Phase I/II Study of Combination Immunotherapy for the Generation of HER-2/Neu (HER2) Specific Cytotoxic T Cells (CTL) in Vivo
Phase: PHASE1
Conditions: HER2/Neu Positive, HLA-A2 Positive Cells Present, Stage IV Breast Cancer, Stage IV Ovarian Cancer
Sponsor: University of Washington

6304p 
 
 
 
 
 
 
 
 
 
 
 
UNIVERSITY OF WASHINGTON 
DEPARTMENT OF MEDICINE 
DIVISION OF ONCOLOGY 
Protocol 118 
 
Phase I/II Study of Combination Immunotherapy for the Generation of HER-2/neu (HER2) 
Specific Cytotoxic T Cells (CTL) in vivo 
 
Current Version Date: 04/06/2016 
Previous Version Date: 11/14/2006 
 
 
Investigators:  
 
Researchers: 
Mary L. Disis, M.D. 
Professor  
Division of Oncology, UW 
Member FHCRC 
(206) 616-1823 
Lupe G. Salazar, M.D. 
Associate Professor 
Division of Oncology, UW 
(206) 616-8503 
William R. Gwin, M.D. 
Acting Instructor 
Division of Oncology, UW 
(206) 221-5956 
Andrew Coveler, M.D. 
Assistant Professor 
Division of Oncology, UW 
(206) 221-7509 
John Liao, M.D. 
Assistant Professor 
GYN/ONC, UW 
(206) 543-3915 
Sasha Stanton, M.D. 
Acting Instructor 
Division of Oncology, UW 
(206) 685-8849 
Doreen Higgins, B.S.N. 
Research Nurse 
Division of Oncology, UW 
(206) 616-9538 
Jennifer Childs, M.P.H. 
Research Coordinator 
Division of Oncology, UW 
(206) 616-2305 
Chara Hokama, B.S. 
Research Coordinator 
Division of Oncology, UW 
(206) 543-3829 
 
 
Emergency (24 hour) pager: Dr. Sasha Stanton (206-797-3524) 
 
IRO received 03/23/21
FHCRC IRB Approval  
04/20/2021 
Document Released Date

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Table of Contents 
 
Page 
 
Synopsis 
1 
 
List of Appendices 
3 
 
List of Abbreviations 
4 
 
List of Figures 
5 
 
Introduction 
6 
 
Background 
6 
 
Rationale 
9 
 
Objectives 
9 
 
Vaccine Preparation 
9 
 
Subject Selection / Inclusion and Exclusion criteria 
11 
 
Experimental Design  
 
Study Design 
12 
 
Sample Size 
12 
 
Outcome Measures 
12 
 
Plan of Treatment 
 
Pre Screen  
12 
 
Initial Evaluation 
13 
 
Monthly Immunizations 
13 
 
Evaluation after Final Vaccine 
13 
 
Long Term Follow up 
13 
 
Evaluation and Management of Toxicity 
13 
 
Dose Modifications 
14 
 
Criteria for Premature Study Termination 
14 
 
 
 
Statistical Consideration 
15 
 
Administrative Considerations 
16 
 
References 
18 
 
Figures 
20 
 
Appendices 
 
 

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1 
SYNOPSIS 
 
Title  
Phase I-II Study of Combination Immunotherapy for the Generation of 
HER-2/neu (HER2) Specific Cytotoxic T Cells (CTL) in vivo 
 
Study Population 
Patients with HER2 overexpressing Stage IV breast cancer who are on 
maintenance trastuzumab alone after being treated with chemotherapy 
and trastuzumab until there is no evidence of disease (NED) or stable 
disease.  Stage IV HER2 overexpressing ovarian cancer patients who 
are on maintenance trastuzumab alone or after being treated with 
chemotherapy and trastuzumab until there is no evidence of disease 
(NED) or stable disease.   
 
Patients must be HLA-A2. 
 
Rationale 
Data from tumor vaccine studies now indicate there might be survival 
advantages for patients who have received tumor-antigen specific 
vaccinations.  Our group has demonstrated a potential survival 
advantage for patients with advanced stage HER2 overexpressing breast 
cancer immunized with a HER2 peptide based vaccine after being 
treated to maximal response or complete remission with standard 
therapy.  Recent studies have demonstrated that “sensitization” of HER2 
overexpressing tumor cells with trastuzumab, in vitro, will enhance the 
function of CTL specific for HER2.  Theoretically, the mechanism of 
trastuzumab’s enhancement of a HER2 specific CTL response might be 
the internalization of the HER2 receptor, degradation of the HER2 
protein, and increased MHC-peptide presentation with a resultant 
increase in CD8+ HER2 specific CTL function.  Thus, combination of 
trastuzumab with HER2 peptide based vaccine designed to elicit CTL in 
the context of HLA-A2 may even further enhance the generation of a 
HER2 specific CTL response and potentially translate into improved 
survival for advanced stage breast and ovarian cancer patients when 
used in the adjuvant setting.   
 
 
This proposal outlines a clinical trial designed to utilize the potential 
synergistic effect between trastuzumab and a HER2 CTL generating 
peptide based vaccine (HER2 CTL vaccine) in order to increase CTL 
precursor frequencies that target HER2 specific epitopes.  Patients will 
be treated to NED or stable disease with chemotherapy and trastuzumab 
or trastuzumab alone and while on maintenance trastuzumab receive 
vaccinations with a HER2 CTL vaccine.   
 
Objectives 
Primary:  
 
1. To evaluate the safety of administering a HER2 CTL peptide-based 
vaccine to Stage IV breast and ovarian cancer patients receiving 
maintenance trastuzumab. 
 
2. To quantify and characterize antigen specific T cells directed 
against HER2 in PBMC of patients after vaccination with a HER CTL 
peptide-based vaccine administered while receiving maintenance 
trastuzumab.   
 

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Secondary: 
 
1. To evaluate overall survival (OS) in Stage IV breast cancer patients 
who complete a vaccination series with a HER2 CTL peptide-based 
vaccine while receiving maintenance trastuzumab. 
 
 
Study Design 
This will be a single arm phase I-II single institution clinical trial in 
patients with HER2 overexpressing Stage IV breast and ovarian cancer 
who are on maintenance trastuzumab alone after being treated with 
chemotherapy and trastuzumab or trastuzumab alone to NED or stable 
disease.  Patients will receive a monthly vaccination for 6 months with 
a HER2 CTL peptide-based vaccine. 
 
Number of Patients 
Twenty subjects will be enrolled.  This will provide statistically 
adequate numbers of subjects for gathering (1) safety data and (2) 
immunologic response data. 
 
Outcome Measures 
 
Primary Endpoints: 
 
 
1. Safety will be assessed using NCI common toxicity criteria. 
 
 
2. Immune response will be defined by cytokine flow cytometry 
(CFC).  Specifically, a positive immune response will be defined as a 
post-vaccination HER2 antigen specific CD8 or CD4 precursor 
frequency measured by CFC of <1:20,000.  For subjects who have 
measurable precursors by CFC at baseline, a positive response will be 
defined as a 2-fold increase in either T cell subset after vaccination.   
 
 
 
HLA-A2 MHC tetramer analysis will be used to demonstrate the 
specificity of the T cells for the HER2 HLA-A2 defined epitopes.  IFN-
gamma secreting PBMC precursor frequency will also be measured by 
ELIspot in order to determine the development of lower precursor 
frequencies not detected by CFC.  ELIspot measurements will not 
define response. 
 
Secondary Endpoints: 
 
 
1. Overall survival for all patients will be followed.  Survival for the 
Stage IV breast cancer patients will be compared to historical control.  
This study will not enroll sufficient numbers of subjects to give 
statistical power to this endpoint.  However, a large difference observed 
between the treatment group and historical control would give 
additional impetus for a phase II study of efficacy.  Although the study 
will enroll both breast and ovarian cancer patients, from our extensive 
experience in combined breast and ovarian cancer vaccine studies for 
HER2, we anticipate approximately 80% of the subjects enrolled will 
be Stage IV breast cancer patients.   

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Appendices 
 
Appendix A  
 ECOG Performance Scale 
 
Appendix B  
 Laboratory Assays for Measuring Immune Response 
 
Appendix C  
 Data Safety Monitoring Plan 
 
Appendix D 
Supplemental HER2 CTL Peptide-Based Booster Vaccine Study 
 

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Abbreviations: 
 
APC  
antigen presenting cells 
BUN 
blood urea nitrogen 
CBC 
complete blood count 
CFC 
cytokine flow cytometry 
CTL 
cytotoxic T lymphocytes 
DC 
dendritic cells 
DLN 
draining lymph node 
DLT 
Dose limiting toxicity 
DSMP 
Data Safety Monitoring Plan 
ECD 
extracellular domain of HER2 
FISH 
fluorescent in situ hybridization 
GM-CSF granulocyte macrophage colony stimulating factor 
HER2 
HER-2/neu 
ICD 
intracellular domain of HER2 
id. 
intradermal 
IFN 
interferon 
IHC 
immunohistochemistry 
LC 
Langerhans cells 
LV 
left ventricular 
MHC 
Major Histocompatability Complex 
NED 
no evidence of disease 
OS 
overall survival 
PBMC 
peripheral blood mononuclear cells 
SI 
stimulation index 
sc 
subcutaneous 
Th 
T helper cells 
UA 
urinanalysis 
UPN 
unique patient number 
 

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List of Figures 
 
Figure 1. 
Patients with HER2 overexpressing cancers can be immunized with the HER2 CTL vaccine. 
 
Figure 2. 
The magnitude of HER2 specific CD8 T cell immunity is dependent on T helper immunity 
 
Figure 3. 
Epitope spreading occurs with active immunization 
 
Figure 4. 
Patients immunized with a HER2 CTL vaccine develop persistent immunity and epitope 
spreading years after active immunizations have ended 
 
Figure 5. 
Overall survival in HER2+ Stage IV breast cancer patients 
 
Figure 6. 
The use of HER2 peptide based vaccines in the adjuvant setting may impact survival in patients 
with advanced stage HER2 overexpressing breast cancer 
 
Figure 7. 
Treatment of HER2+ tumor cells with trastuzumab in vitro enhances lysis of HER2+/A2+ 
tumor cells by T clones specific for p369-377. 

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1. Introduction 
The HER-2/neu (HER2) oncogenic protein is a well defined tumor antigen in patients with breast cancer.  
Monoclonal antibody therapy with agents such as trastuzumab, targeting the HER2 protein, has demonstrated a 
survival benefit for patients with advanced stage breast cancer.  We have completed a phase I study of a HER2 
peptide-based vaccine designed to elicit both T helper (Th) and cytotoxic T cell (CTL) responses.  In an 
evaluation of 20 patients the vaccine was safe, was not associated with toxicity, particularly autoimmune 
toxicity, and was immunogenic.  Patients immunized developed T cell immunity directed against HER2.  
Specifically, HER2 specific CTL from patients post-vaccination lysed HLA-matched HER2 overexpressing 
tumors.  Moreover, the immunization strategy was designed to augment both HER2 specific CD4+ and CD8+ 
immune responses and, indeed, long-lived immunologic memory was created in a substantial number of the 
patients vaccinated.  Since this initial study, standard of care for advanced stage HER2+ breast cancer patients 
includes trastuzumab administered with chemotherapy.  Furthermore, patients with Stage IV HER2 
overexpressing ovarian cancer have received trastuzumab as part of their therapy.  For patient’s who achieve a 
complete remission (CR) or stabilization of disease, treatment with trastuzumab alone continues for several 
months to a year or until progression.  For this reason, a HER2 specific vaccine aimed at preventing relapse in 
stage IV breast cancer patients after standard therapy will have to be given concurrently with trastuzumab.  The 
concurrent administration of trastuzumab with a HER2 CTL vaccine may actually make the vaccine more 
effective in eliciting HER2 specific CTL responses.  The internalization of the HER2 receptor after binding to 
the antibody would potentially increase presentation of processed HER2 proteins in MHC class I molecules.  
Indeed, recent studies have shown that pretreatment of HER2 overexpressing tumor cells with trastuzumab 
makes them more susceptible to lysis by HER2 specific CTL T cell clones. (1) 
 
The proposed clinical trial is a phase I/II study of combination immunotherapy for the generation of HER2 
specific CTL in vivo.  Trastuzumab will be given concurrently with a HER2 peptide based vaccine composed of 
Th epitopes that encompass within their natural sequence HLA-A2 binding epitopes of HER2.  The strategy is to 
administer a vaccine that will stimulate both CD4+ and CD8+ responses simultaneously.  The primary 
objectives of the study are to evaluate the safety of administering a HER2 CTL peptide-based vaccine to Stage 
IV breast and ovarian cancer patients receiving trastuzumab as well as to quantify and characterize antigen 
specific T cell subsets specific to HER2 in PBMC of patients after vaccination.  A secondary objective is to 
evaluate time to overall survival (OS) in patients who complete a vaccination series with a HER2 CTL peptide-
based vaccine while receiving trastuzumab. 
 
2. Background 
A. HER2 is a tumor antigen in human adenocarcinomas.  The HER2 oncogenic protein consists of a 
cysteine rich extracellular ligand binding domain, a short transmembrane domain, and cytoplasmic protein 
tyrosine kinase domain. (2,3)  Binding of ligand to the extracellular domain (ECD) leads to dimerization that 
stimulates the intrinsic tyrosine kinase activity of the receptor and triggers autophosphorylation of specific 
tyrosine residues within the intracellular cytoplasmic domain (ICD).  These phosphorylated residues then serve 
as anchoring sites for signaling molecules involved in the regulation of intracellular signaling cascades (2) and, 
thus, cell growth.  HER2 is a self-protein expressed in a variety of tissues of epithelial origin and plays a 
fundamental role in cellular proliferation and differentiation during fetal development.  In adults, the HER2 gene 
is present as a single copy in normal cells; however, amplification of the gene and resultant protein 
overexpression is seen in various cancers including breast, ovarian, colon, uterine, gastric, prostate, and 
adenocarcinoma of the lung.  Furthermore, the overexpression of HER2 is implicated in the malignant 
transformation of breast cancer (4-6) and is a biologically relevant protein in the pathogenesis of several other 
epithelial-based tumors, for example leading to the development of hormone resistance in prostate cancer. (7)  
Relapse is a significant clinical problem for breast cancer patients whose tumors overexpress HER2. (8) 
 
HER2 is also a tumor antigen.  Patients whose tumors overexpress the HER2 protein can have both low level 
antibody and T cell immunity directed against the protein. (9)  Generating an active immune response directed 
against the HER2 protein has several potential clinical advantages.  Vaccination, if effective, would stimulate 
immunologic memory and could result in the prevention of relapse after standard therapy.   
 

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B. Patients with HER2 over expressing tumors can be safely immunized with HER2 derived peptides 
and generate a HER2 specific CTL response.  The CTL immune response has been considered the primary 
effector arm involved in mediating an anti-tumor immune response.  Experimental vaccination strategies 
designed to stimulate tumor specific CTL responses in vivo vaccinate individuals with tumor cells or viruses 
recombinant for tumor antigens that can infect viable cells so that proteins are expressed inside the cell, 
processed and presented in the class I MHC antigen processing pathway.  An alternative strategy uses soluble 
peptide that is identical or similar to naturally processed peptides present in class I MHC molecules as the 
vaccine along with an adjuvant.  HLA-A2 binding peptide p369-377, derived from the protein sequence of 
HER2 ECD, has been used extensively in clinical trials.  In an initial clinical study, HLA-A2 positive patients 
with metastatic HER2 overexpressing breast, ovarian, or colorectal carcinomas were immunized with 1 mg of 
p369-377 admixed in incomplete Freund’s adjuvant (IFA) every 3 weeks. (10)  Peptide specific CTL were 
isolated and expanded from the peripheral blood of patients after 2 or 4 immunizations.  The CTL could lyse 
HLA matched peptide pulsed target cells but could not lyse HLA matched tumors expressing the HER2 protein.  
More recently, a similar study was performed immunizing patients with p369-377 using GM-CSF as an 
adjuvant. (11)  GM-CSF is a recruitment and maturation factor for skin dendritic cells (DC), Langerhans cells 
(LC) and theoretically provides more efficient presentation of peptide epitopes than standard adjuvants such as 
IFA.  HLA-A2 patients with HER2-overexpressing cancers received 6 monthly vaccinations with 500 mcg of 
HER2 peptide, p369-377 peptide, admixed with 100 mcg of GM-CSF.  The patients had either stage III or IV 
breast or ovarian cancer.  Immune responses to the p369-377 were examined using an IFN-gamma ELIspot 
assay.  Prior to vaccination, the median precursor frequency, defined as precursors/106 PBMC, to p369-377 was 
not detectable.  Following vaccination, HER2 peptide-specific precursors developed to p369-377 in just 2 of 4 
evaluable subjects.  The responses were short-lived and not detectable at 5 months after the final vaccination.  
Immunocompetence was evident as patients had detectable T cell responses to tetanus toxoid and influenza.  
These results demonstrate that HER2 MHC class I epitopes can induce HER2 peptide-specific IFN-gamma-
producing CD8+ T cells.  However, the magnitudes of the responses were low, as well as short-lived.  
Theoretically, the addition of CD4+ T cell help would allow the generation of lasting immunity. 
 
Interestingly, immunizing patients with putative T-helper epitopes from HER2 that have HER2 HLA-A2 
binding motifs embedded in their natural sequence resulted in the generation of peptide-specific CTL capable of 
lysing HER2 overexpressing tumor and immune responses were durable. (12)  In that trial, 19 HLA-A2 patients 
with HER2-overexpressing cancers received the vaccine, twelve patients developed both HER2 specific CD4+ 
and CD8+ T cell responses, and the level of HER2 immunity was similar to immunity to KLH, a foreign protein 
used as a control immunization (Fig. 1).  Importantly, the immune responses were detectable for greater than 1 
year after the final vaccination in selected patients.  These results demonstrate that HER2 MHC class II epitopes 
containing encompassed MHC class I epitopes are able to induce long-lasting HER2-specific IFN-gamma-
producing CD8 T cells.  Indeed, when a comparison was made between the clinical trial that used a HER2 HLA-
A2 binding peptide alone vs. immunizing to achieve CTL using a class II peptide with a class I peptide 
embedded in the natural sequence, the latter immunization strategy was necessary to maintain both high 
magnitude and long lived CD8+ T cell immunity (Fig. 2).  We will evaluate this vaccine, composed of HER2 T 
helper epitopes which encompass HLA-A2 CD8+ epitopes of HER2 in combination with trastuzumab to 
augment HER2 specific CTL T cell immunity in a combination immunotherapy approach.   
 
C. Immunization with a HER2 peptide based vaccine, designed to elicit CTL, is associated with the 
development of immunologic memory and epitope spreading.  Epitope spreading was first described in 
autoimmune disease (13) and has been associated with both MHC Class I- and MHC Class II-restricted 
responses.  An additional finding of the study described above (12) as well as a larger clinical trial vaccinating 
patients with a variety of putative class II binding HER2 epitopes (14) was that epitope spreading was observed 
in 84% of patients and significantly correlated with the generation of HER2 protein-specific T cell immunity 
(p=.03).  Epitope spreading represents the generation of an immune response to a particular portion of an 
immunogenic protein and the natural spread of that immunity to other areas of the protein or even to other 
antigens present in the environment. (15,16)  In this study, epitope spreading reflected the extension of a 
significant T cell immune response to portions of the HER2 protein that were not contained in the patient’s 
vaccine (Fig. 3).  Theoretically, a broadening of the immune response may represent endogenous processing of 

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antigen at sites of inflammation initiated by a specific T cell response or “driver clone”. (17)  The initial immune 
response can create a microenvironment at the site of the tumor that enhances endogenous immune effector cells 
present locally.  These immune cells, e.g. APC and T cells present antigen and create a CTL response to tumor 
antigen present in the body.  It may be that the development of epitope spreading is a better predictor of vaccine 
efficacy than the magnitude of the immune response generated. (18)   
 
We have recently begun to assess the presence of long term immunity in previously immunized patients.  Figure 
4 demonstrates persistent detectable HER2 specific immunity and epitope spreading present in a patient over 4 
years after receiving her last immunization with the HER2 CTL vaccine.  This is one of the first demonstrations 
of persistent long term immunity developing in a cancer patient after the administration of a cancer vaccine.  
The mechanisms responsible for the maintenance of memory T cells are not entirely known.  Studies have 
shown that memory cells are more metabolically active than naïve T cells suggesting that memory cells may be 
continuously signaled by extrinsic factors.  Such signaling most likely involves some degree of contact with 
persistent antigen.  As most of the patients in our vaccine trials are advanced stage patients who have been 
treated to maximal response and invariably have minimal residual disease, it may be that this persistent 
microscopic tumor stimulates the persistence of immunity.  Overall survival in patients with HER2+ Stage IV 
breast cancer is well defined. (19)  A recent trial randomized over 400 stage IV breast cancer patients to receive 
chemotherapy alone vs. chemotherapy plus trastuzumab as primary therapy for their disease.  The median 
overall survival for the patients receiving chemotherapy alone was 20 months and for those receiving the 
combination antibody and chemotherapy was 29 months (Fig. 5).  Patients from the HER2 peptide based 
vaccine studies (12) have now been followed a median of 5 years (Fig. 6).  Six of the 9 (67%) patients who 
completed immunization with the CTL vaccine are still alive.  The 74% overall survival (OS) Kaplan-Meyer 
statistic on this subset is limited because of the small number of patients, however, the historical control data for 
stage IV patients with distant metastasis suggests a benefit to vaccination.  Although HER2 overexpressing 
ovarian cancer patients treated with trastuzumab alone are eligible for this study, based on our previous trials 
enrolling breast and ovarian cancer patients we anticipate over 80% of patients enrolled will be Stage IV breast 
cancer patients.  Thus, immunization of patients with advanced stage disease who have been optimally treated to 
prevent relapse is a clinical goal of vaccination with the HER2 CTL vaccine.   
 
D. Addition of trastuzumab to active immunization with a HER2 CTL peptide based vaccine may result 
in augmented immunity and the generation of a more vigorous CTL response.  Treatment of advanced 
HER2 overexpressing breast cancer has evolved since our first study was initiated.  A common regimen for 
relapsed stage IV HER2 overexpressing breast cancer is treatment with trastuzumab and chemotherapy for 4 
months or until maximal response and trastuzumab monotherapy continued for a year.  Patients with de novo 
stage IV disease often receive a longer cycle of chemotherapy with continued trastuzumab for 1 year.  For this 
reason, vaccines given in the setting of stage IV disease need to be administered concurrent with trastuzumab if 
patients are to be immunized in a minimal residual disease state.  Trastuzumab, when administered prior to or 
concurrent with doxorubicin results in a higher incidence of cardiac toxicity than what is anticipated with 
doxorubicin alone. (20)  For patients with minimal prior exposure to anthracycline the risk of cardiac 
dysfunction, defined as decrease in left ventricular ejection fraction (LVEF), was 1%.  For patients with more 
extensive prior doxorubicin exposure the risk of cardiac dysfunction with trastuzumab was 7%.  If trastuzumab 
was given concurrent with doxorubicin the risk of cardiac dysfunction was 29%.  The etiology of the cardiac 
dysfunction related to trastuzumab administration is unknown, but the temporal relationship to concurrent 
doxorubicin exposure suggests a link with anthracycline induced myocardial injury.  As this will be the first 
study evaluating the concurrent administration of trastuzumab and active immunization targeting HER2 we will 
evaluate any potentially toxicities closely with particular emphasis on cardiac status.   
 
There is a good rationale to suggest that trastuzumab concurrent to vaccination may actually augment HER2 
specific CTL immunity.  Recent studies, by our collaborator, Dr. Helga Bernhard, have demonstrated that 
“sensitization” of HER2 overexpressing tumor cells with trastuzumab, in vitro, will enhance the function of 
CTL specific for HER2.  Lytic activity of HER2 specific CTL derived from unprimed patients has been low, in 
part, because HER2 is a self antigen and T cells may display a low affinity T cell receptor due to induction of 
tolerance. (21-23)  On this basis, the lytic potential of HER2 specific CTL could, theoretically, be improved by 

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further increasing the number of HLA class I bound peptides on tumor cells with the help of trastuzumab, an 
inhibitory antibody against HER2.  Upon binding of trastuzumab, the HER2 receptor is internalized and 
degraded, subsequently inhibiting HER2 mediated signal transduction and tumor cell growth. (24,25)  As 
antibody-induced degradation of HER2 is likely to be accompanied with increased numbers of HER2 peptides 
presented with HLA molecules, Bernhard and colleagues questioned whether trastuzumab-treated tumor cells 
were more susceptible to CTL-mediated lysis.  HER2 reactive CTL clones lysed class I-matched, HER2 
overexpressing tumor cells more efficiently after treatment with trastuzumab (Fig. 7). (1)  Thus, combination of 
trastuzumab with the HER2 CTL vaccine may even further enhance the generation of a HER2 specific CTL 
response and potentially translate into improved survival for advanced stage breast and ovarian cancer patients 
when used in the adjuvant setting.   
 
3. Rationale 
Data from a phase I study of a HER2 CTL peptide-based vaccine demonstrated the safety of the vaccine when 
administered as a single immunotherapeutic agent.  Moreover, in addition to patients developing antigen specific 
T cell responses after vaccination, 5-year follow-up of the Stage IV breast cancer patients enrolled on study 
suggests a potential survival advantage for the patients immunized.  Recent studies have demonstrated that 
“sensitization” of HER2 overexpressing tumor cells with trastuzumab, in vitro, will enhance the function of 
CTL specific for HER2.  Thus, a combination of trastuzumab with the HER2 CTL vaccine may even further 
enhance the generation of a HER2 specific CTL response.  Before moving forward with an efficacy study, 
however, safety of concomitant administration of vaccine and trastuzumab must be demonstrated.  This proposal 
outlines a phase I-II study of a combined immunotherapeutic approach for the generation of HER2 specific CTL 
in vivo.   
 
4. Objectives 
A. Primary objectives 
1. To evaluate the safety of administering a HER2 CTL peptide-based vaccine to Stage IV breast and ovarian 
cancer patients receiving maintenance trastuzumab. 
2. To quantify and characterize antigen specific T cell subsets specific to HER2 in PBMC of patients after 
vaccination with a HER2 CTL peptide-based vaccine while receiving maintenance trastuzumab.   
 
 
B. Secondary objectives 
1.  To evaluate overall survival (OS) in patients who complete a vaccination series with a HER2 CTL peptide-
based vaccine while receiving maintenance trastuzumab. 
 
5. Vaccine Preparation 
A. HER2 CTL peptide-based vaccine  
Synthetic peptides will be used.  The peptides are 14-18 amino acids in length with an amino acid sequence 
identical to the normal HER-2/neu sequence.  These peptides have been evaluated in a previous study (see 
Background) and are covered by BB IND 6524, Sponsor: Mary L Disis, M.D.  The peptides are described 
below:   
 
Table 1.  Peptides composing the HER2 CTL vaccine 
HER2 CTL  
vaccine peptide 
Sequence 
Sequence and location of embedded 
HLA-A2 binding epitope 
p369-384 
KIFGSLAFLPESFDGDPA 
KIFGSLAFL (p369-377) 
p688-703 
RRLLQETELVEPLTPS 
RLLQETELV (p689-697) 
p971-984 
ELVSEFSRMARDPQ 
ELVSEFSRM (p971-979) 
 
B. Peptide synthesis and characterization 
Peptides are manufactured by Multiple Peptide Systems, San Diego, CA. using standard solid phase synthesis 
techniques employing Boc chemistry.  Amino acids and resins for synthesis were purchased from Bachem 
California and the amino acids were analyzed by TLC and polarimetry prior to use.  The peptides were cleaved 
from the solid support using anhydrous hydrogen fluoride and conventional techniques.  Each peptide was 

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purified by HPLC using a Waters Delta-prep system with a gradient of acetonitrile (containing 0.1% 
trifluoroacetic acid) in water (also containing 0.1% trifluoroacetic acid).  The peptides were lyophilized to 
dryness before being passed over an anion exchange column to exchange the trifluoroacetate for acetate.  GMP 
procedures were followed for the manufacture of these sequences. 
 
The peptides were characterized by mass spectrometry to confirm their identity, by HPLC for purity using two 
different elution buffers, and by amino acid analysis for identity and peptide content.  The water content of each 
peptide was determined by the Karl Fischer procedure and the data obtained used to verify the mass balance of 
the peptides.  
 
C. Peptide formulation and stability 
Prior to formulation, the solubility of each peptide in 10 mM sodium acetate buffer (pH 4.0) was determined.  
Three peptides were made up in a 10 mM sodium acetate buffer (pH 4.0).  The stability of each individual 
peptide in the acetate buffer, as well as the peptide mixtures, is monitored by HPLC and mass spectrometry.  
The dissolved peptides are stored at -20˚C prior to use.  Microbial and sterility testing are conducted to ensure 
safety. 
 
D. GM-CSF 
rhuGM-CSF (Sargramostatin, LEUKINE) will be used as the vaccine adjuvant.  GM-CSF is a growth factor that 
supports the survival, clonal expansion, and differentiation of hematopoietic progenitor cells including dendritic 
antigen presenting cells.  The use of GM-CSF in humans has been associated with minor toxicity.  GM-CSF has 
been given s.c. at the same dose and schedule without noted apparent differences in toxicities.  In some studies 
the toxicity profile for s.c. administration has been more favorable than for i.v.  For the current study GM-CSF 
will be used at a total injection dose of 100 mcg mixed prior to injection with HER2 CTL vaccine and injected 
i.d. at the time of immunization.  Local effects at the injection site are not expected with this dose of GM-CSF.  
Recombinant GM-CSF (rhuGM-CSF) is generally well tolerated when administered i.v. or s.c. in doses ranging 
from 50-500 mcg /m2/day.  Severe toxicity is extremely rare in subjects treated with rhuGM-CSF.   
 
Diarrhea, asthenia, rash, and malaise were the only events observed in more than 5% of the rhuGM-CSF group 
compared with the placebo group in phase III controlled studies of subjects undergoing autologous bone marrow 
transplantation.  In uncontrolled phase I/II studies for various indications, the adverse effects report most 
frequently were fever, chills, nausea, vomiting, asthenia, headache and pain in the bones, chest, abdomen, joints, 
or muscles.  Most of these systemic events were mild to moderate in severity and rapidly reversed by the 
administration of analgesics or antipyretics.  Other events reported infrequently were dyspnea, edema, local 
injection site reactions, and rash.  Thrombosis and cardiac arrhythmia have also been reported and there have 
been infrequent reports of tissue sloughing, leukemia progression, congestive heart failure, hepatomegaly, 
intracranial bleeding, and isolated reports of Guillain-Barre syndrome and increased histiocytes.  Neutropenia, 
although usually a pre-existing condition in the subjects receiving rhuGM-CSF, has been reported in association 
with administration.  There was an increased frequency of severe thrombocytopenia in subjects receiving 
concurrent chemotherapy and radiotherapy with rhuGM-CSF.  Eosinophilia and other blood abnormalities may 
occur.  There have been rare reports of: 1) sequestration of granulocytes in the lungs with respiratory symptoms; 
2) a syndrome characterized by respiratory distress, hypoxia, flushing, orthostatic hypotension, and partial loss of 
consciousness; 3) peripheral edema, pericardial or pleural effusions, and capillary leak of fluid; and 4) serious 
allergic or anaphylactic reactions.  
 
Administration of rhuGM-CSF may aggravate fluid retention in subjects with pre-existent edema, capillary leak 
syndrome, or pleural or pericardial effusions.  In some subjects with pre-existing renal or hepatic dysfunction, 
elevation of the serum creatinine or bilirubin and hepatic enzymes has occurred during the administration of 
rhuGM-CSF.  Dose reduction or interruption or rhuGM-CSF administration has resulted in a decrease to 
pretreatment values.  Occasional transient and reversible supraventricular arrhythmia has been reported in 
uncontrolled studies, particularly in subjects with a previous history of cardiac arrhythmia.  Stimulation of 
marrow precursors with rhuGM-CSF may result in a rapid rise in white blood cell count.  Dosing should be 

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stopped if the ANC exceeds 20,000/cm3.  rhuGM-CSF may stimulate the growth of myeloid malignancies, 
therefore caution must be exercised in its use in the malignancies or myelodysplastic syndromes.   
 
rhuGM-CSF is a sterile, white, preservative-free lyophilized powder in 250 mcg vials.  Reconstituted rhuGM-
CSF will be admixed with the vaccine product, HER2 CTL vaccine, and injected at a dose of 100 mcg (0.2 ml 
rhuGM-CSF). 
 
E.  Final vaccine preparation 
The vaccine is vialed and stored at a concentration of 1.9 mg/ml peptide in a volume of 1.1 ml/vial (2.1 mg 
total).  The peptides have been reconstituted in 10mM sodium acetate with 17.8% (v/v) DMSO.  The vaccines 
are stored at -20°C prior to use.  Prior to administration, 150 mcg of rhuGM-CSF (0.3 ml) is added to a single 
vial of the CTL vaccine bringing the total volume in the vial to 1.4 ml.  Thus, the vaccine concentration after the 
addition of adjuvant is 2.1 mg/1.4 ml.  The final dose of peptide vaccine to be delivered is 1.5 mg (500 mcg/each 
peptide) with 100 mcg GM-CSF.  The vaccine will be administered as three intradermal injections, 0.33 ml each, 
within a 5 cm radius in the same draining lymph node basin.   
 
6.  Subject Selection 
A. Inclusion criteria 
1. Subjects must have either Stage IV breast or ovarian cancer in remission or with stable disease on 
trastuzumab monotherapy.   
2. HER2 overexpression by immunohistocytochemistry (IHC) of 2+ or 3+, in the primary tumor or metastasis.  
If overexpression is 2+ by IHC, then patients must have HER2 gene amplification documented by FISH.  
3.  Subjects must be HLA-A2 positive. 
4.  Eligible subjects must have completed appropriate treatment for their primary disease and be off cytotoxic 
chemotherapy and any immunosuppressive agents such as systemic steroids for at least 30 days prior to 
enrollment.  Patients should continue trastuzumab monotherapy throughout the course of this protocol.  
Concurrent hormonal and biphosphanate therapies are allowed.   
5. Subjects must have an ECOG Performance Status Score = 0 or 1 (Appendix A)  
6. Male subjects must agree to contraceptive use during the study period (7 months) and non-menopausal 
female subjects must agree to contraception for the remainder of their childbearing years.  (HER2 is 
overexpressed in normal fetal tissues.  Therefore, induction of long-term immunity to overexpressed HER2 could 
affect the viability of a future fetus.)   
7. Subjects must have a hematocrit > 30, a platelet count > 100,000, and a WBC > 3000/µl.  Laboratory tests 
should be performed within 60 days of enrollment.   
8. Age >18 years 
9. Stable creatinine <  2.0mg/dL, or creatinine clearance > 60ml/min.  Laboratory tests should be performed 
within 60 days of enrollment. 
10. Serum bilirubin < 1.5 mg/dl.  Laboratory tests should be performed within 60 days of enrollment. 
11. SGOT < 2x ULN.  Laboratory tests should be performed within 60 days of enrollment.  
12. Subjects must have recovered from major infections and/or surgical procedures and, in the opinion of the 
investigator, not have a significant active concurrent medical illness precluding protocol treatment or survival. 
13. Patients must have a baseline LVEF measured by MUGA equal to or greater than the lower limit of normal 
for the radiology facility and if there are two consecutive MUGAS performed while on trastuzumab from the 
same radiology facility, there cannot be a decrease in LVEF of > 15% from the original MUGA scan. 
 
B. Exclusion criteria 
1. Subjects cannot be simultaneously enrolled on other treatment studies. 
2. Any contraindication to receiving GM-CSF based vaccine products. 
3. Cardiac disease, specifically restrictive cardiomyopathy, unstable angina within the last 6 months prior to 
enrollment, New York Heart Association functional class III-IV heart failure on active treatment with normalized 
LVEF on therapy, and symptomatic pericardial effusion.   
4. Active autoimmune disease.   
5.  Subjects cannot have an active immunodeficiency disorder, e.g. HIV 

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Subjects must meet all of the listed criteria in order to be eligible for study.  
 
While on study, patients will be followed for disease progression by their primary oncologist according to 
conventional practice standards.  Should patients show evidence of progression, they should receive appropriate 
treatment for their disease through their primary oncologist.  They will be removed from the study if their 
treatment conflicts with the eligibility criteria (e.g., concurrent administration of chemotherapy, immune 
modulators, steroids).   
 
7. Experimental Design 
A. Study design 
This will be a single institution, single arm phase I-II study designed to examine both the safety and the 
immunogenicity of a HER2 CTL peptide-based vaccine when administered concomitantly with trastuzumab to 
patients with Stage IV breast and ovarian cancer.  
 
B. Sample size 
The number of subjects traditionally required to gather preliminary data on the safety of a dose in a phase I 
study is less than the number needed to evaluate immunologic responses.  Since phase I tumor vaccine studies, 
to date, have proven to be relatively free of serious toxicity, the defining endpoint that would move a study 
forward to a more extensive phase II efficacy trial will likely be immunogenicity of the vaccine.  In order to 
have meaningful immunologic data we will accrue a target of 20 subjects unless there is excessive toxicity as 
defined in section 9.0 (see Statistics, Section 11).  Up to 5 additional subjects can be enrolled to replace patients 
who withdraw from study for reasons not associated with toxicity. 
 
C. Outcome measures 
Chemical and clinical parameters will be evaluated at baseline, prior to each vaccination and 1 month following 
the last vaccination to evaluate systemic toxicity (See section 8 for evaluation and management of adverse 
events and section 10 for stopping rules based on adverse events).  Toxicity grading will be evaluated according 
to NCI common toxicity criteria (http://ctep.cancer.gov/forms/CTCAEv3.pdf).   
 
All subjects will undergo immunologic response evaluation by cytokine flow cytometry (CFC) on PBMC 
derived from blood drawn (200 cc) at beginning of study, prior to vaccine # 3, 1 month, 3 months, 6 months and 
12 months following the 6th vaccination or until disease progression (as long immune response is detectable by 
CFC).  Immune response will be defined by CFC.  Specifically, a positive immune response will be defined as a 
post-vaccination antigen specific CD8 or CD4 precursor frequency measured by CFC of < 1:20,000.  For 
subjects who have measurable precursors by CFC at baseline, a positive response will be defined as a 2-fold 
increase in either antigen-specific T cell subset after vaccination.  HLA-A2 MHC tetramer analysis will be used 
to demonstrate the specificity of the T cells for the HER2 specific HLA-A2 epitopes embedded within the 
longer immunizing peptides.  IFN-gamma secreting PBMC precursor frequency will also be measured by 
ELIspot in order to determine the development of low level precursor frequencies (> 1:20,000) not detected by 
CFC.  ELIspot measurements will not be used to define response.  All patients will be vaccinated with tetanus 
toxoid at enrollment to study.  This immunization with a foreign antigen (tt) will serve as a measurable 
vaccinated response for each patient thus confirming immune competence.  
 
Overall survival will be followed and compared to historical control.  This study will not enroll sufficient 
numbers of subjects to give statistical power to this endpoint.  However if large difference are observed between 
the treatment group and historical control, it will give impetus for a sufficiently powered phase II study. 
 
8. Plan of Treatment 
The following section outlines the schedule for evaluation and treatment of subjects prior to and during the 
study.  All blood for laboratory evaluations must be drawn before each immunization.   
 

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A. Prescreen  
Potential subjects will be screened for eligibility by telephone interview with the clinical research staff member 
to insure that their tumor is HER2 positive as described in eligibility and that they are HLA A-2.  Verification 
that the potential subject meets all eligibility criteria will be made by the P.I. prior to scheduling the subject for a 
visit. 
 
B. Initial evaluation 
1. Sign consent form for the vaccine study entitled “Phase I-II Study of Combination Immunotherapy for the 
Generation of HER-2/neu (HER2) Specific Cytotoxic T Cells (CTL) in vivo” 
2. Medical history and complete physical examination.  Clinical evaluation, including vital signs, symptom 
assessment and ECOG scoring.  Pregnancy testing will be performed on female subjects with childbearing 
potential, i.e. B-HCG. 
3. Evaluation of UA (urinalysis by dipstick), CBC (complete blood count with differential and platelet count), 
serum chemistries (electrolytes, AST, ALT, alkaline phosphatase, bilirubin, creatinine and blood urea nitrogen 
(BUN)). 
4. Obtain 10 cc of peripheral blood for the evaluation of a pre-existent HER2 antibody response.  200cc 
peripheral blood to obtain PBMC for T-cell immunity studies.  
5. Tetanus toxoid immunization.   
76. Patients will have a CXR (PA and lateral) as well as an ECG to serve as baseline. (These tests will not be 
used to determine eligibility and will only be repeated should subjects develop pulmonary or cardiac medical 
problems during the course of study.) 
 
C. Monthly immunizations 
1. Post immunization monitoring: The subjects will be observed for at least 60 minutes post immunization.  
Vital signs and local reactions will be assessed by the staff before discharge and at least 60 minutes after 
vaccination.  
2. Monthly vaccination for 6 months.  Vaccinations will be administered i.d. on the upper arm.  If a subject has 
undergone complete axillary node dissection the vaccine will be administered to the contralateral side.  If the 
subject has undergone bilateral axillary node dissections, the vaccine will be administered on the anterior or 
lateral thigh.  As much as possible each dose of vaccine will be administered within the same draining lymph 
node site.   
3. UA, serum chemistry and CBC.  
4. 210 cc blood draw prior to vaccine #3 to evaluate HER2 immune responses. 
 
D. Evaluation after the final vaccine 
1. One month after the final vaccination subjects will undergo a leukapheresis.  If the patient cannot 
undergo leukapheresis for any medical reason, they will undergo a 210 cc blood draw (same as at 
baseline and pre-vaccine #3). Additionally, patients will undergo a blood draw for clinical monitoring of CBC 
(7 cc) and serum chemistries (7 cc). 
2. Three months after the final vaccine subjects will undergo a MUGA and a 210 cc blood draw to obtain 
PBMC for T cell immunity studies.   
3. Six and 12 months after the final vaccine, 210 cc of blood will be drawn for the evaluation of persistent 
antigen specific antibody and T cell immune responses. 
4.  After the final visit with us, patients may ask their regular physician to send us blood samples (about one 
cup) 3, 6 and 12 months after the final vaccine to test for the persistence of an immune response. If this 
cannot be arranged, they will need to come to the University of Washington for follow-up blood draws.  
5.  We may ask subjects to undergo leukapheresis at follow-up visits in lieu of a follow-up blood draw.  This 
will done to recover more lymphocytes for immune function analysis.  This will be determined on a case-by-
case basis by the PI. Subjects will have the option to choose or refuse leukapheresis without affecting their 
continued participation in the study. 

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E. Long term follow-up 
The patient’s primary oncologist will be sent a questionnaire regarding relapse yearly for 5 years after 
completion of study.  The median TTP for stage IV breast cancer patients receiving trastuzumab is 
approximately 25 months.  We will follow patients for OS for 5 years from enrollment.  A comparison of OS to 
historical control will be made for patients with Stage IV breast cancer.   
 
9. Evaluation and Management of Toxicity 
Subjects will be asked to report local reactions (to include pain, tenderness, erythema and induration) at the 
injection site and to report systemic reactions to include but not limited to fever, malaise, myalgia, nausea and 
headache.  Monthly evaluation for toxicity will also include: CBC, UA, serum chemistries and a limited 
physical 
examination. 
 
Toxicity 
grading 
will 
be 
evaluated 
according 
to 
the 
NCI 
criteria;  
http://ctep.cancer.gov/forms/CTCAEv3.pdf  and the Data Safety Monitoring Plan described in Appendix C.   
If a grade 3 or 4 toxicity related to the vaccination is observed in a subject, no further immunizations will be 
administered to that subject.  Grade 2 allergy will not be defined as an excessive toxicity unless the reaction has 
lasted greater than 48 hours.   
 
If the toxicity observed is related to the immune response generated, a regimen of corticosteroids will be 
administered.  The following dose schedule will be used: 
 
 
 
Day 1: 
Intravenous Solu-Medrol at 1 mg/kg 
 
Day 2: 
Intravenous Solu-Medrol at 1 mg/kg 
 
Day 3-4: 
Prednisone at 30 mg BID PO q day 
 
Day 4-5: 
Prednisone at 15 mg BID PO q day 
 
Day 5-6: 
Prednisone at 10 mg BID PO q day 
 
Day 6-7: 
Prednisone at 10 mg PO q day 
 
Day 8-9: 
Prednisone at 5 mg PO q day 
 
All subjects will be monitored closely until the toxicity resolves.  If a patient develops a toxicity that requires 
steroid treatment, no further vaccinations will be administered.   
 
10. Dose modifications 
There will not be any modifications to doses.  There may be allowances for the timing of the administration of 
vaccine to accommodate patient schedules or special circumstances.  For example, if a subject has an acute viral 
illness, the administration of their vaccination may be delayed by as much as 3 weeks.  Such variations in the 
timing of monthly vaccinations will be decided by the Principle Investigator on a case-by-case basis. Subsequent 
vaccinations will be scheduled 30 days from when the previous vaccine was actually administered, not when it 
should have been administered. 
11. Accrual and criteria for premature study termination   
Accrual will be terminated if dose limiting toxicity (DLT) is observed.  DLT is defined as a 5% incidence of 
grade 4 toxicity or a 15% incidence of grade 2 allergy >48 hours or grade 3 or worse toxicity.  Operationally, the 
5th subject enrolled will be a minimum of 1 month from their last vaccination and will be evaluated for toxicity 
with no DLT before further accrual of the next 15 patients. 
 
Stopping rules will become operational if evidence suggests that the true toxicity rate exceeds 5% Grade 4 or 
15% Grade 3 toxicity, where sufficient evidence will be taken to be observed rates of toxicity that correspond to 
one-sided 80% confidence intervals with lower limits greater than 5% or 15%, respectively.  Thus, an incidence 
of Grade 4 toxicity in 1/4 or 2/15 subjects or Grade 3 or worse toxicity in 1/1, 2/5, 3/10 or 4/15 subjects would 
constitute sufficient evidence that the toxicity rates exceed 5% Grade 4 or 15% Grade 3 toxicity.  Tables 2 and 3 
summarize the operating characteristics of these stopping rules, where the probability of stopping the study is 
estimated from 5,000 Monte Carlo simulations. 
 

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Table 2.  Probability of Stopping Trial for Grade 4 Toxicity 
Number of Patients 
True Rate of Event 
Probability of Stopping 
5 
15% 
0.39 
10 
15% 
0.55 
15 
15% 
0.72 
5 
25% 
0.61 
10 
25% 
0.81 
15 
25% 
0.93 
 
Table 3.  Probability of Stopping Trial for Grade 3 or Worse Toxicity 
Number of Patients 
True Rate of Event 
Probability of Stopping 
5 
5% 
0.02 
10 
5% 
0.03 
15 
5% 
0.03 
5 
25% 
0.37 
10 
25% 
0.54 
15 
25% 
0.64 
5 
35% 
0.56 
10 
35% 
0.77 
15 
35% 
0.87 
 
12. Evaluation of immune response  (see Appendix B) 
 
13. Statistical Considerations 
A. Study statistics 
This is a phase I study designed to evaluate (1) the toxicity of a HER2 HLA-A2 specific peptide-based vaccine 
administered concomitantly with trastuzumab and (2) the immunogenicity of HER2 HLA-A2 specific peptide-
based vaccine administered concomitantly with trastuzumab.   
 
The first objective will be achieved by assessing baseline, the type and grade of toxicities and any adverse 
events noted by the investigator.  These data will be tabulated according to the affected body system.  
Descriptive statistics will be used to summarize changes from baseline.   
 
The second objective will be achieved by evaluating the immune response to HER2 in a quantitative fashion 
based on experimental data obtained using methods described in section 12.  A significant immune response in 
an individual patient will be defined as either the development of, in patients with no measurable precursors at 
baseline, precursor frequencies > 1:20,000 as measured by CFC; or as a 2-fold increase in baseline precursor 
frequency in those patients who have a pre existent immune response.  The number of patients who achieve a 
significant immune response after vaccination will be used to assess the immunogenicity of the vaccine.  
Fourteen or more patients out of 20 would need to be successfully immunized for the vaccine to be considered 
immunogenic.  This criterion is based on our hypothesized immunization rate of at least 70%  against the HER2 
antigen, which would justify conducting a Phase II study, designed to evaluate the ability of the immune 
response to protect against recurrence of HER2 overexpressing tumor.  Table 4 shows the probability of 
observing 14 or more successes among 20 subjects for several true rates of success.  
 
 
 
 
 

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Table 4. Probability of observing at least 14 successes in 20 patients 
True rate of success 
Pr (≥14 successes) 
50% 
0.06 
60% 
0.25 
70% 
0.61 
80% 
0.91 
90% 
1.0 
 
Regarding secondary objective, OS in the Stage IV breast cancer patients will be compared to the rate in a 
cohort of historical controls.  This study was not designed to be sufficiently powered for any particular 
hypothesis testing, since time-to-event analyses generally require larger sample sizes than simple two-way 
comparisons.  However, if a large difference is observed between the treatment group and the historical controls, 
then it may give additional impetus for a larger phase II study. 
 
B. Projected gender and ethnic distribution 
The ethnic and gender distribution chart below (Table 5) reflects estimates of race and gender of the population 
to be included in this study.  These estimates are based on the following general statistics of the population in 
the Pacific Northwest and the characteristics of the diseases to be studied.  The population pool from which 
subjects will be drawn is 76% Caucasian, 11% Asian American, 5% African American, 1% American Indian or 
Alaska Native, 6.5% other and 0.6 % Native Hawaiian or other Pacific Islander.  The number of Hispanics 
available for study is 2%.  Patients with HER2 positive cancer will be eligible regardless of their ethnic or racial 
origin.  Although men with breast cancer are not excluded from this study, less than 1% of all breast cancers 
occur in men.  Ovarian cancer is found exclusively in women.  Children are also excluded from the study as the 
long term effects of immunity against a self-antigen, HER2, is unknown.  Furthermore, the incidence of breast 
and ovarian cancer in children is < 1%.   
 
Table 5.  Projected composition of patient population for Protocol 118 
TARGETED/PLANNED ENROLLMENT: Number of Subjects  
Ethnic Category  
Sex/Gender  
Females  
Males  
Total  
Hispanic or Latino  
0 
0 
 
Not Hispanic or Latino  
20 
0 
20 
Ethnic Category Total of All 
Subjects*  
20 
0 
20 
Racial Categories 
American Indian/Alaska Native  
0 
0 
 
Asian  
0 
0 
 
Native Hawaiian or Other 
Pacific Islander  
0 
0 
 
Black or African American  
1 
0 
1 
White  
19 
0 
19 
Racial Categories: Total of All 
Subjects *  
20 
0 
20 
 
14. Administrative Considerations 
A. Institutional review board 
In accordance with federal regulations, an Institutional review board that complies with the regulations in 21CFR 
56 must review and approve this protocol and the informed consent form prior to initiation of the study.  In 
addition the study cannot be instituted without FDA approval of the vaccine formulations. 
 

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B. Consent 
The investigators or their associates must explain verbally and in writing the nature, duration and purpose of the 
study and possible consequences of the treatment.  Subjects must also be informed that they may withdraw from 
the study at any time and for any reason without jeopardizing their future treatment.  In accordance with Federal 
regulations (21 CFR 312) all subjects must sign the IRB approved consent form in the presence of a witness.  
 
C. Reporting adverse events  
Adverse events will be reported using the terminology and grading scale of the NCI’s Cancer Therapy 
Evaluation Program (CTEP) Common Terminology Criteria for Adverse Events (CTCAE), version 3.0.  A copy 
of the CTCAE version 3.0 can be downloaded from the CTEP home page (http://ctep.info.nih.gov).  Guidelines 
for adverse events reporting to the FDA and the NCI are described in the DSMP, Appendix C.  The Monitoring 
Plan (as mandated by the NIH/NCI) Policy and Procedure for this phase I study is described in Appendix C.   
 
D. Specimen Handling 
Upon entry into the study each subject will be assigned a unique patient number (UPN).  All materials collected 
on that subject will be labeled with their UPN.  Lymphocytes, sera and tissue collected will be stored in a liquid 
nitrogen freezer in the laboratory of the Principal Investigator.  

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15. 
References 
1. Meyer zum Buschenfelde C, Hermann C, Schmidt B, Peschel C, Bernhard H: Anti-HER2 monoclonal 
antibody trastuzumab enhances cytolytic activity of class I restricted HER2-specific T lymphocytes against 
HER2-overexpressing tumor cells. Cancer Research 62:2244-2247, 2002 
2. Olayioye MA, Neve RM, Lane HA, Hynes NE: The ErbB signaling network: receptor heterodimerization in 
development and cancer. Embo J 19:3159-3167, 2000 
3. Samanta A, LeVea CM, Dougall WC, Qian X, Greene MI: Ligand and p185c-neu density govern receptor 
interactions and tyrosine kinase activation. Proc Natl Acad Sci U S A 91:1711-1715, 1994 
4. Stark A, Hulka BS, Joens S, Novotny D, Thor AD, Wold LE, Schell MJ, Melton LJ, 3rd, Liu ET, Conway 
K: HER-2/neu amplification in benign breast disease and the risk of subsequent breast cancer. J Clin Oncol 
18:267-274, 2000 
5. Allred D, Clark G, Molina R, Tandon A, Schnitt S, Gilchrist K, Osborne C, Tormey D, McGuire W: 
Overexpression of HER-2/neu and its relationship with other prognostic factors change during the 
progression of in situ to invasive breast cancer. Hum Pathol 23:974-979, 1992 
6. Allred DC, Mohsin SK, Fuqua SA: Histological and biological evolution of human premalignant breast 
disease. Endocr Relat Cancer 8:47-61, 2001 
7. Craft N, Shostak Y, Carey M, CL S: A mechanism for hormone-independent prostate cancer through 
modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 5:280-285, 1999 
8. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of 
relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177-182, 1987 
9. Bernhard H, Salazar L, Schiffman K, Smorlesi A, Schmidt B, Knutson KL, Disis ML: Vaccination against 
the HER-2/neu oncogenic protein. Endocr Relat Cancer 9:33-44, 2002 
10. 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 
11. Knutson KL, Schiffman K, Cheever MA, Disis ML: Immunization of cancer patients with a HER-2/neu, 
HLA-A2 peptide, p369- 377, results in short-lived peptide-specific immunity. Clin Cancer Res 8:1014-
1018, 2002 
12. 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 
13. Lehmann PV, Forsthuber T, Miller A, Sercarz EE: Spreading of T-cell autoimmunity to cryptic 
determinants of an autoantigen. Nature 358:155-157, 1992 
14. Disis ML, Gooley TA, Rinn K, Davis D, Piepkorn M, Cheever MA, Knutson KL, Schiffman K: Generation 
of T-cell immunity to the HER-2/neu protein after active immunization with HER-2/neu peptide-based 
vaccines. J Clin Oncol 20:2624-2632, 2002 
15. el-Shami K, Tirosh B, Bar-Haim E, Carmon L, Vadai E, Fridkin M, Feldman M, Eisenbach L: MHC class I-
restricted epitope spreading in the context of tumor rejection following vaccination with a single 
immunodominant CTL epitope. Eur J Immunol 29:3295-3301, 1999 
16. Vanderlugt CJ, Miller SD: Epitope spreading. Curr Opin Immunol 8:831-836, 1996 
17. Sercarz EE: Driver clones and determinant spreading. J Autoimmun 14:275-277, 2000 
18. Butterfield LH, Ribas A, Dissette VB, Amarnani SN, Vu HT, Oseguera D, Wang HJ, Elashoff RM, 
McBride WH, Mukherji B, Cochran AJ, Glaspy JA, Economou JS: Determinant spreading associated with 
clinical response in dendritic cell-based immunotherapy for malignant melanoma. Clin Cancer Res 9:998-
1008, 2003 
19. Baselga J: Herceptin alone or in combination with chemotherapy in the treatment of HER2-positive 
metastatic breast cancer: pivotal trials. Oncology 61:14-21, 2001 
20. Sparano JA: Cardiac toxicity of trastuzumab (Herceptin): implications for the design of adjuvant trials. 
Semin Oncol 28:20-27, 2001 
21. Fisk B, Blevins TL, Wharton JT, Ioannides CG: Identification of an immunodominant peptide of HER-
2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines. J Exp Med 
181:2109-2117, 1995 

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22. Linehan DC, Goedegebuure PS, Peoples GE, Rogers SO, Eberlein TJ: Tumor-specific and HLA-A2-
restricted cytolysis by tumor-associated lymphocytes in human metastatic breast cancer. J Immunol 
155:4486-4491, 1995 
23. Meyer zum Buschenfelde C, Nicklisch N, Rose-John S, Peschel C, Bernhard H: Generation of tumor-
reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells 
derived from CD34+ hemopoietic progenitor cells. J Immunol 165:4133-4140, 2000 
24. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts C, Carver ME, 
Shepard HM: Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci 
U S A 89:4285-4289, 1992 
25. Hurwitz E, Stancovski I, Sela M, Yarden Y: Suppression and promotion of tumor growth by monoclonal 
antibodies to ErbB-2 differentially correlate with cellular uptake. Proc Natl Acad Sci U S A 92:3353-3357, 
1995 

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Appendix A 
 
ECOG Performance Status Scale 
 
Performance status:  
Patients will be assessed according to the current ECOG performance scale. 
 
 
 
 
 Grade 
Scale  
 
 
 
0 
Fully active: able to carry on all pre-disease performance without 
restriction.   
 
 
1 
Restricted in physically strenuous activity but ambulatory (can walk) and 
able to carry out work of a light or sedentary (sitting) nature, e.g. light 
housework, office work. 
 
 
2 
Ambulatory and capable of self-care but unable to carry out any work 
activities.  Up and about more than 50% of waking hours.  
 
 
3 
Capable of only limited self-care: confined to bed or chair more than 50% 
of waking hours.  
 
 
4 
Completely disabled.  Cannot carry on any self-care.  Totally confined to 
bed or chair.  
 
 
 
      5 
Dead 
 
 

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Appendix B 
 
Laboratory Analysis 
 
 
 
A.  Antigens used in the immunologic assays 
 
Antigen 
Designation 
Assay 
Concentration 
No antigen 
Background control 
All 
NA 
CMV lysate 
Positive control 
CFC* 
2.5 μg/ml 
CMV HLA-A2 pp65 
peptide 
Positive control 
CFC and MHC tetramer 
10 μg/ml 
tt protein 
Positive control 
CFC 
1 μg/ml 
HER2 ICD overlapping 
peptide pools 
Experimental 
CFC 
0.7 μg/ml per peptide 
HER2 ECD overlapping 
peptide pools 
Experimental 
CFC 
0.7 μg/ml per peptide 
Individual HER2 class II 
peptides 
Experimental 
CFC 
10 μg/ml 
Individual HER2 class I 
peptides 
Experimental 
CFC and MHC tetramer 
10 μg/ml 
CEA overlapping peptide 
pool 
Experimental 
(for epitope spreading) 
CFC 
0.7 μg/ml per peptide 
CEA CAP-1 class I 
peptide 
Experimental 
(for epitope spreading) 
CFC and MHC tetramer 
10 μg/ml 
PHA 
Mitogen 
CFC 
2.5 μg/ml 
*  All CFC antigen would be operative for ELIspot analysis 
 
B.  Immunologic analysis of antigen specific T cells 
1.  Detection of HER2 specific T-cell responses by cytokine flow cytometry (CFC).  CFC is a highly quantitative 
method that will enumerate both CD4+ and CD8+ antigen specific T cells.  Either 1 ml of whole blood or 1x106 PBMC 
in 1 ml culture media (EHAA/RPMI, 10% AB serum, penicillin, streptomycin, L-glutamine) is stimulated with no 
antigen (PBS only), 1 μg/ml HER2 peptide mix in overlapping peptide pools, 10 μg individual HER2 CD4+ or CD8+ 
peptides, CMV HLA-A2 pp65 peptide, tt protein, or PHA, concentrations listed above.  2 μg/ml costimulatory 
molecules (CD28 and CD49d cocktail) and 10 mg/ml Brefeldin A are added to the reaction mixture and incubated for 6 
hours at 37°C+5%CO2.  To remove the adherent cells, 2 mM EDTA is added and incubated at room temperature for 15 
minutes.  9 mls 1X FACS lysing solution (BD Biosciences, San Jose, CA) is added and incubated at room temperature 
for 10 minutes to lyse the red cells and fix the PBMC.  The reaction mixture is stored at -80°C freezer overnight.  Cells 
are quickly thawed in a 37°C water bath, centrifuged and 2 mls of FACS permeabilizing solution 2 (BD Biosciences) is 
added.  After a 10 minute incubation in the dark, the cells are washed with 10 mls wash buffer (1X PBS/0.5% BSA).  
Cells are thoroughly resuspended in 650 μl wash buffer and 100 μl aliquoted to 6 microcentrifuge tubes.  Antibodies 
(examples) are added as follows: 
 
 
 
Tube #1:  CD3 cychrome/CD8 FITC/IFN-gamma PE 
 
 
Tube #2:  CD3 cychrome/CD4 FITC/ IFN-gamma PE 
 
 
Tube #3:  CD3 cychrome/CD69 FITC/ IFN-gamma PE 
 
 
 
 
Tube #4:  CD3 cychrome/CD8 FITC/IL-4 PE 
 
 
Tube #5:  CD3 cychrome/CD4 FITC/IL-4 PE 
 
 
Tube #6:  CD3 cychrome/CD69 FITC/IL-4 PE 

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After a 30 minute incubation at room temperature in the dark, the cells are washed twice with wash buffer.  The 
cells are resuspended in 300 μl 1% paraformaldehyde and then analyzed using three color flow cytometry.  % 
responding cells are calculated counting a minimum of 50,000 events (CD8+ or CD4+ T cells).  Antigen specific 
CD8+ or CD4+ IFNγ or IL-4 secreting cells will be reported as % responding cells for a particular population.  A 
precursor frequency based on the total number of CD3+ cells can be calculated from this value.   
 
2.  Detection of HER2 specific T-cell responses by ELIspot.  Briefly, on day 1, PBMC are plated into 96-well 
plates in 6-well replicates in 200 μl of RPMI-1640 containing L-glutamine, penicillin, streptomycin and 10% AB 
serum (T cell medium) in the presence or absence of 10 μg/ml peptide antigen or 1μg/ml TT.  The cells are 
incubated at 370C+5% CO2.  On day 2 (48 hours), 2.5 x 105/well irradiated autologous PBMC and 10 μg/ml 
antigens are added.  Also on day 2, nitrocellulose-backed 96-well plates (NC-plate) are coated with 10 μg/ml 
anti-IFN-gamma antibody in PBS at 50 μl/well.  On day 3 the NC-plate is washed 3X with PBS and blocked for 2 
hours with PBS containing 2% bovine serum albumin followed by 3 washes with PBS.  On day 3, the cells are 
gently re-suspended, pooled, centrifuged, and the media is replaced.  The cells are transferred into the NC-plate 
in a volume of 100 μl/well in T-cell medium.  The NC-plate is incubated at 37oC for a further 20-24 hours 
followed by washing 3X using PBS containing 0.05% Tween-20.  The plate is then incubated for 2.5 hours at 
room temperature in 50 μl/well PBS containing 5μg/ml biotinylated anti-IFN-gamma antibody, washed 3X with 
PBS, and further incubated with 100 μl/well streptavidin-alkaline phosphatase at a dilution of 1:1000 in PBS for 
2 hours at room temperature.  After washing 3X in PBS, the plate is incubated with 100 μl/well AP-colorimetric 
substrate for 20-30 minutes, rinsed with cool tap water, and allowed to completely dry.  Resultant spots are then 
enumerated using a dissecting microscope.  Precursor frequencies are calculated by subtracting the mean number 
of spots obtained from the no antigen control wells from the mean number obtained in the experimental wells.  
Statistical analysis is performed using the Student’s t test (Microsoft Excel 97).  A positive response is defined as 
a precursor frequency that was both significantly (p<0.05) greater than the mean of control no antigen wells and 
detectable (i.e. >1:100,000). 
 
3.  Detection of HLA-A2 restricted HER2 specific responses with MHC tetramers.  200 μl of whole blood is 
mixed with 10 μl HLA-A201 CMV pp65PE/anti-CD8 FITC (Beckman Coulter, San Diego, CA) or HER2 peptide 
specific MHC tetramer (Beckman Coulter, San Diego, CA) and incubated at room temperature for 30 minutes in 
the dark.  1 ml of OptiLyse C (Beckman Coulter) is added and incubated for 10 minutes to lyse the red cells.  1 ml 
of 1X PBS is added and incubated for another 5 minutes.  2 mls 1X PBS is added and samples centrifuged.  Cell 
pellet is resuspended in 500 μl 1% paraformaldehyde.  The labeled cells are analyzed by flow cytometry and at 
least 500,000 events gated on lymphocytes are collected.  
 
C.  Immunologic detection of antigen specific antibodies 
1.  Quantitative ELISA for the detection of HER2 protein specific antibody responses.  96 well Immulon 4 plates 
(Baxter SP, Redmond, WA: Dynex Technologies) are incubated overnight at 4°C with a polyclonal affinity 
purified antibody to HER2 (kindly supplied by Dr. D. Ring) at a concentration of 1µg antibody per ml.  Wells set 
up as a standard curve are incubated with purified human IgG (Sigma BioSciences, St. Louis, MO) in serial 
dilutions ranging from 0.03µg/well to 0.0002µg/well.  After incubation, all wells are blocked with PBS and 1% 
bovine serum albumin (BSA) (Sigma Chemical Co.), 100µl/well for 4 hours at room temperature.  The plate is 
washed with PBS/0.1% Tween and lysates of SKBR3, a human breast cancer cell line which overexpresses the 
HER2 protein, are added in alternating rows with PBS/1% BSA and incubated overnight at 4°C.  After washing, 
the plate is incubated with human sera at the following dilutions: 1:25, 1:50, 1:100, 1:200.  The sera is diluted in 
PBS/1% BSA/1% FBS/25 µg/ml mouse IgG/0.01% NaN3 and then serially into PBS/1% BSA.  5µl of diluted 
sera is added per well and incubated 1 hour at room temperature.  Goat anti-human IgG (gamma) horseradish 
peroxidase (HRP) (Zymed, So. San Francisco, CA) is added to the wells at a 1:50,000 dilution in PBS/1% BSA 
and incubated for 45 minutes at room temperature (Amersham Co., Arlington Heights, IL).  Following the final 
wash, TMB (Kirkegaard and Perry Laboratories, Gaithersburg, MD) developing reagent is added.  Color reaction 
is read at an optical density of 650 nm until the standard curve well containing 0.04 µg reaches 0.3 OD.  The 
reaction is stopped with 1N HCl and the optical density read at 450nm.  The OD of each serum dilution was 
calculated as the OD of the HER2 coated wells minus the OD of the PBS/1% BSA coated wells.  A normal 
human serum is run on each plate as a negative control.  Values are calculated in µg/ml based on the standard 

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curve.  The final value expressed is the mean and standard deviation of quadruplicate dilutions.  All positive 
responses will be evaluated by Western Blot.  A population of 150 volunteer blood donors is run to establish the 
baseline of the assay.  Random normal blood donors (n=10) are run every 100 plates, or quarterly, whichever 
comes first, and the standard curve and antibody values compared to mean and standard deviations of the 
experimental runs.  Validation runs will be tabulated and discussed by the IMC and a quality assurance report 
filed with the data manager.   
 
2.  Quantitative ELISA for the detection of HER2 antibody avidity:  96 well Immulon 4 plates (Baxter SP, 
Redmond, WA: Dynex Technologies) are incubated overnight at 4°C with a polyclonal affinity purified antibody 
to HER2 (kindly supplied by Dr. D. Ring) at a concentration of 1µg antibody per ml.  Wells set up as a standard 
curve are incubated with purified human IgG (Sigma BioSciences, St. Louis, MO) in serial dilutions ranging from 
0.03µg/well to 0.0002µg/well.  After incubation, all wells are blocked with PBS and 1% bovine serum albumin 
(BSA) (Sigma Chemical Co.), 10µl/well for 4 hours at room temperature.  The plate is washed with PBS/0.1% 
Tween and lysates of SKBR3, a human breast cancer cell line which overexpresses the HER2 protein, are added 
in alternating rows with PBS/1% BSA and incubated overnight at 4°C.  After washing, the plate is incubated with 
human sera at the following dilutions: 1:25, 1:50, 1:100, 1:200.  The sera is diluted in PBS/1% BSA/1% FBS/25 
µg/ml mouse IgG/0.01% NaN3 and then serially into PBS/1%BSA.  5µl of diluted sera is added per well in 
duplicate and incubated 4 hours at room temperature.  The plate is washed and incubated with 200 μl/well 8M 
Urea for 3 minutes.  The plate is washed and goat anti-human IgG (gamma) horseradish peroxidase (HRP) 
(Zymed, So. San Francisco, CA) is added to the wells at a 1:50,000 dilution in PBS/1% BSA and incubated for 45 
minutes at room temperature (Amersham Co., Arlington Heights, IL).  Following the final wash, TMB 
(Kirkegaard and Perry Laboratories, Gaithersburg, MD) developing reagent is added.  Color reaction is read at an 
optical density of 650 nm until the standard curve well containing 0.04 µg reaches 0.3 OD.  The reaction is 
stopped with 1N HCl and the optical density read at 450nm.  The OD of each serum dilution was calculated as the 
OD of the HER2 coated wells minus the OD of the PBS/1% BSA coated wells.  Avidity Index is expressed as a 
percentage, where absorbance reading after urea wash is divided with absorbance reading without urea wash 
multiplied by 100. 
 
3.  Quantitative ELISA for the detection of tetanus toxoid antibodies and antibody avidity:  Patients have been 
immunized with a DT at enrollment.  96 well Immulon 4 plates (Dynatech Laboratories) are incubated with tt 
protein (Lederle Labs) at a concentration of 1:1000 diluted in carbonate buffer alternating with rows of PBS/1% 
BSA.  Wells set up as a standard curve are incubated with purified human IgG (Sigma BioSciences, St. Louis, 
MO) in serial dilutions ranging from 0.0217 μg/ml to 2.78 μg/ml overnight at 4°C.  After incubation, all wells 
were blocked with PBS and 1% bovine serum albumin (BSA) (Sigma Chemical Co.), 100 µl/well for 1 hour at 
room temperature.  The protein-coated plate is incubated with experimental sera diluted 1:400, 1:800, 1:1600 and 
1:3200 in duplicate for 2 hours at room temperature.  One set of duplicate is treated with 8M Urea for 5 minutes. 
Goat anti-human IgG (gamma) horseradish peroxidase (HRP) (Zymed, So. San Francisco, CA) are added to the 
wells at a 1:5,000 dilution in PBS/1% BSA and incubated for 45 minutes at room temperature (Amersham Co., 
Arlington Heights, IL).  Following the final wash, TMB (Kirkegaard and Perry Laboratories, Gaithersburg, MD) 
developing reagent is added.  Color reaction is read at an optical density of 650nm until the standard curve well 
containing 0.174 µg/ml reaches 0.3 OD.  More dilute sera will be used if values are at the top or off of the 
standard curve.  The reaction is stopped with 1N HCl and the optical density read at 450nm.  The OD of each 
serum dilution is calculated as the OD of the protein coated wells minus the OD of the PBS/1% BSA coated 
wells.  Values are calculated in µg/ml based on the standard curve.  To calculate the % avidity index, the 
absorbance reading after urea wash is divided with absorbance reading without urea wash and multiplied by 100.  
Random normal blood donors (n=10) are run every 100 plates, or quarterly, whichever comes first, and the 
standard curve and antibody values compared to mean and standard deviations of the experimental runs.  
Validation runs will be tabulated and discussed by the IMC and a quality assurance report filed with the data 
manager. 
 
4.  Quantitative ELISA for the detection of CMV antibodies and antibody avidity:  96 well Immulon 4 plates 
(Dynatech Laboratories) are incubated with CMV lysate (Virusys) at a concentration of 7.5 μg/ml diluted in 
carbonate alternating with rows of PBS/1% BSA.  Wells set up as a standard curve are incubated with purified 

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human IgG (Sigma BioSciences, St. Louis, MO) in serial dilutions ranging from 2.9 to 0.023 μg/ml.  After 
overnight at 4°C, the plate is washed and all wells were blocked with PBS and 1% bovine serum albumin (BSA) 
(Sigma Chemical Co.), 100 µl/well for 1 hour at room temperature.  The protein-coated plate is incubated with 
100 μl sera diluted 1:800, 1:1600, 1:3200 and 1:6400 tested in duplicate for 2 hours at room temperature.  One set 
of duplicate is treated with 8M Urea for 5 minutes.  Goat anti-human IgG (gamma) horseradish peroxidase (HRP) 
(Zymed, So. San Francisco, CA) are added to the wells at a 1:5,000 dilution in PBS/1% BSA and incubated for 45 
minutes at room temperature (Amersham Co., Arlington Heights, IL).  Following the final wash, TMB 
(Kirkegaard and Perry Laboratories, Gaithersburg, MD) developing reagent is added.  Color reaction is read at an 
optical density of 650nm until the standard curve well containing 0.3 µg/ml reaches 0.3 OD.  More dilute sera will 
be used if values are at the top or off of the standard curve.  The reaction is stopped with 1N HCl and the optical 
density read at 450nm.  Values are calculated in µg/ml based on the standard curve.  The values are reported using 
the 1:800 dilution (no urea treatment) with the assay cut-off value of 200 μg/ml.  Results between 100 to 200 
μg/ml will need to be confirmed with western blot.  To calculate the % avidity index, the absorbance reading after 
urea wash is divided with absorbance reading without urea wash and multiplied by 100.  Random normal blood 
donors (n=10) are run every 100 plates, or quarterly, whichever comes first, and the standard curve and antibody 
values compared to mean and standard deviations of the experimental runs.  Validation runs will be tabulated and 
discussed by the IMC and a quality assurance report filed with the data manager.   
 
 
 

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1 
Appendix C 
 
Data and Safety Monitoring Plan (DSMP) 
 
Phase I-II Study of Combination Immunotherapy for the Generation of HER-2/neu (HER2) Specific 
Cytotoxic T Cells (CTL) in vivo 
 
A. Purpose 
To ensure that the Tumor Vaccine Group clinical research staff (Table 1) follow NIH/NCI/CTEP 
guidelines with respect to (1) accurate assessment and timely reporting of adverse drug reactions 
associated with investigational drugs (2) adherence to protocol and (3) accurate reporting of data.  
The DSMP will be attached to all protocols as an appendix and be approved by Human Subjects 
Division Institutional Review Board (IRB) at the University of Washington, Seattle, WA as part of the 
IRB protocol approval process. 
 
The Principal Investigator is responsible for every aspect of the design, conduct and final analysis of 
the protocol.  Regulations defining the responsibilities for assessment and reporting of all adverse 
events (AE), serious AE and unexpected AE are defined by the Code of Federal Regulations:  21 CFR 
312.32 and Common Terminology Criteria for Adverse Events (CTCAE) Version 3.0 published June 10, 
2003 by the Cancer Therapy Evaluation Program (CTEP), a division of the NCI/NIH. A matrix of 
reporting requirements and schedules is in the CTEP web-site at http://ctep.infi.nih/gov. 
 
This clinical study will rely upon the monitoring of the trial by the Principal investigator in conjunction 
with a Research Nurse, a Statistician, and an Independent Study Monitor assigned by the Research 
Trial Office of the University of Washington/Fred Hutchinson Cancer Research Center Cancer 
Consortium.  If an adverse event occurs the Principal Investigator will be notified along with the IRB 
and appropriate federal agencies: FDA, NCI/CTEP and the Clinical Data Update System and/or the 
Adverse Event Expedited Reporting System, (AdEERS).  
  
B. Objectives 
1. To ensure that the Principal Investigator and clinical research staff follow federal and institutional 
regulatory guidelines with respect to timely reporting of adverse reactions associated with investigational drugs.  
2. To provide instructions for completion of adverse events reporting forms as follows:  a) MED WATCH 
FDA form 3500A, or b) Adverse Reaction (ADR) Form for Investigational Agents, or c) the Adverse Event 
Expedited Report for 24 hour phone/fax reporting of adverse events.  
3. To define classification of adverse drug reactions as expected or unexpected. 
4. To define classification of adverse drug reactions as serious or non-serious. 
5. To ensure compliance and accuracy of documentation of adverse drug reactions reportable to the 
FDA/NIH/CTEP. 
6. To ensure compliance and accuracy of reporting and documentation of adverse drug reactions reportable to 
the NCI by the Clinical Data Update System (CDUS, for NCI-funded trials). 
 
C. Adverse Event Reporting Policy and Procedures 
1. Evaluation of adverse events (Table 2): Patients are monitored for the development of end organ damage by 
assessing adverse events with serum chemistries, liver function studies, complete blood counts, urine analysis 
and physical exams performed every month until 30 days after the final vaccine administration.  All adverse 
events for all systems are graded on a scale of 1-5 and attribution is assigned, using the NCI Common 
Terminology Criteria.  If an adverse event occurs during more than one cycle of treatment, then only the most 
severe adverse event is reported. All information pertaining to toxicity is recorded in the source documents.  
2. Definitions of adverse events: 

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a. Adverse Event - any unfavorable and unintended sign (including abnormal laboratory finding), 
symptom or disease temporally associated with the use of a medical treatment or procedure regardless of 
whether it is considered related to the medical treatment or procedure. 
b. Expected Adverse Event - an event that may be reasonably anticipated to occur as a result of the study 
procedure.  
c. Unexpected Adverse Event - any adverse event that is unanticipated.  An event that might have been 
anticipated but is more serious than expected or occurs more frequently than expected, would be considered 
an unexpected adverse event. 
d. Serious Adverse Event - grade 4 or 5 toxicity or any adverse event occurring that results in any of the 
following outcomes: death, a life threatening adverse event, inpatient hospitalization or prolongation of 
existing hospitalization due to the adverse event, a persistent or significant disability/incapacity or a 
congenital anomaly/birth defect. 
e. Life-threatening Adverse Event - the patient was at substantial risk of dying at the time of the adverse 
event or it is suspected that the use or continued use of the product would have resulted in the patient’s 
death. 
3. Scale of scoring adverse events  
a. Grade 1 = mild adverse event 
b. Grade 2 = moderate adverse event 
c. Grade 3 = severe adverse event 
d. Grade 4 = life-threatening or disabling adverse event 
e. Grade 5 = death related to adverse event. 
4. Attribution of adverse event 
a. 5 (definite): the adverse event is clearly related to the investigational agent   
b. 4 (probable): the adverse event is likely related to the investigational agent 
c. 3 (possible): the adverse event may be related to the investigational agent 
d. 2 (unlikely): the adverse event is doubtfully related to the investigational agent 
e. 1 (unrelated): the adverse event is clearly NOT related to the investigational agent. 
5. Adverse event reporting is outlined below by regulatory agency (Table 1): 
 
University of Washington Human Subjects Division - expected adverse events are routinely reported as part of 
the annual renewal.  Unexpected or more serious than expected adverse events are reportable by phone within 
24 hours and followed in writing with a completed Human Subjects Adverse Effect Report 
 
FDA (for trials using an Investigational New Drug (IND) - any unexpected, fatal or immediately life-threatening 
reaction must be reported to the FDA (Center for Biologics) by telephone or facsimile transmission (using 
Medwatch Form 3500A) as soon as possible but no later than seven calendar days after initial receipt of the 
information concerning the event.  All unexpected, serious adverse events are reported in writing (using 
Medwatch Form 3500A) to the FDA (Center for Biologics) within 15 calendar days after initial receipt of the 
information concerning the event.  Yearly written progress reports to the FDA describe all expected or non-
serious unexpected adverse events. 
 
NCI (for NCI-sponsored trials only) - adverse reactions that are expected and grade 1-3 or unexpected and grade 
1 are reportable in a routine report.  Adverse reactions that are unexpected and grade 2 are reportable in writing, 
using the Adverse Event Expedited Report (AdEERS) within 10 days to the NCI for NCI-sponsored trials, via 
the Clinical Trials Evaluation Program (CTEP).  Adverse reactions that are expected and grade 4 or 5, or 
unexpected and grade 3, 4 or 5 are reportable by phone/fax, using the Adverse Event Expedited Report 
(AdEERS) within 24 hours.  Temporary or permanent suspension of an NCI funded clinical trial is to be 
reported to the NCI grant program director responsible for the grant. 
 
6. Procedure for reporting adverse events: 

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3 
a. Identify the classification/attribution of the adverse event as defined above using the Modified Common 
Terminology Criteria for Adverse Events version 3.0 (CTCAE v. 3.0). 
b. After appropriate medical intervention has been instituted, the Principal Investigator or his/her designee 
will be notified within 24 hours. 
c. File appropriate reports immediately by phone/fax with appropriate agencies, as described above. 
d. Notify the patient’s primary physician or referring physician within a medically appropriate timeframe, 
depending on the classification of the adverse event. 
e. Submit written reports to appropriate agencies. 
f. Document the adverse event in the patient’s chart, using a progress note to describe the event and 
treatment, if appropriate.   
g. File copies of all forms/correspondence relating to the adverse event in the patient’s chart. 
 
D.  Clinical Trials Monitoring Operational Procedures (Table 3) 
1. Clinical Data Documentation 
a. Monthly monitoring: 
All patients actively enrolled in the study are seen monthly.  Clinical labs are evaluated each month for the 
development of toxicity (adverse events) related to the vaccine for up to 30 days following the last vaccine.  
Results are recorded on a clinical lab flow sheet that shows lab values over the course of the study.  This 
document becomes part of the patient chart at the end of the study.  Any abnormal lab values are faxed to the 
patient’s physician and followed with a letter.  The Research M.D. or the P.I. sees each study participant 
monthly and the following evaluations completed: toxicity evaluation, physical assessment and adverse events 
summary (these evaluations are part of the source document that is filled out at each visit).  Grade 1 and 2 non-
serious and expected adverse events will be reviewed with the P.I. at a weekly clinical trials review meeting.  
All other adverse events will be reported to the Principal Investigator at the time they become known and 
reported on further as outlined above in Section C. 
 
Each patient chart is audited for completeness one month after enrollment.  Each chart is again audited for 
completeness within one month of the final follow-up visit (30 days after the final vaccination).  This audit will 
be conducted by the Research nurse every 3 months and the results will be presented to the Principal 
Investigator at weekly clinical meetings.  The Research Nurse’s chart audit record form is located in the front of 
each patient chart.  Charts are audited for completeness, legibility, and accuracy.  All source documents must be 
filled out completely.  The Research Nurse will review all charts for subject recruitment and retention, protocol 
adherence, follow-up, data quality, and participant risk versus benefit.  Additionally, all correspondence with 
primary physicians and patients is maintained in the patient chart.  Copies of email communications and 
telephone logs of verbal communications are maintained.  
 
b. Biannual study audit:  
An Independent Study Monitor will be assigned by the Research Trials Office of the FHCRC/University of 
Washington’s Division of Oncology and an independent study audit will be performed two times a year 
(approximately every 6 months).  This audit will be conducted on all patients who have been enrolled on study 
at the time of the audit.  Charts will be formally audited for completeness, accuracy and compliance to protocol.  
10% of all computer data entry will also be reviewed.  
 
2. 
Data validity/integrity 
Systems to insure data integrity have been put into place to provide multiple checks to data entry.  Subject 
eligibility is reviewed by the Research Nurse at screening, by the Research M.D. at enrollment, by the P.I. at a 
weekly meeting, quarterly by the Research Nurse chart audit, and biannually by the Independent Study Monitor.  
Source documents are initially reviewed by the Research Nurse monthly and quarterly for completeness and 
reviewed by the Independent Study Monitor biannually.  All clinical laboratory monitoring data is reviewed in 
real time by the Research M.D. and any abnormalities are assessed.  Any abnormalities are reported to the P.I. at 
weekly meetings.  All subject’s records are kept in the research file and reviewed at quarterly and biannual 

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audits.  All toxicity scoring is reviewed with P.I. at weekly meetings and end of study toxicity scoring is done by 
the research nurse, reviewed by the P.I. and audited by the Independent Study Monitor and at biannual meetings 
as described above.  All research data is checked by the P.I. and reviewed with the technologist or staff scientist 
who generated the data.  Data is taken from the subject’s source documents and entered into a data base which 
links data to subject by a Unique Patient Identifier and is accessible by password code only.  Data entry is made 
by the Study Coordinator and is verified at end of study by the Research Nurse and the P.I. by reviewing all 
source documents and reconciling them to the study database.  10% of all data entry is reviewed by the 
Independent Study Monitor.  At the time of study reports or publications print outs of all data from the data base 
are reviewed by the P.I. 
 
 
 
Table 1.  Protocol 118 Clinical Trials Personnel 
Name 
Role 
Mary L. (Nora) Disis, M.D. 
Principal Investigator 
Lupe Salazar, M.D. 
Research M.D. 
Devon Webster, MD 
Senior Fellow 
Doreen Higgins, BSN 
Research Nurse 
Katherine Guthrie, Ph.D. 
Statistician 
Corazon de la Rosa, BSMT 
Research Lab Manager 
Independent Study Monitor 
for Bi-annual Audits 
 
 
Assigned by Research Trials 
Office FHCRC/University 
of Washington/ Division of 
Oncology 
Jennifer S. Childs, MPH 
Research Coordinator 
Patricia Fintak, MA 
Research Coordinator 
 
 
 
 
Table 2.  Adverse Event Reporting 
 
Regulatory 
Agency 
Expected 
Non-serious  
(Grade 1-2) 
Expected 
Serious  
(Grade 3-5) 
Unexpected 
Non-Serious 
Unexpected 
Serious/Fatal 
UW-IRB 
Annual renewal 
24 hours 
Annual renewal 
24 hours 
FDA 
Yearly progress report 
15 days 
Yearly progress 
report 
ASAP within 7 days-
Medwatch Form 
3500  
NCI/CTEP 
Routine report 
24 hours by 
phone/fax 
(Grade2) In writing 
< 10 days 
24 hours by 
phone/fax-ADR 
Form 
Study Monitor 
Routine report 
24 hours by 
Routine report 
24 hours by 

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phone/fax 
phone/fax 
NOTE: The death of any patient on a clinical trial is considered Serious Adverse Event, regardless of attribution 
and is required to be reported to the IRB, FDA and study sponsor. 
 

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Table 3.  Operational Procedures for Protocol 118 
Study Event 
Specific Action 
Performed By 
Screen 
Telephone screening of interested 
subjects 
Study Coordinator/Research Nurse 
 
Screening checklist 
Study Coordinator/Research Nurse 
Eligibility 
Initial review w/interested subject 
Study Coordinator/Research Nurse 
 
Obtain source documents to 
determine eligibility 
Study Coordinator/Research Nurse 
 
2º review of eligibility 
MD/PI 
 
Weekly meeting w/PI to report 
study status/evaluate eligibility  
Study Coordinator/Research Nurse 
MD 
 
Quarterly evaluation of subject 
eligibility 
 
 
Biannual review  
PI 
MD 
Research Nurse 
Study Coordinator 
Independent Study Monitor 
Consent 
Obtain Informed Consent 
MD/PI 
Monthly Visits 
Blood draw  
Research Nurse 
 
Physical assessment/toxicity 
evaluation  
MD/PI 
 
Vaccinate 
Research Nurse 
 
Monitor for immediate reactions 
Research Nurse 
Source Document 
 
Filled out by Study Coordinator, 
Research Nurse, MD or P.I. 
 
Demographic Data Entry 
Study Coordinator 
 
Monthly review of source 
documents for completeness 
Research Nurse 
 
Quarterly Chart Review 
Research Nurse  
 
Bi-annual audit for data-safety 
monitoring 
Independent study monitor 
Toxicity Monitoring 
Monthly monitoring of adverse 
events with patient at each visit 
Review of non serious and grade 2 
expected adverse events weekly 
MD 
 
P.I. 
 
Quarterly review of adverse events 
Biannual review of documentation 
of adverse events 
Research Nurse/PI 
Independent Study Monitor 
Report of serious (grade 3, 4 or 5)  
or unexpected grade 2 adverse 
event 
See sections B and C of DSMP, 
page 1-4 
Research Nurse and  P.I. 
 
Immunologic Monitoring- 
Research data 
Performed at specific timepoints for 
each subject 
Research Lab Manager & 
Technologist 
 
Review of immunologic data 
PI & MD 
 
Computer entry of immunologic 
data 
Study Coordinator 
Study review with P.I. 
 
 
 
 
Weekly meeting to report study 
status/evaluate eligibility, subject 
recruitment and retention, protocol 
adherence, and adverse event 
evaluations (all grade 1 and 
MD 
Research Nurse  
Study Coordinator 

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Study Event 
Specific Action 
Performed By 
 
 
 
 
expected grade 2). Daily on an as 
needed basis for all unexpected 
grade 2, or grade 3-5 adverse 
events. 
Twice yearly chart audits 
Bi-annual evaluation of subject 
recruitment and retention, protocol 
adherence, adverse events and 
participant risk versus benefit. 
Validation of computer data entry 
from the source document. 
Independent Study Monitor  
Assurance of Data Accuracy and 
Integrity 
Validation of computer data entry 
for clinical data 
Research Nurse 
 
 
Validation of data entry for 
immunologic monitoring-research 
data 
Independent Study Monitor 
P.I. 
Annual reports to:  
IRB 
 
 
Study Coordinator 
FDA 
NCI 
 
P.I.  
P.I 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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8 
Appendix D 
 
Supplemental HER2 CTL Peptide-Based Booster Vaccine Study  
1.  INTRODUCTION 
Vaccinating women with HER2 overexpressed breast cancer against HER2-specific antigens has been shown to 
improve both overall survival in metastatic breast cancer (MBC) and disease free survival (DFS) in women with 
locally advanced non metastatic disease.1,2  Furthermore, vaccination has been shown to both stimulate an 
immune response against the vaccine antigen as well epitope spreading to other antigens within the protein 
(intermolecular spreading) and other tumor-related proteins (intramolecular spreading).1,2  However, immunity 
to the vaccines may wane as patients are greater than 6 months to a year from vaccination.  This loss of 
immunity may explain progressive disease in patients with previous immune control.  To improve prolonged 
immune response, booster vaccinations have been evaluated to re-stimulate HER2-specific immunity after 
completion of a primary vaccination series.  Specifically, using GMCSF and the E75 vaccine, a single booster 
vaccination greater than 6 months after completing the vaccination series was demonstrated to be safe and 
improved immune response.3  We therefore seek to evaluate the immunogenicity of a series of 2 booster 
vaccinations given 6 months apart in patients who have received at least one vaccination with the HER2 
cytotoxic T lymphocyte (CTL) peptide-based vaccine and have no evidence of disease, stable disease, or slowly 
progressive disease.  
Twenty-two HLA-A2 HER2 overexpressing stage IV patients were enrolled in the NCT00194714 trial of six 
HER2 CTL peptide-based vaccines along with concurrent trastuzumab.  Twenty-one patients received 
vaccinations: Fourteen of 21 patients received all six immunizations, five of the patients completed at least three 
immunizations, and two of the patients only completed two immunizations.  The vaccine series was well 
tolerated and had an acceptable safety profile with 99% grade 1 or 2 toxicities, four grade 3 toxicities which 
included injection site reaction, infection, ulceration and fainting.  There was one unrelated grade 4 event of a 
stroke.  Furthermore, the vaccine series was immunogenic with 90% of the patients developing new or increased 
HER2-specific IFN-g immunity as well as epitope spreading to other tumor associated proteins.  The immunized 
patients also had disease response with a median progression free survival (PFS) of 17.7 months at 3 years (with 
33% of patients showing no evidence of disease progression) and median overall survival of 86% at 4 years.  
The immunity induced by the vaccine was followed for up to a year after vaccination in 11 patients.  Forty-six 
percent of patients maintained the same level of immunity, 36% had increased immunity, and 18% had lowered 
immunity after a year.1  In long term follow up, 10 patients are currently living 10 years after completion of the 
initial vaccine series despite having HER2+ MBC.  As an example, one of the patients had 7 years of DFS on 
single agent trastuzumab after the vaccine series and continues to have considerable disease control despite 
slowly progressive disease while remaining on single agent trastuzumab.  These patients’ long disease 
stabilization beyond expected average DFS of 12 months with trastuzumab alone suggests that the immunity 
acquired from vaccination may have contributed to disease stabilization.4,5  We therefore want to evaluate if 
providing additional boosters will possibly increase the anti-HER2 immunity of these previously vaccinated 
patients.  Additionally, we will monitor for safety of the approach.  
Although most cancer vaccine studies have focused on the generation of measurable immunity during active 
immunization, few studies have evaluated the persistence of immunity or the generation of immunologic 
memory.  The development of immunologic memory has been shown to be essential.  Indeed, it is the 
development of immunologic memory to the vaccinating antigen that would be the key to the success of a tumor 
vaccine to protect against relapse.  In this booster study, we will evaluate the persistence of HER2 peptide-based 
specific immunity and also the presence and magnitude of a memory T-cell response after “booster” 
immunization with a HER2 peptide-based vaccine in previously immunized patients. 
 
2.  BACKGROUND 
Immunologic memory, a hallmark of the protective immune response after immunization, is the ability to make 
a faster, stronger, and more effective, immune response to an antigen that has been encountered previously.6  

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Memory T-cells are clones of antigen-specific lymphocytes that are able to be maintained in homeostasis for a 
long time and reactivate when stimulated by a second encounter with an antigen.6  The goal of vaccines, whether 
against infectious or cancer targets, is to develop these memory cells to allow for continued immune 
surveillance.  Antigen-specific immunity can wane over time and booster vaccines can be used to reactivate the 
T-cells, increase memory, and trigger a more robust immune response.  In infectious disease, with each antigen 
challenge (either an infection or booster vaccination) there is a larger increase in effector CD8+ T-cells and a 
higher baseline memory T-cell response.7  Therefore booster cancer vaccines would be expected to also give a 
second antigen challenge increasing both anti-tumor immune response and specific T-cell memory.  Most breast 
cancer vaccine trials have evaluated the immunity that is developed immediately after primary vaccination.8,9  
However, long term follow up from these vaccine trials suggest that this immunity wanes and booster 
vaccinations may reactivate the anti-HER2 immune response and the memory T-cell population.  
A.  HER2 directed immunotherapy is safe when administered concurrently with HER2 CTL peptide 
based vaccination.  As discussed above, patients enrolled to the original HER2 vaccine study were receiving 
concurrent trastuzumab and the combination immunotherapy was well tolerated with no significant cardiac 
toxicity observed.  Over the past 2 years, the first-line treatment of HER2+ MBC has been changing and now 
also includes other HER2 targeted agents.  In 06/2012, the FDA approved the combination of pertuzumab and 
trastuzumab with chemotherapy for the treatment of HER2+ MBC.  This was based on data from the phase III 
CLEOPATRA 
study, 
which 
showed 
improved 
PFS 
with 
the 
combination 
of 
docetaxel 
and 
pertuzumab/trastuzumab versus docetaxel + placebo/trastuzumab.10  Moreover, extensive cardiac surveillance 
conducted in CLEOPATRA did not show increased cardiotoxicity (increased rate of left-ventricular systolic 
dysfunction) in patients receiving concomitant pertuzumab and trastuzumab when compared to concomitant 
placebo and trastuzumab.  As such, it is possible that patients enrolled in the original HER2 peptide vaccine 
study and who are now interested in the booster vaccine study may be receiving maintenance pertuzumab and 
trastuzumab.  Given the acceptable toxicity data of the combined HER2 targeted therapy, we do not anticipate 
any increased cardiac toxicity if pertuzumab and trastuzumab are given concurrently with HER2 peptide booster 
vaccination.  Therefore concurrent use of either trastuzumab or pertuzmab/trastuzumab during booster 
vaccination will be allowed. 
B.  T-cell populations essential for immune efficacy after vaccination.  T-cells go through three stages in 
response to antigen, acute expansion in the lymph node when exposed to antigen, contraction after an acute 
inflammatory response where the antigen-specific T-cell populations decrease, and then development of 
memory T-cells in case of any further exposure to antigen.  Naïve T-cells localized in the lymph node are either 
CD4+ or CD8+ T-cells and, on association with antigen presented in association with MHC class I or II 
receptors, are able to clonally expand into effector T-cells (either CD4+ helper T-cells or CD8+ cytotoxic T-
cells).6  How these T-cells clonally expand is controlled by the cytokine milieu, immune environment, and 
secondary signals from the antigen presenting cell.11,12  After antigen clearance, the number of antigen specific 
CD4+ and CD8+ T-cells rapidly decline believed to be due to decreasing pro-inflammatory cytokines and 
decreased antigen stimulation.6  After contraction of the antigen-specific T-cell populations, a small population 
of memory T-cells remain and these T-cells are called central memory T-cells which can persist for decades 
despite the average lifespan of single memory cells being days to weeks, suggesting that these T-cells are self-
renewing and can persist without antigen present.7  The factors that determine if a cell becomes an effector or a 
memory T-cell remain unclear.  Central memory cells home to lymphoid tissue where they proliferate 
vigorously on re-encountering antigen and produce cytotoxic function slowly.  Effector memory cells on the 
other hand proliferate less well, but produce cytokines rapidly and enter non-lymphoid tissue providing 
immediate protection with re-activation from memory.6  With a second exposure to antigen, either through 
booster vaccination or a second infection, the secondary memory response produces higher numbers of memory 
T-cells.7  Therefore providing booster vaccines should reactivate an acute HER2-specific anti-tumor immune 
response against disease that is present and provide further anti-tumor epitope spreading as well as increase the 
HER2 specific memory T-cell population against further tumor development.  
C.  Rationale for booster vaccination in patients immunized with HER2 CTL peptide.  Booster 
vaccinations have been examined previously in cancer vaccines and a single booster vaccination has the 

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potential to mount an immune response as great if not greater than initial vaccination.13  The booster vaccination 
can also widen the spectrum of CD4+ and CD8+ T-cells against protein specific epitopes.  A booster vaccine in 
HER2+ breast cancer patients has previously been shown to be safe and immunogenic using the HER2-specific 
E75 vaccine along with GMCSF adjuvant.  The peptide-based HER2 vaccine E75 with GMCSF adjuvant was 
given as a single booster in 53 patients who had previously received the E75 vaccine by various schedules in 
previous clinical trials but had not received vaccination for over 6 months.  The investigators found that residual 
CD8+ T-cell immunity existed in 94.4% of patients at 6 months after vaccination but at greater than 6 months 
after vaccination only 48 of patients had residual immunity (p=0.002).  Furthermore, the median residual 
immunity remaining was very low at 0.70% (range 0 to 3.49%).  The booster vaccine was well tolerated with 
only grade 1 and 2 toxicity primarily local injection site reactions and 85% of the patients without E75 specific 
immunity had achieved increased immunity after the booster vaccination.3  We propose to evaluate HER2 
specific immunity and improved memory T-cell response generated using two boosters vaccinations of the 
HER2 peptide vaccine.  Our group has completed a trial using 2 boosters with a DNA vaccine IGFBP2 in 8 
ovarian cancer patients and these boosters were found to be safe and their immunity is currently being evaluated.  
We therefore seek to evaluate a similar booster series with the HER2 peptide vaccine.    
D.  Immune and metabolic activity measured by fluorodeoxyglucose positron emission tomography 
(FDG-PET) imaging.  Changes in SUV quantitated by FDG-PET are a functional measure that is usually 
associated with disease progression, particularly when evaluating breast cancer bone metastases.  In a study of 
28 breast cancer patients with bone dominant disease, higher tumor FDG SUVs predicted a shorter time to 
disease progression (p<0.006).14  However there is an emerging role for PET imaging to evaluate patients for 
immunologic side effects caused by monoclonal checkpoint inhibitor therapy and for measuring immune flare 
after vaccination.15,16  FLT-PET imaging of KLH antigen loaded dendritic cell (DC) vaccines in 3 patients with 
metastatic melanoma to the lymph node showed that only antigen loaded dendritic cells increased PET avidity in 
draining lymph nodes and remained positive up to 3 weeks after vaccination (Aarntzen PNAS 2011).  In another 
study, FDG-PET scans done within 7 days after receiving the 6th vaccine of a DC vaccination series showed 
increased metabolic responses of index lesions (even though new lesions appeared simultaneously) and 
improved survival, indicative of an overall antitumor effect of DC vaccines.17  The authors concluded that it is 
possible that infiltration of inflammatory cells might lead to pseudo-progression in SUV; and thus, PET scans 
may provide a measure to evaluate the activity of immunotherapies over time.  In our experience in a patient 
with HER2-specific adoptive T-cells, a temporal increase in tumor FDG uptake at sites of metastases over a 48 
hour period that resolved by 1 month follow up was associated with significant disease stabilization of 18 
months.  This suggests that acute increases in tumor metabolism as measured by FDG-PET may identify 
inflammation induced by activated T-cells and could potentially indicate a defined level of inflammation needed 
to mediate disease regression in HER2 adoptive T-cell studies (Stanton submitted).  Clinical symptoms of tumor 
flare, such as pain at metastatic sites, after immune modulation have been associated with beneficial clinical 
responses.  After adoptive T-cell therapy for nasopharyngeal carcinoma or breast cancer pain at sites of disease 
and increase in tumor size temporally related to T-cell infusion was correlated with clinical response in specific 
lesions.18,19  Therefore, we will compare baseline FDG-PET imaging prior to the second booster to FDG-PET 
imaging at 48 hours after the second booster vaccine to evaluate if there is any evidence of acute increase in 
tumor metabolism suggestive of activated T-cell infiltration and increased inflammation.   
 
3.  OBJECTIVES 
A. Primary 
1. Evaluate whether HER2 peptide-based booster vaccines can enhance HER2-specific immunity. 
2. Evaluate whether HER2 peptide-based booster vaccines induce increase in memory T-cell populations in 
patients’ peripheral blood. 
3. Evaluate whether HER2 peptide-based booster vaccinations demonstrate acute inflammatory response in 
lymph nodes draining the vaccine site, and in tumor metastasis, by FDG-PET. 
 
 

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B. Secondary 
1. Evaluate the safety of HER2 peptide-based booster vaccines in patients who previously received the HER2 
peptide-based vaccines. 
 
4.  PATIENT SELECTION 
A.  Inclusion criteria 
1. Patients must have received at least 1 vaccination of the HER2 CTL peptide-based vaccine. 
2. Patients must have Stage IV breast cancer with either no evidence of disease, disease that is stable, or slowly 
progressive disease.  
3. Patients taking HER2-targeted monoclonal antibody therapy may remain on the treatment. 
a. HER2-targeted monoclonal antibody therapy is defined as either trastuzumab monotherapy, or 
trastuzumab and pertuzumab combination therapy administered per standard of care. 
4. Patients must be at least 28 days post cytotoxic chemotherapy prior to first vaccine 
5. Patients on bisphosphonates and/or endocrine therapy are eligible and may remain on therapy.   
6. Patients must be at least 28 days post immunosuppressants prior to first vaccine. 
7. Patients must have an ECOG Performance Status Score ≤ 1 (Appendix A of original protocol).  
8. Non-menopausal female subjects who are having sex that can lead to pregnancy must agree to contraception 
for the remainder of their childbearing years.   
9. Laboratory values must be as follows and performed within 60 days of enrollment: 
a. WBC ≥ 1500/mm3 
b. Hgb ≥ 8 g/dl 
c. Platelet count ≥ 75,000/mm3 
d. Serum creatinine ≤ 2.0 mg/dl or creatinine clearance > 60 ml/min 
e. Total bilirubin ≤ 2.5 mg/dl 
f. SGOT ≤ 2.0 times the upper limit of normal  
10. Patients must have recovered from major infections and/or surgical procedures. 
11. Patients receiving a HER2 targeted monoclonal antibody must have adequate cardiac function, as 
demonstrated by normal left ventricular ejection fraction (LVEF) ≥ the lower limit of normal for the facility 
on MUGA scan or echocardiogram (ECHO) within 6 months of enrollment as per standard of care.   
B.  Exclusion criteria 
1. Patients cannot be simultaneously enrolled on other treatment studies. 
2. Any contraindication to receiving GMCSF based vaccine products. 
3. Patients with any of the following cardiac conditions: 
a. Symptomatic restrictive cardiomyopathy 
b. Unstable angina within 4 months prior to enrollment 
c. New York Heart Association functional class III-IV heart failure on active treatment 
d. Symptomatic pericardial effusion 
4. Patients with any clinically significant autoimmune disease uncontrolled with treatment. 
 
 
 

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5.  EXPERIMENTAL DESIGN 
A.  Study design 
This is a supplemental booster vaccine to Protocol 118 (IR# 6304):  Phase I-II Study of Combination 
Immunotherapy for the Generation of HER-2/neu (HER2) Specific Cytotoxic T-Cells (CTL) in vivo. 
B.  Sample size 
Up to 10 patients can be enrolled.  This sample size is not derived based on statistical considerations, but rather 
on the number of potentially eligible patients that we feel can be accrued. 
C.  Booster vaccines 
Patients will receive one HER2 CTL peptide-based vaccine every six months for two vaccines. 
D.  FDG-PET scans 
Patients will undergo FDG-PET prior to their second booster and approximately 48 hours post the second 
booster vaccine to assess for inflammatory changes in lymph nodes draining the vaccine site and any metastatic 
sites. 
E.  Outcome measures 
Primary 
1. Evaluation of whether the HER2 peptide-based boosters enhance HER2-specific immunity through IFN-g 
ELISPOT will be determined 6 months after first and second booster vaccines.  Increased HER2-specific 
immunity will be defined by generating a HER2 protein specific IFN-g precursor augmented over 2 times 
baseline. 
2. Evaluation of whether there is increased memory T-cell populations in the peripheral blood by flow 
cytometry will be evaluated 48 hours after each of the two HER2 peptide-based booster vaccinations.  The 
peripheral memory T-cell populations will be evaluated on PBMC using a flow cytometry panel developed in 
our immune monitoring laboratory.  We will be evaluating CD45R0, CD62, CCR7, CD3, CD4, and CD8 
antibodies for evaluation of memory T-cells. 
3. Evaluation of HER2-specific memory through development of a delayed type hypersensitivity (DTH) 
response at the vaccination site will be determined 48 hours after each booster vaccine.  A positive response 
will be defined as ≥ 10 mm2 induration at the vaccination site.  
4. Evaluation of immune modulation by FDG-PET will be defined as acute inflammatory response evidenced by 
acute increase in FDG avidity in the tumor and/or draining lymph nodes 48 hours after the 2nd booster 
vaccination as compared to baseline FDG-PET prior to the second vaccine. 
Secondary 
1. Safety and systemic toxicity will be determined by chemical and clinical parameters evaluated at various time 
points as described in Section 6.  Toxicity grading will be evaluated per the CTEP CTCAE v3.0 and 
monitoring of adverse events will be done per FDA and NCI guidelines.  CTCAE v.3.0 criteria was used to 
evaluate the original data. 
 
 
 
 
 
 
 
 
 
 
 
 
 

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6. PLAN OF TREATMENT 
The following section outlines the schedule for evaluation and treatment of patients during the study.   
A.  First Visit-Booster Vaccination #1 
1. Sign consent form for the “Supplemental HER2 CTL Peptide-Based Booster Vaccine Study”. 
2. Verification that patient meets eligibility criteria. 
3. Medical history and complete physical examination. 
a. Clinical evaluation, including vital signs and weight, symptom assessment and ECOG scoring. 
4. CBC (complete blood count with differential and platelet count), serum chemistries (electrolytes, AST, 
ALT, alkaline phosphatase, bilirubin, creatinine and blood urea nitrogen (BUN)). 
5. Obtain up to 110 mls peripheral blood for baseline research studies.  
6. Patients of child bearing potential must have a negative urine pregnancy test. 
7. Vaccination will be administered i.d. (intradermal) at the site of previous primary vaccine series. 
8. Post immunization monitoring: The patients will be observed for at least 60 minutes post immunization.  
Vital signs will be done at least 60 minutes post vaccination.   
B. 48 hours after Booster Vaccination #1 
1. Delayed type hypersensitivity (DTH) will be evaluated at the injection site approximately 48 hours (+24 
hours) after the booster vaccine and the DTH response (i.e. induration and erythema) measured in mm2. 
2. Peripheral blood draw to evaluate HER2 specific immunity and T-cell memory (100 mls blood). 
C.  Booster Vaccination #2 (Six months after Booster Vaccination #1) 
1. Complete physical examination. 
a. Clinical evaluation, including vital signs and weight, symptom assessment and ECOG scoring. 
2. Patients of child bearing potential must have a negative urine pregnancy test. 
3. CBC (complete blood count with differential and platelet count), serum chemistries (electrolytes, AST, 
ALT, alkaline phosphatase, bilirubin, creatinine and blood urea nitrogen (BUN)). 
4. Obtain up to 110 mls peripheral blood for research studies, prior to second booster vaccination. 
5. A FDG-PET scan will be performed prior to the second booster vaccination. 
6. Vaccination will be administered i.d. at the site of previous vaccination. 
7. Post immunization monitoring: The patients will be observed for at least 60 minutes post immunization.  
Vital signs will be done at least 60 minutes post vaccination. 
D. 48 hours after Booster Vaccination #2 
1. Delayed type hypersensitivity (DTH) will be evaluated at the injection site approximately 48 hours (+24 
hours) after the booster vaccine and the DTH response (i.e. induration and erythema) measured in mm2. 
2. Peripheral blood draw to evaluate HER2 specific immunity and T-cell memory (100 mls blood). 
3. A repeat FDG-PET scan will be performed to evaluate inflammatory changes in draining lymph nodes. 
E. Six months after Booster Vaccination #2  
1. Peripheral blood draws to evaluate HER2-specific immunity (by ELISPOT) and T-cell memory (210 mls 
blood). 
F. Long Term Follow-up 
A request for records will be sent to the patient’s primary oncologist twice yearly from the last booster vaccine 
for a total of 5 years.   
This information will be put in the study chart which is kept in a locked filing cabinet in a secured building.  
Study charts are kept for 30 years after the close of the study per University of Washington policy.  Information 
requested will include: 
a. 
Most recent laboratory evaluation (CBC, CMP)  

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b. 
Patient’s disease free and overall survival status 
c. 
Recent clinical notes which includes history, imaging reports, and physical exam notes 
 
Notes will be reviewed by study team for vaccine toxicity and followed up with the patient’s primary physician 
if indicated. 
We will look for possible autoimmune symptoms (e.g. unexplained rash, dry eyes, unexplained diarrhea) within 
the above collected records. 
 
7. EVALUATION AND MANAGEMENT OF TOXICITY 
Patients must be off all immunosuppressive treatments such as chemotherapy or systemic steroid therapy a 
minimum of 28 days prior to each booster vaccination.  Evaluation for toxicity will include a physical 
examination and laboratory evaluation as described in Section 5 (Plan of Treatment.).   
 
8. STATISTICS 
This study is descriptive and the statistics will be based on changes from baseline at the various time points as 
described in Section 5.   

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9. REFERENCES 
 
1. 
Disis ML, Wallace DR, Gooley TA, et al: Concurrent trastuzumab and HER2/neu-specific vaccination 
in patients with metastatic breast cancer. J Clin Oncol 27:4685-92, 2009 
2. 
Peoples GE, Gurney JM, Hueman MT, et al: Clinical trial results of a HER2/neu (E75) vaccine to 
prevent recurrence in high-risk breast cancer patients. J Clin Oncol 23:7536-45, 2005 
3. 
Holmes JP, Clifton GT, Patil R, et al: Use of booster inoculations to sustain the clinical effect of an 
adjuvant breast cancer vaccine: from US Military Cancer Institute Clinical Trials Group Study I-01 and 
I-02. Cancer 117:463-71, 2011 
4. 
Jackisch C: HER-2-positive metastatic breast cancer: optimizing trastuzumab-based therapy. Oncologist 
11 Suppl 1:34-41, 2006 
5. 
Schaller G, Bangemann N, Becker C, et al: Therapy of metastatic breast cancer with humanized 
antibodies against the HER2 receptor protein. J Cancer Res Clin Oncol 125:520-4, 1999 
6. 
Kaech SM, Wherry EJ, Ahmed R: Effector and memory T-cell differentiation: implications for vaccine 
development. Nat Rev Immunol 2:251-62, 2002 
7. 
Harty JT, Badovinac VP: Shaping and reshaping CD8+ T-cell memory. Nat Rev Immunol 8:107-19, 
2008 
8. 
Disis ML, Gooley TA, Rinn K, et al: Generation of T-cell immunity to the HER-2/neu protein after 
active immunization with HER-2/neu peptide-based vaccines. J Clin Oncol 20:2624-32, 2002 
9. 
Mittendorf EA, Clifton GT, Holmes JP, et al: Clinical trial results of the HER-2/neu (E75) vaccine to 
prevent breast cancer recurrence in high-risk patients: from US Military Cancer Institute Clinical Trials 
Group Study I-01 and I-02. Cancer 118:2594-602, 2012 
10. 
Swain SM, Kim SB, Cortes J, et al: Pertuzumab, trastuzumab, and docetaxel for HER2-positive 
metastatic breast cancer (CLEOPATRA study): overall survival results from a randomised, double-
blind, placebo-controlled, phase 3 study. Lancet Oncol 14:461-71, 2013 
11. 
Kambayashi T, Laufer TM: Atypical MHC class II-expressing antigen-presenting cells: can anything 
replace a dendritic cell? Nat Rev Immunol 14:719-30, 2014 
12. 
De Koker S, Lambrecht BN, Willart MA, et al: Designing polymeric particles for antigen delivery. 
Chem Soc Rev 40:320-39, 2011 
13. 
Atanackovic D, Altorki NK, Cao Y, et al: Booster vaccination of cancer patients with MAGE-A3 
protein reveals long-term immunological memory or tolerance depending on priming. Proc Natl Acad 
Sci U S A 105:1650-5, 2008 
14. 
Specht JM, Tam SL, Kurland BF, et al: Serial 2-[18F] fluoro-2-deoxy-D-glucose positron emission 
tomography (FDG-PET) to monitor treatment of bone-dominant metastatic breast cancer predicts time 
to progression (TTP). Breast Cancer Res Treat 105:87-94, 2007 
15. 
Koo PJ, Klingensmith WC, Lewis KD, et al: Anti-CTLA4 antibody therapy related complications on 
FDG PET/CT. Clin Nucl Med 39:e93-6, 2014 
16. 
Aarntzen EH, Srinivas M, Punt CJ, et al: Insight into the dynamics, localization and magnitude of 
antigen-specific immune responses by [(18)F]FLT PET imaging. Oncoimmunology 1:744-745, 2012 
17. 
Engell-Noerregaard L, Hendel HW, Johannesen HH, et al: FDG PET scans as evaluation of clinical 
response to dendritic cell vaccination in patients with malignant melanoma. Cancer Immunol 
Immunother 62:17-25, 2013 
18. 
Comoli P, Pedrazzoli P, Maccario R, et al: Cell therapy of stage IV nasopharyngeal carcinoma with 
autologous Epstein-Barr virus-targeted cytotoxic T lymphocytes. J Clin Oncol 23:8942-9, 2005 
19. 
Disis ML, Dang Y, Coveler AL, et al: HER-2/neu vaccine-primed autologous T-cell infusions for the 
treatment of advanced stage HER-2/neu expressing cancers. Cancer Immunol Immunother 63:101-9, 
2014 
 

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SCHEDULE OF EVENTS 
 
 
 
Visit Time Point 
Procedures 
Booster 1 
• 
Sign consent 
• 
Medical history and complete physical examination 
• 
Vitals signs-including weight 
• 
ECOG scoring 
• 
Pregnancy urine dip – if applicable 
• 
Clinical labs: complete blood counts, serum chemistries 
• 
Research blood:  approximately 110 mls 
• 
Symptom assessment 
• 
Booster vaccine #1 
• 
Post-immunization monitoring 
 
Forty-eight hours 
after booster #1 
• 
DTH measurement of vaccine site 
• 
Research blood: approximately 100 mls 
Booster 2: 
Six months post first 
booster vaccine 
• 
FDG-PET scan prior to vaccination 
• 
Complete physical examination 
• 
ECOG scoring 
• 
Pregnancy urine dip – if applicable 
• 
Vitals signs-including weight 
• 
Clinical labs: complete blood counts, serum chemistries 
• 
Research blood prior to vaccination: approximately 110 mls 
• 
Symptom/toxicity assessment 
• 
Booster vaccine #2 
• 
Post-immunization monitoring 
 
Forty-eight hours 
after booster #2 
• 
DTH evaluation 
• 
Research blood: approximately 100 mls 
• 
FDG PET Scan 
Six months after 
booster #2 
• 
Research blood:  approximately 210 mls 
Long-Term Follow-
Up for 5 years 
• 
Every 6 months for 5 years 
• 
Medical documentation request of your oncologist 
• 
During the consent process, and at subsequent visits, patients will be instructed to hydrate 
sufficiently prior to visits requiring large volume blood draws.