Clinical trial NCT02297698
Conditions: Breast Cancer
Keywords: Breast cancer, NeuVax
Sponsor: Cancer Insight, LLC
Start date: 2014-10-01; Completion date: 2021-12-01
Enrollment: 100 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This will be a multi-center, prospective, randomized, single-blinded, placebo-controlled phase II trial of trastuzumab + nelipepimut-S/GM-CSF versus trastuzumab + GM-CSF alone. Our target study population is high-risk HER2-positive breast cancer patients. High-risk HER2-positive breast cancer patients are defined as:

Those with HER2-positive breast cancer, regardless of hormone receptor status, who receive neoadjuvant therapy with an approved regimen that includes trastuzumab and at least four cycles (12 weeks) of taxane-containing chemotherapy, and fail to achieve a pCR.

Those with HER2-positive breast cancer, regardless of hormone receptor status, who undergo surgery as a first intervention and are found to have ≥ 4 positive lymph nodes.

Those with HER2-positive, hormone receptor negative breast cancer who undergo surgery as a first intervention and are found to have 1-3 positive lymph nodes.

Disease-free subjects after standard of care multi-modality therapy will be screened and HLA-typed.

Detailed description:
In this study, the investigators intend to assess the ability of the combination of trastuzumab and the HER2 vaccine nelipepimut-S (administered with the immunoadjuvant GM-CSF) given in the adjuvant setting to prevent recurrences in patients with high-risk HER2-positive breast cancer. High-risk is defined as those patients that do not achieve a pCR after neoadjuvant therapy with an approved regimen that includes trastuzumab and at least four cycles (12 weeks) of taxane-containing chemotherapy or those who undergo upfront surgery and are found to have greater than or equal to four positive lymph nodes regardless of hormone receptor status or 1-3 positive lymph nodes and are hormone receptor negative.

Following surgery, patients will be screened and HLA-typed (consent #1). Nelipepimut-S is a CD8-eliciting peptide vaccine that is restricted to HLA-2+ or HLA-A3+ or HLA-A24+ or HLA-A26+ patients (approximately 80% of the US population). HLA-A2+/A3+/A24+/orA26+ patients who meet all other eligibility criteria will be randomized to receive trastuzumab + nelipepimut-S/GM-CSF or trastuzumab + GM-CSF alone (consent #2). The trastuzumab will be administered to all patients consistent with current standard of care. Patients randomized to the nelipepimut-S/GM-CSF arm will receive vaccinations of nelipepimut-S (1000 mcg) and GM-CSF (250 mcg) administered intradermally every three weeks for six total vaccinations, 30-120 minutes after completion of trastuzumab infusion. The first vaccination will be given with the third dose of maintenance trastuzumab administered as monotherapy optimally, but may be given with later maintenance doses of trastuzumab, provided there are at least six remaining doses of trastuzumab to overlap with the primary vaccine series. Patients randomized to the GM-CSF alone arm will receive inoculations of GM-CSF (250 mcg) administered in an identical manner to those receiving nelipepimut-S/GM-CSF. Patients will be blinded as to whether they are receiving nelipepimut-S/GM-CSF or GM-CSF alone.

Upon completion of the primary vaccination/inoculation series, booster inoculations (same dose and route) will be administered every six months x 4. The first booster inoculation will occur 6 months ± 2 weeks after the completion of the PVS, with subsequent boosters timed every six months + 2 weeks. Boosters will therefore occur at the following time points after completion of the PVS: 6 months ± 2 weeks, 12 months ± 2 weeks, 18 months ± 2 weeks, 24 months ± 2 weeks. Booster inoculations will occur for patients randomized to receive nelipepimut-S/GM-CSF as well as patients randomized to receive GM-CSF alone, and will consist of the same treatment drugs and dosing (i.e., nelipepimut-S/GM-CSF patients will be boosted with nelipepimut-S/GM-CSF while GM-CSF alone patients will be boosted with GM-CSF alone). Patient blinding will be maintained throughout the study.

Subjects will be followed for safety issues, immunologic response and clinical recurrence. Patients will be monitored 48-72 hours after each inoculation for reaction to the inoculation as well as documentation of any adverse effects experienced. Immunologic response will be monitored primarily by in vivo delayed type hypersensitivity (DTH) reactions but also may be documented by other immunologic assays. All patients will be followed for a total of 36 months from the time of initiation of trastuzumab maintenance therapy to document disease-free status.

Arms: arm 1: Patients randomized to this arm will receive vaccinations of nelipepimut-S (1000 μg) and GM-CSF (250 μg) (NeuVax vaccine) administered intradermally every three weeks for six total vaccinations, 30-120 minutes after completion of trastuzumab infusion. The first vaccination will be given with the third dose of maintenance trastuzumab administered as monotherapy optimally, but may be given with later maintenance doses of trastuzumab, provided there are at least six remaining doses of trastuzumbab to overlap with the PVS. Upon completion of the primary vaccination series (PVS), booster inoculations (same dose and route) will be administered every six months x 4. The first booster inoculation will occur 6 months ± 2 weeks after the completion of the PVS, with subsequent boosters timed every six months + 2 weeks. Boosters will therefore occur at the following time points after completion of the PVS: 6 months ± 2 weeks, 12 months ± 2 weeks, 18 months ± 2 weeks and 24 months ± 2 weeks. arm 2: Patients randomized to this arm will receive inoculations of GM-CSF (250 μg) administered in an identical manner to those receiving nelipepimut-S/GM-CSF (NeuVax). Patients will be blinded as to whether they are receiving nelipepimut-S/GM-CSF or GM-CSF alone. Upon completion of the primary vaccination series (PVS), booster inoculations (same dose and route) will be administered every six months x 4. The first booster inoculation will occur 6 months ± 2 weeks after the completion of the PVS, with subsequent boosters timed every six months + 2 weeks. Boosters will therefore occur at the following time points after completion of the PVS: 6 months ± 2 weeks, 12 months ± 2 weeks, 18 months ± 2 weeks and 24 months ± 2 weeks.
Interventions: intervention 1: NeuVax vaccine intervention 2: Trastuzumab intervention 3: GM-CSF
Intervention descriptions: intervention 1: At the time of vaccine administration, a frozen solution of E75 acetate (1.5 mg/mL) is thawed and 1000mcg E75 peptide mixed thoroughly with 250mcg GM-CSF. This constitutes the NeuVax vaccine. Patients randomized to this arm will receive vaccinations of nelipepimut-S/GM-CSF administered intradermally every three weeks for six total vaccinations, 30-120 minutes after completion of trastuzumab infusion. The first vaccination will be given with the third dose of maintenance trastuzumab administered as monotherapy optimally, but may be given with later maintenance doses of trastuzumab, provided there are at least six remaining doses of trastuzumab to overlap with the primary vaccine series. intervention 2: Herceptin will be administered to patients every three weeks as monotherapy for one year, to be given upon completion of standard of care chemotherapy/radiotherapy. The first trastuzumab infusion will be given no sooner than three weeks and no later than 12 weeks after completion of chemotherapy/radiotherapy. Trastuzumab will be dosed at the recommended initial loading dose of 8 mg/kg and at recommended maintenance doses of 6 mg/kg q3wk. intervention 3: For patients randomized to the GM-CSF alone arm, they will receive inoculations of GM-CSF (250mcg) administered intradermally every three weeks for six total vaccinations, 30-120 minutes after completion of trastuzumab infusion. The first injection will be given with the third dose of maintenance trastuzumab administered as monotherapy optimally, but may be given with later maintenance doses of trastuzumab, provided there are at least six remaining doses of trastuzumab to overlap with the primary vaccine series.

Protocol documents:
Prot_SAP_000.pdf: 
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Protocol: 2014-0443 
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Protocol Title: 
Phase II Trial of Combination Immunotherapy 
with nelipepimut-S + GM-CSF (NeuVax™) and 
Trastuzumab in high-risk HER2+ Breast 
Cancer Patients 
 
Study Drugs: 
Nelipepimut-S + GM-CSF (NeuVax™) 
Financial Sponsor: 
Galena Biopharma (to provide NeuVax™) 
Director: 
Cancer Insight, LLC 
IND: 
BB-IND#14919 
 
Principal Investigator (PI): 
Elizabeth A. Mittendorf, MD, PhD 
Associate Professor 
Department of Surgical Oncology 
The University of Texas MD Anderson Cancer 
Center 
1400 Pressler St 
Unit 1484 
Houston, TX 77030 
 
Medical Monitor: 
John Renshaw, MD 
Department of Medicine: 
Hematology/Oncology Services 
Brooke Army Medical Center 
3551 Roger Brooke Drive 
Fort Sam Houston, TX 78234 
 
IND Sponsor/Investigator 
Program Director: 
George E. Peoples, MD 
Cancer Insight, LLC 
110 E. Houston Street, Floor 7 
San Antonio, TX 78205 
 
 
Protocol Version/Date: 
2.1/11 April 2016 
Protocol Number: 
2014-0443 


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Table of Contents 
 
 
Page 
1. INTRODUCTION 
4 
1.1 Disease background 
4 
1.2 HER2-positive breast cancer 
4 
1.3 Nelipepimut-S 
8 
1.4 Combined HER2-directed therapy 
11 
2. OBJECTIVES 
13 
2.1 Primary objectives 
13 
2.2 Secondary objectives 
13 
3. STUDY DESIGN 
14 
3.1 Description of study 
14 
3.2 Rationale for study design 
15 
3.3 Outcome measures 
16 
3.3.1 Primary outcome measures 
16 
3.3.2 Secondary outcome measures 
16 
3.4 Safety considerations 
17 
3.4.1 Trastuzumab 
17 
3.4.2 Nelipepimut-S and GM-CSF 
18 
3.5 Compliance with laws and regulations 
19 
4. MATERIALS AND METHODS 
19 
4.1 Patients 
19 
4.1.1 Patient selection 
19 
4.1.2 Inclusion criteria 
20 
4.1.3 Exclusion criteria 
23 
4.2 Method of treatment assignment 
23 
4.3 Study treatment 
23 
4.3.1 Trastuzumab 
24 
4.3.2 Vaccine 
24 
4.3.2.1 Dosage and preparation 
24 
4.3.2.2 Storage 
25 
4.3.3 Inoculation series administration 
25 
4.3.4 Booster inoculations 
26 
4.4 Study assessments 
26 
4.4.1 Pretreatment assessments 
26 
4.4.2 Assessments during treatment 
27 
4.4.3 Follow-up assessments 
27 
4.4.4 Blood collection and processing 
28 
4.4.5 HLA-A2/A3/A24/A26 testing 
29 
4.4.6 Dextramer assay 
29 
4.4.7 Local reaction 
29 
4.4.8 DTH reaction 
30 
4.5 Discontinuation of protocol-specific therapy 
30 
4.6 Patient discontinuation 
31 


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4.7 Study discontinuation 
32 
4.8 Data collection 
32 
4.9 Statistical methods 
33 
4.9.1 Rationale for study design 
33 
4.9.2 Sample size determination 
33 
4.9.3 Data analysis 
34 
4.9.3.1 Study patient characteristics 
34 
4.9.3.2 Primary efficacy analysis 
34 
4.9.3.3 Secondary efficacy analysis 
35 
4.9.4 Withdrawal 
36 
4.9.5 Missing data 
36 
4.9.6 Interim analysis 
36 
5. ADVERSE EVENTS 
36 
5.1 Adverse event and reporting definitions 
36 
5.2 Reporting of serious adverse events associated with 
37 
this study 
5.2.1 MedWatch 3500A reporting guidelines 
38 
5.3 Reporting requirements for IND holders 
39 
6. INVESTIGATOR REQUIREMENTS 
40 
6.1 Study initiation 
40 
6.2 Study completion 
40 
6.3 Informed consent 
41 
6.4 Institutional review board or ethics committee approval 
41 
6.5 Study monitoring requirements 
42 
6.6 Data safety monitoring plan 
42 
6.7 Study medication accountability 
42 
6.8 Disclosure of data 
43 
6.9 Retention of records 
43 
6.10 Publications 
43 
6.11 Changes to protocol 
43 
 
Trial Appendices 
Appendix A 
Trial schema 
Appendix B 
Vaccination timeline 
Appendix C 
NCI Common Terminology Criteria for Adverse Events, v4.03 
Appendix D 
E75 + GM-CSF and DTH Formulation Plan 
Appendix E 
Leukine package insert 
Appendix F 
ECOG performance status criteria 
Appendix G 
Data Safety Monitoring Plan (free standing document) 
Appendix H 
Adverse event reporting algorithm 
Appendix I 
MedWatch FDA form 3500 link 
Appendix J 
Study Flowchart 
Appendix K 
PI agreement and signature page 
 
1.0 
INTRODUCTION 


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1.1 
Disease background 
 
Breast cancer is the most commonly diagnosed malignancy in women (excluding 
cancers of the skin) and is the second leading cause of cancer mortality. The 
American Cancer Society estimated that there will be over 230,000 new cases of 
breast cancer diagnosed in 2013, with an estimated 40,000 women expected to 
die from this disease annually. 1 Patients with breast cancer are treated with a 
combination of therapies to include surgery, chemotherapy, endocrine therapy, 
targeted monoclonal antibody (mAb) therapy and radiation as dictated by 
characteristics of their tumor and disease stage. 2 Patients with hormone 
receptor (HR) positive tumors are routinely administered endocrine therapy and 
patients with HER2-positive tumors receive trastuzumab, a humanized mAb 
targeting HER2, as a component of their standard of care therapy. Despite 
advances in treatment, approximately 10% of women with breast cancer will 
recur and succumb to their disease by the five-year mark; a number that 
increases to 17% at ten years. 1 
 
1.2 
HER2-positive breast cancer 
 
HER2 is a member of the epidermal growth factor receptor family of receptor 
tyrosine kinases. It is expressed in approximately 70-80% of invasive breast 
cancers and amplified and/or overexpressed (= HER2-positive) in approximately 
20%. 3,4 HER2-positive breast cancers are more aggressive and susceptible to 
recurrence than HER2-normal cancers. HER2 is routinely assessed on breast 
tumors using immunohistochemistry (IHC) to detect protein expression or in situ 
hybridization (ISH) to identify gene amplification.  IHC results are scored as 0, 
1+, 2+ or 3+. ISH reports a ratio of the number of HER2 gene copies to 
chromosome 17 copies.  According to the 2013 update of the American Society 
of Clinical Oncology (ASCO) and College of American Pathologist (CAP) 
guidelines, a tumor is considered HER2-positive if any of the following criteria are 
met: 5 
 
• IHC 3+ 
• Dual-probe ISH ratio (HER2/CEP17) > 2.0 with HER2 copy number of 
>4.0/cell 
• Dual-probe ISH ratio (HER2/CEP17) > 2.0 with HER2 copy number of 
<4.0/cell 
• < 2.0 overall ratio with HER2 copy number of > 6.0/cell 
• Single probe tests average HER2 copy number of > 6.0/cell 
 
Patients with HER2-positive tumors are treated with trastuzumab, a mAb that 
targets the extracellular portion of the HER2 protein. Trastuzumab was first 
evaluated in the metastatic setting where it was shown to provide significant 
clinical benefit as monotherapy and in combination with chemotherapy as first- or 


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second-line therapy. 6-11 Benefit of trastuzumab in the adjuvant setting for early- 
stage breast cancer has also been demonstrated. Four large trials, and several 
smaller studies, conducted in the adjuvant setting showed significant 
improvements in disease-free survival (DFS) (36%-52% reduction in DFS events) 
and overall survival (OS) (33% to 37% reduction in deaths), independent of 
patient age, tumor size, nodal status, or hormone receptor status. 12-16 Two of 
these trials, the National Surgical Adjuvant Breast and Bowel Project (NSABP) B- 
31 and North Central Cancer Treatment Group (NCCTG) N9831 trials were 
conducted in North America. In these two studies, patients with HER2-positive 
breast cancer were randomized to adriamycin and cyclophosphamide followed 
by paclitaxel with or without trastuzumab. Because they were similar in design, 
the trials were analyzed jointly. At the most recent report, after 3.9 years of 
median follow-up, women in the trastuzumab arm had significantly increased 
DFS (p<.001; stratified hazard ratio [HR], 0.52; 95% CI, 0.45 to 0.60) and OS 
(p<.001; stratified hazard ratio [HR], 0.61; 95% CI, 0.50 to 0.75) compared with 
women in the control arm. 12 On the basis of these data and the data from the 
other adjuvant trials, trastuzumab has become a critical component of standard 
therapy for HER2-positive early breast cancer. 
 
More recently, the efficacy of trastuzumab administered in combination with 
chemotherapy in the neoadjuvant (preoperative) setting has been evaluated. In 
the first randomized trial to evaluate this approach, Buzdar et al. reported a 67% 
pathologic complete response (pCR) rate, defined as no residual invasive 
disease in the breast or 
axilla, among patients 
receiving neoadjuvant 
chemotherapy plus 
trastuzumab versus a 
pCR rate of 25% among 
patients receiving 
noadjuvant chemotherapy 
alone (p=.02).17 
Subsequent to this, the 
GeparQuattro,18 NOAH,19 
NeoALTTO,20 
NeoSphere21, American 
College of Surgeons 
Oncology Group 
(ACOSOG) Z1041 22 and 
Cancer and Leukemia 
Group B (CALGB) 40601 
23 trials, have also 
demonstrated improved 
pCR rates among patients 
receiving trastuzumab in 


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addition to chemotherapy (table 1). These trials all used different chemotherapy 
backbones but are consistent in demonstrating high pCR rates in HER2-positive 
patients receiving neoadjuvant chemotherapy plus trastuzumab. 
 
There has been recent interest in using dual HER2-targeted therapy to increase 
pCR rates. In the NeoSphere trial, in addition to the arm adding trastuzumab to 
docetaxel where the pCr rate was 29% (31 out of 107), there was another arm 
that added pertuzumab, a monoclonal antibody that binds to HER2 and inhibits 
dimerization of HER2 with other members of the EGFR family of receptors. The 
addition of pertuzumab to trastuzumab and docetaxel increased the pCR rate to 
46% (49 out of 107). 21 The addition of pertuzumab did not significantly increase 
the rate of grade 3 or higher adverse events (AE). The NeoALTTO and CALGB 
trials evaluated the addition of lapatinib, a HER2-targeted receptor tyrosine 
kinase inhibitor. 20,23 In the NeoALTTO trial, the pCR rate in patients receiving 
trastuzumab plus paclitaxel was 30%; a rate that increased to 51% with the 
addition of lapatinib. 20 In the CALGB trial, the pCR rate for patients receiving 
paclitaxel plus trastuzumab was 40% versus 51% when lapatinib was added. 23 
In both trials, the addition of lapatinib led to increased greater than grade 3 
toxicities including diarrhea, neutropenia and liver enzyme alterations. 20,23 
 
In an initial report from MD Anderson, it was shown that patients achieving a pCR 
have improved outcomes when compared to patients not achieving a pCR after 
neoadjuvant chemotherapy plus trastuzumab. In this study, which included 142 
patients with HER2-positive breast cancer that received neoadjuvant 
chemotherapy with a taxane, anthracycline and concomitant trastuzumab, a pCR 
was achieved in 72 (51%). The 3-year Kaplan-Meier estimates of recurrence- 
free survival (RFS) were 96% (95% CI, 91% to 100%) for patients achieving a 
pCR versus 80% (95% CI, 70.8% to 90.5%) for those not achieving a pCR 
(p=.02). 24 
 
In a subsequent report from MD Anderson that evaluated 229 patients with 
HER2-positive breast cancer, a pCR was achieved in 114 (50%). Compared to 
patients achieving < pCR, those with pCR had higher five-year rates of local 
recurrence-free survival (100% versus 95%, P=0.011), distant metastasis (96% 
versus 80%, P<0.001), RFS (96% versus 79%, P<0.001), and OS (95% versus 
84%, P=0.006). Improvements in RFS and OS were seen with decreasing post- 
treatment stage. Failure to achieve a pCR was the strongest independent 
predictor of recurrence (HR=4.09, 95% CI, 1.67-10.04, P=0.002) and death 
(HR=4.15, 95% CI, 1.39-12.38, P=0.011). 25  These data are consistent with 
those reported from the TECHNO trial, a phase II non-randomized study of 217 
patients with HER2-positive breast cancer who received 6 months of neoadjuvant 
epirubicin and paclitaxel-based chemotherapy and trastuzumab. 26 In this study, 
39% of patients achieved a pCR. Three-year DFS was 88% for patients 
achieving a pCR versus 73% for patients not achieving a pCR (p=.01). Three- 
year OS rates were 96% for patients achieving a pCR versus 86% for patients 


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not achieving a pCR (p=.03). pCR was the strongest prognostic factor for 
relapse and death. Disease-free survival outcomes for patients failing to achieve 
a pCR are summarized in table 2. Taken together, these data show that 
achieving a pCR is a strong predictor of long-term outcome and that novel 
therapeutic strategies are needed for patients failing to achieve a pCR. 
 
Also shown in table 2 are survival outcomes for patients enrolled on the BCIRG- 
006 trial that evaluated trastuzumab in the adjuvant setting. These data 
demonstrate that patients who undergo surgery as an initial intervention and are 
found to have pathologically node-positive disease also represent a high-risk 
population. Data from the combined analyses of the NSABP B-31 and N9831 
adjuvant trastuzumab trials showed that the 5-year recurrence free-survival rates 
were higher for those patients with hormone receptor-positive patients (89.4%) 
compared to those with hormone receptor-negative patients (81.6%).27 
 
Taken together, these data suggest that patients with HER2-positive breast 
cancer that receive neoadjuvant chemotherapy with HER2-targeted therapy as 
well as those that undergo upfront surgery and are found to have ≥ 4 positive 
lymph nodes or 1-3 positive lymph nodes and are hormone receptor negative are 
a high-risk group in which novel therapeutic strategies are needed. 


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1.3 
Nelipepimut-S 
 
Interest in the development of cancer vaccines increased after advances in the 
molecular characterization of human tumors led to the identification of tumor- 
associated antigens (TAA) that can be recognized by T lymphocytes. TAAs 
expressed by tumors can elicit a very specific immune response; therefore, a 
vaccine is appealing in that it represents a nontoxic therapeutic modality with 
great specificity. HER2 is one such TAA. Several peptides capable of inducing 
CD8+ T cells (also referred to as CTL) have been described from the HER2 
protein including E75 (Nelipepimut-S), a 9 amino acid peptide derived from the 
HER2 protein’s extracellular domain (aa: 369-377:KIFGSLAFL), which is the 
most studied in laboratory and clinical investigation (reviewed by Mittendorf EA, 
et al.28). 
 
Initial studies investigating E75 in vitro and in an in vivo animal model showed 
that it was capable of inducing a peptide-specific, CTL-mediated immune 
response.29,30 This then led to a number of clinical trials including phase I/II 
studies conducted by our group evaluating E75+GM-CSF in the adjuvant setting. 


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The initial trial, which enrolled node-positive breast cancer patients with tumors 
expressing any degree of HER2 (immunohistochemistry [IHC] 1+, 2+ or 3+), was 
a phase I two-stage safety trial designed with escalating doses of E75 peptide in 
the initial stage followed by schedule alterations in the second stage.31 A trial 
enrolling high-risk node-negative patients was designed to delineate optimal 
biologic dosing. Both trials transitioned to phase II with disease recurrence as 
the primary efficacy endpoint. Patients in both studies had received standard 
therapy including surgery, chemotherapy, and radiation therapy as appropriate 
and were without evidence of disease at the time of enrollment. Because of the 
human leukocyte antigen (HLA)-restriction of the E75 peptide, patients were HLA 
typed after enrollment and then placed in either the treatment arm (HLA-A2+ or 
A3+ patients) or the observation group (HLA-A2- or A3- patients). The goals of 
these trials were to document safety, immunogenicity and clinical efficacy of the 
E75 vaccine. Because the node-positive and node-negative trials were run 
concurrently and the protocols were identical except for overlapping 
doses/schedules, the results were merged for analysis.32 
 
At the time of an initial planned analysis of the combined results of the trials, a 
total of 171 patients were enrolled; 90 patients received the E75 vaccine while 81 
were in the observation arms.32 Combined toxicity was minimal with local 
reactions being grade 1 (81%) and grade 2 (19%). Systemic toxicity was grade 0 
(12%), 1 (71%), 2 (14%), and 3 (2%) (Figure1). The most common systemic 
toxicities were bone pain, flu-like symptoms and fatigue which are attributable to 
the GM-CSF. There were no grade 4 or 5 toxicities. All patients demonstrated in 
vitro immunologic responses and in vivo delayed type hypersensitivity (DTH) 
responses post-vaccination. At a median follow-up of 20 month, the recurrence 
rate seen in the vaccinated patients was 6% compared to 15% for patients in the 
observation group (p=0.04). In light of these encouraging data, the trial was 
continued and follow-up was 
extended to five years. With 
additional follow-up, late 
recurrences were seen in the 
vaccination arms of the trials 
which correlated with 
decreased levels of E75- 
specific CTL. A voluntary 
booster program was initiated 
and vaccinated patients ≥ 6 
months from completion of their 
primary vaccination series 
(PVS) were eligible for 
participation. Patients enrolled 
onto the trial after initiation of 
the booster program were 
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receive booster inoculations. An initial report of the booster inoculations showed 
them to be safe and effective in stimulating E75-specific immunity in patients who 
had waning levels of E75-specific CTL six months following completion of their 
PVS.33 When the length of follow-up for the trials was extended, additional 
analyses were incorporated to include evaluation of DFS at 24 and 60 months. 
With 60 month follow-up completed in all patients, we recently reported the final 
5-year follow-up results. Among the 187 patients (108 HLA-A2/A3+ vaccinated; 
79 HLA-A2/3- controls), the 60-month DFS were 90% for vaccinated patients 
versus 80% for controls (p=.08), a 50% reduction in the relative risk of 
recurrence.34 Because the trials began as dose- and schedule-finding trials, not 
all patients received the dose that was eventually determined to be optimal 
(1000µg E75 + 250µg GM-CSF). When outcomes were evaluated by dosing, the 
DFS rate was 95% for those who 
received the optimal dose (p=.05 
versus unvaccinated controls) 
(Figure 2). When evaluated by 
whether patients received a 
booster inoculation, the DFS rate 
in the 53 boosted patients was 
96% versus 83% in vaccinated 
patients that did not receive a 
booster inoculation (p=.04). 
 
E75 has been licensed to Galena 
Biopharma which has secured the 
nonproprietary drug name 
nelipepimut-S. NeuVax 
(nelipepimut-S + GM-CSF) is 
currently being evaluated in a 
multicenter, multinational, 
prospective, randomized, double- 
blind, controlled phase III study 
(NCT01479244; PI: Mittendorf). 35 
The trial is enrolling NP breast 
cancer patients with HER2 1+/2+ tumors that are HLA-A2/3+ and are disease 
free after standard of care therapy. Patients are randomized to receive 
nelipepimut-S+GM-CSF or GM-CSF alone. The primary endpoint is 36-month 
DFS. 
 
 
Subsequently, E75 has also been found to bind to HLA-A24 and HLA-A26. 
Patients expressing these additional alleles have been included in another 
ongoing trial of NeuVax. Comparative clinical results between A2 and/or A3- 
positive patients and A24 and/or A26-positive patients has not been completed 
yet. 


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1.4 
Combined HER2-directed therapy 
 
Nelipepimut-S and trastuzumab have been shown to have clinical benefit in 
patients with HER2-positive breast cancer. Although these agents share the 
same target, their mechanisms of action are different; therefore, the concept of 
combining these two immunotherapies is novel and worthy of investigation. 
Nelipepimut-S works by stimulating a HER2-specific CTL response. There have 
been multiple mechanisms of action described for trastuzumab including 
decreased signaling by prevention of HER2 dimerization, increased endocytic 
destruction of the HER2 receptor, and inhibition of extracellular domain shedding. 
36 In addition, multiple immune-mediated mechanisms of action have been 
described. Trastuzumab is a human IgG antibody with a conserved Fc portion. 
Early in vitro and in vivo reports support an important role for antibody- 
dependent cell-mediated cytotoxicity (ADCC) mediated by natural killer cells 
which have an Fc receptor (FcR) that binds the Fc portion of trastuzumab.37-39 
More recently, an important role for adaptive immunity in mediating anti-HER2 
therapy effects has been shown. Using a Rag-1-/- mouse that lacks T cells and B 
cells but does have FcR+ innate cells, Park et al. showed that HER2-targeted 
antibody treatment had minimal impact on tumor growth, suggesting that T cells 
are required for antibody activity.40 Consistent with this, when wild-type mice 
were administered a CD8-depleting antibody, the mice showed minimal initial 
responses to antibody therapy then relapsed quickly. In a small study of patients 
with HER2-positive breast cancer receiving trastuzumab, Taylor et al. 
demonstrated generation of a HER2-specific CD4+ T cell response in 6 of 10 
evaluable patients. 41 This T cell response is in part due to increased antigen 
uptake through FcR on dendritic cells present in the tumor microenvironment 
resulting in increased antigen presentation for recognition by T cells. In that 
same study, investigators showed anti-HER2 antibodies in approximately 60% of 
27 evaluable patients during treatment and found that these anti-HER2 humoral 
responses significantly increased during therapy and were associated with 
improvements in clinical response.41 Data presented by Knutson et al. at the 
2013 annual meeting of the American Society of Clinical Oncology showed that 
patients treated on the trastuzumab arm of the NCCTG 9831 trial developed 
HER2-specific antibody responses.42 Cox modeling suggested that larger 
increases in antibody responses were associated with improved DFS (HR=0.23, 
p=.04). There is also data demonstrating an inverse relationship between HER2 
and HLA class I expression suggesting that down-regulation of HER2 may 
increase antigen presentation by upregulating HLA molecules.43 Thus a 
complementary approach to immune-mediated therapy in breast cancer may 
provide synergism resulting in improved clinical outcomes. 
 
We have completed preclinical testing of the combination of trastuzumab with 
HER2-targeted vaccines.44 HER2-expressing tumor cells were incubated 
overnight with trastuzumab in concentrations of 10μg/mL and 50μg/mL. 


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Cytotoxicity of both pretreated and untreated tumor cells was measured using 
peripheral blood mononuclear cells (PBMC) from healthy HLA-A2+ donors as well 
as from patients vaccinated with nelipepimut-S. Cytotoxicity by peptide-specific 
CTL from non-vaccinated patients was increased by 5.6% (10μg /mL) and 15.3% 
(50μg /mL) (p=.002) in pretreated cells over untreated cells. Using PBMC from 
nelipepimut-S-vaccinated patients, peptide-specific cytotoxicity was 34% in 
untreated cells compared to 40% (10μg /mL) (p=.04) and 41% (50μg /mL) 
(p<.05) in pretreated cells. These findings suggest a synergism may exist with 
the combination of trastuzumab and vaccination. 
 
The safety of the combination of trastuzumab and HER2-derived peptide 
vaccines has been evaluated in early clinical trials. The administration of 
trastuzumab concurrently with a HER2 CD4+ helper T cell-eliciting vaccine 
composed of multiple peptides (one of which contains the E75 epitope) to 
patients with HER2-positive breast cancer (n=22) was found to be safe with over 
99% of toxicities limited to grade 1 or 2.45 Median left ventricular ejection fraction 
(LVEF) was unchanged pre- to post-treatment, with three patients (15%) 
experiencing asymptomatic decreases in LVEF. Our group recently completed 
enrollment to a phase I trial evaluating the combination of trastuzumab and a 
CTL-eliciting HER2 peptide vaccine administered concurrently in the adjuvant 
setting to patients with HER2-positive breast cancer. All patients (n=18) have 
completed their vaccination series and we have not seen any cardiac toxicity with 
combination therapy during vaccine dose escalation. Finally, in preliminary 
results from our group’s ongoing phase II trial evaluating AE37 (HER2-derived 
CD4+ helper T cell eliciting vaccine) or GP2 (HER2-derived CTL-eliciting 
vaccine), no cardiac toxicity has been found in HER2-positive patients 
randomized to receive the vaccine after trastuzumab.46 
 
These early clinical trials have shown the promise of efficacy in combination 
therapy as well. Disis and colleagues demonstrated prolonged increases in 
peptide-specific immunity after vaccination, as well as epitope-spreading within 
the HER2 protein and to other non-HER2 tumor antigens.47 In our studies, we 
investigated sequential therapy with trastuzumab followed by HER2 vaccination 
in the adjuvant setting. In our phase I/II studies of nelipepimut-S that enrolled 
patients with any degree of HER2 expression, 11 patients with HER2-positive 
tumors were vaccinated after standard of care trastuzumab. In our ongoing 
phase II study evaluating a second CTL-eliciting HER2-derived vaccine 
(GP2+GM-CSF) which has the same mechanism of action as nelipepimut-S, we 
have enrolled 78 HER2-positive patients who received standard of care 
trastuzumab; 44 were vaccinated after standard of care trastuzumab, 34 were 
randomized to no vaccine. Combining the patients from these two studies, after 
a median follow-up of 36 months, the DFS rate is 84% in the 34 patients 
randomized to no vaccine – comparable with reported rates of similarly staged 
and treated patients. In contrast, for the 55 patients who were vaccinated after 
completing trastuzumab therapy, the DFS rate is 100% (p=.012; unpublished 


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data) (Figure 3). 
 
These encouraging data suggest 
that synergism between 
trastuzumab and HER2 CTL- 
eliciting vaccines warrant further 
investigation. We believe that 
the patients with high-risk HER2- 
positive breast cancer defined as 
those who receive a neoadjuvant 
chemotherapy regimen that 
contains trastuzumab and who 
fail to achieve a pCR or those 
that undergo upfront surgery and 
are found to be node positive (≥ 
4 positive lymph nodes 
regardless of hormone receptor 
status or 1-3 positive lymph 
nodes and are hormone receptor negative) represent the ideal population in 
which to further investigate this strategy. 
 
2.0 
OBJECTIVES 
 
In this study, we intend to assess the ability of the combination of trastuzumab 
and the HER2 vaccine nelipepimut-S (administered with the immunoadjuvant 
GM-CSF) given in the adjuvant setting to prevent recurrences in patients with 
high-risk HER2-positive breast cancer. High-risk is defined as those patients that 
do not achieve a pCR after neoadjuvant therapy with an approved regimen that 
includes trastuzumab and at least four cycles (12 weeks) of taxane-containing 
chemotherapy or those who undergo upfront surgery and are found to have ≥ 4 
positive lymph nodes regardless of hormone receptor status or 1-3 positive 
lymph nodes and are hormone receptor negative. 
 
2.1 
Primary objective 
 
The primary objective is to compare invasive DFS between the two treatment 
groups from time of initiation of trastuzumab maintenance therapy (trastuzumab 
monotherapy) to time of invasive local, regional or distant recurrence, new 
primary, or death due to any cause. 
 
2.2 
Secondary objectives 
 
• Distant recurrence-free survival (DRFS). Time from initiation of 
trastuzumab maintenance therapy to distant recurrence, or death due 
to any cause (continuing follow-up on patients with local or regional 


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recurrence) 
• Assess local and systemic toxicities using standard NCI-CTCAE 
criteria V 4.03 
• Evaluate in vivo and in vitro immune responses 
 
 
 
3.0 
STUDY DESIGN 
 
3.1 
Description of study 
 
This will be a multi-center, prospective, randomized, single-blinded, placebo- 
controlled phase II trial of trastuzumab + nelipepimut-S/GM-CSF versus 
trastuzumab + GM-CSF alone (Appendix A). The vaccine to be used in this 
study, nelipepimut-S, is investigational and will be used in combination with 
trastuzumab under the Investigational New Drug (IND) application BB-IND 
#14919. The Sponsor/Investigator of this IND is Dr. George E. Peoples. 
 
Our target study population is high-risk HER2-positive breast cancer patients. 
High-risk HER2-positive breast cancer patients are defined as: 
 
• Those with HER2-positive breast cancer, regardless of hormone 
receptor status, who receive neoadjuvant therapy with an approved 
regimen that includes trastuzumab and at least four cycles (12 weeks) 
of taxane-containing chemotherapy, and fail to achieve a pCR. 
• Those with HER2-positive breast cancer, regardless of hormone 
receptor status, who undergo surgery as a first intervention and are 
found to have ≥ 4 positive lymph nodes. 
• Those with HER2-positive, hormone receptor negative breast cancer 
who undergo surgery as a first intervention and are found to have 1-3 
positive lymph nodes. 
 
Following surgery, patients will be screened and HLA-typed (consent #1). 
Nelipepimut-S is a CD8-eliciting peptide vaccine that is restricted to HLA-A2+ or 
HLA-A3+ or HLA-A24+ or HLA-A26+ patients (approximately 80% of the US 
population). HLA-A2+/ /A3+/A24+/orA26+ patients who meet all other eligibility 
criteria will be randomized to receive trastuzumab + nelipepimut-S/GM-CSF or 
trastuzumab + GM-CSF alone (consent #2). The trastuzumab will be 
administered to all patients consistent with current standard of care. Patients 
randomized to the nelipepimut-S/GM-CSF arm will receive vaccinations of 
nelipepimut-S (1000 μg) and GM-CSF (250 μg) administered intradermally every 
three weeks for six total vaccinations, 30-120 minutes after completion of 
trastuzumab infusion. With prior approval from the Principal Investigator, the 
vaccine may be administered 15 minutes after completion of the trastuzumab 


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infusion. The first vaccination will be given with the third dose of maintenance 
trastuzumab administered as monotherapy (Appendix B) optimally, but may be 
given with later maintenance doses of trastuzumab, provided there are at least 
six remaining doses of trastuzumab to overlap with the primary vaccine series 
(PVS). Patients randomized to the GM-CSF alone arm will receive inoculations 
of GM-CSF (250 μg) administered in an identical manner to those receiving 
nelipepimut-S/GM-CSF. Patients will be blinded as to whether they are receiving 
nelipepimut-S/GM-CSF or GM-CSF alone. 
 
Upon completion of the PVS, booster inoculations (same dose and route) will be 
administered every six months x 4 (Appendix B). The first booster inoculation 
will occur 6 months ± 2 weeks after the completion of the PVS with subsequent 
boosters timed every six months + 2 weeks. Boosters will therefore occur at the 
following time points after completion of the PVS: 6 months ± 2 weeks, 12 
months ± 2 weeks, 18 months ± 2 weeks and 24 months ± 2 weeks. Booster 
inoculations will occur for patients randomized to receive nelipepimut-S/GM-CSF 
as well as patients randomized to receive GM-CSF alone, and will consist of the 
same treatment drugs and dosing (i.e. nelipepimut-S/GM-CSF patients will be 
boosted with nelipepimut-S/GM-CSF while GM-CSF alone patients will be 
boosted with GM-CSF alone). Patient blinding will be maintained throughout the 
study. 
 
Patients will be followed for safety issues, immunologic response and clinical 
recurrence. Patients will either return to their study site or be contacted by phone 
for questioning regarding any systemic and local toxicity during the initial 48-72 
hours after inoculation. If they return to their study site, the local reaction at the 
inoculation sites, will be examined and measured. For patients that do not return 
to the study site, a tool will be given and instructions provided to measure the 
local reaction. Immunologic response will be monitored primarily by in vivo 
delayed type hypersensitivity (DTH) reactions but also may be documented by 
other immunologic assays. All patients will be followed for a total of 36 months to 
document disease-free status. 
We plan to enroll 100 patients (50 in each treatment arm randomized 1:1) at a 
planned accrual rate of 5 patients per month (approximately 0.5-1enrollment per 
study site per month). With accrual beginning in approximately September 2014, 
enrollment of the last patient would be expected in approximately September 
2016 followed by a three-year follow-up period. The duration of the trial is 
expected to be five years. 
 
3.2 Rationale for study design 
 
In HER2-positive breast cancer patients who receive neoadjuvant chemotherapy 
plus trastuzumab, the 3-year Kaplan-Meier estimates of RFS are 95.7% for 
patients that achieve a pCR versus 80.1% for patients that do not achieve a pCR 


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(p=.02). 24 For HER2-positive breast cancer patients that undergo surgery as a 
first intervention and are found to be node-positive, 5-year DFS rates are 72% for 
those with 4 or more positive lymph nodes and 78% for those with 1-3 positive 
lymph nodes. 15 Based on preliminary data, the combination of trastuzumab plus 
a CTL-eliciting HER2-derived peptide vaccine virtually eliminated recurrences. 
This current study will add to these data by investigating the combination of 
trastuzumab and nelipepimut-S+GM-CSF in an adequately powered randomized 
clinical trial. 
 
3.3 
Outcome measures 
 
3.3.1 Primary outcome measure 
 
Disease state will be determined by the patients’ own physicians at the individual 
study sites during their routine follow-up screening. This will occur for all enrolled 
patients, regardless of randomization, approximately every three months for the 
first 24 months after completion of primary therapies and every six months 
thereafter with clinical exam, laboratory (CBC and CMP) and radiographic 
surveillance as indicated. Evaluation of the patients’ labs will be performed 
approximately every 3 months during the primary series and approximately every 
6 months during the booster phase to monitor for potential toxicity from the study 
drug combination.  This follow-up strategy is standard of care for these patients. 
If records are not available, patients, or their referring physicians, will be 
contacted to discern their disease status. All enrolled patients, regardless of 
randomization will be followed for clinical recurrence for three years from the date 
of initiation of trastuzumab maintenance therapy. The primary outcome measure 
of the trial is invasive DFS. 
 
3.3.2 Secondary outcome measures 
 
Secondary outcome measures include determining DRFS from initiation of 
trastuzumab maintenance therapy, assessing local and systemic toxicities, and 
evaluating immune responses. 
 
Distant recurrence free survival will be assessed as part of the patient’s disease 
state determined by their physician at the individual study sites during routine 
follow-up screening (see 3.3.1 Primary outcome measure). Determination of 
DRFS will allow for continued follow-up on patients with local or regional 
recurrence. 
 
Standard local and systemic toxicities will be collected and graded per the NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 graded 
toxicity scale (Appendix C). For both the inoculations during the PVS and the 
booster inoculations, the patient will be monitored for 30 minutes with vital signs 
taken as needed. Patients will either return to their study site or be contacted by 


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phone for questioning regarding any systemic and local toxicity during the initial 
48-72 hours after inoculation. If they return to their study site, the local reaction 
at the inoculation sites will be examined and measured. For patients that do not 
return to the study site, a tool will be given and instructions provided to measure 
the local reaction. Serious AEs will be reported as described in Section 5. 
 
Immune responses will be primarily documented using the delayed type 
hypersensitivity (DTH) reaction and using the dextramer assay to enumerate 
peptide-specific CTL. Each of these measurements will be performed regardless 
of randomization. Detailed descriptions of these assays/tests are described in 
Sections 4.4.6 and 4.4.8. DTH reactions will be measured prior to initiation of the 
vaccination series, one month ± 1 week after completion of the PVS, and one 
month ± 1 week after the final booster inoculation. Dextramer measurements will 
be performed prior to initiating the vaccination series as well as one month ± 1 
week after completion of the PVS. Additionally, these assays may be performed 
pre- and post-each booster.  Alternatively, these assayed time points may also 
be performed all at once on frozen and banked cells. 
 
3.4 
Safety considerations 
 
3.4.1 Trastuzumab 
 
If trastuzumab is discontinued by the treating oncologist for any indication to 
include those listed below, the patient will be taken off treatment, but continued in 
the study for disease follow-up. 
 
Cardiac. Cardiac evaluation with MUGA or ECHO will be performed per 
standard of care dictated by the treating oncologist. 
 
Management of symptomatic cardiac changes. Patients who develop 
signs and symptoms of congestive heart failure will be taken off treatment, 
but continued in the study for disease follow-up. 
 
Management of asymptomatic decreases in LVEF. Patients experiencing 
asymptomatic absolute decrease in LVEF < 20 percentage points from 
baseline, will continue on study as long as 1) the LVEF remains within the 
normal limits of the institution, and 2) their treating oncologist continues 
trastuzumab treatment. Patients with an asymptomatic decrease in LVEF 
of ≥ 20 percentage points (even if within the normal limits of the institution) 
or an ejection fraction below the range of normal limits, will be taken off 
treatment, but remain in the study for disease follow-up. 
 
Hematologic Toxicity and Neutropenic Infections. In prior clinical trials, an 
increased incidence of anemia was observed in patients receiving trastuzumab 
plus chemotherapy compared with patients receiving chemotherapy alone. The 


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majority of these anemia events were mild or moderate in intensity and 
reversible; none resulted in discontinuation of trastuzumab therapy. 
 
In the clinical trials, the per-patient incidences of moderate to severe neutropenia 
and of febrile neutropenia were higher in patients receiving trastuzumab in 
combination with myelosuppressive chemotherapy as compared to those who 
received chemotherapy alone. In the post-marketing setting, deaths due to 
sepsis in patients with severe neutropenia have been reported in patients 
receiving trastuzumab and myelosuppressive chemotherapy, although in 
controlled clinical trials (pre- and post-marketing), the incidence of septic deaths 
was not significantly increased. The pathophysiologic basis for exacerbation of 
neutropenia has not been determined. 
 
Secondary acute leukemia or myelodysplastic syndrome has been reported in 4 
of approximately 1200 patients who participated in trastuzumab clinical trials. 
Patients treated with chemotherapeutic agents are known to be at increased risk 
for secondary leukemia. The observed incidence of leukemia among 
trastuzumab-treated patients appears to be consistent with the expected 
incidence of leukemia among patients treated with chemotherapy for metastatic 
breast cancer. Therefore, the contribution of trastuzumab to the etiology of acute 
leukemia or myelodysplastic syndrome in these cases is unclear. 
 
Management of Hematologic Toxicities. Care should be taken to carefully 
monitor the patient’s hematologic status throughout the course of the trial. 
Use of hematopoietic growth factors to ameliorate hematologic toxicity is 
at the discretion of the physician investigator and should be in accordance 
with the American Society of Clinical Oncologists (ASCO) guidelines. 
 
3.4.2 Nelipepimut-S and GM-CSF 
 
In our previous trials evaluating Nelipepimut-S + GM-CSF, approximately 18% of 
patients experienced robust local reactions (>100mm) or greater than or equal to 
grade 3 local or systemic reactions greater than or equal to grade 2 
hypersensitivity reactions. 32 
 
Dose Reductions of Vaccine. Toxicities observed with the vaccine are 
attributable to the GM-CSF. For patients experiencing robust local 
reactions (>100mm x 100mm) or greater than or equal to grade 3 local or 
systemic reactions greater than or equal to grade 2 hypersensitivity 
reactions, the dose of GM-CSF will be reduced by 50% for subsequent 
inoculations. If subsequent reactions occur necessitating dose reduction, 
the GM-CSF dose will continue to be reduced serially by 50%. For 
example, GM-CSF dose would first be reduced from 250 mcg to 125 mcg, 
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dose reduction of GM-CSF is inadequate in limiting reactions, the 
nelipepimut-S dose (if applicable) will then be reduced by 50%. 
 
Dose reductions will continue for booster inoculations in the same manner 
as performed in the PVS, except for patients who underwent multiple dose 
reductions in the primary vaccine series, who will begin booster 
inoculations with an initial GM-CSF dose of 125 mcg. 
 
In our prior trial, approximately 5% of patients developed delayed urticarial 
reactions (generalized urticaria 10-14 days after booster inoculation). 
This was not seen during the PVS. The majority of these patients were 
readily treated with anti-histamines or oral steroids and most continued 
with subsequent booster inoculations. If dose reduction is performed for 
hypersensitivity reactions (generalized urticaria), antihistamines may be 
used before or after subsequent inoculations. If urticaria persists with the 
use of anti-histamines and oral steroids, or intravenous steroids are 
required, or hospitalization is required, or in the opinion of the Principal 
Investigator, further inoculations will be discontinued. 
 
3.5 
Compliance with laws and regulations 
 
This study will be conducted in accordance with current U.S. Food and Drug 
Administration (FDA) Good Clinical Practices (GCPs), and local ethical and legal 
requirements. 
 
4.0 
MATERIALS AND METHODS 
 
4.1 
Patients 
 
Patients over the age of 18 years with a diagnosis of HER2-positive breast 
cancer who are receiving neoadjuvant therapy with an approved regimen that 
includes trastuzumab and do not achieve a pathologic complete response or who 
undergo upfront surgery and are found to be pathologically node-positive will be 
targeted. Patients treated with neoadjuvant therapy must receive an approved 
regimen that includes trastuzumab and at least four cycles (12 weeks) of taxane- 
containing chemotherapy. These patients will be approached after their surgical 
pathology report is available and confirms residual disease. For patients who 
undergo upfront surgery, they will be approached after their surgical pathology 
report confirms node-positive disease. For clarity, standard of care pertuzumab 
is allowed.. Patients will be recruited from medical and surgical oncology and/or 
hematology/oncology clinics at the individual study sites. All patients will be 
properly consented. 
 
4.1.1 Patient selection 


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Potentially eligible patients, specifically patients with high-risk HER2-positive 
breast cancer will be identified by staff in the medical and surgical and/or the 
hematology/oncology clinics at the individual study sites. High-risk HER2- 
positive is defined as those that receive neoadjuvant therapy with an approved 
regimen that includes trastuzumab and at least four cycles (12 weeks) of taxane- 
containing chemotherapy and fail to achieve a pCR or those who undergo 
surgery as a first intervention and are found to be pathologically node-positive. 
For clarity, standard of care pertuzumab is allowed. 
 
A research nurse and/or study coordinator will approach these patients about 
being in the trial and will introduce the trial to the prospective patient. If the 
patient is interested and appears eligible, the nurse will arrange to counsel the 
patient. The nurse will thoroughly screen the patient for inclusion and exclusion 
eligibility criteria. If the patient remains interested and eligible, informed consent 
will be obtained. 
 
Written informed consent will be obtained from all study participants. Prospective 
participants will be provided with a copy of the consent form to read. The 
Principal Investigator (PI) or individuals who are authorized by their institution to 
obtain informed consent and who are familiar with the study, will explain the 
study and review the consent form with the patient. Patients will be given ample 
time to ask and have all questions answered prior to signing the consent form. 
 
Once written informed consent is obtained, the patient will be HLA-typed. They 
will be asked for 6-8 cc of blood for HLA-A2/A3/A24/A26 screening to be 
performed in a CLIA-certified laboratory at MD Anderson. Only HLA-A2/ HLA- 
A3/HLA-A24 or HLA-A26 positive patients will be enrolled in the trial. Those 
patients who are not HLA-A2,A3, A24, or A26 positive will be excluded. Patients 
will be counseled prior to obtaining consent that lack of HLA-A2,A3, A24, or A26 
positivity will result in exclusion. 
 
4.1.2 Inclusion criteria 
 
Patients will be included in the study based on the following criteria: 
 
• 18 years or older 
• Eastern Cooperative Oncology Group (ECOG) performance status 0,1 
• AJCC stage I – III non-inflammatory, HER2-positive (according to 
ASCO-CAP guidelines 5) breast cancer 
• Completed neoadjuvant therapy with an approved regimen that 
includes trastuzumab and at least four cycles (12 weeks) of taxane- 
containing chemotherapy and underwent surgery with final pathology 
showing evidence of residual disease in the breast or axilla (residual 


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ductal carcinoma in situ or microinvasive disease not eligible) or 
underwent surgery as a first intervention and was found to be 
pathologically node-positive: ≥ 4 positive lymph nodes (pN2 or pN3) 
regardless of hormone receptor status or 1-3 positive lymph nodes 
(pN1) if hormone receptor negative. Patients with micrometastases 
(pN1mi) are not eligible. For clarity, standard of care pertuzumab is 
allowed. 
• Completed an approved regimen of neoadjuvant or adjuvant therapy 
with an approved regimen that includes trastuzumab and at least four 
cycles (12 weeks) of taxane-containing chemotherapy with plan for 
completion of one year of trastuzumab therapy. For clarity, standard of 
care pertuzumab is allowed. 
• Completed appropriate surgical therapy to include: 
• Total mastectomy and axillary staging with sentinel lymph node 
dissection or axillary lymph node dissection (level I/II). Patients 
with a positive sentinel lymph node must have undergone a 
completion axillary lymph node dissection. 
• Breast conserving surgery (BCS) and axillary staging with sentinel 
lymph node dissection or axillary lymph node dissection. Patients 
undergoing surgery as a first intervention with a positive sentinel 
lymph node must have undergone a completion axillary dissection 
level I/II unless they had clinically node negative T1-T2 tumors and 
fewer than 3 involved lymph nodes. Patients receiving neoadjuvant 
chemotherapy that have a positive sentinel lymph node must have 
undergone a completion axillary lymph node dissection. 
• Completed or receiving appropriate radiation therapy if indicated: 
• For patients undergoing total mastectomy surgery as a first 
intervention, post-mastectomy radiation to the chest wall, 
infraclavicular and supraclavicular areas is required for patients 
with ≥ 4 positive lymph nodes. Radiation to the internal mammary 
lymph nodes is not required per protocol but is allowed at the 
discretion of the patient’s treating radiation oncologist. For patients 
with 1-3 positive lymph nodes, post-mastectomy radiation to the 
chest wall, infraclavicular, supraclavicular, and internal mammary 
areas is not required per protocol but is allowed at the discretion of 
the patient’s treating radiation oncologist. 
• For patients undergoing breast conserving surgery (BCS) as a first 
intervention, whole breast irradiation with or without a boost, and 
radiation to the infraclavicular and supraclavicular areas is required 
for patients with ≥ 4 positive lymph nodes. Radiation to the internal 


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mammary lymph nodes is not required but is allowed at the 
discretion of the patient’s treating radiation oncologist. For patients 
with 1-3 positive lymph nodes, whole breast irradiation with or 
without a boost is required. Radiation to the infraclavicular, 
supraclavicular, and internal mammary areas is not required per 
protocol but is allowed at the discretion of the patient’s treating 
medical oncologist. 
• For patient’s undergoing mastectomy after neoadjuvant 
chemotherapy post-mastectomy radiation to the chest wall, 
infraclavicular and supraclavicular areas is required for patients 
presenting with clinical N2 or N3 disease or with ≥ 4 positive lymph 
nodes identified pathologically at the time of surgery. Radiation to 
the internal mammary lymph nodes is not required per protocol but 
is allowed at the discretion of the patient’s treating radiation 
oncologist. For patients with 0-3 positive lymph nodes identified 
pathologically, post-mastectomy radiation to the chest wall, 
infraclavicular, supraclavicular and internal mammary areas is not 
required per protocol but is allowed at the discretion of the patient’s 
treating radiation oncologist. 
• For patient’s undergoing BCS after neoadjuvant chemotherapy, 
whole breast irradiation with or without a boost is required. For 
patients with clinical N2 or N3 disease or with ≥ 4 positive lymph 
nodes identified pathologically at the time of surgery, radiation to 
the infraclavicular and supraclavicular areas is required. Radiation 
to the internal mammary lymph nodes is not required per protocol 
but is allowed at the discretion of the patient’s treating radiation 
oncologist. For patients with 0-3 positive lymph nodes identified 
pathologically, radiation to the infraclavicular, supraclavicular and 
internal mammary areas is not required per protocol but is allowed 
at the discretion of the patient’s treating radiation oncologist. 
• HLA-A2A3, A24, or A26 positive 
• LVEF >50%, or an LVEF within the normal limits of the institution’s 
specific testing (MUGA or ECHO) 
• Adequate organ function as determined by the following laboratory 
values: 
• ANC ≥ 1,000/μL 
• Platelets ≥ 75,000/μL 
• Hgb ≥ 9 g/dL 


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• Creatinine ≤ 1.5 x upper limit of normal (ULN) of institution’s range 
or Creatinine clearance ≥ 50% 
• Total bilirubin ≤ 1.5 ULN of institution’s range 
• ALT and AST ≤ 1.5 ULN of institution’s range 
• For women of child-bearing potential, agreement to use adequate birth 
control (abstinence, hysterectomy, bilateral oophorectomy, bilateral 
tubal ligation, oral contraception, IUD, or use of condoms or 
diaphragms) 
 
• Signed informed consent 
 
4.1.3 Exclusion criteria 
 
Patients will be excluded from the study based on the following criteria: 
• 
AJCC Stage IV breast cancer 
• 
NYHA stage 3 or 4 congestive heart failure 
• 
Immune deficiency disease or known history of HIV, HBV, HCV 
• 
Receiving immunosuppressive therapy including chronic steroids, 
methotrexate, or other known immunosuppressive agents 
• 
Pregnancy (assessed by urine HCG) 
• 
Breast feeding 
• 
Any active autoimmune disease requiring treatment, with the 
exception of vitiligo 
• 
Active pulmonary disease requiring medication to include multiple 
inhalers (>3 inhalers including one containing steroids) 
• 
Involved in other experimental protocols 
 
4.2 
Method of treatment assignment 
 
If patients meet all inclusion criteria and none of the exclusion criteria and agree 
to participate, they will continue in the study. Patients will be randomly assigned 
by a designated Cancer Insight staff member using a computer-generated 
randomization table to receive trastuzumab + nelipepimut-S/GM-CSF or 
trastuzumab + GM-CSF alone. Randomization between the two treatment 
groups will occur in a 1:1 allocation ratio using an institutional balancing 
algorithm. Patients will be blinded as to whether they are receiving trastuzumab 
+ nelipepimut-S/GM-CSF or trastuzumab + GM-CSF alone. 
 
4.3 
Study treatment 


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4.3.1 Trastuzumab 
 
All patients will receive trastuzumab as per current standard practice. 
Trastuzumab will be administered as per institutional protocol. 
 
4.3.2 Vaccine 
 
4.3.2.1 Dosage and preparation 
 
The E75 vaccine (nelipepimut-S; as 1.5 mg/mL E75 acetate solution) and GM- 
CSF (as 250 mcg lyophilized Leukine) will be supplied by Galena Biopharma. 
Galena Biopharma also provides DTH skin test kits, each of which contains 1.5 
mg/mL E75 acetate solution. 
 
GM-CSF is a potent cytokine that stimulates granulocytes (neutrophils, 
eosinophils and basophils) and monocytes, but not lymphocytes, erythrocytes, or 
platelets. Recombinant human GM-CSF is a 14.6 kDa globular protein consisting 
of 128 amino acids containing 2 intramolecular disulfide bonds and 2 potential N- 
linked glycosylation sites. Known adverse effects of GM-CSF include bone pain, 
allergic reactions, lethargy, malaise, anorexia, skin rashes, flushing, fever, and 
chills. At doses higher than planned in this study, weight gain may be seen along 
with breathing difficulties, blood clots, and collections of fluid around the heart or 
lungs, dyspnea, thromboembolic phenomena, and pleural and pericardial 
effusion. 
 
Leukine® (sargramostim) is a recombinant human granulocyte-macrophage 
colony stimulating factor (rhu GM-CSF) produced by recombinant DNA 
technology in a yeast (S. cerevisiae) expression system. Biological potency is 
expressed in International Units (IU) as tested against the World Health 
Organization (WHO) First International Reference Standard. The specific activity 
of GM-CSF is approximately 2.8 x 106 IU/mL. See GM-CSF (Leukine® 
(sargramostim)) package insert and labeling (Appendix E) for additional details. 
The E75 vaccine acetate drug substance is a 9-amino acid peptide produced by 
solid-phase peptide synthesis. Nelipepimut-S acetate drug product is 
manufactured by Oso Biopharmaceuticals Manufacturing, Albuquerque, NM as a 
1.5 mg/mL solution in 1 mL water for injection (WFI) in a 2-mL glass vial. Each 
mg of Nelipepimut-S acetate is equivalent to 0.94 mg of Nelipepimut S peptide. 
Hence, the vaccine concentration is equivalent to 1.41 mg/mL Nelipepimut-S 
peptide. The control is WFI. 
 
Prior to administration, 1,065 mcg (0.8 mL) of the E75 acetate or control is mixed 
by the pharmacist or research nurse with 1 mL of reconstituted lyophilized 
Leukine® GM-CSF (reconstituted as 250 mcg/mL in solution). Immediately after 
mixing, the study drug is withdrawn into four 1 mL syringes and within 6 hours of 


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mixing, a total of 1.6 mL is administered as 4 intradermal injections to deliver a 
total of 1,000 mcg of E75 peptide vaccine (1,065 mcg of E75 acetate). The 
control vial contains 1 mL WFI. The detailed guidance for mixing E75 (or control) 
solution from lyophilized Leukine® 250μg and test drug (E75 solution or control) 
is stated in Appendix D. 
 
For DTH skin test, E75 acetate solution (1.5mg/1mL vial) is supplied in DTH skin 
test kit. Detailed mixing instruction for DTH skin test prep is stated in Appendix D. 
If dose reduction of GM-CSF is required based on >100 mm x 100 mm 
induration, and/or ≥Grade 3 toxicity at the injection site after the previous dose, 
the liquid Leukine® vial is diluted with an additional 1.0 mL WFI, using aseptic 
technique, and mixed gently by rolling the Leukine vial between your hands. 
Allow any foaming to dissipate before continuing. Do not invert the vial. 
Subsequently, 1 mL is withdrawn and discarded and the remaining 1 mL is used 
for mixing following the same methodology above to make a 1.6 mL Leukine + 
E75 mixture. Further dilutions (approximately 1:1) may be required if >10 cm 
erythema and induration and/or ≥ Grade 3 toxicity are observed on subsequent 
doses, the details of which will be provided in study instructions. 
 
4.3.2.2 Storage 
 
For detailed storage instruction of the vaccine and its components (E75 acetate 
in WFI and GM-CSF) please follow the instructions in the Investigator’s Brochure. 
Stability data support storage of E75 acetate in WFI and control to E75 acetate in 
WFI at -20°C (± 5°C) or 5°C (± 3°C). Store study drug (E75 acetate in WFI, 
control to E75 acetate in WFI and GM-CSF) as per appropriate storage 
temperature designated on clinical carton label. 
 
4.3.3 Inoculation series – administration 
 
Patients will receive 4 intradermal inoculations on the anterior or medial side of 
the same thigh. The general area of inoculation will be at a location midway 
between the inguinal ligament and the knee preferably, but may be given in the 
arm. 
 
The 1.6 mL by volume vaccine (or GM-CSF alone) will be administered 
intradermally in four equal inoculums at four different sites in a square 
configuration 5 cm from each other. Inoculations will be given every three weeks 
and will be administered in the same lymph node draining area (same arm or 
leg). Timing of the inoculation series with trastuzumab treatment will occur as 
outlined in Section 3.1 (see also Appendix B). The dose will be 1000 mcg of E75 
peptide and 250 mcg of GM-CSF. The research nurse coordinator will administer 
the inoculations sterilely in the vaccine or clinical laboratory facility located at 
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pregnancy test will be performed before each inoculation. If this test is positive at 
any time, the patient will be discontinued from the study. 
 
4.3.4 Booster inoculations 
 
After completion of the six-inoculation PVS, patients will then receive a total of 
four booster inoculations to be administered, respectively, at 6, 12, 18, and 24, 
months +/- 2 weeks from the date of completion of the PVS (Appendix B). One 
booster inoculation will be administered at each time point noted above ± two 
weeks and administered in the same extremity as the primary series. Booster 
inoculations will consist of the same intervention as each patient received during 
their regular inoculation series. Patients enrolled to the E75/GM-CSF vaccine 
arm of the trial will receive four consecutive booster inoculations of 1000 mcg 
E75 peptide + 250 mcg GM-CSF, and patients enrolled to the GM-CSF alone 
arm will receive four consecutive booster inoculations of 250 mcg GM-CSF only. 
 
For patients with childbearing potential, a urine pregnancy test will be performed 
before each booster inoculation. If this test is positive at any time, the patient will 
be discontinued from the study. 
 
4.4 
Study assessments 
 
Signed, IRB-approved informed consent must be obtained from patients prior to 
any pretreatment assessments. 
 
4.4.1 Pretreatment assessments 
 
All patients must have a Complete Blood Count (CBC) and Comprehensive 
Metabolic Panel (CMP) within two months of trial initiation and, for female patients, 
a urine pregnancy test (upon consent to study), for screening and immediately 
prior to any vaccine inoculations or DTH. If the pregnancy test is positive, the 
patient will be excluded from the study. Women who have had a hysterectomy, 
bilateral oophorectomy, tubal ligation, documented absence of menses for two 
years, or FSH hormonal laboratory results that verify menopause, will not be 
required to have pregnancy testing. For patients who have a complete metastatic 
evaluation (CBC, LFTs, CXR, chest and abdomen CT, bone scan, and PET scan) 
available, all studies will be screened, but these studies are not required for 
enrollment. Results of cardiac evaluation with MUGA or ECHO will also be 
screened. Disease-free status will be assured by the patients’ primary 
treating/referring physician. Overall health screen will be assessed utilizing the 
ECOG performance status grading system (Appendix F). 
 
To ensure eligibility, each patient will be HLA-typed as described in Section 
4.4.5). They will be asked for 6-8 cc of blood for HLA-A2/A3/A24/A26 screening. 
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All patients regardless of randomization may be assessed for baseline 
immunologic responses prior to the first inoculation. Specimen handling, 
processing, and assays are described in Sections 4.4.4 and 4.4.6. 
 
All patients will be tested for pre-inoculation DTH reaction against nelipepimut-S 
as described in Section 4.4.8. Reactions will be read 48-72 hours after 
placement of the peptide. 
 
4.4.2 Assessments during treatment 
 
All patients will have cardiac assessment performed according to standard 
practice (approximately every 3 months) as dictated by their treating oncologist. 
Either MUGA or ECHO is acceptable but should be consistently used for any 
given patient. Results of cardiac testing will be reviewed by the research nurses 
or study coordinators and any abnormal results will be reviewed by the study 
investigators. Per standard practice, all patients will have CBC and CMP 
monitoring approximately every 3 months during the primary series and 
approximately every 6 months during the booster phase. Any laboratory 
evaluations or imaging studies ordered by the treating oncologist as part of 
standard practice will be reviewed by the research nurses or study coordinators 
and any abnormal results will be reviewed by the study investigators. 
 
For both the PVS and the booster inoculations, the patient will be monitored for 
30 minutes after the inoculation with vital signs taken as needed. 
 
Patients will either return to their study site or be contacted by phone for 
questioning regarding any systemic and local toxicity during the initial 48-72 
hours after inoculation. If they return to their study site, the local reaction at the 
inoculation sites will be examined and measured. For patients that do not return 
to the study site, a tool will be given and instructions provided to measure the 
local reaction. 
 
The NCI CTCAE version 4.03 graded toxicity scale (Appendix C) will be utilized 
to assess local and systemic toxicity. 
 
Blood samples will be obtained from all patients, regardless of randomization, to 
determine induction of peptide-specific immune responses as described in 
Sections 4.4.4 and 4.4.6. 
 
4.4.3 Follow-up assessments 
 
The clinical endpoint of recurrence will be determined by patients’ own 
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physical examination every 3-6 months for all patients and yearly mammogram 
for patients undergoing breast conserving therapy (per ASCO guidelines). 48 
Additional laboratory and radiographic surveillance will be performed as indicated 
and directed by the patients treating physicians. The determination of recurrence 
will be per standard practice by the treating physicians and communicated to the 
study investigators on a routine basis. Documentation of the recurrence will be 
obtained. Whenever possible, pathologic confirmation of recurrence will be 
obtained. However, a patient will also be considered to have a recurrence if 
highly suggested on radiographic evaluation and oncologic treatment for the 
recurrence is initiated by the treating physician. Documentation of the latter will 
be obtained. 
For the purposes of this trial, the invasive DFS will be calculated 
from the date of initiation of trastuzumab maintenance therapy until date of last 
follow-up or recurrence. If records are not available, patients, or their referring 
physicians, will be contacted to discern their disease status and every effort will 
be made to obtain documentation. All enrolled patients, regardless of 
randomization, will be followed for clinical recurrence for up to three years from 
the date of initiation of trastuzumab maintenance therapy. 
 
4.4.4 Blood collection and processing 
 
Multiple blood draws will be required for this trial. All blood tubes will be labeled 
only with the patients’ unique study number. Approximately 6-8 cc will be drawn 
for HLA-typing and approximately 70cc for each Immunologic assessment (pre 
DTH #1 and #2, prior to each booster and 1 month ± 1 week after each booster 
and at study completion 36 months after initiation of trastuzumab maintenance 
therapy). Thus a total of approximately 800cc of blood will be drawn over the 
three year course of the study. De-identified patient blood samples showing only 
the unique study ID number will be sent from study sites via overnight delivery to 
the laboratory of the Principal Investigator, Dr. Elizabeth Mittendorf, at MD 
Anderson where immunologic response assays (Section 4.6) will be performed. 
At no point will MD Anderson personnel have access to patient identifiers. Blood 
not used to perform the dextramer assay (Section 4.6) will be frozen and stored 
in the laboratory of Dr. Elizabeth Mittendorf under the unique protocol number 
and identifier for up to five years for additional immunologic studies related to this 
protocol (for example, to repeat assays or perform new immunologic assays that 
do not exist at present but may become available) as needed and then 
destroyed. No genetic testing will be performed on this material. Study 
participants will not be contacted in the future for additional use of these stored 
blood specimens. If study participants want their blood specimens removed from 
storage and destroyed, they may do so by contacting the PI or research nurse at 
any time. Additionally, any stored blood may also be utilized to assess new 
generations of vaccines. 
 
For blood draws performed for immunologic assessment, 10cc are collected into 
a BD Vacutainer Rapid Serum tube (BD, Franklin Lakes, NJ) which contain a clot 


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activator and silicone coated interior.  After centrifugation, serum will be 
collected, aliquoted in 1cc vials and frozen.  The remaining 60cc are collected 
into BD Vacutainer CPT Cell Preparation tubes which contain an anticoagulant 
(sodium heparin or sodium citrate) with FICOLL HYPAQUE density gradient fluid 
and a polyester gel barrier.  The density gradient fluid and the gel barrier allow 
for the separation of PBMC from the red blood cells by a single step 
centrifugation process. The PBMC fraction will be collected by centrifugation and 
suspended in RPMI-1640 (GIBCO, Invitrogen Corporation, Carlsbad, CA) with 
10% FCS (Gemini Bio-Products, West Sacramento, CA) and antibiotics. 
 
4.4.5 HLA-A2/A3/A24/A26 testing 
 
HLA testing will be performed at the CLIA certified human flow cytometry lab at 
MD Anderson. The expression of HLA-A2 by the patients is confirmed by 
staining PBMC with anti-HLA-A2 monoclonal antibody, (clone BB7.2), directly 
conjugated to phycoerythrin (PE) or fluorescein-isothiocyanate (FITC) (BD 
Biosciences) at 4ºC for 60 minutes. The expression of HLA-A3, A24, and A26 by 
the patients is confirmed by staining PBMC with the appropriate biotinylated-anti 
HLA-allele monoclonal antibody (One Lambda Inc.) for 45 minutes after which 
the cells are washed and incubated for an additional 15 minutes with 
streptavidin-PE. After incubation the cells are washed and analyzed on a flow 
cytometer. 
 
4.4.6 Dextramer assay 
 
Fresh PBMC will be stained with aqua live/dead stain (Invitrogen) and the 
following antibodies: CD8 APC-H7 (BD Biosciences), CD3 PE Cy7 (BD 
Biosciences), E75-APC-conjugated dextramer (Immudex), and the following 
pacific blue conjugated lineage antibodies: CD14 (BD Biosciences), CD16 (BD 
Biosciences), and CD19 (Biolegend). Cells will be analyzed on a Canto flow 
cytometer (BD Biosciences). The frequency of E75-specific CD8+ T cells will be 
determined as the percentage of cells that are alive, lineage-, CD3+ CD8+ and 
E75-dextramer+. 
 
4.4.7 Local reaction 
 
Patients will be assessed for evidence of in vivo immunologic response by 
evaluation of the injection site 48-72 hours after each inoculation. The injection 
site reaction will be measured using the sensitive ball point pen method if the 
patient returns to the study site. A digital photograph of the local reaction may be 
taken to document the reaction and for future reporting purposes.  It will in no 
way be visually identifiable as that patient. Photographs will be labeled with the 
patient’s study number and will not include the face or distinguishing birthmarks 
or tattoos.  They will be electronically stored on a password protected computer 
in a locked office in the vaccine or clinical laboratory facility at each site. Per 


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FDA regulations, each patient’s photograph will be kept for two years after 
submission of a New Drug Application (NDA) and then destroyed. If the patient 
does not return to the study site, they will be contacted by phone 48-72 hours 
after inoculation for questioning regarding the reaction at the injection sites. 
 
4.4.8 DTH reaction 
 
Regardless of randomization, a pre--inoculation series DTH response will be 
assessed of all patients with 100 mcg of nelipepimut-S (without GM-CSF) 
injected intradermally at a site on the back or anterior thigh (opposite side from 
the vaccination site). A post-inoculation series DTH response will be assessed 
one month ± 1 week after completion of the PVS. The DTH reaction will be 
measured at 48-72 hours using the sensitive ball point pen method and 
compared between pre-inoculation and post-inoculation time points. 
Additionally, a DTH responses will be assessed 1 month ± 1 week after the fourth 
booster. The low dose of peptide used in the DTH test is not expected to induce 
a long-term immune response in control patients; however, any response that is 
induced is expected to be transitory in nature. A digital photograph of the local 
reaction will be taken to document the reaction and for future reporting purposes. 
It will be labeled with the patient’s study number, will not visually identify the 
patient, and will not include the face or distinguishing birthmarks or tattoos. 
Photographs will be electronically stored on a password protected computer in a 
locked office in the vaccine or clinical laboratory facility at each site. As per FDA 
regulations, each patient’s photograph will be kept for two years after submission 
of a NDA and then destroyed. These results will also be recorded in the immune 
response database. 
 
4.5 
Discontinuation of protocol-specific therapy 
 
Protocol-specific therapy may be discontinued for any of the following reasons: 
 
• Progressive disease 
 
• Unacceptable toxicity as described in the protocol 
 
o Please refer to sections 3.4 (Safety Considerations) for the 
management of cardiac issues (symptomatic and asymptomatic), 
hematologic toxicity and neutropenic infections, as well as robust 
local reactions and delayed urticarial reactions. Adverse events that 
do not respond to either dose modification (as described) or clinical 
management of the reaction will result in discontinuation from the 
study. 
o Please refer to section 4.6 (Patient Discontinuation) for a list of 
severe adverse reactions warranting discontinuation from the study. 


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• Patient election to discontinue therapy (for any reason) 
• Physician’s judgment 
 
4.6 
Patient discontinuation 
 
Those patients who display significant reactions (i.e. severe anaphylactic 
reaction immediately after vaccine administration) or toxicities will be 
discontinued from the study as determined by the PI. They will be followed by 
the study investigator until resolution of the adverse event. 
 
Inoculations will be immediately halted if any serious adverse reactions occur to 
include: death, life-threatening adverse drug experience (i.e., severe 
anaphylactic reaction immediately after vaccine administration), inpatient 
hospitalization or prolongation of existing hospitalization, a persistent or 
significant disability/incapacity, a congenital anomaly/birth defect, or other 
important medical events that may not result in death, be life-threatening, or 
require hospitalization but which, when based upon appropriate judgment of the 
PI, be determined to jeopardize the patient or require medical or surgical 
intervention to prevent an outcome listed above. Any death or grade 4 adverse 
drug experience found at least possibly related to the experimental vaccine will 
result in suspension of patient enrollment to the study. If the SAE is determined 
to be unrelated to the study drug, inoculations may be re-started if the patient 
desires to continue and it is safe for them to do so as determined by the PI and 
Sponsor. 
 
For robust local, systemic > Grade 3, and > Grade 2 hypersensitivity reactions, 
as evaluated under CTCAE v4.03, will result in dose reduction or discontinuation 
as discussed (section 3.4.2). 
 
Patients may withdraw from the study at any time and for any reason. A patient 
may be asked to withdraw from the study by the PI if they are not compliant with 
the timing of the inoculation series, observation period, or return visits to monitor 
for study-associated toxicities. Additionally, if the PI determines that it is no 
longer safe for a patient to continue in the trial for any reason, they may be 
withdrawn. 
 
Because it is not known whether these inoculations might harm an unborn child, 
patients who are pregnant, plan on becoming pregnant, or who are breast- 
feeding will not be enrolled into the study. Women of childbearing potential will 
take a urine pregnancy test before starting this study and prior to each 
inoculation; a positive test result will terminate the patient’s participation in the 
study. Patients will be counseled to avoid becoming pregnant while participating 
in this study, and that in order to prevent pregnancy they should either have no 
sexual relations or use a reliable type of birth control. They will be counseled 


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that with the exception of hysterectomy, bilateral oophorectomy, or tubal ligation, 
birth control methods are not totally effective in preventing pregnancy, and that 
the only ways to completely avoid the risk of the vaccine or immunoadjuvant 
alone to an unborn baby are (1) avoid becoming pregnant, or (2) do not receive 
these inoculations. Patients will be counseled to avoid becoming pregnant for at 
least 6 months after receiving the inoculations as pregnancy within this time after 
inoculation administration may be a risk to an unborn baby. 
 
If a patient is discontinued from the study for an adverse event or pregnancy, 
they will continue to be followed for resolution of adverse event and clinical 
recurrences unless the patient withdraws consent for further study evaluation. 
The reason for any premature discontinuation of a patient from the study will be 
recorded on the appropriate Case Report Form. 
 
 
4.7 
Study discontinuation 
 
The IND sponsor Dr. George E. Peoples, the financial sponsor Galena 
Biopharma, the Data Safety Monitoring Board, and the Principal Investigator 
have the right to terminate this study at any time. Reasons for terminating the 
study may include the following: 
 
• The incidence or severity of AEs in this or other studies indicates a 
potential health hazard to patients. While vaccine related toxicity has 
been shown to be low, and the safety profile of Herceptin is known, the 
safety of the concurrent use is yet to be proven. As such, toxicities will be 
carefully monitored per treatment group in real time. If at any point, the 
level of toxicity, particularly cardiac toxicity, becomes statistically higher in 
the vaccinated group, the study will be stopped. 
• Patient enrollment is unsatisfactory 
• Data recording are inaccurate or incomplete 
• Study protocol not followed 
 
4.8 
Data collection 
 
Basic demographic, pathologic, and relevant clinical information will be gathered 
on each patient and entered into an Electronic Data Capture (EDC) database. 
The URL for the EDC system is 
https://cancerinsight.eclinicalhosting.com/OpenClinica. It can be accessed from 
any web browser. User name and password will be generated by the Data 
Manager of Cancer Insight and sent via email to appropriate site personnel prior 
to enrolling the first patient. Clinical nurses at the site will be provided with 
Source Document Flow Sheets to capture data at enrollment and for each study 


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visit. Although some data fields on the Flow Sheets will not be entered into EDC, 
they must be captured on the Flow Sheets for monitoring purposes. 
 
All data .must be submitted within 72 hours of the data collection visit. Data entry 
will begin with the patient’s randomization with enrollment date to be date of 
signed informed consent. Edit checks will fire in real time as data is being 
entered in the EDC system to ensure quality data is provided. In addition to edit 
checks, queries will be generated as Discrepancy Notes from the Data Manager 
and Monitors. Sites will have ten business days to update a query. It is the 
responsibility of the coordinator at each site to ensure that data has been 
submitted. Cancer Insight quality assurance personnel or the sponsor’s 
representative will perform an audit of the site-specific Flow Sheets and will 
match them against source documents to ensure the quality of data coming to 
the Data Manager in the EDC per the internal monitoring plan. 
 
The database, hosted by OpenClinica, resides in a SAS 70 Type II data center 
and meets ISO 17799 standards for information security. The EDC system is 
HIPAA and 21 CFR part 11 compliant with robust audit logs, controlled user 
access and electronic signature/password management. In addition to the site 
user, the Data Manager, the Monitors, the study PI and Sponsor/Sponsor 
representative have access to the database. Each user is assigned a role which 
grants limited access and functionalities dependent upon that specific role. 
 
4.9 
Statistical methods 
 
4.9.1 Rationale for study design 
 
In patients with HER2-positive breast cancer who receive neoadjuvant 
chemotherapy plus trastuzumab, the 3-year Kaplan-Meier estimates of RFS are 
95.7% for patients that achieve a pCR versus 80.1% for patients that do not 
achieve a pCR (p=.02).24 For HER2-positive breast cancer patients that undergo 
surgery as a first intervention and are found to be node-positive, 5-year DFS 
rates are 72% for those with 4 or more positive lymph nodes and 78% for those 
with 1-3 positive lymph nodes. 15 Based on preliminary data, the combination of 
trastuzumab plus a CTL-eliciting HER2-derived peptide vaccine virtually 
eliminated recurrences. This study will investigate the combination of 
trastuzumab and nelipepimult-S+GM-CSF in an adequately powered randomized 
clinical trial. 
 
4.9.2 Sample size determination 
 
The primary objective of this study is to compare DFS between the two treatment 
groups. The study will have a 3 year follow-up period. We anticipate a 3-year 
DFS of 80% in patients randomized to receive trastuzumab without the vaccine 
versus 95% in patients randomized to receive trastuzumab + nelipepimut-S+GM- 


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CSF (estimates are based on data from previous studies, section 1.2 and table 
2).24-26 In order to detect a DFS difference of 0.15 using a one-sided alpha level 
of 0.05 with 80% power, assuming an accrual time of 2 years, follow-up time of 3 
years and 5% loss to follow-up (2.5% in each group), 100 patients (50 in each 
arm) are required. The power computation was determined using PASS 
software package version 2005. 
 
4.9.3 Data analysis 
 
The intention-to-treat (ITT) population will include all patients who were randomly 
assigned.  ITT analysis will be performed with each randomized patient 
evaluated in the treatment arm to which randomized, regardless of actual 
treatment received. The per-protocol (PP) population is a subset of the ITT 
population. Patients may be excluded from the PP population for the following 
reasons and others as determined prior to database lock: violations of eligibility 
criteria, development of other malignancies, early recurrences occurring prior to 
completion of the entire PVS, receiving alternative disease-directed therapy 
without evidence of recurrence, or major deviation from prescribed vaccination 
schedule (to include boosters).  Additionally, with the inclusion of HLA-A24 
and/or A26-positive patients, the ITT and PP analysis will be performed with and 
without these patients. 
 
 
4.9.3.1 Study patient characteristics 
 
Demographic characteristics of all patients in the ITT and PP populations will be 
summarized including age, race, disease histology, tumor size, estrogen 
receptor/progesterone receptor status, grade, nodal status, AJCC clinical stage, 
surgery extent, and other disease-directed therapies to include chemotherapy, 
radiation therapy, and hormonal therapy. Continuous variables will be 
summarized using the number of patients, mean, standard deviation, median, 
minimum, and maximum; and categorical variables will be summarized using the 
frequency count and the percentage of patients in each category. Differences 
between treatment groups will be determined using a chi-square test for 
categorical variables and a t-test for continuous variables. 
 
4.9.3.2 Primary efficacy analysis 
 
The primary efficacy endpoint of the trial is invasive DFS. Invasive DFS will be 
defined as the time from the date of initiation of trastuzumab maintenance 
therapy to the date of invasive local, regional or distant recurrence, new primary, 
death due to any cause or last follow-up date if patient had no evidence of 
disease. Invasive DFS estimates will be determined using the Kaplan-Meier 
method and differences between treatment groups will be assessed using the 


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log-rank test. In addition, Cox proportional hazards regression models will be fit 
to determine the association between DFS and treatment group and covariates 
of interest. The primary efficacy analysis will be performed on the ITT population 
as primary and the PP population as secondary. 
 
Because it is anticipated that 50% of patients will be enrolled at MD Anderson, 
the primary efficacy analysis will also be conducted by site for the ITT and PP 
populations in order to explore the possibility of a treatment by investigative site 
interaction. Investigative sites that individually represent fewer than ten patients 
will be combined for this exploratory analysis; geographic region will be 
substituted if the average number of patients per site is fewer than ten. 
4.9.3.3 Secondary efficacy analysis 
Safety 
The incidence of treatment-emergent AEs, SAEs, severe AEs, AEs related 
to study drug (defined as possibly, probably, or definitely related to study 
drug), and AEs which lead to study discontinuation will be summarized by 
treatment group.  Differences between treatment groups will be 
determined using Fisher’s exact tests. 
 
A formal interim analysis for safety will be performed after the midpoint 
(50th patient) of enrollment and randomization. 
 
Time to AE onset will be estimated using the Kaplan-Meier method and 
treatment differences will be determined using the log-rank test. In 
addition, duration of any AE with >10% incidence, total number of AEs, 
SAEs, and related AEs will be summarized and treatment group 
differences assessed using a t-test or Wilcoxon rank sum test, depending 
on the distribution of the data. 
 
Immunologic response 
 
Immunologic responses will be determined using DTH reactions (in vivo) 
and dextramer assays (in vitro). With respect to the in vivo immunologic 
response as assessed by DTH reaction, the treatment groups will be 
compared with respect to the percentage with a positive post-vaccination 
DTH (defined as greater than pre-vaccination DTH) as well as absolute 
DTH reaction sizes within the groups. Expression percentages will be 
compared using a chi-square test while continuous outcomes such as 
mean DTH response will be compared using a paired t-test (within 
treatment group) and a two-sample t-test (between treatment groups). For 
the in vitro response evaluated by the dextramer assay, the number of 
peptide-specific CTL will be determined at multiple time points (see 
Section 4.4.4) during the trial. Changes from pre-vaccination levels to 


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each time point will be computed and evaluated using a repeated 
measures mixed-effects model with terms for treatment group, time point, 
and treatment group-by-time point interaction. . 
 
4.9.4 Withdrawal 
 
Patients may withdraw or be discontinued from the study as discussed in Section 
4.6. Patients who do withdraw or are discontinued will be included in the efficacy 
analyses unless a patient withdraws consent to participate. In the instance of a 
patient withdrawing consent, any data collected will be excluded from analysis. 
 
4.9.5 Missing data 
 
Every reasonable attempt will be made to recover any missing data. If any data 
for the primary efficacy measure remains missing that data point will be excluded 
from analysis for that patient. 
 
4.9.6 Interim analysis 
 
No interim analysis is currently planned. 
 
5.0 
ADVERSE EVENTS 
 
Reporting of adverse events will be performed in accordance with the Data 
Safety Monitoring Plan (Appendix G) and Sections 5.2 and 5.3 below. 
 
5.1 
Adverse event and reporting definitions 
 
With the occurrence of an adverse event, the first concern will be for the safety of 
the patient. Investigators are required to report to the IRB, medical monitor, 
clinical research organization (CRO) Quality Assurance Officer or her designee 
and CRO Regulatory Affairs Officer any serious adverse event, whether 
expected or unexpected, and which is assessed by the investigator to be 
reasonably or possibly related to or caused by the vaccine components. Elective 
surgeries resulting in hospitalization and unrelated to the study agents will not be 
reported as SAEs, (breast reconstruction, etc.). All events meeting the outlined 
criteria will be reported for the time period beginning with any amount of 
exposure to vaccine components through the protocol-defined follow-up period. 
The Regulatory Affairs Officer will then report the event to the 
Sponsor/Investigator. The IND Sponsor/Investigator or Sponsor/Investigator’s 
representative will report these to the FDA and Galena Biopharma. Serious 
criteria, definitions, and guidance for reporting follow. 
 
An adverse event (AE) is any untoward medical occurrence in a patient 
participating in an investigational study or protocol regardless of causality 


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assessment. An adverse event can be an unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, syndrome or disease 
associated with or occurring during the use of an investigational product whether 
or not considered related to the investigational product. 
 
Serious adverse events (SAE) are AEs occurring at any dose which meet one or 
more of the following serious criteria: 
 
• Results in death (i.e. the AE caused or led to death) 
• Is life-threatening (i.e. the AE placed the patient at immediate risk of 
death; it does not apply to an AE which hypothetically might have caused 
the death if it were more severe) 
 
• Requires or prolongs inpatient hospitalization (i.e. the patient was 
admitted to the hospital or extended hospitalization due to the event) 
 
• Is disabling (i.e. the AE resulted in a substantial disruption of the patient’s 
ability to carry out normal life functions) 
 
• Results in a congenital anomaly/birth defect (i.e., an adverse outcome in a 
child or fetus of a patient exposed to the study drug prior to conception or 
during pregnancy) 
 
• Does not meet any of the above serious criteria but may jeopardize the 
patient and may require medical or surgical intervention to prevent one of 
the outcomes listed above 
 
Expected AEs are those AEs that are listed or characterized in the current 
Investigator Brochure. 
 
Unexpected AEs are those not listed in the current Investigator Brochure. This 
includes AEs for which the specificity or severity is not consistent with the 
description in the Investigator Brochure. For example, under this definition, 
hepatic necrosis would be unexpected if the Investigator Brochure only referred 
to elevated hepatic enzymes or hepatitis. 
 
5.2 
Reporting of serious adverse events associated with this study 
 
Each site PI will within 24 hours of notification of the event report all related or 
unrelated serious AEs occurring in patients enrolled at their respective study site 
to the Internal Review Board (IRB) of their site (see AE Reporting Algorithm, 
Appendix H). This will be accomplished by submitting an AE report memorandum 
to the IRB per the IRB’s site-specific standard operating procedures. The site PI 
will also, within 24 hours of notification of the event, forward a copy of the serious 


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adverse event report (CRF Form 3.1(g)) to the CRO Quality Assurance (QA) 
Officer or her designee, and to the CRO Regulatory Affairs Officer. In addition, 
the site PI will also follow the same reporting for all hypersensitivity events of 
grade 2 or greater in the primary series. 
 
The QA Officer or her designee will then forward the report to the 
Sponsor/Investigator. The Sponsor/Investigator or sponsor’s representative will 
review the serious AE to determine the need for expedited reporting to the FDA. 
If expedited reporting is required, the site nurse coordinator will be notified by the 
QA Officer or her designee to complete a FDA Form 3500 (MedWatch) 
(Appendix I). The Regulatory Affairs Officer will then submit the final report to the 
Sponsor/Investigator or Sponsor/Investigator’s representative who will report 
them to the FDA. 
 
 
5.2.1 FDA Form 3500A (MedWatch) reporting guidelines 
 
In addition to completing appropriate patient demographic and suspect 
medication information, the report should include the following information within 
the Event Description (Section 5) of the FDA Form 3500A form (MedWatch): 
 
• Protocol description (and number, if assigned) 
• Description of event, severity, treatment, and outcome if known 
• Supportive laboratory results and diagnostics 
• Investigator’s assessment of the relationship of the AE to each 
investigational product and suspect medication 
 
Follow-up information: 
 
Additional information may be added to a previously submitted report by any of 
the following methods: 
 
• Adding to the original FDA Form 3500A (MedWatch) report and 
submitting as follow-up 
 
• Adding supplemental summary information and submitting it as follow-up 
with the original FDA Form 3500A (MedWatch) 
 
• Summarizing new information and faxing it with a cover letter including 
patient identifiers (i.e. D.O.B. initial, patient number), protocol description 
and number, if assigned, brief adverse event description, and notation 
that additional or follow-up information is being submitted. (The patient 


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identifiers are important so that the new information is added to the 
correct initial report.) 
 
The CRO, IND sponsor or Galena Biopharma may contact the reporter for 
additional information, clarification, or current status of the patient for whom an 
AE was reported. For questions regarding SAE reporting, you may 
contact the Quality Assurance (QA) Officer. 
Study Drug Relationship: 
The Investigator will determine which events are associated with the use of study 
drug. For reporting purposes, an AE should be regarded as possibly related to 
the use of vaccine components if the Investigator believes: 
 
• There is a clinically plausible time sequence between onset of the AE 
and administration of vaccine components; and/or 
 
• There is a biologically plausible mechanism for vaccine components to 
cause or contribute to the AE; and 
 
• The AE cannot be attributed solely to concurrent/underlying illness, other 
drugs, or procedures. 
 
5.3 
Reporting requirements for IND holders 
 
For Sponsored IND Studies, there are some additional reporting requirements for 
the FDA in accordance with the guidance set forth in 21 CFR § 600.80. Events 
meeting the following criteria need to be submitted to the FDA as expedited IND 
Safety Reports according to the following guidance and timelines: 
 
7 Calendar-Day Telephone or Fax Report: The Sponsor is required to notify the 
FDA of any fatal or life-threatening AE that is unexpected and assessed by the 
investigator to be possibly related to the use of the investigational product. An 
unexpected AE is one that is not already described in the Investigator Brochure 
for the vaccine. Such events are to be reported by the sponsor to the FDA and 
within 7 calendar days of first learning of the event. 
 
15 Calendar-Day Written Report: The Sponsor is also required to notify the FDA 
and all participating investigators, in a written IND Safety Report, of any serious, 
unexpected AE that is considered reasonably or possibly related to the use of the 
study agents. An unexpected AE is one that is not already described in the 
Investigator Brochure for vaccine. 
 
• Written IND Safety Reports should include an Analysis of Similar Events 
in accordance with regulation 21 CFR § 312.32. All safety reports 


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previously filed with the IND concerning similar events should be analyzed 
and the significance of the new report in light of the previous, similar 
reports commented on. 
 
• Written IND safety reports with Analysis of Similar Events are to be 
submitted to the FDA and all participating investigators within 15 calendar 
days of first learning of the event. The FDA prefers these reports on a 
FDA Form 3500A (MedWatch) but alternative formats are acceptable (e.g. 
summary letter). 
 
6.0 
INVESTIGATOR REQUIREMENTS 
 
6.1 
Study initiation 
 
Before the start of this study, the following documents must be on file with the 
sponsor, Galena Biopharma, or a representative: 
 
• Original U.S. FDA Form 1572 for each site (for all studies conducted 
under U.S. Investigational New Drug [IND] regulations), signed by the 
Principal Investigator. The names of any sub-investigators must appear 
on this form. Investigators must also complete all regulatory 
documentation as required by local and national regulations. 
 
• Current curriculum vitae of the Principal Investigator 
• Written documentation of IRB approval of protocol and informed consent 
document 
 
• A copy of the IRB-approved informed consent document 
• A signed Clinical Research Agreement 
6.2 
Study completion 
 
The following materials are requested by Galena Biopharma when a study 
is considered complete or terminated: 
 
• A summary, prepared by the Principal Investigator, of the study, and/or a 
study manuscript, and/or a study abstract submitted to scientific 
conferences. 
 
 
 
6.3 
Informed consent 


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An informed consent template will be provided, and the final IRB-approved 
document must be provided to the sponsor for regulatory purposes. The informed 
consent document must be signed by the patient or the patient’s legally 
authorized representative before his or her participation in the study. The case 
history for each patient shall document that informed consent was obtained prior 
to participation in the study. A copy of the informed consent document must be 
provided to the patient or the patient's legally authorized representative. If 
applicable, it will be provided in a certified translation of the local language. 
Signed consent forms must remain in each patient’s study file and must be 
available for verification by study monitors at any time. 
 
 
6.4 
Institutional review board or ethics committee approval 
 
This protocol, the informed consent document, and relevant supporting 
information must be submitted to the IRB for review and must be approved 
before the study is initiated. The study will be conducted in accordance with U.S. 
FDA, applicable national and local health authorities, and IRB requirements. 
 
The Principal Investigator is responsible for keeping the IRB apprised of the 
progress of the study and of any changes made to the protocol as deemed 
appropriate, but in any case the IRB must be updated at least once a year. The 
Principal Investigator must also keep the IRB informed of any significant adverse 
events. Investigators are required to promptly notify their respective IRB of all 
adverse drug reactions that are both serious and unexpected. This generally 
refers to serious adverse events that are not already identified in the Investigator 
Brochure and that are considered possibly or probably related to the molecule or 
study drug by the investigator. Some IRBs may have other specific adverse 
event requirements that investigators are expected to adhere. Investigators must 
immediately forward to their IRB any written safety report or update provided by 
Galena Biopharma (e.g., IND safety report, Investigator Brochure, safety 
amendments and updates, etc.). 
 
 
Ethics and Regulatory Considerations 
The protocol will be reviewed and approved by the IRB or Independent Ethics 
Committee (IEC) of each participating center prior to study initiation. A list of 
IRB/IEC members should be obtained by the investigator and provided to the 
sponsor and sponsor representative. Any documents that the IRB/IEC may need 
to fulfill its responsibilities, such as protocol amendments and/or information from 
the sponsor will be submitted to the IRB/IEC. The IRB/IEC’s written unconditional 
approval of the study protocol and the informed consent form will be in the 
possession of the investigator and the sponsor before the study is initiated. The 
IRB/IEC’s unconditional approval statement will be transmitted by the investigator 
or designee to the sponsor/sponsor representative prior to shipment of study 


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drug supplies to the site. This approval document must refer to the study by 
exact protocol title and protocol version number/date and must identify the 
documents reviewed and the date of review. 
 
Protocol modifications or changes may not be initiated without prior written 
IRB/IEC approval except when necessary to eliminate immediate hazard to the 
patients. Such modifications will be submitted to the IRB/IEC and written 
verification that the modification was submitted and IRB/IEC 
acknowledgement/approval should be obtained and transmitted to the 
Sponsor/Investigator or Sponsor/Investigator’s representative. The IRB/IEC must 
be informed by the principal investigator of any changes or revisions of informed 
consent form or other documents originally submitted for review; serious and/or 
unexpected adverse experiences occurring during the study; any new information 
that may affect adversely the safety of the patients or the conduct of the study; 
annual updates and/or request for re-approval; and when the study has been 
completed. 
 
6.5 
Study monitoring requirements 
 
Site visits may be conducted by authorized Sponsor representative or Galena 
Biopharma representatives to inspect study data, patient’s medical records, and 
CRFs in accordance with current U.S. GCPs and the respective local and 
national government regulations and guidelines (if applicable). 
 
The Principal Investigator will permit authorized representatives of 
Sponsor/Investigator, Galena Biopharma, the U.S. FDA, and the respective 
national or local health authorities to inspect facilities and records relevant to this 
study. 
 
6.6 
Data Safety Monitoring Plan 
 
A Data Safety Monitoring Plan (DSMP) (Appendix F) describing the CRO internal 
monitoring plan includes data safety and integrity and site initiation/QA 
monitoring, as well as external monitoring plan - the Data Safety Monitoring 
Board (DSMB) charter and responsibilities. 
 
6.7 
Study medication accountability 
 
The study drug will be provided by Galena Biopharma. The recipient will 
acknowledge receipt of the drug by returning the INDRR-1 form indicating 
shipment content and condition. Damaged supplies will be replaced. Accurate 
records of all study drug dispensed from and returned to the study site should be 
recorded by using the institution’s drug inventory log or the NCI drug 
accountability log. All partially used or empty containers should be disposed of at 
the study site according to institutional standard operating procedure. Return 


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unopened, expired, or unused study drug with the Inventory of Returned Clinical 
Material form as directed by Galena Biopharma. 
 
6.8 
Disclosure of data 
 
Patient medical information obtained by this study is confidential, and disclosure 
to third parties other than those noted above is prohibited. Upon the patient’s 
permission, medical information may be given to his or her personal physician or 
other appropriate medical personnel responsible for his or her welfare. Data 
generated by this study must be available for inspection upon request by 
representatives of the U.S. FDA, national and local health authorities, Galena 
Biopharma, and the IRB for each study site, if appropriate. 
 
6.9 
Retention of records 
 
U.S. FDA regulations (21 CFR §312.62[c]) require that records and documents 
pertaining to the conduct of this study and the distribution of investigational drug, 
including CRFs, consent forms, laboratory test results, and medication inventory 
records, must be retained by the Principal Investigator for two years after 
marketing application approval. If no application is filed, these records must be 
kept two years after the investigation is discontinued and the U.S. FDA and the 
applicable national and local health authorities are notified. The sponsor will 
notify the Principal Investigator of these events. For studies conducted outside 
the United States under a U.S. IND, the Principal Investigator must comply with 
U.S. FDA IND regulations and with the record retention policies of the relevant 
national and local health authorities. 
 
6.10 
Publications 
 
The investigator must agree to send to the Sponsor/Investigator or 
Sponsor/Investigator’s representative, for review all manuscripts, abstracts and 
presentations using data from this study prior to their submission. The 
Sponsor/Investigator or Sponsor/Investigator’s representative reserves the right 
to delete from such materials any part or parts deemed to be confidential or 
proprietary. 
 
6.11 
Changes to protocol 
 
The protocol may not be modified without written approval of the 
Sponsor/Investigator or Sponsor/Investigator’s representative, or the study 
director. All changes to the protocol must be submitted to the FDA, the 
overseeing IRB, and local IRB/IEC. Additionally, changes must be approved by 
overseeing IRB prior to their implementation. Documentation of IRB/IEC approval 
must be sent to the Sponsor/Investigator or Sponsor/Investigator’s 
representative, and the Study Director immediately upon receipt. Any changes 


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and modifications to the informed consent language must be reviewed and 
approved by the Sponsor/Investigator or Sponsor/Investigator’s representative, 
and the Study Director prior to submission to the local IRB. 


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Immunotherapy for Breast Cancer Patients: Safety and Immunologic Data from a 
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Appendix A 
 
Trial Schema 
 
 
 


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51 
 
 
 
Appendix B 
 
Vaccination Timeline 
 
 
 
 


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Appendix C 
 
NCI Common Terminology Criteria for Adverse Events, v4.03 
obtained from http://ctep.cancer.gov/forms/CTCAEv4.03.pdf 
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06 14_QuickReference_8.5x11.pdf 


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Appendix D 
 
 
 2014-0443 
Phase II Trial of combination immunotherapy with nelipepimut-S + GM-CSF 
(NeuVax™) and trastuzumab in high-risk HER2+ breast cancer patients 
 
Dosing Preparation Instructions 
 
 
Dosing Summary: 
The study treatment is 1000 mcg of E75 peptide (nelipepimut-S) mixed with 250 mcg of 
lyophilized Leukine® (sargramostim, GM-CSF) or placebo (water for injection) mixed with 
Leukine, administered in four separate 0.4 mL intradermal injections. 
 
The E75 peptide is provided as 1.5 mg/mL of E75 acetate (contains a 1.42 mg/mL solution of the 
E75 peptide). Using aseptic technique, 0.8 mL of E75 acetate solution (1136 mcg E75 peptide) is 
added to the re-constituted Leukine. Taking into account vial losses, this procedure will yield a 
total dose of approximately 1010 mcg of E75 peptide in a volume of 1.6 mL. 
 
Stability: 
Once prepared, administer the E75 peptide/placebo and lyophilized Leukine mixture within 6 
hours. 
 
Materials: 
 
1. 
Randomized Test Drug kit with Patient ID number. This kit contains either a single 1.0 
mL vial of E75 acetate solution (1.5 mg/mL) or a single 1.0 mL vial of placebo. 
2. 
Lyophilized Leukine kit with Patient ID number. This kit contains a single vial of 
lyophilized Leukine (250 μg). 
3. 
Ancillary Supplies: 7-1mL TB syringes, 5-3/8 inch 26G needles, 4-2 inch 21G needles, 
2-3 mL luer lock syringes. 
 
Dose Preparation: 
 
NOTE: DO NOT INVERT ANY STUDY DRUG VIALS DURING PREPARATION 
 
1. 
Remove cap from vial of SWFI and wipe septum with alcohol prep pad. 
2. 
Remove cap from vial of lyophilized Leukine (250 μg) and wipe with alcohol prep pad. 
3. 
Attach a 2 inch 21G needle to a 1.0 mL TB syringe and draw up 1.0 mL from the vial of 
SWFI. 
4. 
Using the needle and syringe from step #3, insert the needle into the Leukine vial. 


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• Angle the needle to the side of the Leukine vial and slowly expel 1.0 mL SWFI into 
the lyophilized Leukine. 
• DO NOT INVERT VIAL at any time. Roll the vial slowly between your hands to 
mix, taking care to be sure all powder is dissolved in the vial. 
• Reconstitution of lyophilized Leukine should take approximately 2 minutes. DO NOT 
SHAKE OR VORTEX. 
5. 
Remove cap from vial of Test Drug (E75 acetate, 1.5 mg/mL or placebo) and wipe 
septum with alcohol prep pad. 
6. 
Attach a 2 inch 21G needle to a 1.0 mL TB syringe. Leaving vial upright (do not invert), 
insert the 2 inch needle all the way down to the bottom of the vial and withdraw 0.8 mL 
into the syringe. 
7. 
Using the needle and syringe from step #6, slowly inject the 0.8 mL of Test Drug into the 
reconstituted Leukine vial. 
8. 
Keeping the vial upright, slowly roll the Leukine/Test Drug vial between your hands. 
Allow any foaming to dissipate before continuing. 
9. 
Attach a 2 inch 21G needle to a 3.0 mL syringe and draw 2.0 mL of air into the syringe. 
Inject 2.0 mL of air into the upright Leukine/Test Drug vial. DO NOT INVERT THE 
VIAL. Insert the 2 inch needle down to the bottom of the vial and slowly withdraw the 
entire contents of the vial making sure all liquid is removed. 
10. 
Using the needle and syringe containing the Leukine/Test Drug from step #9, top-fill four 
1.0 mL TB syringes with 0.4 mL of the Leukine/Test Drug in each syringe. 
11. 
Attach a new sterile 3/8 inch 26G needle to each syringe containing 0.4 mL of 
Leukine/Test Drug. Appropriately label each syringe and dispense syringes for 
intradermal administration of study drug. The syringes must be used within 6 hours of 
mixing. 
 
 
 
 
 
Guidance for DTH Preparation from E75 Acetate Solution 1.5mg/1ml Vial 
 
To Prepare the DTH skin test, withdraw 0.07 mL of the 1.5mg/1.0mL E75 
acetate solution. 
The DTH skin test is administered intradermally using 0.1 mL (100 μg) of the E75 
acetate solution. 
 
Supplies: All DTH skin test kits will contain an active vial, which contains 1.0 mL 
of E75 acetate in WFI at 1.5mg/mL. Some DTH kits will contain a second vial, 
which is a control vial containing 1.0 mL of Water For Injection (WFI). If the DTH 
kit contains the WFI discard the WFI in the kit per your institution’s policy. 


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E75 acetate solution preparation: 
 
1. Remove cap from 1.5 mg/1mL E75 acetate solution and wipe with alcohol 
prep pad 
2. Open 1 mL syringe with needle attached and uncap the needle 
3. Draw > 0.07 mL air into the syringe and inject into the E75 acetate solution 
vial 
4. Withdraw 0.07 mL of E75 acetate solution into the syringe 
5. Remove the syringe and the needle from the vial 
6. Drawback syringe plunger to capture hub volume into the syringe 
7. Remove and discard needle 
8. Attach a new, sterile needle to the syringe containing 0.07 mL (100 ug) 
E75 acetate solution for intradermal administration. 


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Appendix E 
 
Leukine package insert 


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Appendix F 
 
ECOG Performance Status Criteria 
 
Score 
Description 
0 
Fully active, able to carry on all pre-disease performance 
without restriction 
1 
Restricted in physically strenuous activity but ambulatory 
and able to carry out work of a light or sedentary nature, 
e.g., light housework, office work 
2 
Ambulatory and capable of all self-care but unable to carry 
out any work activities. Up and about more than 50% of 
waking hours. 
3 
Capable of only limited self-care, confined to bed or chair 
more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any self-care. Totally 
confined to bed or chair. 


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Appendix G 
 
Data Safety Monitoring Plan 
This is a free standing document 


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Appendix H 
 
Adverse Events Reporting Algorithm 
 
 


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Appendix I 
 
MedWatch FDA Form 3500 Link 
 
http://www.fda.gov/downloads/Safety/MedWatch/DownloadForms/ucm082728.pdf 


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APPENDIX J 
 
Study Flowchart 
 
 
 
 
 
Days –60 to –1 
 
 
Week 1 
(Day 0) 
 
 
 
DTH Pre/Post (6) 
vaccines 2 
Every 3 
Weeks 
Every 3 
Months 
 
 
 
Every 6 
Months 
 
 
 
If Clinically 
Indicated 
 
 
Final Visit 
in 36th month 
Hematology (CBC, diff., platelets)1 
X 
 
 
x 
X1 
 
 
Chemistry panel (CMP) 
X 
 
 
X 
X1 
 
 
HLA blood draw for typing 
During study screening process/patient identification process. 
Immunological Samples2 
 
 
X2 
X2 
 
X2 
Trastuzumab administration (q3 weeks) 
 
 
 
X 
 
 
 
DTH administration3 
 
 
1, 23 
 
 
 
Vaccine administration4 
 
 
 
X4 
 
 
 
Booster inoculations 5 
 
 
 
X5 
 
 
Post-Booster Immunological Samples2 
 
 
 
X2 
 
 
Complete medical history (treating physician) 
X 
 
 
 
X 
 
Complete physical exam (treating physician, based on SOC) 
X 
 
 
X 
X 
X 
 
Clinical assessment-Study nurse visit4,5,10 
 
X 
X4 
X4 
X5 
 
X10 
Weight, height (treating physician) 
X 
 
 
 
X 
 
ECOG performance status 
 
X 
 
 
 
 
Toxicity evaluation9,10 
 
 
X9 
X9 
X9 
X9 
X10 
Vital signs9 
 
 
X9 
X9 
X9 
 
 
Cardiac Assessment (MUGA scan or Echo)6 
 
 
 
X 
 
X6 
 
Chest X-ray (AP, lateral)/EKG 
 
 
 
 
X 
 
Urine pregnancy test (if applicable)7 
 
X7 
X7 
X7 
X7 
 
 
Tumor assessment (treating physician)8 
X8 
 
 
 
X 
 
Disease status monitoring8,10 
X8 
 
 
X8 
X8 
X8 
X10 
 
 
1 Per standard practice, all patients will have CBC/CMP monitored approximately every 3 
months during the primary series and approximately every 6 months during the booster 
phase. 
 
 
2Immunological samples will be drawn pre DTH#1 and #2, prior to each of 4 boosters, 1 
month (+/-1 week) after each booster, and at study completion 36 months after initiation 
of Trastuzumab maintenance therapy. 
 
3DTH #1 given prior to initiation of primary vaccine series, one month (+/- 1 week) after 
completion of primary vaccine series, and one month (+/-1 week) after the final booster 
inoculation. 
 
4Vaccine inoculation administered every 3 weeks for 6 inoculations during trastuzumab 
treatment schedule. Vaccine inoculations to begin with 3rd dose of maintenance 
trastuzumab infusion, optimally, but may be given with later maintenance doses of 
trastuzumab, provided there are at least six remaining doses of trastuzumab to overlap 


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with the PVS. Vaccine inoculations given within 30-120 minutes after completion of 
infusion. (With prior approval from the Principal Investigator, the vaccine may be 
administered 15 minutes after completion of trastuzumab infusion). 
 
5Booster inoculations will be given after initial vaccine series, every 6 months x4, 
beginning 6 months (+/- 2 weeks) after the completion of the Primary Vaccine Series 
Subsequent boosters will be given at 12, 18, and 24 months (+/- 2 weeks), after the 
completion of the PVS. 
 
6All patients will have cardiac assessment performed according to standard practice as 
dictated by their treating oncologist. Results of cardiac testing will be reviewed by the 
research nurses or study coordinators and any abnormal results will be reviewed by the 
study investigators. 
 
7Urine HCG waived if hysterectomy, bilateral oopherectomy, tubal ligation, documented 
absence of menses for two years, or FSH lab results verifying menopause. 
 
8Disease status will be determined by patients’ own physicians at the individual study 
sites during routine follow-up. This will include physical examination every 3-6 months 
for all patients and yearly mammogram for patients undergoing breast conserving 
therapy (per ASCO guidelines) 
 
9Toxicity evaluation done during 30 minutes observation after vaccine administration with 
vital signs taken as needed and during return visit or by phone call 48-72 hours after 
inoculation. Delayed adverse event reporting per protocol. 
 
10Final study assessment in month 36 by study nurse with toxicity assessment and 
disease status update 


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Appendix K 
 
PROTOCOL TITLE: 
Phase II Trial of Combination Immunotherapy 
with nelipepimut-S + GM-CSF (NeuVax™) and 
Trastuzumab in high-risk HER2+ Breast 
Cancer Patients (2014-0443) 
 
STUDY DRUG: 
Nelipepimut-S + GM-CSF (NeuVax™) 
 
PRINCIPAL INVESTIGATOR: 
Elizabeth A. Mittendorf, MD, PhD 
 
PROGRAM DIRECTOR 
SPONSOR/INVESTIGATOR: 
George E. Peoples, MD, FACS 
 
PROTOCOL VERSION/DATE: 
2.1/11 April 2016 
 
INVESTIGATOR’S AGREEMENT / INVESTIGATOR’S SIGNATURE PAGE 
I have read the protocol described above. I have fully discussed the objectives of 
this trial and the content of this protocol with the Sponsor or Sponsor’s 
representative. I understand that the information in this protocol is confidential 
and should not be disclosed, other than to those directly involved in the execution 
or the ethical review of the trial, without written authorization from Cancer Insight. 
It is, however, permissible to provide information to a patient in order to obtain 
consent. I agree to conduct this trial according to the protocol and to comply with 
its requirements, subject to ethical and safety considerations and guidelines, and 
to conduct the trial in accordance with all applicable regulations, and guidelines 
as stated in the protocol and other information supplied to me. I understand that 
the Sponsor may decide to suspend or prematurely terminate the trial at any 
time, for whatever reason. Such a decision will be communicated to me in 
writing. Conversely, should I decide to withdraw from execution of the 
trial, I will communicate my intention immediately, in writing to the Sponsor. 
 
Signed:  
Date:   
 
 
Investigator’s Name and Address: