VOLUME
22
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NUMBER
10
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MAY
15
2004
JOURNAL OF CLINICAL ONCOLOGY
O R I G I N A L
R E P O R T
Effect of Dose on Immune Response in Patients
Vaccinated With an HER-2/neu Intracellular Domain
Protein–Based Vaccine
Mary L. Disis, Kathy Schiffman, Katherine Guthrie, Lupe G. Salazar, Keith L. Knutson, Vivian Goodell,
Corazon dela Rosa, and Martin A. Cheever
From the Tumor Vaccine Group, Oncology, University of Washington; Fred
Hutchinson Cancer Research Center;
and Corixa Corporation, Seattle, WA.
Submitted September 3, 2003; accepted
March 2, 2004.
Supported by grants from the Cancer
Research Treatment Foundation and
the National Institutes of Health (NIH),
National Cancer Institute grant No. U54CA090818 (M.L.D), by NIH training
grant No. T32 (HL07093; L.G.S.), and
by a fellowship from the Department of
Defense Breast Cancer Program
(DAMD 17-00-1-0492; K.L.K.). Patient
care was conducted through the Clinical Research Center Facility at the University of Washington, which is supported through NIH grant No. MO1-RR00037.
Authors’ disclosures of potential conflicts of interest are found at the end of
this article.
Address reprint requests to Mary L.
Disis, MD, Tumor Vaccine Group, Oncology, Box 356527, University of
Washington, Seattle, WA 98195-6527;
e-mail: [email protected].
A
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T
A
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Purpose
To evaluate the safety of an HER-2/neu intracellular domain (ICD) protein vaccine and to estimate
whether vaccine dose impacts immunogenicity.
Patients and Methods
Twenty-nine patients with HER-2/neu– overexpressing breast or ovarian cancer and with no evidence of
disease after standard therapy received a low- (25 ␮g), intermediate- (150 ␮g), or high-dose (900 ␮g)
HER-2/neu ICD protein vaccine. The vaccine was administered intradermally, monthly for 6 months, with
granulocyte-macrophage colony-stimulating factor as an adjuvant. Toxicity and both cellular and humoral
HER-2/neu–specific immunity was evaluated.
Results
The vaccine was well tolerated. The majority of patients (89%) developed HER-2/neu ICD-specific T-cell
immunity. The dose of vaccine did not predict the magnitude of the T-cell response. The majority of
patients (82%) also developed HER-2/neu–specific immunoglobulin G antibody immunity. Vaccine dose
did not predict magnitude or avidity of the HER-2/neu–specific humoral immune response. Time to
development of detectable HER-2/neu–specific immunity, however, was significantly earlier for the highversus low-dose vaccine group (P ⫽ .003). Over half the patients retained HER-2/neu–specific T-cell
immunity 9 to 12 months after immunizations had ended.
Conclusion
The HER-2/neu ICD protein vaccine was well tolerated and effective in eliciting HER-2/neu–specific T-cell
and antibody immunity in the majority of breast and ovarian cancer patients who completed the vaccine
regimen. Although the dose of vaccine did not impact the magnitude of T-cell or antibody immunity
elicited, patients receiving the highest dose developed HER-2/neu–specific immunity more rapidly than
those who received the lowest dose.
J Clin Oncol 22:1916-1925. © 2004 by American Society of Clinical Oncology
© 2004 by American Society of Clinical
Oncology
0732-183X/04/2210-1916/$20.00
INTRODUCTION
DOI: 10.1200/JCO.2004.09.005
The HER-2/neu oncogenic protein is a tumor
antigen in patients with breast and ovarian
cancer.1 Multiple investigations have shown
that some cancer patients, whose tumors overexpress the HER-2/neu oncoprotein, have
both detectable antibody and T-cell immunity
directed against HER-2/neu.2-4 Furthermore,
experiments in rodents suggest that the HER2/neu protein, particularly the intracellular
domain (ICD), is a tumor rejection antigen.
The generation of an immune response specific for neu ICD will provide animals some
1916
R
protection from a tumor challenge,5 and T
cells specific for the ICD will mediate an antitumor response when infused into animals
bearing neu-overexpressing tumors.6 For
these reasons, several vaccine strategies have
been developed to target the HER-2/neu oncogene and are being evaluated for safety and
immunogenicity in phase I clinical trials.
One of the most common methods of
immunizing cancer patients against weakly
immunogenic proteins expressed in their
tumors is via peptide vaccination. Peptide
vaccines are composed of fragments of the
tumor antigen specifically designed to elicit
HER-2/neu ICD Protein–Based Vaccine
HLA class I (cytotoxic T cell) and/or HLA class II (T helper)
antigen-specific T-cell responses. Peptide vaccines targeting
the HER-2/neu oncogenic protein have been studied extensively in phase I studies and have been found to be both safe
and immunogenic.7-9 However, there are disadvantages to
the use of peptides as vaccine antigens. Development of
peptide vaccines necessitates some preclinical assessment as
to the most useful immune response desired, and the choice
of epitope must be made before clinical studies.10 Furthermore, peptides are designed for exogenous binding in major
histocompatability antigen molecules; therefore, HLA restrictions are of utmost importance in predicting vaccine efficacy.
Finally, HLA class I–specific epitope vaccines, in particular,
may require a concomitant T helper response to promote a
durable cytotoxic T-cell response.11 Whole protein vaccines
are composed of both HLA class I and class II epitopes and are
not subject to specific HLA restrictions to predict efficacy.
However, little is known about the use of protein-based vaccines to elicit immunity to self tumor antigens, and unlike the
case of peptide-based vaccines,12 appropriate immunogenic
doses of tumor protein vaccines have not been defined.
The study described here is the first clinical trial of a
recombinant HER-2/neu ICD protein– based vaccine designed to stimulate HER-2/neu–specific immunity in patients with HER-2/neu– overexpressing breast and ovarian
cancers. The goal of the study was to determine the safety of
the approach as well as assess the immunogenicity of the
vaccine. Patients were vaccinated in the adjuvant setting
after optimal cytoreductive therapy. Recently, the clinical
application of cancer vaccines is being focused on patients
with minimal disease. Extrapolating from application of
active immunization in infectious diseases, vaccines, such
as those targeting chicken pox or influenza, are given in the
absence of infection and have only limited efficacy if administered after exposure to the pathogen. The goal of vaccination against cancer may be to elicit significant immunologic
memory capable of rapidly expanding an antigen-specific
T-cell population in the presence of low levels of antigen
encountered during cancer onset or early relapse. Indeed,
vaccination against cancer may likely only be effective when
disease is either absent or below the limit of detection.13
Investigation of recombinant protein vaccines in infectious
disease models suggests that the dose of protein administered affects the antigen-specific immune response elicited.
Studies were undertaken to evaluate the effect of dose of a
recombinant HER-2/neu ICD protein vaccine on the incidence and magnitude of HER-2/neu–specific cellular and
antibody immunity generated with active immunization.
PATIENTS AND METHODS
Vaccine Formulation and Administration
The HER-2/neu ICD construct consisted of amino acids 676
to 1255 of the full-length HER-2/neu. The first 20 amino acids
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were unrelated to the ICD including an 8⫻ histidine tag followed
by a Ser-Ser-Gly linker and an enterokinase site. For this reason,
proteins derived from Leishmania major and Mycobacterium tuberculosis were constructed in the same fashion and purified in a
similar manner as the HER-2/neu ICD vaccine to be used as
negative controls in evaluating immunogenicity. The HER-2/neu
ICD protein was produced in Escherichia coli (pet 28 vector;
Novagen, Madison, WI; BLRplyS strain) as inclusion bodies by the
Alberta Research Council (Edmonton, Canada). The inclusion
body pellets were purified, and the final product was frozen at
⫺20°C until formulated (Corixa Corp, Seattle, WA). Disulfide
bond formation was minimized by first reducing with 10 mmol/L
dithiothreitol before purification followed by a second reduction
before dialysis in the final buffer. The material was then stored at
⫺20°C to prevent further oxidation. The material was not chemically modified. The molecular weight of the final product was
approximately 80 kd. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis of the final product showed only one band
at 80 kd. The final formulation consisted of 500 ␮g HER-2/neu
ICD in 1 mL of 20 mmol/L Tris (pH 9.0), with 5% mannitol and
0.05% Tween-80. Patients were enrolled sequentially to receive
intradermal vaccinations monthly for 6 months with 25 ␮g (low
dose), 150 ␮g (intermediate dose), or 900 ␮g (high dose) of
HER-2/neu ICD protein admixed with 100 ␮g of granulocytemacrophage colony-stimulating factor (supplied by Immunex
Corp, Seattle, WA) as an adjuvant. The vaccines were administered to the same draining lymph node site for each patient. All
subjects received a tetanus toxoid (tt) booster vaccination, as a
positive control, within 2 weeks before beginning immunization with the HER-2/neu vaccine. Blood was obtained for assessment of immunologic responses and toxicity evaluations at
baseline, 1 month after the first, third, fifth, and sixth vaccinations, 2 to 3 months after completion of the sixth vaccination,
and then, if possible, every 3 months for 1 year.
Patient Population
Patients with stage II, III, or IV HER-2/neu– overexpressing
breast cancer with no evidence of disease after standard therapy or
stage IIC or III HER-2/neu– overexpressing ovarian cancer in
complete remission were eligible for enrollment onto a phase I
study of a HER-2/neu ICD protein– based vaccine between April
2000 and January 2001. The study was approved by both the
United States Food and Drug Administration and the University
of Washington Institutional Review Board. To be enrolled, patients had to meet the following criteria: documentation of HER2/neu overexpression in their primary tumor or metastasis;
Karnofsky performance status ⱖ 90%; WBC count ⱖ 3,000/mL
and platelet count more than 100,000/mL; normal renal and hepatic function; fully treated for their disease by conventional standards and in clinical complete remission; and off chemotherapy
and other immune-suppressive drugs, such as steroids, for a minimum of 30 days before starting the vaccination series. The clinical
characteristics of the patient population are listed in Table 1.
Evaluation of T-Cell Immunity After Vaccination
Antigen-specific T-cell proliferation was measured using a
modified limiting dilution assay that has been previously described.12 Results are reported as a stimulation index (SI), defined
as the mean of 24 experimental wells divided by the mean of 24
control wells. Phytohemagglutinin incubated with patient T cells
at a concentration of 5 ␮g/mL was used as a positive control to
assess the ability of T cells to respond to mitogen and resulted in an
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Disis et al
Table 1. Patient Characteristics for Patients at Each Dose Level
Dose of HER-2/neu ICD
Protein (No. of patients)
Characteristics
Diagnosis
Breast
Stage II
Stage III
Stage IV (CR)
Ovarian IIC
Total patients per dose
Age, years
Median
Range
Time from last chemotherapy, months
Median
Range
Low
Intermediate
High
9
7
2
0
1
10
10
8
1
1
0
10
9
4
3
2
0
9
52
39-76
50
41-63
46
35-54
9
2-26
8
4-28
6
3-34
Abbreviations: ICD, intracellular domain; CR, complete remission.
SI greater than 2 in all cases (data not shown). Patients’ peripheralblood mononuclear cells were evaluated for immunologic response against the following antigens; recombinant HER-2/neu
domain proteins, extracellular domain (ECD) and ICD (1 ␮g/
mL),9 tt (5 ␮g/mL) evaluated as a vaccinated control antigen, and
proteins derived from L major and M tuberculosis (1 ␮g/mL)
served as negative control protein antigens for an E coli and vectorrelated response, having been produced under identical manufacturing methods as the HER-2/neu ICD protein. In addition, because the negative control proteins were manufactured in an
identical vector as the HER-2/neu construct, it is likely any immunogenicity elicited by the components involved in purification,
such as histidine, would also be detected to these constructs. A
reference population of 30 age-matched volunteer donors was
used to establish baseline response to the HER-2/neu antigens.9
The mean and standard deviation (SD) of this population was 1.2
⫾ 0.3 ␮g/mL for HER-2 ICD and 0.9 ⫾ 0.4 ␮g/mL for HER-2
ECD. A subset of 10 volunteer donors was assessed for the control
proteins, and the mean and SD of this population was 1.0 ⫾ 0.2
␮g/mL for L major and 0.8 ⫾ 0.2 ␮g/mL for M tuberculosis. An SI
of more than 2 to antigen defined a positive response (mean and
two SDs greater than the reference population). If subjects had an
SI of greater than 2 to the HER-2/neu ICD before vaccination, a
postvaccination T-cell response was defined as positive if the value
was twice the prevaccine value. Patients with a pre-existent immune response to the HER-2/neu protein had to meet this requirement to be included as responders in the analysis.
Evaluation of Antibody Immunity After Vaccination
Antibody immunity to HER-2/neu protein was assessed as
previously described using a human breast cancer cell line that
overexpresses HER-2/neu as a source of HER-2/neu protein.14
Some patients did have pre-existent detectable antibody immunity to the HER-2/neu protein and were only considered to have
responses if they boosted the antibody level to twice the baseline
value. Pre- and postvaccination samples for each patient were
analyzed simultaneously on the same plate. A positive sample was
defined as an antibody concentration greater than the mean of a
volunteer blood donor population ⫾ two SDs (0.20 ⫾ 0.40 ␮g/
mL, n ⫽ 200). The specificity of the assay was 78%, and the
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sensitivity was 90%. The assay was linear over the range of antibody concentrations tested and had a correlation coefficient of
0.99. On the basis of repeated measures of 20 volunteer and 20
experimental sera over a 6-month period, the intra- and interassay
coefficients of variation were 15% and 9%, respectively. Positive
responses were verified by Western blot analysis.15 Antibody responses to E coli were also measured as a control. Briefly, 96-well
Immulon 4HBXmicrotiter plates (Dynex Technologies, Inc,
Chantilly, VA) were coated with E coli lysates diluted (1:50) in
carbonate buffer, alternating rows with carbonate buffer alone.
Diluted purified human immunoglobulin (Ig) G (Sigma Chemical
Co, St Louis, MO) provided a standard curve on each plate.
Treated plates were incubated overnight at 4°C. All wells were then
blocked with 100 ␮L/well of 10% phosphate-buffered saline
(PBS)-1% bovine serum albumin solution (Sigma) and incubated
at room temperature with agitation for 1 to 2 hours. Plates were
washed four times with 10% PBS and 0.5% Tween-20 before the
addition of patient sera at dilutions of 1:100, 1:200, 1:400, and
1:800. Sera were diluted with 10% PBS, 1% FCS, 1% bovine serum
albumin, and 25 ␮g/mL mouse IgG (Sigma), 50 ␮L/well, and
incubated at room temperature on a rocker for 1 hour. After
serum incubation, plates were washed four times as described
above, and goat antihuman IgG-horseradish peroxidase conjugate
(diluted 1:100,000; Zymed Laboratories, San Francisco, CA) was
added, 50 ␮L/well, and incubated for 45 minutes at room temperature. After a final wash, TMB (Kirkegaard and Perry Laboratories,
Gaithersburg, MD) developing reagent was added (75 ␮L/well),
and color reaction was read at 640 nm until the well containing the
standard at a concentration of 0.16 ␮g/mL read 0.3 optical density
(OD). The reaction was then stopped with 75 ␮L/well of 1 N HCl
and read at 450 nm. Pre- and postimmunization samples were
assayed on the same plate, and patients were considered to have
boosted E coli protein–specific antibodies if the mean of four
replicates of their postvaccine sample was two SDs above the mean
of four replicates of their prevaccine sample.
Evaluation of Antibody Avidity After Vaccination
Assessment of HER-2/neu–specific antibody avidity is based
on a modification of previously described methods.16 Two-fold
serial dilutions of serum samples were plated in duplicate and
incubated for 3 hours at room temperature. After four washes, 200
␮L of wash buffer (described earlier) containing 8 mol/L urea was
added to one of the duplicate wells and incubated at room temperature for 3 minutes. All wells were washed four times. For the
detection of antibody binding the plates, goat antihuman IgG
conjugated with horseradish peroxidase (diluted 1:10,000; Zymed
Laboratories) was added for 45 minutes at room temperature.
After washing, 3,3⬘,5,5⬘-tetramethylbenzidine substrate solution
(Kirkegaard & Perry Laboratories) was added, and the reaction
was allowed to proceed for 3 to 5 minutes and was stopped by
adding 75 ␮L of 1 N HCl per well. For a given patient serum
dilution, the avidity index (AI) was calculated using the following
formula: (change of OD with urea/change of OD without urea) ⫻
100; and the AI was expressed as a percentage. An identical assay
was developed to evaluate tt-specific antibodies, with the modification of tt (1 ␮g/mL) plated in alternating rows with carbonate
buffer. An AI less than 30% is considered low avidity, 31% to 50% is
considered intermediate avidity, and greater than 51% is considered
high avidity.16 Avidity is reported here at the 1:25 dilution of sera.
JOURNAL OF CLINICAL ONCOLOGY
HER-2/neu ICD Protein–Based Vaccine
Statistical Analysis
To estimate the significance of differences in median immune
responses, the data were compared using a two-tailed Student’s t
test, with a level of significance set at 0.05 (GraphPad InStat
v.3.05; GraphPad Software, San Diego, CA). The probability of
developing detectable immunity was summarized using a cumulative incidence estimate.17
RESULTS
The HER-2/neu ICD Protein–Based Vaccine Was
Well Tolerated at All Doses
Twenty-nine subjects were enrolled onto the trial; 10
were enrolled at the lowest vaccine dose (25 ␮g), 10 were
enrolled at the intermediate vaccine dose (150 ␮g), and nine
were enrolled at the highest vaccine dose (900 ␮g) of HER2/neu ICD protein. Twenty-seven of the 29 subjects enrolled completed all six vaccinations; two subjects who were
enrolled at the lowest dose withdrew from study because of
progressive disease after receiving only the first vaccination.
The ages of patients, stages of disease, and time from completing prior chemotherapy were similar for the subjects in
each group (Table 1). Adverse events are reported on all 29
subjects, and immune responses are reported on the 27
subjects who completed six vaccinations (eight for the lowdose, 10 for the intermediate-dose, and nine for the highdose group).
The vaccine was well tolerated at all doses. There were
no grade 2, 3, or 4 adverse events. Three subjects, one at each
dose level, reported grade 1 fatigue; three subjects, two in
the low-dose group and one in the high-dose group, reported grade 1 myalgia; and 24 subjects, seven in the lowdose group, eight in the intermediate-dose group, and nine
in the high-dose group, experienced grade 1 local injection
site erythema.
Majority of Patients Immunized With an HER-2/
neu ICD Protein–Based Vaccine Developed HER2/neu–Specific T-Cell Immunity
The majority of patients (24 of 27 patients, 89%) who
completed all six vaccines developed HER-2/neu ICD–
specific T-cell immunity with active immunization regardless of the vaccine dose. Eight (100%) of eight patients who
received the lowest dose of HER-2/neu ICD protein (Fig
1A), nine (90%) of 10 patients who received the intermediate dose (Fig 1B), and seven (78%) of nine patients who
received the highest dose of the vaccine (Fig 1C) developed
T-cell immunity to the HER-2/neu ICD protein domain.
All responses obtained during the duration of the trial for
each group are shown. Figure 2 demonstrates the temporal
development of immunity to the ICD during the time of
active immunization and immediately after immunization
in the low-dose (Fig 2A), intermediate-dose (Fig 2B), and
high-dose (Fig 2C) populations. Two patients in the highdose group had a pre-existent immune response to the
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HER-2/neu ICD protein (one patient had an SI of 3.5 and
one had an SI of 4.4). Both patients boosted immunity
during the course of vaccination, one at a distant time point.
None of the patients developed a significant immune response to the L major or M tuberculosis proteins, indicating
the specificity of the HER-2/neu ICD response to the HER2/neu protein and not to any potentially contaminating
proteins residual to the vaccine production process (Fig 1).
All patients developed significant immunity to tt, the control vaccination (Fig 1). Those patients with a pre-existent
detectable T-cell response to tt boosted immunity over
twice baseline after the control immunization, indicating
the immune competence of the study population. Only one
(4%) of 27 patients developed immunity to the HER-2/neu
ECD, which demonstrates epitope spreading (Fig 1).9
Dose of Protein in the Vaccine Does Not Predict
the Magnitude of HER-2/neu–Specific T-Cell
Immunity Elicited With Active Immunization
The magnitude of the T-cell response to HER-2/neu
ICD after immunization was similar at all doses (Fig 3). The
median SI for patients receiving the low-dose vaccine was
3.4 (range, 2.2 to 9.2), the median SI for patients receiving
the intermediate-dose vaccine was 4.4 (range, 1.4 to 15.8),
and the median SI for patients receiving the high-dose
vaccine was 5.7 (range, 1.6 to 15.7). There was no statistical
difference in the median responses of each group (low v
intermediate, P ⫽ .41; intermediate v high, P ⫽ .79; and low
v high, P ⫽ .32).
Higher Dose of a HER-2/neu ICD Protein–Based
Vaccine Was Associated With Earlier
Development of Detectable HER-2/neu–Specific
T-Cell Immunity
Although the dose of HER-2/neu ICD protein– based
vaccine was not associated with a difference in the magnitude of HER-2/neu–specific T-cell or antibody immunity,
the higher dose vaccine was associated with a more rapid
development of detectable immunity (Fig 4). The percentage of patients achieving a HER-2/neu ICD–specific T-cell
response, as defined by an SI of more than 2.0, is shown over
the time of active immunization as a cumulative incidence
curve for each of the dose groups (Fig 4). The time to the
development of a detectable T-cell immune response was
significantly earlier for the group receiving the higher dose
vaccine compared with the lowest dose vaccine (P ⫽ .003).
HER-2/neu–Specific T-Cell Immunity Persists
After Active Immunizations Have Ended
The majority of the patients were able to be observed
for persistent immunity after their vaccine regimen was
completed. There was no difference in the persistence of
immunity between any of the dose groups. Figure 5 shows
HER-2/neu ICD T-cell immunity persisting as long as 1 year
from the end of active immunization. At 1 month after the
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Disis et al
Fig 1. The majority of patients who were immunized developed T-cell immunity. T-cell responses, (A) low-dose, (B) intermediate-dose, and (C) high-dose
groups. Preimmunization (E) and maximal response during immunization (F). The dashed line is the mean and two standard deviations of the reference
population for all antigens except tt. SI, stimulation index; ICD, intracellular domain; ECD extracellular domain; tt, tetanus toxoid; Leish, Leishmania major; Mtb,
Mycobacterium tuberculosis.
vaccinations were finished, 18 (72%) of 25 assessable patients had detectable HER-2/neu ICD T-cell immunity
(median SI, 2.6; range, 1.0 to 7.6). Six months after the end
of immunizations, 12 (55%) of 22 assessable patients had
detectable HER-2/neu ICD T-cell immunity (median SI,
1.9; range, 0.5 to 15.7). Finally, between 9 months and 1 year
after the end of the vaccine regimen, eight (53%) of 16
assessable patients still had detectable HER-2/neu ICD
T-cell immunity (median SI, 2.6; range, 0.4 to 14.0).
Majority of Patients Immunized With an
HER-2/neu ICD Protein–Based Vaccine Develop
HER-2/neu–Specific Antibody Immunity
The majority of patients (22 of 27 patients, 82%) who
completed all six immunizations developed HER-2/neu–
specific IgG antibody immunity. Results are shown as the
HER-2/neu antibody level determined 1 month after the
1920
last vaccine. Figure 6A demonstrates that five (63%) of eight
patients in the low-dose group developed HER-2/neu antibody immunity. One patient had a pre-existent HER-2/
neu–specific antibody response (3.0 ␮g/mL, which boosted
to 6.0 ␮g/mL after vaccination). Ten (100%) of 10
intermediate-dose patients developed new HER-2/neu–
specific IgG antibody immunity (Fig 6B). Finally, seven
(78%) of nine patients developed HER-2/neu–specific antibody immunity after receiving the highest dose of vaccine
(Fig 6C). One patient had a significant HER-2/neu antibody
response of 6.0 ␮g/mL before receiving any immunizations,
which decreased over the course of immunization to 0.9
␮g/mL (a negative response). No patient had boosted antibody immunity to E coli lysate during the course of the
immunizations. The mean E coli antibody response (⌬OD
at 450 nm) at a 1:5000 sera dilution before immunization
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HER-2/neu ICD Protein–Based Vaccine
Fig 2. Intracellular domain T-cell immunity over the time of active immunization. T-cell immunity as stimulation index (SI) for each patient at each time. (A)
Low-dose, (B) intermediate-dose, and (C) high-dose groups. The dashed line is the mean and two standard deviations of the reference population.
was 0.264. The mean E coli antibody response (⌬OD at 450
nm) after the end of active immunizations at a sera dilution
of 1:5000 was 0.379 (P ⫽ .111). HER-2/neu–specific antibody responses were documented by Western blot analysis,
and examples are shown in Figure 7. Figure 7A shows antibodies directed against HER-2/neu, which were immunoprecipitated from SKBR3, a human breast cancer cell line
that overexpresses HER-2/neu, in a breast cancer patient
who developed HER-2/neu antibodies detected by enzymelinked immunosorbent assay.15 Figure 7B is a similar immunoblot from a patient who did not develop detectable
antibodies after active immunization.
The dose of protein in the vaccine did not predict the
magnitude or avidity of HER-2/neu–specific antibody immunity elicited with active immunization. In the low-dose
group, the median antibody response was 1.6 ␮g/mL
(range, 0 to 9.3 ␮g/mL); in the intermediate-dose group, it
was 2.5 ␮g/mL (range, 1.5 to 7.6 ␮g/mL); and in the highdose group, it was 2.3 ␮g/mL (range, 0.58 to 5.42 ␮g/mL).
There was no statistical difference in the median response
between any group (low v intermediate, P ⫽ .41; intermediate v high, P ⫽ .29; or low v high, P ⫽ .96). Of the 22
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patients who developed HER-2/neu–specific antibody immunity, 17 (77%) of 22 demonstrated low-avidity antibody
immunity (range, 0% to 29%; Fig 3D), three (14%) of 22
demonstrated intermediate-avidity antibody immunity
(range, 38% to 43%), and two (9%) of 22 demonstrated
high-avidity antibody immunity (64% and 65%). The
intermediate- and high-avidity responses were detected in
patients at all dose levels (Fig 6D). All patients who completed the study had high-affinity tt antibodies (data not
shown). The patients with intermediate- and high-avidity
HER-2/neu–specific antibodies were able to be observed for
persistence of T-cell immunity over time, and all five patients had persistent HER-2/neu–specific T-cell immunity
at 12 months after vaccination.
DISCUSSION
The study described here is the first report of a clinical trial
evaluating an HER-2/neu protein– based vaccine and is also
one of the first evaluations of the effect of the dose of a
tumor protein antigen vaccine on the immune response
elicited in cancer patients. The data presented demonstrate
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Disis et al
Fig 3. Dose of protein did not predict magnitude of HER-2/neu–specific
T-cell immunity elicited. Maximal intracellular domain (ICD) responses are
shown for the low, intermediate, and high doses. Solid bars denote median
value. The dashed line is the mean and two standard deviations of the
reference population. SI, stimulation index.
the following: the majority of patients who were immunized
developed HER-2/neu protein–specific T-cell immunity,
and the dose of the vaccine did not impact the magnitude of
the antigen-specific T-cell response; the majority of patients
who were immunized developed HER-2/neu protein–specific antibody immunity, and the dose of the vaccine did not
impact the magnitude or avidity of the antigen-specific
antibody response; the highest dose of vaccine was associated with earlier development of detectable HER-2/neu–
specific T-cell immunity; and tumor antigen–specific immunity persisted after active immunization had ended.
The HER-2/neu ICD protein– based vaccine elicited
both cellular and humoral immunity in the majority of
patients. Preclinical development of HER-2/neu protein–
based vaccines indicates that the ICD may be a better immunogen for the generation of HER-2/neu–specific immunity than the ECD. The HER-2/neu oncogenic protein is a
Fig 4. Higher dose was associated with earlier development of detectable
HER-2/neu–specific T-cell immunity. Time (months) to development of
detectable HER-2/neu–specific T-cell immunity for the percentage of patients receiving the highest dose (solid black line), intermediate dose (gray
dashed line), and low-dose (black dotted-dashed line). ICD, intracellular
domain.
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Fig 5. HER-2/neu–specific T-cell immunity persists after immunizations
have ended. T-cell immunity 1, 6, and 9 to 12 months after immunizations.
The dashed line depicts the mean and two standard deviations of the
reference population response. Solid bars denote the median value. SI,
stimulation index.
self protein, and in animal models, vaccination using the
whole protein or the ECD portion of neu to stimulate
immunity was found to be ineffective secondary to immunologic tolerance.18,19 Rodents immunized with the ICD,
however, can develop significant neu-specific T-cell responses.5 Inability to immunize to the ECD may be a result
of the extracellular nature of this portion of the HER-2/neu
protein, and thus, previous exposure to the immune system
results in tolerance. Because the ICD is sequestered from the
immune system by its intracellular location, it is potentially
more immunogenic. Furthermore, T-cell immunity directed against the HER-2/neu ICD has been shown to mediate an antitumor effect in animal models of neuoverexpressing breast cancer.5,8 Of note, although the T-cell
immunity detected was specific for HER-2/neu ICD, because of the amount of clinical material available for analysis, it is unknown what component of that response is
CD8⫹ versus CD4⫹ HER-2/neu–specific T cells. Peptidebased vaccines are specifically designed to elicit cellular
immunity.10 Vaccines formulated from protein, however,
are successful in eliciting both humoral and cellular immunity.20 Studies in animals have demonstrated that rejection
of HER-2/neu– overexpressing tumors after vaccination
may require both tumor-specific T cells and antibodies.21
Although antibodies to the ICD protein may not have the
direct antitumor effects associated with antibodies specific
for the ECD, such as trastuzumab, antibodies binding to
ICD protein present in a tumor bed or shed by dying cells
may serve to amplify the cellular immune response via
induction of antibody-dependent cell-mediated cytotoxicity. Thus, immunizing cancer patients, whose tumors overexpress HER-2/neu, with an HER-2/neu protein– based
vaccine may be an effective method to elicit both tumorJOURNAL OF CLINICAL ONCOLOGY
HER-2/neu ICD Protein–Based Vaccine
Fig 6. The majority of patients who were immunized developed antibody immunity. HER-2/neu antibodies before (E) and after (F) immunization: (A) low dose,
(B) intermediate dose, and (C) high dose. Dashed line is the mean and two standard deviations of controls. (D) Antibody avidities (䡵) of dose groups (1:25
dilution). IgG, immunoglobulin G; ICD, intracellular domain.
www.jco.org
1923
Disis et al
Fig 7. Antibody immunity to HER-2/neu. (A) Immunoblot of a positive response. Lanes A and B are post- and prevaccination sera against immunoprecipitated
HER-2/neu, respectively; lane C is the molecular weight marker. (B) Immunoblot of a negative response. Lane A is the molecular weight marker; lane B is
preimmunization sera, and lane C is postimmunization sera against immunoprecipitated protein.
specific T-cell and antibody immunity in the majority of
vaccinated individuals.
Potentially, the dose of a protein-based vaccine can
impact the development of an antigen-specific immune
response in many ways. First, some studies in infectious
disease systems have suggested that higher doses of recombinant protein vaccines are associated with more robust
antigen-specific T-cell responses.22 A trial performed in
animals, immunizing against a herpesvirus, demonstrated
higher herpes-specific T-cell proliferative responses in
those animals receiving the 40-␮g versus the 20-␮g dose of
recombinant protein-based vaccine. In the study reported
here, the magnitude of the T-cell response was not dependent on the dose. Second, several trials of infectious disease
vaccines have suggested that higher doses of protein result
in more subjects developing antigen-specific immunity. An
evaluation of a recombinant human papillomavirus protein
vaccine in volunteers suggested that a 533-␮g dose was
superior to a 128-␮g or 26-␮g dose in generating interferon
gamma–producing human papillomavirus–specific T cells
in the greatest number of vaccinated subjects.23 Similarly,
higher doses of a modified tick-borne encephalitis vaccine
resulted in a greater number of seroconversions in volunteers.24 Unlike these foreign antigen vaccines, the dose of
the HER-2/neu ICD protein– based vaccine did not influence the number of patients developing detectable immunity. The lowest dose and highest dose were equally effective
in stimulating HER-2/neu–specific antibody and T-cell responses. Finally, key to the development of any vaccine is
the ability of immunization to elicit immunologic memory.
Memory responses are particularly important for cancer
vaccines that may have the greatest clinical utility in pre1924
venting disease relapse months to years after primary therapy has been completed. The development of T-cell memory responses after active immunization has been associated
with higher antigen doses in vaccines.25 Similarly, highavidity antigen-specific antibody immunity, a potential
predictor of immunologic memory, has been associated
with high-dose vaccines. In the current study, there was no
difference in the persistence of T-cell immunity after active
immunizations had ended in the low-, intermediate-, or
high-dose groups receiving the HER-2/neu ICD vaccine.
Furthermore, intermediate- and high-avidity HER-2/neu–
specific antibody responses were found in all dose groups.
The dose of HER-2/neu ICD protein– based vaccine
did impact the length of time needed to develop a detectable
HER-2/neu–specific T-cell response. HER-2/neu–specific
T-cell immunity could be detected within 2 months of
starting the vaccine regimen in the majority of patients
receiving the highest dose of vaccine compared with 6
months in the majority of patients receiving the lowest dose
vaccine. The more rapid induction of measurable immunity
with the highest dose of protein may have importance in
the clinical application of cancer vaccines. For example,
if a vaccine was being developed for a cancer that was
associated with rapid time to relapse after primary therapy, a higher dose of a protein vaccine may allow a more
rapid induction of potentially protective immunity. Furthermore, for vaccine strategies applied in the neoadjuvant setting where definitive therapy cannot be delayed
for the extended period of time needed for multiple
immunizations, higher dose protein-based vaccines may
allow induction of tumor antigen–specific immunity in a
shorter period of time. Recombinant protein-based vacJOURNAL OF CLINICAL ONCOLOGY
HER-2/neu ICD Protein–Based Vaccine
cines are much more expensive to formulate than
peptide- or plasmid-based vaccines. Therefore, it is important to choose clinical situations where the higher,
more expensive dose would be preferred to the lower, less
expensive dose.
HER-2/neu, particularly the ICD portion of the protein, is a biologically relevant tumor antigen that has
become a model for testing a variety of vaccination strategies from peptides,7,9 to dendritic cells,26 to plasmidbased vaccines.27 Immunizing cancer patients with recombinant HER-2/neu protein has several advantages
when compared with other vaccine formulations; the
vaccine is immunogenic across all HLA types, can elicit
immunity in the majority of patients vaccinated, can
stimulate both tumor-specific cellular and humoral immunity, and will result in the development of immunologic memory. Detailed evaluations of the immunogenicity of cancer vaccines in the setting of phase I trials will
REFERENCES
1. Disis ML, Knutson KL, Schiffman K, et al:
Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer. Breast Cancer Res Treat 62:245-252, 2000
2. Scardino A, Gross DA, Alves P, et al:
HER-2/neu and hTERT cryptic epitopes as novel
targets for broad spectrum tumor immunotherapy. J Immunol 168:5900-5906, 2002
3. Bremers AJ, Andreola S, Leo E, et al: T cell
responses in colorectal cancer patients: Evidence for class II HLA-restricted recognition of
shared tumor-associated antigens. Int J Cancer
88:956-961, 2000
4. Disis ML, Pupa SM, Gralow JR, et al: High
titer HER-2/neu protein specific antibody immunity can be detected in patients with early stage
breast cancer. J Clin Oncol 15:3363-3367, 1997
5. Foy TM, Bannink J, Sutherland RA, et al:
Vaccination with Her-2/neu DNA or protein subunits protects against growth of a Her-2/neuexpressing murine tumor. Vaccine 19:25982606, 2001
6. Knutson KL, Disis ML: Expansion of
HER2/neu-specific T cells ex vivo following immunization with a HER-2/neu peptide-based vaccine. Clin Breast Cancer 2:73-79, 2001
7. Zaks TZ, Rosenberg SA: Immunization
with a peptide epitope (p369-377) from HER-2/
neu leads to peptide-specific cytotoxic T lymphocytes that fail to recognize HER-2/neu⫹ tumors.
Cancer Res 58:4902-4908, 1998
8. Knutson KL, Schiffman K, Disis ML: Immunization with a HER-2/neu helper peptide
vaccine generates HER-2/neu CD8 T-cell immunity in cancer patients. J Clin Invest 107:477-484,
2001
9. Disis ML, Gooley TA, Rinn K, et al: Generation of T-cell immunity to the HER-2/neu protein
www.jco.org
better define the clinical utility and application of tumorspecific immunization.
■ ■ ■
Acknowledgment
We are grateful for the nursing care of Donna Davis,
BSN, and the assistance in manuscript and data preparation
by Chalie Livingston and Robert Schroeder. We also thank
all the patients who agreed to participate in this study.
Authors’ Disclosures of Potential
Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for
drugs or devices used in a study if they are not being evaluated
as part of the investigation. Owns stock (not including shares
held through a public mutual fund): Martin A. Cheever,
Corixa. Served as an officer or member of the Board of a
company: Martin A. Cheever, Corixa.
after active immunization with HER-2/neu
peptide-based vaccines. J Clin Oncol 20:26242632, 2002
10. Disis ML, Knutson KL, McNeel DG, et al:
Clinical translation of peptide-based vaccine trials: The HER-2/neu model. Crit Rev Immunol
21:263-273, 2001
11. Knutson KL, Schiffman K, Cheever MA, et
al: Immunization of cancer patients with a HER2/neu, HLA-A2 peptide, p369-377, results in
short-lived peptide-specific immunity. Clin Cancer Res 8:1014-1018, 2002
12. Disis ML, Grabstein KH, Sleath PR, et al:
Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian
cancer using a peptide-based vaccine. Clin Cancer Res 5:1289-1297, 1999
13. Finn OJ: Cancer vaccines: Between the
idea and the reality. Nat Rev Immunol 3:630-641,
2003
14. Ward RL, Hawkins NJ, Coomber D, et al:
Antibody immunity to the HER-2/neu oncogenic
protein in patients with colorectal cancer. Hum
Immunol 60:510-515, 1999
15. Disis ML, Calenoff E, McLaughlin G, et al:
Existent T cell and antibody immunity to HER-2/
neu protein in patients with breast cancer. Cancer Res 54:16-20, 1994
16. Goldblatt D, Vaz AR, Miller E: Antibody
avidity as a surrogate marker of successful priming by Haemophilus influenzae type b conjugate
vaccines following infant immunization. J Infect
Dis 177:1112-1115, 1998
17. Gooley TA, Leisenring W, Crowley J, et al:
Estimation of failure probabilities in the presence
of competing risks: New representations of old
estimators. Stat Med 18:695-706, 1999
18. Disis ML, Gralow JR, Bernhard H, et al:
Peptide-based, but not whole protein, vaccines
elicit immunity to HER-2/neu, an oncogenic selfprotein. J Immunol 156:3151-3158, 1996
19. Bernards R, Destree A, McKenzie S, et al:
Effective tumor immunotherapy directed against
an oncogene-encoded product using a vaccinia
virus vector. Proc Natl Acad Sci U S A 84:68546858, 1987
20. Casal J, Tarrago D: Immunity to Streptococcus pneumoniae: Factors affecting production and efficacy. Curr Opin Infect Dis 16:219224, 2003
21. Reilly RT, Machiels JP, Emens LA, et al:
The collaboration of both humoral and cellular
HER-2/neu-targeted immune responses is required for the complete eradication of HER-2/
neu-expressing tumors. Cancer Res 61:880-883,
2001
22. Martina BE, van de Bildt MW, Kuiken T, et
al: Immunogenicity and efficacy of recombinant
subunit vaccines against phocid herpesvirus type
1. Vaccine 21:2433-2440, 2003
23. de Jong A, O’Neill T, Khan AY, et al:
Enhancement of human papillomavirus (HPV)
type 16 E6 and E7-specific T-cell immunity in
healthy volunteers through vaccination with TACIN, an HPV16 L2E7E6 fusion protein vaccine.
Vaccine 20:3456-3464, 2002
24. Ehrlich HJ, Pavlova BG, Fritsch S, et al:
Randomized, phase II dose-finding studies of a
modified tick-borne encephalitis vaccine: Evaluation of safety and immunogenicity. Vaccine 22:
217-223, 2003
25. Kundig TM, Bachmann MF, Oehen S, et al:
On the role of antigen in maintaining cytotoxic
T-cell memory. Proc Natl Acad Sci U S A 93:
9716-9723, 1996
26. Brossart P, Wirths S, Stuhler G, et al:
Induction of cytotoxic T-lymphocyte responses
in vivo after vaccinations with peptide-pulsed
dendritic cells. Blood 96:3102-3108, 2000
27. Disis M, Shiota F, McNeel D, et al: Soluble
cytokines can act as effective adjuvants in plasmid DNA vaccines targeting self-tumor antigens.
Immunobiology 207:179-186, 2003
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