Concurrent Trastuzumab and HER2/neu-Specific Vaccination in Patients With Metastatic Breast Cancer

Article (PDF Available)inJournal of Clinical Oncology 27(28):4685-92 · September 2009with41 Reads
DOI: 10.1200/JCO.2008.20.6789 · Source: PubMed
Abstract
The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of combination therapy consisting of concurrent trastuzumab and human epidermal growth factor receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic breast cancer. Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and overall survival were collected. Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range during combination therapy. Although many patients had pre-existing immunity specific for HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude of the T-cell response generated was significantly inversely correlated with serum transforming growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached. Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity and results in prolonged, robust, antigen-specific immune responses in treated patients.
Concurrent Trastuzumab and HER2/neu-Specific
Vaccination in Patients With Metastatic Breast Cancer
Mary L. Disis, Danelle R. Wallace, Theodore A. Gooley, Yushe Dang, Meredith Slota, Hailing Lu,
Andrew L. Coveler, Jennifer S. Childs, Doreen M. Higgins, Patricia A. Fintak, Corazon dela Rosa,
Kathleen Tietje, John Link, James Waisman, and Lupe G. Salazar
From the Tumor Vaccine Group, Center
for Translational Medicine in Women’s
Health, University of Washington; Fred
Hutchinson Cancer Research Center,
Seattle, WA; and Breastlink Medical
Group, Long Beach, CA.
Submitted October 23, 2008; accepted
March 2, 2009; published online ahead
of print at www.jco.org on August 31,
2009.
Supported by grants from Gateway
Foundation (Cancer Research Treat-
ment Foundation; M.L.D.) and National
Institutes of Health (NIH) Grant No.
K23CA100691 (to L.G.S.). Patient care
was conducted through the Clinical
Research Center Facility at the Univer-
sity of Washington (NIH Grant No. UL1
RR025014).
Authors’ disclosures of potential con-
flicts of interest and author contribu-
tions are found at the end of this
article.
Clinical Trials repository link available on
JCO.org.
Corresponding author: Mary L. Disis,
MD, Tumor Vaccine Group, Center for
Translational Medicine in Women’s
Health, 815 Mercer St, 2nd Floor, Box
358050, University of Washington,
Seattle, WA 98195-8050; e-mail:
ndisis@u.washington.edu.
The Acknowledgment is included in
the full-text version of this article,
available online at www.jco.org.
It is not included in the PDF version
(via Adobe® Reader®).
© 2009 by American Society of Clinical
Oncology
0732-183X/09/2728-4685/$20.00
DOI: 10.1200/JCO.2008.20.6789
ABSTRACT
Purpose
The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of
combination therapy consisting of concurrent trastuzumab and human epidermal growth factor
receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic
breast cancer.
Patients and Methods
Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab
therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded
according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed
by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and
overall survival were collected.
Results
Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients
experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range
during combination therapy. Although many patients had pre-existing immunity specific for
HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity
could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/
neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude
of the T-cell response generated was significantly inversely correlated with serum transforming
growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median
overall survival in the study population has not been reached.
Conclusion
Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity
and results in prolonged, robust, antigen-specific immune responses in treated patients.
J Clin Oncol 27:4685-4692. © 2009 by American Society of Clinical Oncology
INTRODUCTION
A potential application of cancer vaccines is in the
prevention of tumor recurrence or progression in
patients with minimal residual disease. Vaccines will
need to be coadministered with primary therapy or
given after optimal treatment. Human epidermal
growth factor receptor 2 (HER2)/neu is a tumor
antigen and vaccine target in breast cancer. With the
prolonged use of trastuzumab in the treatment of
most HER2/neu-positive breast cancers, evaluation
of the potential additive toxicity of the combina-
tion of trastuzumab and HER2/neu vaccination
is warranted.
The vaccine used in this trial was designed to
elicit HER2/neu-specific T-helper (Th) immunity.
1
Vaccine-induced CD4
Th1 cells may traffic to the
tumor, secrete inflammatory cytokines such as in-
terferon gamma (IFN-
), and activate local antigen-
presenting cells (APC) enhancing cross-priming at
the tumor site.
2
Via cross-priming, tumor-specific
CD8
T cells can be elicited.
3
Finally, antigen-
specific CD4
T cells can enhance and sustain
tumor-specific T-cell immunity over time.
Data presented here suggest that combination
trastuzumab and HER2/neu vaccine therapy is safe
and generates robust and persistent tumor-specific
T-cell immunity.
PATIENTS AND METHODS
Patient Population
After informed consent, patients were enrolled in
this trial approved by the United States Food and Drug
JOURNAL OF CLINICAL ONCOLOGY
ORIGINAL REPORT
VOLUME 27 NUMBER 28 OCTOBER 1 2009
© 2009 by American Society of Clinical Oncology
4685
Administration and the University of Washington Human Subjects Division.
Enrollment criteria were as follows: stage IV breast cancer in complete remis-
sion (CR) or stable disease (SD) on trastuzumab; documented HER2/neu
overexpression via immunohistochemistry or fluorescent in situ hybridiza-
tion; HLA-A2
; and a left ventricular ejection fraction (LVEF) in the normal
range (Table 1). Twenty-two patients were enrolled, and 21 patients received
vaccinations. Fourteen of 21 patients completed all six immunizations; five of
21 patients completed at least three immunizations, a sufficient number to
immunize; and two of 21 patients received fewer than three immunizations.
4
Clinical data are presented on 21 patients. Immunologic data for at least one
immunizing peptide and protein are presented on patients who had baseline
and at least one additional evaluation of immunity assessed (n 19).
Study Design
Serum and peripheral-blood mononuclear cells (PBMCs) were collected
before, at midpoint, and at 1, 3, 6, and 12 months after immunization. The
sample size was chosen based on the following: if no toxicities were seen, the
probability of such an occurrence would be at least 90% if the true toxicity rate
was 10% or less.
T-Cell Responses
IFN-
enzyme-linked immunosorbent spot assay was performed as pre-
viously described.
5
Ten
g/mL of immunizing peptides were used: p369
through 384 (KIFGSLAFLPESFDGDPA) derived from the extracellular do-
main (ECD), p688 through 703 (RRLLQETELVEPLTPS) from the trans-
membrane domain, and the intracellular domain (ICD) derived p971 through
984 (ELVSEFSRMARDPQ; denoted as 15 [eg, “X.”15] or 9 [eg, “X.”9] amino
acids in length).
1,6
Each antigen was assessed in six replicates of 2 10
5
/well.
One
g/mL of overlapping peptide pools (15 amino acids overlapping by 11
amino acids) for the HER2/neu ICD or ECD, 1
g/mL of recombinant human
p53, insulin-like growth factor binding protein 2 (IGFBP-2; Sigma-Aldrich, St
Louis, MO), and topoisomerase II-
(Topogen, Columbus, OH) were also
used. Tetanus toxoid (0.5 U/mL) and phytohemaglutinin (2.5 ug/mL) were
positive controls. All samples for each patient were cryopreserved, then thawed
and analyzed simultaneously to ensure comparability.
7,8
Validation studies
demonstrated that the assay is linear and precise between 2.0 and 3.5 10
5
PBMCs/well, with a detection limit of 1:100,000. Data are calculated estimates,
and some results are considered below the level of reproducible detection. Ten
age-matched volunteer female donors were evaluated as controls. Data are
presented on individuals as a calculated 1/frequency of IFN-
–secreting cells
in 10
6
PBMCs and discussed as the ratio of responding cells to PBMCs. In
summary analyses, data are presented as IFN-
spots per well (SPW) corrected
for background or described as the number of spots per 10
6
PBMCs postvac-
cination minus the number of spots prevaccination.
Patients were considered to have pre-existing immunity if, at baseline,
the mean antigen-specific SPWs were statistically different (P .05) from no
antigen wells. Patients were considered to have increased response if the
current SPW was greater than 2 standard deviations (SD) above the previous
value, remained the same if the mean SPW was within 2 SD of the previous
value, and decreased if the mean SPW was greater than 2 SD below the
previous value. Two SD is equivalent to a P value of .05 in that there is a 95%
probability that the values are statistically significant.
9
Cytolytic function of generated T-cell lines
5
was evaluated (Cytotox 96,
Promega, Madison, WI). The HLA-A2 transfected human HER2/neu-
expressing breast cancer cell line, SKBR3-A2, was plated at 10
4
cells/well in
triplicate. T cells, expanded after stimulation with immunizing peptide, were
added in an effector/target ratio of 40:1. Nontransfected SKBR3 cells were
controls. After incubation, supernatants were analyzed per manufacturer’s
specifications. Percent specific lysis was calculated as: ([experimental release
spontaneous releases of cytotoxic T-lymphocyte cells and target cells]/[maxi-
mum release spontaneous release of target cells]) 100.
Serum Transforming Growth Factor Beta Levels
Levels were measured, in triplicates, by enzyme-linked immunosorbent
assay (eBioscience, San Diego, CA). The concentration of human transform-
ing growth factor beta (TGF-ß) was calculated from a curve of serially diluted
human recombinant TGF-ß. The change in TGF-ß levels is described as the
value of TGF-ß postvaccination minus the value of TGF-ß prevaccination in
picograms per milliliter.
T-Regulatory Cell Levels
Evaluation was performed as previously described.
10
Data are expressed
as the percentage of FOXP3
CD4
cells among all CD4
CD3
T cells.
Statistical Analysis
Differences in median immune responses were assessed using a two-
tailed Mann-Whitney test, with a level significance set at .05. The relationship
between magnitude of immunity and serum TGF-ß levels was assessed using
Pearson’s product moment correlation. Pre- versus postimmunization data
were compared using a paired t test (two-tailed). Kaplan-Meier curves were
Table 1. Patient Characteristics
Characteristic
No. of
Patients %
Intent to treat 22
Received treatment 21
Age, years
Median 49
Range 33-76
Time from metastatic diagnosis, months
Median 18
Range 7-76
Disease status
Complete remission 11 52
Stable 10 48
Time from last chemotherapy, months
Median 4
Range 1-61
No. of prior chemotherapy regimens
2629
2-3 12 57
4314
Time on trastuzumab before study entry, months
Median 13
Range 3-85
LVEF at time of study entry
Median 61
Range 46-72
ER status
Positive 13 62
Negative 8 38
PR status
Positive 9 43
Negative 12 57
Prior hormonal therapy
Yes 15 71
No 6 29
Concurrent hormonal therapy with vaccination
Yes 7 33
No 14 67
HER2 status by IHC
2 524
3 15 71
Unknown 1 5
HER2 status by FISH, n 12
Median 5.18
Range 2.05-12.34
Abbreviations: LVEF, left ventricular ejection fraction; ER, estrogen receptor;
PR, progesterone receptor; IHC, immunohistochemistry; FISH, fluorescent in
situ hybridization.
Disis et al
4686 © 2009 by American Society of Clinical Oncology
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generated to show the probability of overall survival (OS) and progression-free
survival (PFS), where OS was defined as the time elapsed between beginning
vaccinations and death or last follow-up, and PFS was defined as the time
elapsed between first vaccine and the earliest of death, disease progression as
reported by the patients’ primary physicians, or last contact. Data for patients
without death (for OS) or death as a result of progression (for PFS) were
censored at the date of last known status. Differences in survival curves based
on immunologic response were assessed by the Gehan-Breslow-Wilcoxon test.
Analyses were performed with GraphPad InStat v.5.01 (GraphPad Software,
San Diego, CA).
RESULTS
HER2/neu Th Peptide Vaccine Administered
Concurrently With Trastuzumab Was Well Tolerated
and Did Not Result in Additional Cardiac Toxicity
Table 2 details 573 adverse events. The majority of toxicities were
grade 1 or 2 (99%). There were four grade 3 toxicities, three possibly
related to the treatment: injection-site reaction, fainting, and ulcer-
ation. There was one nonrelated grade 4 event, a stroke. The median
LVEF before treatment was 61% (range, 46% to 72%) and post-
treatment was 61% (range, 45% to 66%). Three patients (15%) had a
decrease in LVEF to less than normal values on study. None developed
symptoms of left ventricular dysfunction. Cardiac toxicities were
grade 1 and 2.
HER2/neu Th Peptide Vaccine Administered
Concurrently With Trastuzumab Stimulates or
Boosts HER2/neu-Specific Immunity in the Majority
of Patients
The median peptide-specific T-cell response before the first vac-
cine was a frequency of less than 1 antigen-specific cell in 75,000
PBMCs (range, 1:400,000 to 1:77,000; Fig 1A). Ninety percent of
patients developed new or augmented immunity. The maximal re-
sponse to p369.15 was a median frequency of one in 4,121 PBMCs
(range, 1:323 to 1:2,000,000; P .0015 compared with prevaccina-
tion), the maximal response to p688.15 was one in 4,152 PBMCs
(range, 1:307 to 1:3,000,000; P .0012), and the maximal response to
p971.15 was one in 2,086 PBMCs (range, 1:266 to 1:86,960; P .0066;
Fig 1A). Ten (53%) of 19 patients had pre-existing immunity to any of
these peptides. Sixteen patients (84%) significantly augmented immu-
nity, three (16%) did not augment, and none had a decrease in
peptide-specific immunity with immunization. The percentage of
responding patients for the peptides is shown (Fig 1B).
Table 2. Adverse Events
Adverse Event
AE Possibly, Probably, or Definitely Related
No. % of All AEs No. % of All Related AEs
Most common
Injection site reaction 64 11 64 17
Fatigue 42 7 38 10
Myalgias 42 7 33 9
Headache 41 7 27 7
Lymphopenia 33 6 28 7
Leukocytes, total WBCs 29 5 27 7
Pruritus/itching 21 4 19 5
Nausea 20 3 13 3
Rigors/chills 20 3 16 4
Diarrhea 16 3 5 1
AE grading
1 508 89 337 88
260104311
34131
41000
50000
Cardiac AEs
Palpitations 9 45 2 29
Hypertension 5 25 0 0
Left ventricular systolic dysfunction 3 15 3 43
Chest tightness 1 5 0 0
Supraventricular and nodal arrhythmia 1 5 1 14
Other 1 5 1 14
AE grading
11260114
2 8 40 6 86
30000
40000
50000
Abbreviation: AE, adverse event.
For AE grading, percent is shown (not % of all AEs or % of all related AEs).
Concurrent Trastuzumab and HER2/neu vaccination
www.jco.org © 2009 by American Society of Clinical Oncology 4687
Peptides were derived from both the HER2/neu ECD and ICD.
Of note, seven (64%) of 11 patients had significant pre-existing im-
munity to the ECD (median, 1:17,729; range, 1:660 to 1:3,000,000),
and six (32%) of 19 patients had significant pre-existing immunity to
the ICD (median, 1:309,524; range, 1:226 to 1:3,000,000; Fig 1A).
Sixty-nine percent of patients developed new or augmented immu-
nity: 37% to ECD, 53% to ICD, and 21% to both. The maximal
response to the ECD was a median of one cell in 2,312 PBMCs (range,
1:678 to 1:1,000,000; P .3017 compared with baseline) and to the
ICD was one cell in 9,677 PBMCs (range, 1:232 to 1:3,000,000; P
.0894; Fig 1A). Five patients did not augment, and none had a signif-
icant decrease in domain immunity with immunization. The percent-
age of responding patients is shown (Fig 1B).
Vaccination Can Elicit Tumor-Specific Cytotoxic
T Cells
Embedded within the native sequence of the Th peptides are
HLA-A2 binding motifs: p369.9, p688.9, and p972.9.
1,5
The maximal
response to class I peptides was a median frequency to p369.9 of
1:10,200 (range, 1:1,032 to 1:3,000,000; P .0030 compared with
baseline), to p689.9 was 1:10,050 (range, 1:1,039 to 1:3,000,000;
P .1720), and to p971.9 was 1:5,659 (range, 1:845 to 1:3,000,000;
P .0126; Fig 2A). T-cell cultures were established for five patients
who had excess PBMCs available. The resultant T-cell lines could
specifically lyse HER2/neu-positive/HLA-A2–positive breast cancer
cells (range, 7% to 70% lysis; Fig 2B).
Seven (37%) of 19 patients had pre-existing immunity to these
HLA-A2 peptides. Overall, 14 patients (74%) significantly augmented
the class I HER2/neu peptide-specific immune response, four patients
(21%) did not augment, and one patient had a decrease in immunity
A
B
Baseline and Maximal Cellular Immune Response
ELISPOT (-1/specific cell
frequency)
p369.15 p688.15 p971.15 ECD ICD
0
20
40
60
80
100
HER-2/neu Antigens
Patients with Response (%)
1.0 x 10
1
1.0 x 10
2
1.0 x 10
3
1.0 x 10
4
1.0 x 10
5
1.0 x 10
6
1.0 x 10
7
Pre 369.15
Max 369.15
Pre 688.15
Max 688.15
Pre 971.15
Max 971.15
Pre ECD
Max ECD
Control ECD
Pre ICD
Max ICD
Control ICD
Fig 1. A human epidermal growth factor receptor 2 (HER2)/neu T-helper peptide
vaccine administered concurrently with trastuzumab stimulates or boosts HER2/neu-
specific immunity in the majority of patients. (A) Prevaccine (Pre) and maximal (Max)
responses 1/frequency (Y axis) tested antigens (X axis). Connected points: mean and
SE of six replicates with median bar. Data derived from 10 controls are shown for
extracellular domain (ECD) and intracellular domain (ICD). (B) Percent HER2/neu-
specific immunity after vaccination. Blue, percent increased; yellow, percent un-
changed. ELISPOT, enzyme-linked immunosorbent spot assay.
A
B
C
Baseline and Maximal Cellular Immune Response
ELISPOT (-1/specific cell
frequency)
p369.9 p689.9 p971.9
0
20
40
60
80
100
HER-2/neu Antigens
Patients With Response (%)
1.0 x 10
1
1.0 x 10
2
1.0 x 10
3
1.0 x 10
4
1.0 x 10
5
1.0 x 10
6
1.0 x 10
7
Pre 369.9
Max 369.9
Pre 689.9
Max 689.9
Pre 971.9
Max 971.9
0 20 40 60 80 100
SK-A2
SK
SK-A2
SK
SK-A2
SK
SK-A2
SK
SK-A2
SK
1181211818118141180711822
Patients and Targets
Specific Lysis
(effector:target ratio of 40:1)
Fig 2. Vaccination can elicit tumor-specific cytotoxic T cells. (A) Prevaccine
(Pre) and maximal (Max) responses 1/frequency (Y axis) HLA-A2 peptides (X
axis). Connected points: mean and SE of six replicates with median bar. (B)
Percent lysis: SKBR3 (blue), SKBR3-A2 (yellow) with SE of four replicates. (C)
Percent human epidermal growth factor receptor 2 (HER2)/neu peptide-
specific immunity. Blue, percent increased; Yellow, percent unchanged.
ELISPOT, enzyme-linked immunosorbent spot assay.
Disis et al
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© 2009 by American Society of Clinical Oncology
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OURNAL OF CLINICAL ONCOLOGY
to the peptides with immunization. Percentage of responding patients
for the HLA-A2 peptides is shown (Fig 2C).
Vaccination-Induced or Enhanced Epitope Spreading
Was Observed in the Majority of Patients and Was
Associated With a Decrease in Serum TGF-ß
p98.15 and p776.15 are native epitopes of HER2/neu, immuno-
genic, and not included in the vaccine formulation.
11
Development of
immunity to these epitopes demonstrates intramolecular epitope
spreading, which was elicited or augmented in the majority of patients
(Fig 3A).
6
The median maximal T-cell response to nonimmunizing
epitopes was a frequency to p98.15 of one in 7,558 PBMCs (range,
1:1,205 to 1:3,000,000; P .0055 compared with prevaccination) and
to p776.15 of one in 2,183 PBMCs (range, 1:527 to 1:40,000; P .0006;
Fig 2A). Nine (47%) of 19 patients had pre-existing immunity to these
peptides. Fourteen patients (74%) significantly augmented the im-
mune response, five patients (26%) did not augment, and none sig-
nificantly decreased immunity to these peptides with immunization.
We have identified several immunogenic breast cancer–associated
proteins and questioned whether new or augmented immunity to
IGFBP-2, p53, and topoisomerase II-
were stimulated with vaccina-
tion (ie, intermolecular epitope spreading).
10,12
Seven patients had
sufficient PBMCs available for this analysis. All patients had a pre-
existing immune response to at least one antigen, and all seven devel-
oped new or augmented immunity to at least one of the antigens (Fig
2B). The postvaccination median response to IGFBP-2 was a fre-
quency of one in 5,405 PBMCs (range, 1:1,993 to 1:150,000; P .0973
compared with prevaccination), postvaccination median response to
p53 was one in 6,793 PBMCs (range, 1:1,061 to 1:109,091; P .1282),
and postvaccination median response to topoisomerase II-
was one
in 3,659 PBMCs (range, 1:1,575-1:8,219; P .0111; Fig 2B). Five
patients (71%) augmented immunity to IGFBP-2, six patients (86%)
augmented immunity to p53, and all patients tested augmented im-
munity to topoisomerase II-
. Two patients had a significant decrease
in a pre-existing immune response to IGFBP-2 with immunization.
The multiple specificities of IFN-
secreting T cells induced by
vaccination led us to question whether these T cells, which could
potentially traffic to tumor, might impact the immunosuppressive
environment that has been described in breast cancer.
13,14
TGF-ß has
been shown to be elevated in the serum of patients with breast cancer
and is associated with T-cell dysfunction.
15-17
The greater the magni-
tude of the intramolecular epitope spreading T-cell response, the
greater the decrease in serum TGF-ß (r 0.614; P .0003; Fig 3C).
There was weak correlation between the magnitude of the tetanus
toxoid response, evaluated as a control, and change in TGF-ß levels
(r 0.016; P .93; Fig 3D).
A
C
Baseline and Maximal Cellular Immune Response
Change in IFNγ Spots/10
6
PBMC
ELISPOT (-1/specific cell
frequency)
Change in Serum TGFβ (pg/ml)
1.0 x 10
1
1.0 x 10
2
1.0 x 10
3
1.0 x 10
4
1.0 x 10
5
1.0 x 10
6
1.0 x 10
7
Pre 98.15
Max 98.15
Pre 776.15
Max 776.15
B
Breast Cancer–Associated Antigens
ELISPOT (-1/specific cell
frequency)
1.0 x 10
3
1.0 x 10
4
1.0 x 10
5
1.0 x 10
6
1.0 x 10
7
1.0 x 10
8
Pre IGFBP2
Post IGFBP2
Pre p53
Post p53
Pre topolla
Post topolla
-500 500 1,000 1,500
-4,000
-3,000
-2,000
-1,000
1,000
2,000
D
Change in IFNγ Spots/10
6
PBMC
Change in Serum TGFβ (pg/ml)
-500 500 1,000 1,500
-4,000
-3,000
-2,000
-1,000
1,000
2,000
Fig 3. Vaccination-induced epitope spreading occurred in the majority of patients and was associated with a decrease in serum transforming growth factor beta (TGF-
)
levels. (A) Prevaccine (Pre) and maximal (Max) intramolecular epitope spreading (IMS) 1/frequency (Y axis) peptides (X axis). (B) Pre-Max intermolecular epitope
spreading (Y axis) antigens (X axis). Connected points: mean and standard deviation of six replicates with median bar. (C) X axis, change in magnitude of IMS response
(interferon gamma [IFN-
] spots per well/10
6
peripheral-blood mononuclear cells [PBMCs]). (D) X axis, change in magnitude of tetanus toxoid response. Y-axis; change
in TGF-ß. ELISPOT, enzyme-linked immunosorbent spot assay.
Concurrent Trastuzumab and HER2/neu vaccination
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© 2009 by American Society of Clinical Oncology 4689
Treg levels were measured before and after immunization in
eight patients. The median percent Treg was 1.64% before immuni-
zation (range, 0.33% to 7.33%) and 1.32% 1 month after vaccines
(range, 0.41% to 5.08%; P .60). At 1 year after immunization, the
median Treg level was 1.11% (range, 0.24% to 4.91%; P .61 com-
pared with preimmunization).
HER2/neu-Specific Immunity Can Persist and Even
Increase After Active Immunizations Have Ended
Figure 4 shows the OS and PFS of the study population from the
time of first vaccination. The median follow-up among survivors was
36 months (range, 21 to 49 months). The median PFS was 17.7
months, and the Kaplan-Meier estimate of PFS was 33% at 3 years.
The median OS has not been reached; the Kaplan-Meier estimate of
OS is 86% at 4 years.
Although the generation of a new or augmentation of a pre-
existing immune response to either immunizing peptides or protein
(P .11) or to epitope spreading peptides (P .47) was not associated
with survival, the magnitude of immunity generated tended to be
higher in surviving patients. All 10 patients who had T-cell responses
greater than the median to HER2/neu immunizing peptides and asso-
ciated protein (P .08) or to peptides associated with intramolecular
epitope spreading (P .09) were survivors as compared with patients
with responses less than the median.
We monitored immunity to HER2/neu-related antigens after the
end of immunizations in 11 patients (ECD, n 6). Five patients
(46%) maintained the same level of immunity to p369.15 in long-term
follow-up as compared with the maximal response achieved during
active immunization. Two (18%) decreased immunity and four
(36%) continued to augment immunity (median, 1:4,121; range,
1:788 to 1:3,000,000). Seven patients (64%) maintained the same level
of immunity to p688.15. One patient (9%) decreased compared with
the maximal response achieved, and three patients (27%) continued
to augment immunity to p688.15 (median, 1:12,876; range, 1:307 to
1:3,000,000). Five patients (46%) maintained the same level of immu-
nity to p971.15. Two patients (18%) decreased and four patients
(36%) continued to augment immunity (median, 1:5,236; range,
1:266 to 1:222,222).
Persistent immunity to the HER2/neu ECD and ICD was as-
sessed. Five (83%) of six patients maintained the same level of immu-
nity to the ECD in follow-up. One patient (17%) had decreased and
none continued to augment immunity to the ECD (median, 1:4,625;
range, 1:803 to 1:11,321). Six (55%) of 11 patients maintained immu-
nity to the ICD. Two (18%) decreased and three (27%) continued to
augment immunity (median, 1:6,024; range, 1:232 to 1:3,000,000).
Finally, intramolecular epitope spreading was maintained in five pa-
tients (46%) as evidenced by immunity to p776.15. Two patients
(18%) significantly decreased and four (36%) continued to augment
immunity (median, 1:2,667; range, 1:527 to 1:300,000).
DISCUSSION
Concurrent administration of trastuzumab and a HER2/neu-specific
vaccine is tolerated in patients with metastatic breast cancer (MBC),
and significant pre-existing immunity to HER2/neu can be boosted
and maintained with immunization. Moreover, epitope spreading
elicited with vaccination may modulate systemic mediators of tumor-
induced immune suppression.
Trastuzumab-related cardiac damage is generally reversible once
the drug is stopped.
18
After immunization, however, the T-cell re-
sponse is not as easily “turned off.” As long as antigen is present, T-cells
will clonally expand, further augmenting immunity, as occurred in a
third of our patients. A study of long-term trastuzumab use in MBC
reported that approximately 25% of patients experience some cardiac
event.
18
In our trial, 15% of patients had an asymptomatic drop in left
ventricular function below the normal range, although given the lim-
ited sample size, we cannot rule out the possibility that the true rate is
higher than the historical rate.
An unexpected finding was the number of patients treated with
trastuzumab who had significant pre-existing immunity to HER2/neu
and other antigens. Although we do not have an assessment of HER2/
neu immunity before starting trastuzumab, investigations by our
group suggest only 10% of trastuzumab-naïve patients would have
measurable cellular immunity.
19
Only one study has shown that en-
dogenous HER2/neu-specific humoral and T-cell immunity could be
elicited with trastuzumab treatment.
20
Despite the presence of immu-
nity to HER-2/neu at the start of immunization, most responses could
be boosted to greater levels with vaccination, and in patients with no
pre-existing immunity, robust T-cell responses could be generated.
Priming with trastuzumab and boosting with an HER2/neu vac-
cine may generate levels of immunity more robust than vaccination
alone. Indeed, the magnitude of the response achieved after immuni-
zation in this trial seemed to be greater than what we have historically
observed. The patients in this study were comparable to the patients in
our initial study both demographically and clinically.
1
There were no
statistically significant differences in the two populations in age, dis-
ease status at time of vaccination, hormone receptor status, or number
of chemotherapy regimens before vaccination. Our initial study did
not include trastuzumab, as the drug was not in widespread use at the
time, and the median peptide-specific T-cell response after vaccina-
tion was one in 16,129 PBMCs.
1
In this current study, the median
T-cell response to all HER2/neu antigens was 1:1,838 PBMCs, a log-
fold increase. Moreover, in our initial study, none of the patients
continued to augment immunity after vaccinations had ended.
1
Here,
nearly one third of patients augmented immunity, only a minority
10 20 30 40 50
20
0
40
60
80
100
Time (months)
Survival (%)
OS
PFS
Fig 4. Overall survival (OS) and progression-free survival (PFS) of immunized
patients. Kaplan-Meier curves of the percent OS and PFS in months from the
time of first vaccine (n 21).
Disis et al
4690
© 2009 by American Society of Clinical Oncology
J
OURNAL OF CLINICAL ONCOLOGY
demonstrated a diminution in response, and the remainder main-
tained the same level of HER2/neu immunity long term as during
active immunization.
Vaccination induced epitope spreading. Epitope spreading is as-
sociated with autoimmune disease and enhances tissue destruc-
tion.
21,22
Some studies suggest that antibody-dependent cell-mediated
cytotoxicity, by which APC presentation of new antigens is increased,
is responsible for epitope spreading.
23,24
Others suggest that antigen-
homing Th1-cells deliver cytokines, such as interferon, to the tumor,
which activate local APC-enhancing cross-priming.
2,22
The presence
of immunity to HER2/neu and other antigens before vaccination
suggests that antibody-dependent cell-mediated cytotoxicity me-
diated by trastuzumab may have initiated cross-priming at the
tumor site. The observation that increasing numbers of Th1
antigen-specific T cells, associated with epitope spreading and
elicited via vaccination, decrease serum TGF-ß indicate that traf-
ficking T cells may also be effectively modulating the tumor micro-
environment. Theoretically, a decrease in serum TGF-ß could
facilitate the continued augmentation and persistence of tumor-
specific immunity.
Is HER2/neu-specific immunity induced by vaccination related
to clinical outcome? Although the study was not designed to address a
clinical end point, OS was assessed to gather additional data on the
potential therapeutic efficacy of the combination.
25
The current ob-
served results are encouraging in light of the historical results for
patients with pretreated HER2/neu-positive MBC. Recent phase II
studies using trastuzumab and various chemotherapy agents in the
salvage setting have demonstrated that 40% to 60% of patients are able
to achieve either complete or partial responses with second- or third-
line trastuzumab-containing regimens. The median PFS and OS
ranged from 7 to 12 and 18 to 23 months, respectively.
26-28
Combina-
tion therapy with trastuzumab and an HER2/neu-specific vaccine
warrants further evaluation as a therapeutic regimen.
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS
OF INTEREST
Although all authors completed the disclosure declaration, the following
author(s) indicated a financial or other interest that is relevant to the subject
matter under consideration in this article. Certain relationships marked
with a “U” are those for which no compensation was received; those
relationships marked with a “C” were compensated. For a detailed
description of the disclosure categories, or for more information about
ASCO’s conflict of interest policy, please refer to the Author Disclosure
Declaration and the Disclosures of Potential Conflicts of Interest section in
Information for Contributors.
Employment or Leadership Position: None Consultant or Advisory
Role: Mary L. Disis, VentiRx Pharmaceuticals (C) Stock Ownership:
None Honoraria: None Research Funding: Mary L. Disis,
GlaxoSmithKline Expert Testimony: None Other Remuneration: None
AUTHOR CONTRIBUTIONS
Conception and design: Mary L. Disis, Theodore A. Gooley,
Lupe G. Salazar
Financial support: Mary L. Disis
Administrative support: Mary L. Disis, Jennifer S. Childs, Patricia A.
Fintak, Kathleen Tietje
Provision of study materials or patients: Mary L. Disis, Jennifer S.
Childs, Patricia A. Fintak, John Link, James Waisman, Lupe G. Salazar
Collection and assembly of data: Mary L. Disis, Danelle R. Wallace,
Yushe Dang, Meredith Slota, Hailing Lu, Andrew L. Coveler, Jennifer S.
Childs, Doreen M. Higgins, Patricia A. Fintak, Corazon dela Rosa
Data analysis and interpretation: Mary L. Disis, Danelle R. Wallace,
Theodore A. Gooley, Yushe Dang, Meredith Slota, Hailing Lu,
Lupe G. Salazar
Manuscript writing: Mary L. Disis, Danelle R. Wallace, Lupe G. Salazar
Final approval of manuscript: Mary L. Disis, Danelle R. Wallace,
Kathleen Tietje, John Link, James Waisman, Lupe G. Salazar
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    • "For example, Chuang et al. treated OVA-expressing tumors with an oncolytic VacV encoding the same antigen after priming with a DNA vaccine [95]. Given that other boosting strategies have successfully utilized pre-existing antitumor immunity as a platform for boosting [96], it has even been suggested that in some cases the tumor alone may serve as sufficient priming for boosting by oncolytic vaccines [97]. While this represents an exciting prospect it is likely that endogenous responses, if present, would not be of sufficient magnitude or quality to mediate appreciable therapeutic benefit in many cases. "
    [Show abstract] [Hide abstract] ABSTRACT: The past decade has seen considerable excitement in the use of biological therapies in treating neoplastic disease. In particular, cancer immunotherapy and oncolytic virotherapy have emerged as two frontrunners in this regard with the first FDA approvals for agents in both categories being obtained in the last 5 years. It is becoming increasingly apparent that these two approaches are not mutually exclusive and that much of the therapeutic benefit obtained from the use of oncolytic viruses (OVs) is in fact the result of their immunotherapeutic function. Indeed, OVs have been shown to recruit and activate an antitumor immune response and much of the current work in this field centers around increasing this activity through strategies such as engineering genes for immunomodulators into OV backbones. Because of their broad immunostimulatory functions, OVs can also be rationally combined with a variety of other immunotherapeutic approaches including cancer vaccination strategies, adoptive cell transfer and checkpoint blockade. Therefore, while they are important therapeutics in their own right, the true power of OVs may lie in their ability to enhance the effectiveness of a wide range of immunotherapies.
    Full-text · Article · Aug 2016
    • "However, in light of the compelling evidence regarding potential synergy, this has led us to question whether the early recurrences may have been prevented with concurrent vaccination and trastuzumab. In a study of 21 patients with stage IV HER2-positive breast cancer being treated with trastuzumab, Disis et al. showed that concurrent vaccination with a vaccine designed to elicit HER2- specific T-helper immunity was safe [17]. Our group has completed a phase I study of the concurrent administration of trastuzumab and GP2+GM-CSF in early stage HER2- positive breast cancer patients. "
    [Show abstract] [Hide abstract] ABSTRACT: GP2 is a HER2-derived, HLA-A2+ restricted peptide. Phase I studies showed GP2 administered with GM-CSF to be safe and immunogenic. Here we report the primary analysis of a prospective, randomized, multicenter phase II adjuvant trial conducted to determine the vaccine's efficacy. The trial enrolled HLA-A2+, clinically disease-free, node-positive and high-risk node-negative breast cancer patients with tumors expressing HER2 (immunohistochemistry[IHC] 1+-3+). Patients were randomized to GP2+GM-CSF versus GM-CSF alone. Disease-free survival (DFS) was analyzed in intention-to-treat (ITT) and per-treatment cohorts; pre-specified subgroup analyses were performed for patients with IHC 3+ or FISH+ disease. The trial enrolled 180 patients; 89 received GP2+GM-CSF and 91 received GM-CSF alone. The groups were well-matched for clinicopathologic characteristics. Toxicities have been minimal. The Kaplan-Meier estimated 5-year DFS rate in the ITT analyses was 88% (95% CI:78-94%) in vaccinated vs. 81% (95% CI:69-89%) (P = 0.43) in control patients after a 34 month median follow-up. In the per-treatment analysis, the estimated 5-year DFS rates were 94% (95% CI:83-98%) and 85% (73-92%) (P = 0.17). In IHC 3+/FISH+ patients, the estimated 5-year DFS rate was 94% (82-98%) in vaccinated patients (n = 51) vs. 89% (71-96%) in control patients (n = 50), (P = 0.86) in the ITT analyses and 100% vs. 89% (71-96%) in vaccinated vs. control patients in the per-treatment analyses (P = 0.08). While the overall ITT analysis did not demonstrate benefit to vaccination, this trial confirmed that the GP2 vaccine is safe and suggests that vaccination may have clinical activity, particularly in patients with HER2 overexpression who received the full vaccine series (ie per-treatment group).
    Article · Jul 2015
    • "One possible explanation for these observations is that helper peptides unrelated to tumor antigens may be ineffective in guiding effector CD8 T cells within the tumor [61]. These observations are in line with previous reports by using tumor-derived helper peptides such as HER2-neu and hTERT [62,63]. Hence, a hTERT-derived helper peptide vaccine called GV1001 has been evaluated in many cancers [64]. "
    [Show abstract] [Hide abstract] ABSTRACT: Nowadays, immunotherapy represents one promising approach for cancer treatment. Recently, spectacular results of cancer immunotherapy clinical trials have confirmed the crucial role of immune system in cancer regression. Therapeutic cancer vaccine represents one widely used immunotherapy strategy to stimulate tumor specific T cell responses but clinical impact remains disappointing in targeting CD8 T cells. Although CD8 T cells have been initially considered to be the main protagonists, it is now clear that CD4 T cells also play a critical role in antitumor response. In this article, we discuss the role of tumor antigen-specific CD4 T cell responses and how we can target these cells to improve cancer vaccines.
    Full-text · Article · Jun 2015
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