Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence for Patients With HER-2 Driven High Risk Invasive Breast Cancer
Phase: PHASE1
Conditions: Breast Cancer
Sponsor: H. Lee Moffitt Cancer Center and Research Institute

 
 
Multisite Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence for 
Patients with HER-2 Driven High Risk Invasive Breast Cancer 
 
NCT02063724 
 
Version 2.0 
March 1, 2017 

Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence for Patients with HER-2 Driven High Risk Invasive Breast Cancer 
Version v2 03/01/2017 
 
 
 
 
Multisite Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence 
for Patients with HER-2 Driven High Risk Invasive Breast Cancer 
Co-Principal Investigator 
 
 
Co-Principal Investigator 
Brian J Czerniecki, MD, PhD, Moffitt Cancer Center 
 
 
Investigators 
 
 
 
 
 
 
 
 
Cardiology 
 
 
 
Radiology 
 
 
 
Pathology 
 
 
Biostatistics 
 
 
Director Vaccine Preparation 
 
 

Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence for Patients with HER-2 Driven High Risk Invasive Breast Cancer 
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Table of Contents 
1.0 PURPOSE .............................................................................................................................................................. 1 
2.0 DURATION OF STUDY ...................................................................................................................................... 1 
3.0 SUBJECT RECRUITMENT ................................................................................................................................ 1 
4.0 LOCATION OF THE TRIAL .............................................................................................................................. 1 
5.0 BACKGROUND .................................................................................................................................................... 2 
5.1 THE PROBLEM ................................................................................................................................................... 2 
5.2 THE SOLUTION................................................................................................................................................... 3 
5.3 PRELIMINARY DATA .......................................................................................................................................... 3 
6.0 RESEARCH DESIGN ....................................................................................................................................... 10 
6.1 IDENTIFICATION OF SUBJECTS ........................................................................................................................... 10 
6.2 PRE-ENROLLMENT SCREENING .......................................................................................................................... 11 
6.3 CONSENT PROCESS ........................................................................................................................................... 12 
6.4 PREPARATION OF THE VACCINE ........................................................................................................................ 12 
6.4.1 Subject pheresis ......................................................................................................................................... 12 
6.4.2 Elutriation .................................................................................................................................................. 13 
6.4.3. Cell culture and preparation of dendritic cell vaccine (Table 1) ............................................................. 13 
6.4.4 Quality assurance of DC1 .......................................................................................................................... 16 
6.4.5 Pharmacologic Information and Preparation of Reagents ...................................................................... 17 
6.4.6 Administration of the Vaccine ................................................................................................................... 18 
6.5 POST-VACCINE PROCEDURES ........................................................................................................................... 23 
6.6 CLINICAL MONITORING .................................................................................................................................... 24 
6.7 TOXICITY AND ADVERSE EVENT REPORTING ................................................................................................... 27 
6.8 IMMUNOLOGIC TESTING ................................................................................................................................... 30 
6.10 DATA STORAGE ................................................................................................................................................ 34 
7.0 POTENTIAL RISKS ........................................................................................................................................... 34 
8.0 CONSENT PROCEDURE .................................................................................................................................. 35 
9.0 PROTECTION OF SUBJECTS ......................................................................................................................... 35 
10.0 POTENTIAL BENEFITS ................................................................................................................................. 36 
11.0 EXCEPTIONS AND DEVIATIONS ............................................................................................................... 36 
12.0 RISK/BENEFIT RATIO ................................................................................................................................... 36 
13.0 REFERENCES .................................................................................................................................................. 37 

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1.0 Purpose 
 
We have demonstrated that administration of HER-2/neu pulsed DC1 in patients with DCIS and 
early invasive breast cancer (IBC) results in strong immunologic responses as well as apparent 
clinical responses during 4 and 6 week neoadjuvant vaccination. Patients with locally advanced 
breast cancer that have significant nodal disease have significant risk of systemic recurrence. In 
addition, those patients with recurrence after chemotherapy and trastuzumab have significant risk 
of additional local or systemic recurrence and may be resistant to trastuzumab. Those patients 
with HER-2 2+ that are FISH negative that are often ER positive with significant nodal disease 
are also at high risk for recurrence despite chemotherapy and anti-estrogen therapy. These 
patients are not classically treated with trastuzumab but may still respond to HER-2 directed 
therapy but also have diminished immunity to HER-2. All these patients have little additional 
therapy options. We hypothesize that adding DC1 vaccines pulsed with HER-2, will reduce the 
risk of recurrence and diminish deaths in these high risk patients. The primary goals of this trial 
will be to determine the safety and immune activity of HER-2 pulsed DC1 vaccine in patients 
with high risk HER-2pos breast cancer with significant risk of systemic recurrence. We will also 
explore the possibility of determining whether circulating tumor cells can be used as surrogate to 
assess response to vaccination. 
 
2.0 Duration of Study 
 
This study will enroll 18 evaluable subjects over 1.5 years averaging 10 subjects per year. 
Evaluable implies that a patient received all 6 vaccinations and underwent leukapheresis after the 
vaccinations for immune response evaluation. 
 
Fifteen subjects were enrolled at the Rowan Breast Center. Approximately 4 subjects will receive 
their treatment at follow up at Moffitt Cancer Center while remaining subjects will continue to be 
followed at Rowan Breast Center.  
 
 
3.0 Subject Recruitment 
 
Women with advanced breast cancer with ≥ 4 or more positive nodes with classic HER-2pos or 
HER-2 2+ FISH negative, or those with local or systemic recurrence that are rendered NED by 
any means will be recruited from the clinical practices of the Rena Rowan Breast Center at the 
Hospital of the University of Pennsylvania and the Breast Center Moffitt Cancer Center. 
 
 
4.0 Location of the Trial 
 
The subjects will be recruited and followed in the Rowan Breast Center and the Moffitt Cancer 
Center. Mammograms will be performed at either site. Leukapheresis will be performed in the 
Apheresis Infusion Blood Donation Center on the 3rd Floor of Ravdin and at Moffitt Cancer 
Center.  Elutriation will be performed in the FDA approved facility in the Blood Bank on 3 
Ravdin and at Moffitt Cancer Center.  The vaccines will either be prepared in the FDA approved 
facility, under the direction of 
, at the Abramson Family Cancer Research 
Institute on 6 Maloney, or the Vaccine Production Facility in the M2GEN building on the 
McKinley campus at Moffitt Cancer Center under the direction of 
 Vaccinations 

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will be administered in the Clinical Research Center 1 Dulles Building at Penn or at Moffitt 
Cancer Center.. 

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5.0 Background 
 
5.1 The Problem 
 
Most breast cancer patients, especially those with high-risk HER-2 positive or triple negative breast 
cancers, live with significant fear of recurrence-even when initial therapy appears  successful.  
Patients with positive lymph nodes or patients with locally advanced HER-2 positive or triple 
negative cancer treated with neoadjuvant therapy that have residual disease found at surgery, both 
have significant risk of systemic recurrence5. Tragically, there is little to offer these patients in the 
way of recurrence risk reduction that does not come with significant toxicity or other deleterious side- 
effects. Even those patients with intermediate expression of HER-2 (2+) are at significant risk for late 
recurrence6. In addition, solid evidence now suggests HER-2 expressing stem cells and disseminated 
tumor cells can be found in the marrow and metastatic deposits in patients whose primary tumor does 
not express significant HER-27. Even amongst HER-2 negative patients EGFR (HER-1) or HER-3 
pathways are active and phosphorylated HER-2 proteins are identified8. These numerous patient 
populations are therefore in great need of new therapies to prevent outgrowth of distant disease, 
provided physicians and scientists can develop technologies to eliminate these remaining dormant 
cancer stem cells that will ultimately reactivate, leading to significant mortality. 
 
Role of Receptor Tyrosine Kinases in Breast Development and Tumorigenesis 
To explain why these molecules are important in breast cancer development is to understand their  
role in normal breast development. These RTK molecules are critically involved in multiple levels of 
breast development 9. HER-1 and HER-2 are expressed in early breast bud development especially 
Her-1 (EGFR) which is required for duct elongation at puberty. HER-2 and HER-3 play particularly 
important roles in duct elongation and branching during pregnancy and invasion of mammary pads. 
Hence they are critically involved in normal breast development and lactation. Mutation or assault 
from carcinogens may cause these molecules to at later time become over-expressed or aberrantly 
expressed in breast tumor tissue. The expression of these molecules could be a particularly critical 
site for interactions with viruses, other pathogens, and chronic inflammation causing damage or 
mutation and over- expression of the HER members. There are examples of identified mutations in 
HER-1, HER-2 and HER-3 in all phenotypes of breast cancer 10, 11. 
 
Several murine models have been identified where over-expression of HER family members leads to 
invasive breast cancer development in mature mice12. Over-expression of these molecules appears to 
be sufficient to cause breast cancer development. Evidence is fairly abundant in human tissues that 
these molecules are expressed in early human breast cancers. We have demonstrated that HER-2 
expression in DCIS is associated with finding invasive disease suggesting HER-2 may be progression 
factor 13, 14. HER-3 is also expressed in DCIS and we have found that its expression increases with 
grade of DCIS. HER-1 is least expressed in early breast cancer except in triple negative DCIS and 
few other instances. HER-1 is expressed in about 30% of the DCIS lesions in BRCA1 mutation 
carriers going along with its expression in triple negative breast cancer. Taken together these HER 
family members seem to play an important role in normal breast development and when mutated or 
over-expressed play a dominant role in early breast cancer development. 
 
Immune Response Impacts Breast Cancer 

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Evidence has demonstrated that infiltration of breast tumors with CD8 T cells results in prolonged 
survival15. There is also evidence that accumulation of CD4 and CD8 T cells in regional nodes is 
associated with better outcomes as well16. Counteracting this there is an accumulation of 
macrophages, immature myeloid suppressor cells and regulatory T cells that inhibit the immune 
response and promote growth and invasion of tumor cells 17, 18. Chemotherapies such as 
cyclophosphamide and docetaxol may eliminate some of the inhibitory cells enhancing immune 
responses19. There is evidence that combinations of chemotherapy and immune therapies with 
antibodies may increase responses such as seen with trastuzumab20. Adding immune therapies 
especially through vaccines with chemotherapy may enhance the immune response as the inhibitory 
cells are diminished thus filling the immune space with tumor specific lymphocytes. We will take 
advantage of chemotherapy to open space in the immune repertoire and vaccinating to add specific 
anti-HER T cells to eliminate residual microscopic tumor deposits. 
 
5.2 The Solution 
 
The HER family of RTK provide critical signaling for development of breast cancer. HER-1 (EGFR) 
HER-2 and HER-3 have all been shown critical in normal breast development as well as breast 
tumorigenesis and invasion 8. These molecules are over-expressed or mutated in a significant fraction 
of early and late breast cancer lesions 13, 21. These are particularly dominant in HER- 2 and triple 
negative breast cancers 22.There is evidence that therapeutic antibodies like trastuzumab and 
pertuzumab, combined with chemotherapy, can have dramatic impacts in eliminating these primary 
tumors 23. However, there is also evidence of the molecules being involved in resistance to  treatments 
24. Patients with locally advanced HER-2/neu and triple-negative cancers are at significant risk of 
systemic recurrence, especially when complete pathologic response is not obtained with neoadjuvant 
chemotherapy. We believe that targeting these pathways with a cellular immune response  
(particularly CD4 T cells) may prevent or eliminate dormant tumor cells remaining after therapy. Our 
preliminary data outlined below highlights an opportunity to potentially increase complete responses 
and help prevent recurrence in these patients with significant risk of systemic failure following 
standard therapy. Dendritic cell cancer vaccines combined with chemotherapy may increase complete 
responses giving breast cancer specific immune cells greater opportunity to function while the  
immune  repertoire  is  being  shifted  by chemotherapy to  anti-breast  cancer  response  and  offer the 
chance to test secondary prevention of breast cancer in high risk settings. There is a need to determine 
whether this ICAIT DC1 can activate CD4 and CD8 T cells prior to or in combination with 
chemotherapy with or without added trastuzumab. This would be a tremendous opportunity to  
improve outcomes for these high risk patients. 
 
5.3 Preliminary Data 

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Experience with the vaccine to prevent recurrence: DC1 induces durable Th1-polarized anti- 
HER-2 responses with apparent clinical activity. A large body of novel and well-founded 
preclinical and clinical data justifies and supports our expandable platform for breast cancer 
prevention. We have completed a phase-I clinical trial with 27 subjects to vaccinate against HER- 
2/neu over-expressing early breast cancer (Ductal carcinoma in situ-DCIS) or DCIS with 
microinvasion3,4, and  are currently undertaking  a  second  phase  I/II study to  extend  these findings. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subjects diagnosed with HER-2/neupos DCIS were vaccinated four times at weekly intervals prior to 
scheduled surgical resection of the tumor. The vaccines consisted of HER-2 peptide-pulsed  
autologous DC specially activated with IFN-γ and a clinical-grade toll-like receptor agonist (LPS), 
resulting in high-level IL-12 and Th1 chemokine secretion ( DC1). The peptides used to pulse ICAIT 
DC consisted of 6 HLA class II promiscuous binders, as well as two HLA class I binding peptides (for 
HLA-A2.1pos subjects). A summary of trial results is shown in the composite Figure 1 A-G. Briefly, 
Immunohistochemical staining and comparison of pre-vaccine biopsy with post-vaccine surgical 
specimen showed that vaccination induced heavy influx of CD4pos helper-T cells around DCIS (Fig. 
1A), as well as CD8pos CTL and CD20 B cell (not shown). Immunohistochemical staining of HER- 
2/neu expression by DCIS in the pre-vaccine biopsy (“PRE”) compared with the post-vaccine surgical 
specimen (“POST”) indicated marked reduction of HER-2 expression in half of the subjects (Fig. 1B). 
The left panels of “B” show photo microscopy of stained DCIS sections from one representative 
subject (note loss of brown HER-2 staining in “POST” specimen) while the right panels show 
composite data of HER-2 reductions from 8 sample subjects as well as unvaccinated controls. No such 
reductions were observed in an unvaccinated contemporaneous control group (Fig. 1B) suggesting  
that vaccination could eliminate HER-2-overexpressing tumor cells. A comparison of estimated tumor 
size determined radiographically prior to vaccination with the actual tumor removed during surgery 
indicated reductions in the area of disease for about half of the subjects, with results for five of these 
clinically-responsive individuals shown (Fig. 1C). In fact, five of 27 subjects had no detectable DCIS 
at  the time  of surgery,  suggesting vaccine-induced elimination of  DCIS  for some  cases.  For HLA- 
A2.1pos subjects, we routinely demonstrated the capacity of post-vaccination CD8pos T cells to directly 
recognize and secrete IFN-γ in response to HER-2pos breast cancer cell lines in vitro. Results from a 
representative subject are shown (Fig.1D). This is important since CD4pos and CD8pos T  cell  
infiltration in IBC is associated with long-term survival.  The immunity generated by this   vaccination 

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regimen proved to be extremely long-lived. Seventeen of twenty-seven subjects voluntarily provided 
additional blood samples 8-48 months post-vaccination. All subjects showed long-term CD4 T cell 
immunity to vaccine peptides by ELISpot. Anti-peptide results for subjects 1, 2 and 6 (taken 48, 48 
and 43 month’s post-vaccination, respectively) without prior stimulation are shown (Fig. 1E). Note 
that the long-term recall responses to vaccine peptides are often superior in magnitude to the recall 
response against tetanus toxoid. Despite the fact that this vaccine regimen was not optimized for 
antibody induction, we nonetheless demonstrated in some patients’ sera a vaccine-induced increase in 
complement-dependent lysis of HER-2/neupos tumor lines in vitro (Fig. 1F), a property not seen with 
trastuzumab. Although antibody titers appeared fairly low (not shown), these data provide strong 
encouragement that antibody responses are present and ripe for dramatic improvement. Of utmost 
importance in this completed trial and a second follow-on trial now in progress is that we have a 
composite complete pathologic response rate of about 35% in ERneg HER-2pos (i.e. hormone- 
independent) DCIS patients compared with 4% for ERpos HER-2neg (Fig. 1G). Since pathologic CR in 
breast cancer is associated with decreased risk of recurrence and deaths, it is not surprising that we 
have observed no breast cancer events in the patients that are ERneg HER-2pos compared to the group 
that is ERpos HER-2pos (Fig. 1G) we are highly confident that this Th1-polarizing DC vaccine will be 
associated with diminished secondary recurrence of DCIS and IBC. We wish to determine whether 
these vaccines following chemotherapy can induce immune responses and ultimately decrease 
recurrence in high risk node positive patients. 
 
Clinical experience with DC1 vaccines: The vaccines are readily produced from all 80 subjects thus 
treated with early breast cancer. There is abundant IL-12 production. There is 85-90% induction of 
CD4 and CD8 T cells that recognize HER-2 post vaccination. None of these patients had immediately 
had chemotherapy or trastuzumab. The side effects of vaccine included grade 1 and 2 fevers, chills, 
fatigue, injection site soreness, headache. There were <10% of patients with asymptomatic declines in 
MUGA scan all within normal range above 50%. There was no one that developed heart failure 
clinically or by cardiac ejection. Any grade 1 or 2 changes in cardiac ejection fraction were reverted to 
baseline in repeat scan 30 days later. Thus the vaccines were considered safe and well tolerated. 
 
Evidence for CD4 T cells that recognize extracellular domain HER-1 and HER-3 in early breast 
cancer: The majority of DCIS patients even prior to vaccination have evidence of CD4 T cells that 
secrete INF-γ when stimulated with extracellular domain (ECD) HER-1 (Fig 2 left graph). Those 
patients that do not have anti-HER-1 response developed a response to HER-1 following vaccination 
against HER-2 (Fig 2 right graph). There were only a few patients with evidence of HER-3 CD4 T  
cell responses prior to vaccination but the majority developed suggestion of weak CD4 T cell 
responses beginning after vaccination (Fig 3). These data support that following HER-2 vaccination 
using DC1 there is evidence that immune responses against HER-1 and HER-3 can be generated and 
are ripe for the improvements proposed in this application. 

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3. Patients deemed to require anti-estrogen therapy for treatment of their breast cancer can 
continue anti-estrogen therapy during vaccinations 
4. Women of childbearing age with a negative pregnancy test documented prior to enrollment. 
5. Patients with ECOG Performance Status Score of 0 or 1 (Appendix D). 

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6. Women of childbearing potential must agree to use a medically acceptable form of birth 
control (medically accepted methods: birth control pills, condoms and spermicidal lubricants, 
intrauterine device, and diaphragm) during their participation in the study. 
7. Subjects who have voluntarily signed a written Informed Consent in accordance with 
institutional policies after its contents have been fully explained to them. 
 
Exclusion Criteria 
 
1. Pregnant or lactating females. 
2. Subjects with positive HIV or hepatitis C at baseline 
3. Subjects with coagulopathies, including thrombocytopenia with platelet count <75,000,   INR 
> 1.5 and partial thromboplastin time > 50 sec 
4. Subjects with major cardiac illness MUGA < 50% EF. 
5. Subjects with pre-existing medical illnesses or medications which might interfere with the 
study as determined by PI. 
 
6.3 Pre-enrollment screening 
 
A. Tumor Characteristics 
 
1. A core biopsy or excisional biopsy demonstrating IBC. 
2. A standard clinical HER-2/neu immunohistochemical stain and FISH test will be 
performed using the Hercept test (Dako) on specimens obtained by core biopsy material 
or resected tumor. 
3. A standard clinical ER, PR test. 
 
B. Baseline patient evaluation 
 
1. All subjects will have a baseline history and physical exam. 
 
2. Laboratory work-up will consist of: 
a) CBC w/ differential 
b) PT/PTT 
c) electrolytes-Na/K/Cl/CO2/BUN/Cr/glucose 
d) liver function tests 
e) urinalysis 
f) Beta-HCG serum (for all pre and perimenopausal women) 
g) EKG 
h) HLA typing (to be obtained as part of study) 
i) MUGA or Echocardiogram (Must have 50% EF)  
j) Blood for immune analysis ( 6 tubes) 
 
3. Baseline radiologic evaluation 
a)  Mammogram up to six months prior to consenting to participate in the study will be 
acceptable. (Unilateral or bilateral if bilateral mastectomies mammogram not needed) 

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b) CT scans of the chest abdomen and pelvis or PET/CT within 60 days of 
enrollment 
 
C. Performance of the Pre-screening tests 
 
All of the pre-screening tests will be obtained by the patient prior to first treatment. 
 
1. Histopathologic Testing of Tumor 
 
Subjects will have undergone core or resection of primary tumor prior to enrollment in this 
protocol to confirm diagnosis of IBC. The Hercept (Dako) antibody will be used to analyze the 
expression of HER-2/neu on the IBC. Conventional HER-2 3+ 10% of the cells will be 
considered classic HER-2 positive or 2+ with FISH amplification 2 fold increase in FISH 
amplicons. For HER-2 2+ FISH negative, ten percent of the cells staining at least 2+ will be 
considered positive for HER-2/neu expression. 
 
 
6.1 Preparation of the vaccine 
 
Preparation of the vaccine is a multi-step process starting with leukapheresis and countercurrent 
centrifugal elutriation to obtain the relevant monocyte pool. This monocyte pool will then be 
treated with GM-CSF then pulsed with HER-2/neu peptides and finally matured with INF-, and 
LPS for conversion into type 1 dendritic cells (DC1).When occurring at University of 
Pennsylvania a study monitor will oversee quality assurance of vaccine production. When 
occurring at Moffitt Cancer Center, all standard institutional quality assurance policies will be 
followed. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6.1.1 Subject Leukapheresis 
 
Subjects will first undergo leukapheresis to obtain lymphocyte and monocyte fractions. The 
leukapheresis will be performed in the pheresis facilities at the University of Pennsylvania or at 
Moffitt Cancer center by certified personnel. Subjects will be apheresed 15-20 liters whole blood 
on a Gambro Spectra or equivalent apparatus. We expect to be able to generate 1-5x1010 PBMC. 
In the event a subject  has poor peripheral access, the option of having an internal jugular line 

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placed by the interventional radiology division will be offered as an alternative.   At the time of    
the apheresiscollection, a tube of serum will be drawn and tested for a baseline  anti-HER-2/neu 
response.  This will then be compared with a specimen of serum that will be taken and tested 
post vaccine.  
 
For subjects in whom leukapheresis did not yield sufficient number of cells to make all 6 
vaccines and 3 booster vaccines (1.8x10^9 monocytes), an additional apheresis collection will be 
undertaken to yield enough cells to manufacturer the remaining vaccines. . 
 
Subjects may additionally undergo leukapheresis (15-20 liters) at the completion of the first 6 
vaccines to obtain adequate numbers of the T lymphocyte population for immunologic analysis, as 
well as to obtain lymphocyte and monocyte fractions to produce 3 booster vaccines. For subjects 
in whom the initial and second leukapheresis did not yield sufficient number of cells to make all 6 
vaccines and 3 booster vaccines, an additional pheresis may be undertaken. At the completion of 
pheresis all IV access will be removed. 
 
Subjects will have 5mL of peripheral blood drawn at multiple time points during the vaccine 
schedule (pre vaccine, post six vaccines, pre booster 1, pre booster 2, pre booster 3, and 
completion of all vaccinations) to monitor immunologic responses to the administered vaccines. 
Subjects will also have the option to undergo additional leukapheresis (10-15 liters) at the 
completion of the 6 vaccines or completion of the three boosters to obtain T lymphocyte 
population for additional immunologic analysis. 

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6.1.2 Elutriation 
 
To separate the lymphocyte and monocyte fractions, the apheresis product obtained will then be 
subject to countercurrent centrifugal elutriation on a Model J-6M centrifuge (Beckman 
Instruments) equipped with a JE-5.0 elutriation rotor operating at 1725g and 20oC) or equivalent 
apparatus. The apheresis product is loaded onto the elutriator at 120cc/minute flow rate, then 
fractions are collected using stepwise increases in flow rates to obtain lymphocyte-rich (120  and 
140 cc/minute), transitional (150 cc/minute) and monocyte-rich (180 and 190 cc/minute) 
fractions. A final monocyte-rich fraction is collected at zero rpm (rotor off). Lymphocyte- rich 
fractions are then further purified with density gradient centrifugation using Ficoll-Hypaque to 
remove the red blood cells. 
 
The monocyte fraction is washed, counted and aliquoted out into portions for individual 
vaccines and cryopreserved for use at a later date.  
 
6.4.3. Cell culture and preparation of dendritic cell vaccine (Table 1) 
 
All elutriation will be performed in the FDA-approved facilities in the blood bank.  All work  here 
in the production of the monocytes will be performed following strict guidelines for sterility and 
will be done with Good Laboratory Practices. Ms. Mary Sell, Supervisor in the Cell Processing 
Center will oversee elutriation at University of Pennsylvania and Dr. Linda Kelly will oversee 
elutriation at Moffitt Cancer Center. The vaccine activation will take place in the FDA approved 
facility in the Clinical Cell and Vaccine Production Facility (CVPF) in Maloney 6 of the Hospital 
of the University of Pennsylvania, or at the Cell Therapy Facility (CTF) at Moffitt Cancer Center, 
under Good Manufacturing Practices. The CVPF and CTF are both accredited by the Foundation 
for the Accreditation of Cellular Therapy (FACT). 
, will oversee vaccine 
production and 
 will be responsible for release of vaccine for clinical delivery at 
University of Pennsylvania. 
 has 20 year’s experience with preparing clinical grade DC 
vaccines. 
 will oversee DC1 vaccine manufacturing at the Moffitt Cancer Center. 

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For those subjects transferring from University of Pennsylvania and receiving vaccines at Moffitt 
Cancer Center, elutriated product will be shipped cryopreserved and monitored using standard 
shipping methods already in place at the Cell Therapy Facility at the Moffitt Cancer Center. Those 
elutriated monocytes will be stored cryopreserved and activated into DC as described below. Any 
subjects newly enrolled at Moffitt Cancer Center will have elutriated product generated in the Cell 
Therapy Laboratory at Moffitt Cancer Center.  Any subject receiving vaccine or boost at Moffitt 
cancer center will be a registered patient of Moffitt Cancer Center. 
Monocytes will be washed 2 times in Hanks Buffer. Monocytes will be cultured with GM-CSF, 
IL-4, IFN-, and LPS for conversion into DC1. Monocytes at concentrations of 3x106 cells/ml 
will be cultured in serum free medium (Gibco BRL, Grand Island, NY) with 50 ng/ml (250 
IU/ml) rhGM-CSF, and IL-4 1000 u/ml (R&D Systems, Minneapolis, MN) and 20 ug/ml HER- 
2/neu peptides will be added to the culture on the morning of day 2. If, after thawing the 
cryopreserved cells, there is less than 85% cell viability, the exact number of cells needed for 
vaccine production will be cultured for one extra day in rhGM-CSF and vaccine administration 
will be moved back one day. Recombinant HER-2/neu peptides 3 ECD p42-56 
(HLDMLRHLYQGCQVV), 
p98-114 
(RLRIVRGTQLFEDNYAL), 
p328- 
345(TQRCEKCSKPCARVCYGL) and 3 ICD p776-790 (GVGSPYVSRLLGICL), p927-941 
(PAREIPDLLEKGERL)  and  p1166-1180  (TLERPKTLSPGKNGV)  (American  Peptide    Co, 
Sunnyvale, CA) will be pulsed on day 2. The peptides will be pulsed in separate wells 2-3 wells 
per peptide to avoid competition for binding in the MHC. The evening of day 2 IFN- (1000 
u/ml) will be added. Six – eighteen hours later LPS (10 ng/ml) will be added and the cells 
cultured an additional 2-8 hours. For HLA-A2+ subjects, one hour prior to harvest, pulse DC1 in 
addition to above with 50ug/ml of HER-2/neu class I binding peptides into one well of each ECD 
and ICD with two different peptides (369-377, 689-697). For HLA-A3+ subjects, one hour prior 
to harvest, pulse DC1 in addition to above with 50ug/ml of HER-2/neu class I binding peptide 
into one well of each ECD and ICD with one peptide (369-377). In the event that HLA-A tissue 
typing results that have not been completed in time for the HLA-A2 peptide addition, will have 
the peptides added to the culture under the principle that if said patient is not HLA-A2+ or HLA- 
A3+ the peptides will not be able to bind properly and be washed away during the harvest of the 
vaccine.  The cells will then be harvested from the plates, washed three times with Hanks  
Buffer, counted and pooled. Viability will be determined by trypan blue exclusion.   About 2    to 
2.5 million DC1 from each peptide specificity will be pooled so that a total vaccine number of 
about 1.0-2.0 x 107 cells resuspended in 20cc sterile saline. From this 20cc 5cc will be removed 
for gram stain, sterility testing, endotoxin testing and FACS analysis. These tests will be 
performed with this 5cc solution. The remainder of the cells, about 10-20 million cells, will be 
centrifuged and the pellet resuspended in 1ml for administration. The final vaccine solution will 
be labeled with the subject’s name, date and time prior to release. The vaccine will be 
administered shortly after preparation. Long term, > 4 hour, storage of the vaccine is not 
anticipated. Short term storage of the vaccine at 40C will be accommodated for 4-6 hours after 
which the vaccine will be discarded if not used. The vaccine prepared will be stored in the 
administration syringe 1cc tuberculin syringe at 40C and will be directly administered to the 
subject nodes.  A 22 or 23 gauge needle will be used for injection to prevent cell lysis. 
 
A sample of each vaccine will be sent for endotoxin testing, gram stain, and Bac Tec and broth 
sterility cultures. An aliquot  of the clinical product will be submitted to the Department of 
Microbiology or to the Cell Therapy Quality Control Lab at Moffitt Cancer Center and they will  
prepare a smear for gram stain testing. In addition, two tubes (or BacTAltert blood culture 
bottles at Moffitt) will be inoculated and this will be sent to the Department of Microbiology or 
to the Cell Therapy Quality Control Lab for sterility testing A negative gram stain of the 

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required in order to clear the vaccine for subject administration. The endotoxin test will be 
performed according to Good Laboratory Practices in the CVPF. The results of endotoxin 
testing and microbiology cultures will be checked when available and if not meeting 
specifications the vaccine will be discarded. An endotoxin level of less than 5 EU/kg body 
weight will be accepted. Sterility testing will be performed in accordance with CFR 610.12 and 
a specification of no growth will be required. A final quality analysis certificate will be issued for 
each dose of vaccine administered. (Appendix B). Additionally, cell viability of the prepared 
vaccine will be determined using trypan blue staining. The results will be available prior to 
vaccine administration. Cell viability of >70% will be required for release of vaccine. If this 
cutoff is not met, then the vaccine will be re-made. In addition, a sample of each vaccine will 
also be removed for FACS analysis of cell surface markers to confirm conversion of monocytes 
to DC. Since the activity of the vaccine does not depend on phenotype the vaccine will not be 
held for phenotype but will serve as an internal evaluation of the process of vaccine preparation 
(see below). 
The requirements for vaccine release then are: 
Viability 
>70% 
gram stain result 
no organisms 
endotoxin test 
<5EU/kg subject weight 
culture and sensitivity 
no growth final culture 
 
Since the culture test will become available after vaccine administration the following 
contingency plan will be in place for a positive culture: 
 Notification of both the primary attending physician and the subject. 
 Identification of the responsible microorganism and sensitivity testing. 
 A description of additional subject monitoring as required by the circumstances of the 
contamination incident. 
 Initiate an investigation of the event and take corrective actions to prevent future 
recurrences. The scope of the investigation and proposed corrections should include: 
o Evaluation of your current procedures for collecting and processing the 
autologous peripheral blood product to determine the step at which contamination 
could be introduced. 
o Development of changes in the procedures that will assist in preventing sterility 
lapses in the future. 
o Development of appropriate testing to ensure that revised changes to the 
procedures produce a sterile product. This should include sterility testing at 
various points in the preparation of the product. 
 Report the incident as an adverse event to the agency within 15 working days. 
 
The first vaccine may be made from freshly collected and elutriated or cryopreserved cells. 
Subsequent vaccinations will be made from cryopreserved aliquots. At the University of 
Pennsylvania, once thawed, these cells  will be washed with medium twice prior to their 
placement in culture.At Moffitt Cancer Center, an individual vial will be removed from liquid 
nitrogen and allowed to thaw to room temperature.  Once vial is fully thawed, the vaccine will be 
transferred from the vial into a syringe for injection.  The thawed vaccine will be stored at a cool 
temperature, around 4 degrees C. 

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6.4.4 Quality assurance of DC1 
 
Surface markers on monocytes cells after treatment will be analyzed by FACS to in select 
products to determine whether they have converted to dendritic cells. After culture activation a 
small quantity of DC will be harvested for FACS analysis. Cells will be stained with FITC 
labeled antibodies against human cell surface markers CD14 and PE labeled antibodies CD80, 
CD86 and CD83. Two color analyses will be performed on a Beckman FACscan with  
appropriate isotype matched control antibodies. Dead cells will be gated using  propridium 
iodide. This facility is available through the Abramson Cancer Center at the University of 
Pennsylvania. The presence of the monocytic marker CD14, costimulatory molecule B7.1 and 
B7.2, MHC Class I and II and the expression of accessory molecules, CD40 and CD54 will be 
assessed using fluorescent multicolor flow cytometry. The conversion from monocytes to 
dendritic cells will be confirmed if the cells are CD80, CD86 and CD83 positive and have 
downregulated CD14. Flow cytometry will not be available for analysis until after the vaccine 
administration. The most useful measure of DC1 function will be a sample and will be analyzed 
for IL-12 production by standard ELISA. We utilize ELISA reagents purchased from Pharmigen 
according to the manufacturer’s recommendation to measure secreted IL-12. 
 
Table 1: Preparation of Dendritic Cell Vaccines: Summary of Procedure 
1. Suspend monocytes in serum free medium (SFM) to concentration of 3x106cells/ml. Plate 1cc 
of cell suspension/well in 64 well plate for a total of 12-18 wells (60-80 at Moffitt). Cells plated 
in numbers higher than actual dose to compensate for 50-75% retrieval rate with harvesting. 
 
2. If monocytes cryopreserved, thaw, add 20 cc Hanks Buffer and spin at 1200 rpm for 5 
minutes at 220C. Aspirate supernatant and resuspend pellet in 10 cc medium.  Remove 20µl for 
cell count. Use trypan blue to exclude dead cells from cell count.  Add 10 cc Hanks Buffer and 
spin again at 1200 rpm for 5 minutes at 220C. Resuspend to concentration of 3x 106 cells/ml in 
SFM and plate as above.   
3. Add rhGM-CSF for final concentration of 50 ng/ml or 250 IU/ml and IL-4 1000 u/ml (see IV 
Preparation of Reagents). Culture overnight.  If, after thawing the cryopreserved cells, there is 
less than 85% cell viability, the exact number of cells needed for vaccine production will be 
cultured for one extra day in rhGM-CSF and vaccine administration will be moved back one day. 
4. The next morning, add HER-2/neu peptides for a final concentration of of 20µg/ml 3 ECD - 
56, p98-114, p328-345 and 3 ICD p776-790, p927-941 and p1166-1180  These will be pulsed 
in separate wells. 
 
 

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5. In the afternoon, add IFN- 1000 u/ml, culture cells at 37o C and 5% CO2 overnight for 14 hours. 
 
6. The following morning, add LPS (10 ng/ml)  
 
7. Incubate 2-8 hours. 
 
8. For HLA-A2+ subjects, one hour prior to harvest, pulse DC1 in addition to above with 50 ug/ml 
HER-2/neu class I binding peptides into one well of each ECD and ICD with two different peptides 
(369-377 and 689-697). For  HLA-A3+ subjects, one hour prior to harvest, pulse DC1 in addition to 
above with 50ug/ml of HER-2/neu class I binding peptide into one well of each ECD and ICD with 
one peptide (369-377).  In the event that HLA-A tissue typing results have not been completed in 
time for the HLA-A2 peptide addition, will have the peptides added to the culture under the principle 
that if said patient is not HLA-A2+ or HLA-A3+ the peptides will not be able to bind properly and 
be washed away during the harvest of the vaccine.      
 
9. Harvest cells into 50cc Falcon tubes.  Maximum 20 cc cell volume and add 20 cc Hanks Buffer 
and spin at 1200 rpm for 5 minute @ 220C. Repeat this step, wash the cells again. 
 
10. Aspirate supernatant. Remove 20µl for cell count to estimate % cell death. Pool 2-2.5 million 
cells per each peptide and respin cells at 1200 rpm, 220C for 5 minutes.  Resuspend in 20cc sterile 
saline.  Remove 12-18 million cells, about 15cc, for vaccine preparation and from the 5cc remaining 
the tests of endotoxin, sterility testing, gram stain and FACS (see 6.5.4 Phenotypic Analysis of DC1) 
will be obtained.  The vaccine component will be centrifuged and resuspended to 1cc with a total 
cell number 1.0-2.0 x 107 cells.  
 
11. Label with subjects name, date and time. Release for administration if cell viability >70% and 
gram stain no organisms seen Endotoxin testing must be less than 5 EU/kg body mass for 
administration.  At University of Pennsylvania, some of the tested material will be reserved so that, 
in the event a contaminant is discovered in the process of sterility testing, we will have individual 
components to test so to determine the exact point of contamination. 
 
12. Remake the vaccine if cell viability <70%, positive gram stain, or endotoxin level >5EU/kg. At 
Moffitt the vial will be removed from liquid nitrogen and allowed to thaw at room temperature.  
Once vial is fully thawed the vaccine will be removed from the vial and transferred into a syringe for 
injection.  The vaccine will be kept at a cool temperature, around 4 degrees C, until administration 
 
 
 
 
 
6.4.5 Pharmacologic Information and Preparation of Reagents 
 
A. HER-2/neu ECD and ICD peptides 
 
Recombinant HER-2/neu peptides 3 ECD p42-56 (HLDMLRHLYQGCQVV), p98-114 
(RLRIVRGTQLFEDNYAL), p328-345(TQRCEKCSKPCARVCYGL) and 3 ICD p776-790 
(GVGSPYVSRLLGICL),        p927-941        (PAREIPDLLEKGERL)        and         p1166-1180 
(TLERPKTLSPGKNGV) will be made at American Peptide Co. Sunnyvale, CA. These peptides 

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will be >95% pure and certificate of analysis will be provided for each of the 15mers. The 
peptides will be stored lyophilized and reconstituted in sterile PBS for use. Sterility tests will be 
performed on a sample of each shipment of peptides delivered to the University of Pennsylvania.  
 
B. HER-2/neu MHC class I binding peptides 
 
Three MHC class I binding peptides 369-377, 971-979, and 688-695 will be synthesized at 
United Biochemical Research, Seattle, WA or BACHEM, Torrance, CA. These peptides will be 
>95% pure and certificate of analysis  will  be provided for each of the 14mers.  The peptides  
will  be  stored lyophilized  and 

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reconstituted in sterile PBS for use. These peptides will be used in addition to the above peptides 
for the subjects that are HLA-A2+ and HLA-A3+. 
 
C. Cytokines 
 
1.   GM-CSF (NSC# 613795) 
 
Granulocyte macrophage colony stimulation factor (GM-CSF, Leukine, Sargramostim, Immunex 
Co, Seattle, WA) is available as a sterile, white, preservative free lyophilized powder in vials 
containing 250 µg GM-CSF (1.25 x 107 Units) or 500 µg GM-CSF (2.5 x 107 Units) with 40 mg 
mannitol, USP, 10 mg sucrose, USP and 1.2 mg tromelhamine, USP. Each dosage form is 
supplied individually or in a package of 10 single use vials.       GM-CSF should be reconstituted 
with 1cc sterile water, USP (without preservative). Reconstituted GM-CSF is a clear, odorless, 
isotonic solution, pH 7.4±0.3.  The single use vials should not be reused. 
 
GM-CSF is further diluted in 0.9% NaCl, USP (or Hanks, at Moffitt) to a concentration 1 µg/ml 
and used in cultures at a final dose of 10 ng/ml (50 IU/ml). 
 
 
 
2.  rhIFN-
rhIFN- will be purchased from Actimmune (Brisbane, CA) or R&D Systems, Minneapolis,  and 
provided as a lyophilized powder. It will be stored according to manufacturer’s recommendation, 
reconstituted in serum free media when appropriate, and diluted in sterile aliquots for addition to 
cultures. 
 
3.   LPS (NSC # 67801) 
 
At University of Pennsylvania, Clinical Grade LPS will be obtained from Dr. Anthony Suffredini 
at the NIH supplied in sterile saline. It will be aliquotted in sterile containers and added to the 
cultures as specified. It will be stored at 4o C. At Moffitt Endotoxin Reference Standard LPS will 
be obtained from the USP and provided as lyophilized powder.  It will be stored according to 
manufacturer’s recommendation, reconstituted in sterile water, and diluted in sterile aliquots for 
addition to cultures.   
 
4.  IL-4 
 
Recombinant human IL-4 will be purchased from R&D and will be stored frozen until  
aliquotted. A certificate of analysis will be obtained. It will be added to cultures at 1000 units /ml 
final concentration. 
 
 
 
The Investigational Pharmacy at the University of Pennsylvania Hospital or the Cell 
Therapy Facility at Moffitt will order and aliquot and disperse all clinical reagents 
including peptides, GM-CSF, IL-4, INF-γ, and LPS. 

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6.4.6 Administration of the Vaccine 
 
A. Selection of Lymph nodes for vaccination 
 
Ultrasound will be used to identify non-pathologic lymph nodes in the groin as first choice or 
axilla as second choice. Non-pathologic nodes by ultrasound criteria have a fatty hilum and   will 

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be selected; two nodes per vaccine preferentially 1 node per groin. If a patient has lymphedema  
or prior groin dissection on a single side then the contralateral groin will be used or alternatively 
the axilla. Often we only are able to identify 1-2 nodes in the groin by ultrasound.  Nodes  
without a fatty hilum that have evidence of pathologic disease will be excluded from injection. 
 
B. Administration of the initial Vaccine 
 
Vaccines will be labeled (for example, with the subjects study ID, study initials, date and time) 
prior to release. The patient will be seen by the PI or designee, and Research Coordinator and a 
history and physical exam will be performed. Ultrasound (US) guided intranodal delivered 
vaccines will be administered in the NIH Clinical Research Center at Penn or the Clinical 
Research unit at Moffitt Cancer  Center by a radiologist or sonographer experience  in  
ultrasound  guidance  and   the 
principal investigators or his/her designee. Each dose will consist of between 1.0-2.0 x 107 cells 
and will be injected into 1-2 different normal groin lymph nodes or axillary nodes. 
 
C. Post vaccine assessment 
 
Subjects will be administered the vaccine and observed.  All subjects with vaccine related AEs will 
be followed until the AE has resolved to baseline. Vital signs will be obtained prior to the 
vaccination and again 15 minutes after the dose is given. Subjects with no noted complications will 
be then discharged. The anticipated complication could be fever. If an adverse reaction is observed, 
the subject observation time will be revised appropriately. A physician familiar with the study will 
be readily available in the event of any complication and such subjects will be transferred to an 
appropriate care unit for further monitoring and treatment Similarly any discharged subject who 
develops symptoms will be promptly re-evaluated and admitted for care if inpatient hospitalization 
is deemed necessary by the principal investigator or his/her designee. 
 
D. Vaccine Schedule 
 
The vaccine schedule is meant to be weekly; however the first 6 vaccines may be given 
anywhere between 8-21 days apart. 
 
E. Vaccine Tolerance 
 
DC vaccination protocols on humans to date have not demonstrated serious toxicities and we 
anticipate that subjects on this protocol will also tolerate vaccination without incident. 
Specifically, our own HER-2/neu peptide pulsed DC1 protocol using this same regimen has been 
well tolerated. We have therefore designed this vaccination trial as a largely out-patient based 
protocol. However, should significant toxicities be noted this trial as a result of the additional 
breast vaccinations, the trial will be amended to increase the period of patient observation 
accordingly. 
 
F. Administration of Second Vaccine 
 
Between 8 and 21 days post vaccine 1 the subject will return for the second vaccination. Again a 
history and physical exam will be taken.  Any changes in history or physical will be documented. 

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Ultrasound (US) guided intranodal delivered vaccines will be administered in the NIH Clinical 
Research Center at Penn or the Clinical Research unit at Moffitt Cancer Center by a radiologist  
or sonographer experience in ultrasound guidance and the principal investigators or his/her 
designee. Each dose will consist of between 1.0-2.0 x 107 cells and will be injected into 1-2 
different normal groin lymph nodes or axillary nodes. 
 
G. Post vaccine assessment 
 
Subjects will be administered the vaccines and observed. All subjects with vaccine related AEs 
will be followed until the AE has resolved to baseline. Vital signs will be obtained just prior to 
the vaccination and again 15 minutes after the dose is given. Subjects with no noted 
complications will then be discharged. The anticipated complication could be fever. If an 
Adverse Event is observed, the monitoring schedule will be revised appropriately. A physician 
familiar with the study will be readily available in the event of any complication and such 
subjects will be transferred to an appropriate care unit for further monitoring and treatment. 
Similarly any discharged patient who develops symptoms will be promptly re-evaluated and 
admitted for care if inpatient hospitalization is deemed necessary by the PI or his/her designee. 
 
H. Administration of  Third Vaccine 
 
Between 8 and 21 days post vaccine the patient will return for the third vaccination. Again a 
history and physical exam will be taken. Any changes in history or physical will be documented. 
Ultrasound (US) guided intranodal delivered vaccines will be administered in the NIH Clinical 
Research Center at Penn or the Clinical Research unit at Moffitt Cancer Center by a radiologist  
or sonographer experience in ultrasound guidance and the principal investigators or his/her 
designee. Each dose will consist of between 1.0-2.0 x 107 cells and will be injected into 1-2 
different normal groin lymph nodes or axillary nodes. 
 
I. Post vaccine assessment 
 
Subjects will be administered the vaccine and observed All subjects with vaccine related AEs will 
be followed until the AE has resolved to baseline. Vital signs will be obtained prior to the 
vaccination and again 15 minutes after the dose is given.  Subjects with no noted complications 
will then be discharged.  The anticipated complication could be fever.  If an Adverse Event is 
observed, the subject observation time will be revised appropriately .A physician familiar with the 
study will be readily available in the event of any complication and such subjects will be 
transferred to an appropriate care unit for further monitoring and treatment as necessary.  Similarly 
any discharged subject who develops symptoms will be promptly re-evaluated and admitted for 
care if inpatient hospitalization is deemed necessary by the PI or his/her designees. 
 
J. Administration of Fourth Vaccine 
 
Between 8 and 21 days post vaccine the patient will return for the fourth vaccination. Again a 
history and physical exam will be taken. Any changes in history or physical will be documented. 
Ultrasound (US) guided intranodal delivered vaccines will be administered in the NIH Clinical 
Research Center at Penn or the Clinical Research unit at Moffitt Cancer Center by a radiologist 

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or sonographer experience in ultrasound guidance and the principal investigators or his/her 
designee. Each dose will consist of between 1.0-2.0 x 107 cells and will be injected into 1-2 
different normal groin lymph nodes or axillary nodes 
 
K. Post vaccine assessment 
 
Subjects will be administered the vaccines and observed.  All subjects with vaccine related AEs 
will be followed until the AE has resolved to baseline. Vital signs will be obtained prior to the 
vaccination and again 15 minutes after the dose is given.  Subjects with no noted complications 
will then be discharged.  The anticipated complication could be fever.  If an Adverse Event is 
observed, the subject observation time will be revised appropriately. A physician familiar with the 
study will be readily available in the event of any complication and such subjects will be 
transferred to an appropriate care unit for further monitoring and treatment.  Similarly any 
discharged subject who develops symptoms will be promptly re-evaluated and admitted for care if 
inpatient hospitalization is deemed necessary by the PI or his/her designee. 
 
L. Administration of Fifth Vaccine 
 
Between 8 and 21 days post vaccine the patient will return for the fifth vaccination. Again a 
history and physical exam will be taken. Any changes in history or physical will be documented. 
Ultrasound (US) guided intranodal delivered vaccines will be administered in the NIH Clinical 
Research Center at Penn or the Clinical Research unit at Moffitt Cancer Center by a radiologist  
or sonographer experience in ultrasound guidance and the principal investigators or his/her 
designee. 
Each dose will consist of between 1.0-2.0 x 107 cells and will be injected into 1-2 different 
normal groin lymph nodes or axillary nodes 
 
M. Post vaccine assessment 
 
N. Subjects will be administered the vaccines and observed.  All subjects with vaccine related AEs will be 
followed until the AE has resolved to baseline. Vital signs will be obtained just prior to the vaccination 
and again 15 minutes after the dose is given.  Subjects with no noted complications will then be 
discharged.  The anticipated complication could be fever.  If an Adverse Event is observed, the subject 
observation time will be revised appropriately. A physician familiar with the study will be readily 
available in the event of any complication and such subjects will be transferred to an appropriate care 
unit for further monitoring and treatment.  Similarly any discharged subject who develops symptoms 
will be promptly re-evaluated and admitted for care if inpatient hospitalization is deemed necessary by 
the PI or his/her designee.Administration of Sixth Vaccine 
Between 8 and 21 days post vaccine the patient will return for the sixth vaccination. Again a 
history and physical exam will be taken. Any changes in history or physical will be documented. 
Ultrasound (US) guided intranodal delivered vaccines will be administered in the NIH Clinical 
Research Center at Penn or the Clinical Research unit at Moffitt Cancer Center by a radiologist  
or sonographer experience in ultrasound guidance and the principal investigators or his/her 
designee. 
Each dose will consist of between 1.0-2.0 x 107 cells and will be injected into 1-2 different 
normal groin lymph nodes or axillary nodes. 

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O. Post vaccine assessment 
 
Subjects will be administered the vaccines and observed.  All subjects with vaccine related AEs 
will be followed until the AE has resolved to baseline. Vital signs will be obtained prior to the 
vaccination and again 15 minutes after the dose is given.  Subjects with no noted complications 
will then be discharged.  The anticipated complication could be fever.  If an Adverse Event is 
observed, the subject observation time will be revised appropriately. A physician familiar with the 
study will be readily available in the event of any complication and such subjects will be 
transferred to an appropriate care unit for further monitoring and treatment.  Similarly any 
discharged subject who develops symptoms will be promptly re-evaluated and admitted for care if 
inpatient hospitalization is deemed necessary by the PI or his/her designees. 
 
P. Administration of Additional Boost Vaccines. 
 
After the subject has completed 6 vaccines, they will undergo leukapheresis to obtain  
lymphocyte and monocyte fractions, if needed. 
 
Q. Administration of First Boost Vaccine 
 
Approximately 3 months from completion of the induction vaccines, subjects will receive 3  
boost vaccines at approximately 3 month intervals. Again a history and physical exam will be 
taken. Any changes in history or physical will be documented. Ultrasound (US) guided  
intranodal delivered vaccines will be administered in the NIH Clinical Research Center at Penn  
or the Clinical Research unit at Moffitt Cancer Center by a radiologist or sonographer experience 
in ultrasound guidance and the principal investigators or his/her designee. Each dose will consist 
of between 1.0-2.0 x 107 cells and will be injected into 1-2 different normal groin lymph nodes or 
axillary nodes 
 
R. Post vaccine assessment 
 
Subjects will be administered the vaccine and observed.  All subjects with vaccine related AEs will 
be followed until the AE has resolved to baseline. Vital signs will be obtained prior to the 
vaccination and again 15 minutes after the dose is given.  Subjects with no noted complications 
will then be discharged.  The anticipated complication could be fever.  If an Adverse Event is 
observed, the subject observation time will be revised appropriately. A physician familiar with the 
study will be readily available in the event of any complication and such subjects will be 
transferred to an appropriate care unit for further monitoring and treatment.  Similarly any 
discharged subject who develops symptoms will be promptly re-evaluated and admitted for care if 
inpatient hospitalization is deemed necessary by the PI or his/her designee. 
 
S. Administration of Second Boost Vaccine 
 
Approximately 3 months from completion of the first boost vaccine Subjects will be 
administered 2nd boost vaccine. Subjects will first have blood drawn for immune assessment of 
the boost this will be 5 green top and 1 serum tube. Again a history and physical exam will be 

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taken. Any changes in history or physical will be documented. Ultrasound (US) guided  
intranodal delivered vaccines will be administered in the NIH Clinical Research Center at Penn  
or the Clinical Research unit at Moffitt Cancer Center by a radiologist or sonographer experience 
in ultrasound guidance and the principal investigators or his/her designee. Each dose will consist 
of between 1.0-2.0 x 107 cells and will be injected into 1-2 different normal groin lymph nodes or 
axillary nodes. 
 
T. Post vaccine assessment 
 
Subjects will be administered the vaccines and observed.  All subjects with vaccine related AEs 
will be followed until the AE has resolved to baseline. Vital signs will be obtained prior to the 
vaccination and again 15 minutes after the dose is given.  Subjects with no noted complications 
will then be discharged.  The anticipated complication could be fever.  If an Adverse Event is 
observed, the subject observation time will be revised appropriately. A physician familiar with the 
study will be readily available in the event of any complication and such subjects will be 
transferred to an appropriate care unit for further monitoring and treatment.  Similarly any 
discharged subject who develops symptoms will be promptly re-evaluated and admitted for care if 
inpatient hospitalization is deemed necessary by the PI or his/her designee. 
 
U. Administration of Third Boost Vaccine 
 
Approximately 3 months from completion of the second boost vaccine Subjects will be 
administered 3rd boost vaccine. Again a history and physical exam will be taken. Any changes  
in history or physical will be documented. Ultrasound (US) guided intranodal delivered vaccines 
will be administered in the NIH Clinical Research Center at Penn or the Clinical Research unit at 
Moffitt Cancer Center by a radiologist or sonographer experience in ultrasound guidance and the 
principal investigators or his/her designee. Each dose will consist of between 1.0-2.0 x 107 cells 
and will be injected into 1-2 different normal groin lymph nodes or axillary nodes 
 
V. Post vaccine assessment 
 
Subjects will be administered the vaccine and observed.  All subjects with vaccine related AEs will 
be followed until the AE has resolved to baseline. Vital signs will be obtained prior to the 
vaccination and again 15 minutes after the dose is given.  Subjects with no noted complications 
will then be discharged.  The anticipated complication could be fever.  If an Adverse Event is 
observed, the subject observation time will be revised appropriately. A physician familiar with the 
study will be readily available in the event of any complication and such subjects will be 
transferred to an appropriate care unit for further monitoring and treatment.  Similarly any 
discharged subject who develops symptoms will be promptly re-evaluated and admitted for care if 
inpatient hospitalization is deemed necessary by the PI or his/her designee. 
 
6.5 Post-Vaccine Procedures 
 
A. In vitro Immunologic testing 

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Subjects will have 5mL of peripheral whole blood drawn at multiple time points during the 
vaccine schedule (pre vaccine, post six vaccines, pre booster 1, pre booster 2, pre booster 3, and 
completion of all vaccinations) to monitor immunologic responses to the administered vaccines. 
Subjects will also have the option to undergo additional leukapheresis (10-15 liters) at the 
completion of the 6 vaccines or completion of the three boosters to obtain T lymphocyte 
population for additional immunologic analysis. The leukapheresis will be performed in the 
pheresis facilities at the University of Pennsylvania by certified personnel or at Moffitt Cancer 
Center, if needed. Attempts will first be made to obtain peripheral access for the pheresis 
protocol.  Subjects will be pheresed 15-20 liters whole blood on a Gambro Spectra or equivalent 
apparatus.  We expect to be able to generate 1-5x1010 PBMC. 
 
 
B. Post vaccine laboratory studies 
 
In addition to the blood which will be drawn for the above analyses, an extra tube of serum will 
be drawn to test the subject’s anti-HER-2/neu response. After boost vaccines additional 5 tubes 
of whole blood will be obtained for immune analysis. These results will be compared with the 
immune response results that will have been obtained prior to vaccination. An alternative post 
vaccine leukapheresis can be performed as alternative to blood draws. 
 
1. After receiving their vaccines and prior to receiving boost vaccine series, subject will 
have the following completed:  
 
a) CBC w/ differential 
b) PT/PTT 
c) electrolytes-Na/K/Cl/CO2/BUN/Cr/glucose 
d) liver function tests 
e) urinalysis 
f) MUGA or Echocardiogram (Compare to first study) 
 
In addition to the blood drawn for the above analyses, 5 tubes of whole blood will be obtained for 
immune analysis and tube of serum will be drawn to test subjects anti-HER-2/neu response.  These 
results will be compared with the immune response results that will have been obtained prior to 
vaccination. 
 
 
In the event of abnormal symptoms, laboratory monitoring may be done earlier. The laboratory 
studies and intervals at which they are done will be determined by the PI on a case by case basis. 
 
If significant decline in EF was evident in pre/post vaccine MUGA scan or ECHO, another 
MUGA scan or ECHO may be performed 1 month post-vaccine, as determined by the PI. 
Subjects will be closely monitored by the PI, who will determine the need for additional MUGA 
scan(s) during the 6 month post-vaccine monitoring period on a case by case basis. 
 
C. Long-term Follow-up 
 
After completing the 6 month post-vaccine monitoring period, all subjects will be seen yearly. 
Mammograms will be obtained at yearly visits if patients have remaining breasts. For 

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mastectomy subjects a yearly mammogram of the contralateral breast. This will continue for 5 
years. 
 
6.6 Clinical Monitoring 
 
A. Monitor  
 
At University of Pennsylvania, monitoring will be conducted by the PI and the Research 
Coordinators  in accordance with all applicable regulations.  Additionally, the PI and RC will meet 
with the Medical Monitoring Committee as needed to discuss the study and request additional 
monitoring support as necessary.   
 
At Moffitt Cancer Center, regulatory documents and case report forms will be monitored internally 
according to Moffitt Cancer Center Monitoring Policies. Monitoring will be performed regularly by 
the MCC Clinical Monitoring Core for accuracy, completeness, and source verification of data 
entry, validation of appropriate informed consent process, reporting of SAEs, and adherence to the 
protocol, Good Clinical Practice (GCP) guidelines, and applicable regulatory requirements. 

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. 
 
B. Initial Assessment Monitoring 
 
This serves as early detection and will help identify trouble areas in the study, staff or site early 
enough in the study to allow corrections which will benefit the study long term. The PI and the 
RC will meet to review the study data and processes after each treatment (9 total) for the first  
two subjects. During the initial assessment monitoring, the PI will review all AE/SAE and lab 
abnormalities with the Radiologist, Medical Oncologist and/or Pathologist (as appropriate). 
AE/SAEs will be recorded on a study specific Adverse Event form and all SAEs will be reported 
to the IRB, DSMC, Safety Monitoring Committee, GCRC, FDA, and NIH in accordance with all 
applicable regulations. All errors will be addressed and corrected at that time and training and 
study design related issues will be resolved in a timely manner. 
 
C. Ongoing Monitoring 
 
During the course of the trial, safety and data quality monitoring will be performed in an on- 
going manner by the PI, RC and other applicable study staff. The RC will monitor the data 
quality every 6 week (s). This includes ensuring that all source documents exist for the data on 
the case report forms, ensuring all fields are completed appropriately, all corrections are done 
according to GCPs and any inconsistencies/deviation are documented.  The PI will review the  
lab results for each patient on an on-going basis and will document his review by signing and 
dating the lab report. The PI will also review all Adverse Event forms in real-time to ensure 
appropriateness of the data and timeliness of reporting. All errors will be addressed at the time 
the monitoring takes place and when the PI reviews the safety data. The PI will also ensure that 
all renewals are received in a timely manner and all correspondences are documented. Issues 
related to staff training, protocol design, CRF design and monitoring frequency will be 
documented along with the appropriate corrective actions. Any issues uncovered will be 
addressed and corrected immediately. 
 
Moffitt Cancer Center 
 
Regulatory documents and case report forms will be monitored internally according to 
Moffitt Cancer Center Monitoring Policies. Monitoring will be performed regularly by the 
MCC Clinical Monitoring Core for accuracy, completeness, and source verification of data 
entry, validation of appropriate informed consent process, reporting of SAEs, and 
adherence to the protocol, Good Clinical Practice (GCP) guidelines, and applicable 
regulatory requirements. 
 
 
D. Data Quality 

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At University of Pennsylvania the  RC will make sure that all CRFs are completed, all changes to 
CRFs are made in accordance with regulations, source documents exist for all data points, the 
consent/re-consent process is clearly documented in the source records, investigational agent 
accountability is maintain proactively and response is clearly documented. Paper CRFs will be 
utilized until electronic CRFs are available. After the first 1/3 of the subjects are enrolled, the RC 
and PI will  collectively review the charts of 3 randomly selected subjects to ensure compliance 
with regulations and this plan. The collective review will again occur with a review of 3 subjects 
drawn from the next 1/3 of accrual and 3 subjects drawn from the final 1/3 of accrual. 
 
E. Safety Monitoring Committee 
 
At University of Pennsylvania 
, in the Department of Gynecology, 
 
 in the Department of Medical Oncology will be the 
Safety Monitoring Committee for this study. Neither 
 is 
directly involved in the trial. None of these physicians have any clinical trials in association with 
Dr. Czerniecki or 
. The Safety Monitoring Committee will meet every six months. 
Two weeks  prior to this meeting each committee member will receive a report of all AEs and 
safety data prepared by the research coordinator. The committee will review all AEs including 
grading, toxicity assignments, all other safety data and activity data observed in the ongoing 
clinical trial at this meeting. They may recommend reporting of adverse events and relevant 
safety data not previously reported and may recommend suspension or termination of the trial. 
The initial meeting of this committee will be six months after the first patient is enrolled. The 
final meeting will be six months after the final subject is vaccinated. 
 
F. Study Changes 
 
All changes to the protocol and informed consent will be submitted to the IRB, NIH, FDA and 
CTSRMC in the form of a formal amendment for approval prior to conducting the study under 
the proposed changes. If said changes potentially affect the subject’s willingness to participate, 
each subject will be re-consented. Any changes to this monitoring plan will be submitted to the 
DSMC for approval prior to implementing the changes. In the event of an early study  
suspension, the PI will immediately notify the IRB, NIH, FDA and CTSRMC. 
 
G. Monitoring Log 
 
At University of Pennsylvania a  monitoring log will be completed and maintained to document 
monitoring by the PI and RC. Moffitt Cancer Center will follow institutional procedures for 
monitoring of subjects that transfer care to the Center and are newly enrolled at the Center. 
 
H. Auditing 
 
This study will be audited by the Department of Compliance and Monitoring (DOCM) on behalf 
of the DSMC in accordance with the NCI approved Institutional Data Safety and Monitoring  
Plan (DSMP). 
 
Moffitt Cancer Center Protocol Monitoring Committee (PMC); 

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The PMC meets monthly and reviews accrual, patterns and frequencies of all adverse events, 
protocol violations and when applicable, internal audit results. 
 
 
Monitoring by the DSMC will be at the following milestones: within 2 weeks of the sixth, or 
final, vaccination. Two weeks after each boost vaccine and two weeks after completion of post 
vaccine studies. Monitoring will be conducted on the first three subjects enrolled. The frequency 
of monitoring of additional subjects (e.g. every other or every third) will be determined after the 
review of the first three subjects. The frequency of monitoring is subject to change if the DOCM 
feels it is necessary. The DOCM will be informed when each subject is enrolled, at which time 
the monitoring visits will be scheduled with the research coordinator. 
 
Moffitt Cancer Center Protocol Monitoring Committee (PMC): 
The PMC meets monthly and reviews accrual, patterns and frequencies of all adverse events, 
protocol violations and when applicable, internal audit results. 
 
6.7 Toxicity and Adverse Event Reporting 
 
A. General Toxicity 
 
The HER-2/neu protein is expressed by the epithelial cells of normal breast, skin, 
gastrointestinal, respiratory, reproductive and urinary tissues. The HER-2/neu gene is not over- 
amplified in these normal cells and a level of protein expression is thus expected to be 
significantly lower than the gene over-amplification and protein over-expression seen in DCIS. 
In animal models of successful immunity to HER-2/neu protein using HER-2/neu ECD and ICD 
peptides, no evidence of autoimmunity has been noted. Additionally, in DC trials of melanoma 
and prostate, no clinical evidence of autoimmunity directed against normal tissues sharing the 
targeted tumor antigen has been discovered. Therefore, although the risk is presumed to be 
minimal, the possibility of autoimmune disease directed at these normal tissues cannot be 
entirely excluded. 
 
This vaccine has not been previously administered to individuals who have been treated with 
Herceptin. Recent trials of Herceptin, the monoclonal antibody therapy to HER-2/neu ECD, have 
noted approximately 5% risk of cardiac toxicity leading to heart failure. The mechanism appears 
to be related to presence of HER-2/neu on cardiac cells. The levels of HER-2/neu expression by 
cardiac myocytes, however, are extremely low, detectable only by PCR analysis. 
 
Subjects on this trial will have cardiac evaluation of MUGA or echocardiogram to determine 
their ejection fraction. Subjects will also be closely monitored for signs/symptoms of heart 
failure. In our first trial there was no evidence of clinically relevant cardiac failure. Less than 8% 
of subjects had asymptomatic declines in ejection fractions post-vaccine, thought all EFs were 
still within normal range. There have been no long term cardiac toxicities noted as a result of this 
vaccine. Additionally, Herceptin does not cause long term cardiac toxicity issues. We do not 
expect the cardiac toxicity of prior Herceptin treatment and treatment with the vaccine to be 
additive. 
 
It has been demonstrated that fetuses express HER-2/neu during the third trimester and there is 
the possibility of spontaneous abortion as a result of anti-HER-2/neu T cell responses against the 

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fetus.  Therefore women of childbearing years will be made aware of this possibility. 

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B. Toxicity Definitions 
 
Toxicity will be graded according to the NCI-CTCAE version 4.02 
 
C. Toxicity Definition for Leukapheresis 
 
Grade 1: Requirement for oral calcium supplementation either during or just after procedure. 
Severe bruising requiring treatment such as warm compresses or anti-inflammatory medication 
for a brief 1 week duration. 
 
Grade 2: Prolonged requirement for calcium administration greater than 24 hours, infection at 
the intravenous site requiring treatment or intervention with antibiotics or surgery. 
 
Grade 3: Alteration in vital signs such as severe hypotension requiring hospitalization, 
hospitalization secondary to infection. 
 
D. Dose Limiting Toxicity (DLT) 
 
Since the agent being administered is a biologic agent prepared from the subjects own cells as 
opposed to a single drug we will lump the toxicity together as that caused by the process of 
preparation of the DC vaccine. A subject that develops any grade 3 or greater toxicity per the 
NIH Cancer Therapy Evaluation Program Common Toxicity Criteria version 4.03 except for 
fever greater than 40oC for <12 hours will be taken off protocol.  A continuous monitoring rule 
for 
dose-limiting toxicity will be followed during the trial, as described in section 6.11 Statistical 
Considerations. 
 
Grade 2 allergic reaction: generalized urticaria 
Greater than or equal Grade 3 allergic reaction 
Greater than or equal Grade 2 autoimmune reaction 
Greater than or equal Grade 3 cardiac toxicity 
Any Greater than or equal Grade 3 hematologic and non-hematologic toxicity. 
Greater than or equal Grade 2 toxicity for leukapheresis 
 
E. Adverse Events 
 
Adverse events will be graded according to NCI Common Toxicity Criteria (version 4.03 
seehttps://evs.nci.nih.gov/ftp1/CTCAE/About.html.  A copy is enclosed in the Appendix. 
 
1. Adverse Event Reporting 
 
All adverse events will whether or not associated with the study agents be recorded on the CRF. 
Adverse Events will be reported to the IRB, Data Safety and Monitoring Committee (DSMC), 
Moffitt Cancer Center Protocol Monitoring Committee (PMC);Safety Monitoring Committee, 
GCRC, FDA, and NIH.  The information collected includes: 
 
•The time of onset of any new adverse event or worsening of a previous adverse event 

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•The specific type of adverse reaction in standard medical terminology 
•The duration of the adverse event 
•The grade of the adverse event 
•The attribution of the adverse event to the study reagents 
•Description of action taken in treating the adverse event and/or change in study administration 
of dose 
 
NCI Adverse Event Reporting for Local, Investigator-Initiated Phase I Trials 
 
UNEXPECTED EVENT 
EXPECTED EVENT 
Grade 2-3 
Attribution of 
Possible, Probable 
or Definite 
Grades 4 and 5 
Regardless of 
Attribution 
Grades 1-3 
Grades 4 and 5 
Regardless of 
Attribution 
Grade 2 – Expedited 
report to within 10 
working days 
Grade 3 – Report by 
phone/fax within 24 
hrs. Expedited 
reports to follow 
within 10 working 
days. 
Grade 1 – adverse 
event reporting not 
required 
Report by phone 
within 24 hrs. 
Expedited report to 
follow within 10 
working days 
NOTE: this includes 
deaths within 30 
days of the last dose 
of treatment. 
Adverse Event 
Expedited Reporting 
is NOT required. 
Report by phone 
within 24 hrs. 
Expedited report to 
follow within 10 
working days. 
NOTE: This 
includes deaths 
within 30 days of 
the last dose of 
treatment. 
 
Adverse events will be reported to the IRB in accordance with their SOPs.  
 
 
2. Serious Adverse Event Reporting 
 
Deaths that are unforeseen and indicate that other participants or others are at increased risk will 
be reported to the IRB at the University of Pennsylvania, the Data Safety and Monitoring 

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Committee (DSMC), Moffitt Cancer Center Protocol Monitoring Committee (PMC); Safety 
Monitoring Committee, GCRC, FDA, and NIH within 24 hours of the time the investigator 
becomes aware of the event. Deaths that occur that do not meet the above definition will be 
reported within 72 hours of the time the investigator becomes aware of the event. 
 
Serious Adverse Events will be reported to the IRB, Data Safety and Monitoring Committee 
(DSMC), Safety Monitoring Committee, Moffitt Cancer Center Protocol Monitoring Committee 
(PMC) GCRC, FDA, and NIH. 
 
 SAEs will be reported to the IRB promptly within the IRB’s guidelines 
 
All on-site SAEs will be sent to Abramson Cancer Center’s Data Safety and Monitoring 
Committee (DSMC) and Moffitt Cancer Center Protocol Monitoring Committee (PMC) according 
to the following guidelines: all grade 3 or higher events within ten days of knowledge, all 
unexpected deaths within 24 hours of knowledge, all others deaths within 30 days of knowledge. 
 
All SAE’s to the Safety Monitoring Committee and Moffitt Cancer Center Protocol Monitoring 
Committee (PMC) biannually in a written report including all AE’s which will be distributed by 
intramural mail and email. 
 
All SAE’s observed at University of Pennsylvania will be reported  to the GCRC promptly, but 
within three days with a written report of the incident via intramural mail and email. 
 
Serious Adverse Events that are not “unexpected” will be reported to the FDA annually in the 
Annual Safety Report. SAEs that are drug-related and unexpected will be reported to the FDA 
within 15 calendar days of discovery of the event via IND safety report. SAEs that are drug- 
related, unexpected and life-threatening or fatal will be reported to the FDA within 7 calendar 
days of discovery of the event via IND safety report by telephone or fax. 
SAEs will be reported to the NIH promptly, but within three days with a written report via email 
and mail. 
 
 
 
6.8 Immunologic Testing 
 
To ascertain the immunologic efficacy of the prepared vaccines, several parameters will be 
investigated at the initiation and then again at the conclusion of the four vaccine regimen. These 
include in vitro sensitization assays to determine the development of HER-2/neu CD4+ and CD8+ 
T  cell  precursors.    For  each  subject,  immunologic  evaluations  on  pre  and  post vaccination 
samples will be conducted simultaneously to minimize discrepancies in technique and culture 
conditions. 
 
To assess the in vivo immunologic effects of our DC vaccine, CD4+ T and CD8+ T cells will be 
assessed before and after the vaccine protocol for qualitative evidence of HER-2/neu 
sensitization.   In addition T cells from the sentinel node will be tested post vaccination.   If   this 

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Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence for Patients with HER-2 Driven High Risk Invasive Breast Cancer 
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Administration of HER-2/neu pulsed DC may result in several potential complications. First the 
administration of activated DC may cause transient hypotension from release of cytokines. This 
is a theoretic possibility and has not been observed in mouse models or human trials 
administering DC alone without IL-2 or other cytokines. The second possible complication 
includes the induction of autoimmune reactions to normal epithelial cells expressing the HER- 
2/neu antigen. However, in animal models of successful immunity to HER-2/neu protein using 
HER-2/neu ECD and ICD peptides, no evidence of autoimmunity has been noted. Additionally, 
in DC trials of melanoma and prostate, no clinical evidence of severe autoimmunity directed 
against normal tissues sharing the targeted tumor antigen has been discovered. To date, in our 
own trial using peptide pulsed DC1s for vaccination in subjects with DCIS, the protocol has been 
well tolerated with toxicities of grade 1 fever, chills, headache, fatigue, and injection site 
tenderness. 
 
Recent data from studies of Herceptin, a monoclonal antibody targeting HER-2/neu, have 
reported a small incidence of cardiomyopathy likely related to low level expression of HER- 
2/neu on cardiac cells. The risk of this complication is estimated at 5%. This toxicity is a result 
of an antibody and it is anticipated that with low level expression that T cells will be able to 
discriminate against the heart. In addition since the heart cells are MHC class II negative CD4+ 
T cells would not interact with these cardiac cells. 
 
This vaccine has not been previously administered to individuals who have been treated with 
Herceptin. Subjects on this trial will have cardiac evaluation of MUGA or echocardiogram to 
determine their ejection fraction. Subjects will also be closely monitored for signs/symptoms of 
heart failure. In our first trial there was no evidence of clinically relevant cardiac failure. Less 
than 8% of subjects had asymptomatic declines in ejection fractions post-vaccine, thought all 
EFs were still within normal range. There have been no long term cardiac toxicities noted as a 
result of this vaccine. Additionally, Herceptin does not cause long term cardiac toxicity issues. 
We do not expect the cardiac toxicity of prior Herceptin treatment and treatment with the vaccine 
to be additive. 
 
It has been demonstrated that fetuses express HER-2/neu during the third trimester and there is 
the possibility of spontaneous abortion as a result of anti-HER-2/neu T cell responses against the 
fetus.  Therefore women of childbearing years will be made aware of this possibility. 
 
8.0 Consent Procedure 
 
The nature of the study and the procedures to be performed will be reviewed with the subjects by 
the Principal Investigator or his/her designee. The subjects will be given ample time to ask 
questions and decide on participation in the study. A consent form will be signed and kept in the 
research file and a copy on the patient’s chart. Any patient given vaccine or boost at Moffitt will 
be re-consented using Moffitt Cancer Center consent form. 
 
9.0 Protection of Subjects 
 
Subject records and data sheets will be kept in individual research files. The subject’s chart will 
document participation in the study with the protocol name and number.  Under no circumstance 

Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence for Patients with HER-2 Driven High Risk Invasive Breast Cancer 
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will any medical record or donor information be issued to persons outside this study unless 
deemed medically indicated. Donor confidentiality will be maintained in any manuscripts or 
documents that are prepared for scientific publication. All routine safe medical practices applied 
to subjects with IBC will be used to keep this protocol as safe as possible for the subjects treated. 
Once IND officially moved to Moffitt Cancer Center and IRB approvals noted records of the  
trial and data will be transported to Moffitt Cancer Center using secured Aegis Fortress 
encryption drive to regulatory site at Moffitt Cancer Center. 
 
10.0 Potential Benefits 
 
For subjects enrolled in this trial there is no evidence to suggest any likelihood of benefit. The 
subjects enrolled have significant risk of systemic recurrence and targeting HER-2/neu may 
reduce the risk of subsequent recurrence and if this study is safe and generates immune responses 
would lead to additional trials to test for reducing recurrence. 
 
11.0 Exceptions and Deviations 
 
 
There will be no exceptions to eligibility, contraindicated treatment/therapies/interventions or safety 
tests. 
 
An exception is a one time, intentional action or process that departs from the IRB and CTSRMC 
approved study protocol, intended for one occurrence. If the action disrupts the study progress, such 
that the study design or outcome (endpoints) may be compromised, or the action compromises the 
safety and welfare of study subjects, advance documented approval will be requested from the IRB, 
DSMC, Moffitt Cancer Center PMC and Safety Monitoring Committee. 
 
A deviation is a one time, unintentional action or process that departs from the IRB and DSMC 
approved study protocol, involving one incident and identified retrospectively, after the event 
occurred. If the impact on the protocol disrupts the study design, may affect the outcome (endpoints) 
or compromises the safety and welfare of the subjects, the deviation will be reported to the DSMC 
and Safety Monitoring Committee within 5 business days and to the IRB within 10 business days. 
 
 
12.0 Risk/Benefit Ratio 
 
In breast cancer, there remain a significant proportion of subjects who will not benefit from the 
current adjuvant therapies and relapse with metastatic disease. Immunotherapy offers a new 
approach to adjuvant therapy and one that may even be applied in prevention of breast cancer. 
Therefore, the immunologic information gathered from studies such as this one have the  
potential to impact large numbers of subjects with breast cancer or at high risk for breast cancer. 
The recent DC trials for melanoma and prostate have demonstrated that such vaccines can be 
administered safely without adverse effects. Therefore, with minimal risk to the  subjects  
enrolled in this study, information will be gathered that can determine the feasibility of a whole 
new approach for breast cancer care. 

Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence for Patients with HER-2 Driven High Risk Invasive Breast Cancer 
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13.0 References 
 
1. 
Teng MW, Swann JB, Koebel CM, Schreiber RD, Smyth MJ. Immune-mediated dormancy: an 
equilibrium with cancer. J Leukoc Biol. Oct 2008;84(4):988-993. 
2. 
Kalinski P, Mailliard RB, Giermasz A, et al. Natural killer-dendritic cell cross-talk in cancer 
immunotherapy. Expert Opin Biol Ther. Oct 2005;5(10):1303-1315. 
3. 
Czerniecki BJ, Koski GK, Koldovsky U, et al. Targeting HER-2/neu in early breast cancer 
development using dendritic cells with staged interleukin-12 burst secretion. Cancer Res. Feb 15 
2007;67(4):1842-1852. 
4. 
Sharma A, Koldovsky U, Xu S, et al. HER-2 pulsed dendritic cell vaccine can eliminate HER-2 
expression and impact ductal carcinoma in situ. Cancer. Sep 1;118(17):4354-4362. 
5. 
Untch M, Fasching PA, Konecny GE, et al. Pathologic complete response after neoadjuvant 
chemotherapy plus trastuzumab predicts favorable survival in human epidermal growth factor 
receptor 2-overexpressing breast cancer: results from the TECHNO trial of the AGO and GBG 
study groups. J Clin Oncol. Sep 1;29(25):3351-3357. 
6. 
Rossi V, Sarotto I, Maggiorotto F, et al. Moderate Immunohistochemical Expression of HER-2 
(2+) Without HER-2 Gene Amplification Is a Negative Prognostic Factor in Early Breast Cancer. 
Oncologist.17(11):1418-1425. 
7. 
Becker S, Becker-Pergola G, Fehm T, Wallwiener D, Solomayer EF. Her2 expression on 
disseminated tumor cells from bone marrow of breast cancer patients. Anticancer Res. May-Jun 
2005;25(3B):2171-2175. 
8. 
Sassen A, Rochon J, Wild P, et al. Cytogenetic analysis of HER1/EGFR, HER2, HER3 and HER4 
in 278 breast cancer patients. Breast Cancer Res. 2008;10(1):R2. 
9. 
Eccles SA. The epidermal growth factor receptor/Erb-B/HER family in normal and malignant 
breast biology. Int J Dev Biol.55(7-9):685-696. 
10. 
Wang SE, Narasanna A, Perez-Torres M, et al. HER2 kinase domain mutation results in 
constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine 
kinase inhibitors. Cancer Cell. Jul 2006;10(1):25-38. 
11. 
Siegel PM, Muller WJ. Mutations affecting conserved cysteine residues within the extracellular 
domain of Neu promote receptor dimerization and activation. Proc Natl Acad Sci U S A. Aug 20 
1996;93(17):8878-8883. 
12. 
Ursini-Siegel J, Schade B, Cardiff RD, Muller WJ. Insights from transgenic mouse models of 
ERBB2-induced breast cancer. Nat Rev Cancer. May 2007;7(5):389-397. 
13. 
Roses RE, Paulson EC, Sharma A, et al. HER-2/neu overexpression as a predictor for the 
transition from in situ to invasive breast cancer. Cancer Epidemiol Biomarkers Prev. May 
2009;18(5):1386-1389. 
14. 
Harada S, Mick R, Roses RE, et al. The significance of HER-2/neu receptor positivity and 
immunophenotype in ductal carcinoma in situ with early invasive disease. J Surg Oncol. 
Oct;104(5):458-465. 
15. 
Mahmoud SM, Paish EC, Powe DG, et al. Tumor-infiltrating CD8+ lymphocytes predict clinical 
outcome in breast cancer. J Clin Oncol. May 20;29(15):1949-1955. 
16. 
Kohrt HE, Nouri N, Nowels K, Johnson D, Holmes S, Lee PP. Profile of immune cells in axillary 
lymph nodes predicts disease-free survival in breast cancer. PLoS Med. Sep 2005;2(9):e284. 
17. 
Allinen M, Beroukhim R, Cai L, et al. Molecular characterization of the tumor microenvironment in 
breast cancer. Cancer Cell. Jul 2004;6(1):17-32. 
18. 
Artacho-Cordon A, Artacho-Cordon F, Rios-Arrabal S, Calvente I, Nunez MI. Tumor 
microenvironment and breast cancer progression: a complex scenario. Cancer Biol Ther. Jan 
1;13(1):14-24. 
19. 
Taieb J, Chaput N, Schartz N, et al. Chemoimmunotherapy of tumors: cyclophosphamide 
synergizes with exosome based vaccines. J Immunol. Mar 1 2006;176(5):2722-2729. 
20. 
Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination with chemotherapy 
versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro- 
oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet. 
Aug 28;376(9742):687-697. 

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21. 
Vaught DB, Stanford JC, Young C, et al. HER3 is required for HER2-induced preneoplastic 
changes to the breast epithelium and tumor formation. Cancer Res. May 15;72(10):2672-2682. 
22. 
Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JM. Expression of the HER1-4 family of 
receptor tyrosine kinases in breast cancer. J Pathol. Jul 2003;200(3):290-297. 
23. 
Gianni L, Pienkowski T, Im YH, et al. Efficacy and safety of neoadjuvant pertuzumab and 
trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer 
(NeoSphere): a randomised multicentre, open-label, phase 2 trial. Lancet Oncol. Jan;13(1):25-32. 
24. 
Frogne T, Benjaminsen RV, Sonne-Hansen K, et al. Activation of ErbB3, EGFR and Erk is 
essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant. 
Breast Cancer Res Treat. Mar 2009;114(2):263-275. 
25. 
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. Jan 9 
1987;235(4785):177-182. 
26. 
Bauer KR, Brown M, Cress RD, Parise CA, Caggiano V. Descriptive analysis of estrogen receptor 
(ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, 
the so-called triple-negative phenotype: a population-based study from the California cancer 
Registry. Cancer. May 1 2007;109(9):1721-1728. 
27. 
Dent R, Trudeau M, Pritchard KI, et al. Triple-negative breast cancer: clinical features and 
patterns of recurrence. Clin Cancer Res. Aug 1 2007;13(15 Pt 1):4429-4434. 
28. 
Duru N, Fan M, Candas D, et al. HER2-Associated Radioresistance of Breast Cancer Stem Cells 
Isolated from HER2-Negative Breast Cancer Cells. Clin Cancer Res. Nov 16. 
29. 
Simon R, Nocito A, Hubscher T, et al. Patterns of her-2/neu amplification and overexpression in 
primary and metastatic breast cancer. J Natl Cancer Inst. Aug 1 2001;93(15):1141-1146. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Pilot Phase I HER-2 Pulsed DC Vaccine to Prevent Recurrence for Patients with HER-2 Driven High Risk Invasive Breast Cancer 
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Appendix 1 – Schedule of Assessments 
 
Procedures 
Baseline
/ Pre 
Vaccine 
Vaccin
e 1 
Vaccine 
2 
Vaccine 
3 
Vaccine 
4 
Vaccine 
5 
Vaccine 
6 
Booster 
1 
Booster 
2 
Booster 
3 
EOT 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
Tumor Biopsy1 
X 
 
 
 
 
 
 
 
 
 
 
Biomarkers2 
X 
 
 
 
 
 
 
 
 
 
 
Medical Hx 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Vital Signs 
X 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
X3 
 
Physical Exam 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
 
 
 
 
 
 
 
 
 
 
CBC w/diff 
X 
 
 
 
 
 
 
X 
 
 
 
PT/PTT 
X 
 
 
 
 
 
 
X 
 
 
 
CMP 
X 
 
 
 
 
 
 
X 
 
 
 
Urinalysis 
X 
 
 
 
 
 
 
X 
 
 
 
Pregnancy 
Test 
X 
 
 
 
 
 
 
 
 
 
 
EKG 
X 
 
 
 
 
 
 
 
 
 
 
HLA 
X 
 
 
 
 
 
 
 
 
 
 
LVEF4 
X 
 
 
 
 
 
 
X 
 
 
 
Immune 
Assays 
X 
 
 
 
 
 
 
X 
X 
X 
 
ECOG PS 
X 
 
 
 
 
 
 
 
 
 
 
Mammogram 
X 
 
 
 
 
 
 
 
 
 
 
CT/PET 
X 
 
 
 
 
 
 
 
 
 
 
Ultrasound 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
AE 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Leukapheresis 
X 
 
 
 
 
 
X5 
 
 
X5 
 
 
 
 
Procedures 
FOLLOW UP 
 
 
 
 
 
6 mos. 
Y1 
Y2 
Y3 
Y4 
Y5 
Consent 
 
 
 
 
 
 
Tumor Biopsy1 
 
 
 
 
 
 
Biomarkers2 
 
 
 
 
 
 
Medical Hx 
X 
 
 
 
 
 
Vital Signs 
X 
 
 
 
 
 
Physical Exam 
X 
 
 
 
 
 
Height 
 
 
 
 
 
 
Weight 
 
 
 
 
 
 
CBC w/diff 
X 
 
 
 
 
 
PT/PTT 
X 
 
 
 
 
 
CMP 
X 
 
 
 
 
 
Urinalysis 
 
 
 
 
 
 
Pregnancy 
Test 
 
 
 
 
 
 
EKG 
 
 
 
 
 
 
HLA 
 
 
 
 
 
 
LVEF4 
 
 
 
 
 
 
Immune 
Assays 
X 
 
 
 
 
 
ECOG PS 
 
 
 
 
 
 
Mammogram 
 
X 
X 
X 
X 
X 
CT/PET 
 
 
 
 
 
 
Ultrasound 
 
 
 
 
 
 
AE 
 
 
 
 
 
 
Leukapheresis 
X5 
 
 
 
 
 
1. 
Core biopsy or excisional biopsy demonstrating IBC.  
2. 
Biomarkers collected: ER, PR, HER-2/neu and FISH 
3. 
Collect VS prior to injection and 15 (+/- 5 min) post injection 
4. 
Subject is closely monitored and the need for additional LVEF during the 6 month post-vaccine monitoring period is determined on 
case-by-case basis.  
5. 
Optional leukapheresis, done only if needed