
80
10
10
10
10
10
10
55
54
90
74
93
9
10 ␮g
1
2
3
4
5
20
2560
320
40
320
9
9
10
11
11
40
40
40
20
20
10
11
10
10
10
3 ␮g
1
2
3
4
5
IgG FACS
(% DMS 79 cells pos)
Abbreviations: pos, positive; Pre, prevaccination; Post, postvaccination; NP, not performed.
induces an IgM antibody response against fucosyl GM1 and
tumor cells expressing fucosyl GM1, which is comparable with
the response induced by fucosyl GM1-KLH conjugate originating from bovine thyroid tissues (7). This was confirmed by
complement-dependent cytotoxicity against SCLC cells, which
was at least as potent as that seen after immunization with the
bovine-derived fucosyl GM1.
Unexpectedly, the IgG response to vaccination with the
synthetic conjugate was lower than seen with the bovine-derived
conjugate. Only 1 of 6 patients at the highest dose had an IgG
antibody titer of 1:80 or greater, compared with 7 of 10 patients
immunized with the naturally derived conjugate. Neither vaccine induced IgG antibodies with frequent reactivity to SCLC
cells by FACS (0 of 6 were positive with the synthetic fucosyl
GM1; 3 of 10 were marginally positive with the bovine-derived
fucosyl GM1), and we have been unable to demonstrate antibody-dependent cellular cytotoxicity against SCLC with sera
from either trial (data not shown); therefore, the apparent superiority of the bovine-derived conjugate may be of little consequence.
Our observation with carbohydrate epitopes in a variety of
glycolipids and glycoproteins has been that, even when hightiter, vaccine-induced IgG antibodies are demonstrated by
ELISA, cell-surface reactivity demonstrable by FACS is rarely
seen. This is probably a consequence of the relatively low
affinity of these IgG antibodies, which is substantially augmented in the pentameric configuration of IgM antibodies. It is
likely that, as a consequence of the carbohydrate and autoantigen status of their targets, affinity maturation of these antibodies
does not occur. The difference between the IgG ELISA reactivity in the two trials is probably a consequence of the structural
difference between the two antigens. Although the hexasaccharide primary fucosyl GM1 epitope is the same in both, synthetic
fucosyl GM1 lacks the ceramide tail that is present on natural
fucosyl GM1 (Fig. 1) used both for vaccine construction in the
initial trial and as target in ELISA assays for both trials. It may
be that a portion of the epitope recognized by these IgG antibodies includes the proximal portion of the sphingolipid tail.
The use of an ineffective dose could also explain the findings.
On the basis of the hypothesis that a broader immune
response would more likely result in immunologic activity
Fig. 2 IgM antibody ELISA responses in six SCLC patients vaccinated
with fucosyl GM1-KLH plus QS-21 at the first dose level (30 ␮g).
Arrows, the time of vaccinations.
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Clinical Cancer Research 6099
Fig. 3 IgM FACS results before and after immunization
with synthetic fucosyl GM1KLH plus QS-21 for the six
patients who received the
30-␮g fucosyl GM1 dose. Target, DMS-79 SCLC cell line.
Percentage positive cells (mean
fluorescence intensity)
KLH, induced IgM antibodies to NP-polySA in six of six
patients, and these cross-reacted with unmodified polySA in all
but one case (20). IgG antibodies to NP-polySA were observed
in five patients, but these did not cross-react with polySA. The
other antigens, GM2 and Globo H, have demonstrated immunogenicity in melanoma, breast, and prostate cancer trials (9, 21,
22). Preclinical data further support the use of this “tetravalent”
vaccine (23). Ten SCLC cell lines were tested with monoclonal
against this heterogeneous tumor, we plan to combine the synthetic fucosyl GM1 vaccine with vaccines against three other
antigens that are prevalent in SCLC—GM2, and Globo H, and
polysialic acid (polySA; ref. 1). PolySA is a negatively charged
side chain on the neural cell adhesion molecule with highly
selective expression in SCLC (1, 17, 18). PolySA was shown to
be a poor immunogen (19). However, vaccination with polySA,
modified by N-propionylation (NP-polySA) and conjugated to
Table 3
Time to progression (TTP) and survival from start of vaccination
Initial stage
Response to
chemotherapy
Time from
diagnosis
to vaccination (mo)
TTP (mo)
Site of
progression
Survival (mo)
30 ␮g
1
2
3
4
5
6
Limited
Extensive
Extensive
Extensive
Limited
Limited
Partial
Partial
Partial
Partial
Complete
Complete
11
8
7
5
8
5
5
Unknown
2
2
NA
5
Lung
Unknown
Liver
Lung
NA
Adrenal
21
28
13
21
32*
19
10 ␮g
1
2
3
4
5
Limited
Extensive
Limited
Limited
Limited
Complete
Complete
Complete
Complete
Complete
10
11
12
13
7
Unknown
NA
Unknown
NA
5
Unknown
NA
Unknown
NA
Bone
22*
31*
16*
20
11
3 ␮g
1
2
3
4
5
Limited
Extensive
Extensive
Limited
Limited
Complete
Complete
Partial
Complete
Complete
12
7
5
8
5
2
2
1
NA
4
Lung
Brain
Lung
NA
Lung
9*
6
3
18*
10
Dose and
patient no.
Abbreviations: NA, not applicable.
* Patient remains alive or was lost to follow-up.
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6100 Vaccination with Synthetic Fucosyl GM1-KLH Conjugate
antibodies against seven target antigens individually or pooled
in different combinations. No single monoclonal antibody
bound to more than 6 or 4 of the 10 cell lines by FACS or CDC.
However, combining monoclonal antibodies against GM2, fucosyl GM1, Globo H, and polySA resulted in strong reactivity
against 8 of 10 cell lines tested by flow cytometry and 9 of 10
cell lines tested by CDC. The addition of mAbs against GD2,
GD3, and sLea increased reactivity only slightly. A randomized
phase II trial testing this polyvalent vaccine in patients who have
completed usual initial therapy for SCLC will begin next year.
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Downloaded from clincancerres.aacrjournals.org on April 28, 2017. © 2004 American Association for Cancer
Research.
Vaccination of Patients with Small-Cell Lung Cancer with
Synthetic Fucosyl GM-1 Conjugated to Keyhole Limpet
Hemocyanin
Lee M. Krug, Govind Ragupathi, Chandra Hood, et al.
Clin Cancer Res 2004;10:6094-6100.
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