Clinical trial NCT02636582
Conditions: Breast Ductal Carcinoma In Situ
Sponsor: National Cancer Institute (NCI)
Start date: 2016-06-14; Completion date: 2023-05-17
Enrollment: 43 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This phase II trial studies how well nelipepimut-S plus GM-CSF vaccine therapy or sargramostim works in treating patients with breast cancer. Vaccines made from peptide or antigen and/or a person's white blood cells mixed with tumor proteins may help the body build an effective immune response to kill tumor cells that express breast cancer antigens. It is not yet known whether nelipepimut-S plus GM-CSF vaccine or sargramostim is more effective in treating patients with breast cancer.

Detailed description:
PRIMARY OBJECTIVE:

I. Evaluate for nelipepimut-S-specific cytotoxic T lymphocyte (CTL; cluster of differentiation \[CD\]8+ T cell) response in patients receiving NeuVax (nelipepimut-S plus GM-CSF \[sargramostim\]) compared to patients receiving GM-CSF alone (control).

SECONDARY OBJECTIVES:

I. Toxicity profile and frequency of adverse events in women with ductal carcinoma in situ (DCIS) of the breast receiving nelipepimut-S vaccine as compared to women receiving GM-CSF alone.

II. Presence of DCIS at resection. III. Difference in HER2 expression in the biopsy and the surgical specimen excised post-vaccination.

IV. Histologic responses:

IVa. Degree of lymphocyte infiltration determined on hematoxylin and eosin (H\&E) stained slides and immune infiltration as determined by multiplex immunofluorescence staining for markers including but not limited to CD3, CD4 and CD8.

IVb. Immune infiltrates in normal tissue maximally distant from the tumor (in mastectomy samples).

OUTLINE: Patients are randomized to 1 of 2 arms.

ARM I: Patients receive nelipepimut-S plus GM-CSF vaccine intradermally (ID) on days 0 and 14 and then undergo surgery on day 28.

ARM II: Patients receive sargramostim ID on days 0 and 14 and then undergo surgery on day 28.

After completion of study treatment, patients are followed up at 1 and 3 months.

Arms: arm 1: Patients receive nelipepimut-S plus GM-CSF vaccine ID on days 0 and 14 and then undergo surgery on day 28. arm 2: Patients receive sargramostim ID on days 0 and 14 and then undergo surgery on day 28.
Interventions: intervention 1: Laboratory Biomarker Analysis intervention 2: Nelipepimut-S Plus GM-CSF Vaccine intervention 3: Sargramostim intervention 4: Surgical Procedure
Intervention descriptions: intervention 1: Correlative studies intervention 2: Given ID intervention 3: Given ID intervention 4: Undergo surgery

Protocol documents:
Prot_SAP_000.pdf: 
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COVER PAGE 
 
DCP Protocol #: 
MDA2014-04-02  
 
Local Protocol #: 2016-0164 (MD Anderson), 16-710 (DFCI), 17F.278 (TJU) 
 
VADIS Trial: Phase II trial of Nelipepimut-S Peptide Vaccine in Women with DCIS of the Breast 
 
 
Consortium Name: 
 
University of Texas MD Anderson Early Phase Clinical Research 
Consortium 
 
Name of Consortium Principal  
Powel H. Brown, MD, PhD 
Investigator: 
 
Professor and Chair, Department of Clinical Cancer Prevention 
 
 
University of Texas MD Anderson Cancer Center 
 
 
1515 Holcombe Boulevard, Unit 1360 
Houston, TX 77030-4009 
Telephone: (713) 792-4509 
Fax: (713) 794-4679 
 
 
phbrown@mdanderson.org 
 
MD Anderson Consortium 
 
Lana A. Vornik, MHA, MS 
Administrative Director: 
 
Associate Director, Res Planning and Development 
 
 
Department of Clinical Cancer Prevention 
 
 
University of Texas MD Anderson Cancer Center 
 
 
1515 Holcombe Boulevard, Unit 1360 
Houston, TX 77030-4009 
Telephone: (713) 792-9594 
Fax: (713) 745-4230 
 
 
lavornik@mdanderson.org 
 
MD Anderson Consortium 
 
Saba Abutaseh, BS 
Lead Org Coordinator: 
 
Program Manager 
 
 
Department of Clinical Cancer Prevention 
 
 
University of Texas MD Anderson Cancer Center 
 
 
1515 Holcombe Boulevard, Unit 1360 
Houston, TX 77030-4009 
Telephone: 713-745-8181 
Fax: (713) 745-4230 
 
 
sgabutas@mdanderson.org 
 
Organization Name: 
 
 
Dana Farber Cancer Institute 
Protocol Principal Investigator: 
Elizabeth A. Mittendorf, MD, PhD 
Robert and Karen Hale Distinguished Chair in Surgical Oncology 
Director, Breast Immuno-Oncology Program 
Dana-Farber/Brigham and Women’s Cancer Center 
450 Brookline Ave, Suite 1220 
Boston, MA 02215 
Telephone: 617-582-9980 
Fax: 617-632-2495 
emittendorf@bwh.harvard.edu  


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Investigator: 
 
Judy E. Garber, MD MPH 
Director, Center for Cancer Genetics and Prevention 
Medical Oncology 
 
 
450 Brookline Ave 
 
 
Boston, MA 02215  
 
 
Telephone: (617) 632-5770  
 
 
Fax: (617) 632-4088 
 
 
Judy Garber@dfci.harvard.edu 
 
 
Organization:  
 
 
University of Texas MD Anderson Cancer Center 
Investigator: 
Isabelle Bedrosian, MD, FACS 
Professor 
Breast Surgical Oncology 
 
 
1400 Pressler Blvd, Unit 1434 
Houston, TX 77030 
Telephone: (713) 563-1872 
Fax: (713) 794-5720 
ibedrosian@mdanderson.org 
 
 
Investigator:  
 
 
Powel H. Brown, MD, PhD 
 
 
 
 
 
Professor and Chair, Department of Clinical Cancer Prevention 
 
 
1515 Holcombe Boulevard, Unit 1360 
Houston, TX 77030-4009 
Telephone: (713) 792-4509 
Fax: (713) 794-4679 
 
 
phbrown@mdanderson.org 
 
 
Investigator:  
 
 
COL (ret) George E Peoples, MD, FACS 
Director, Cancer Vaccine Development Program  
and Cancer Insight, LLC 
Professor, Surgery, Uniformed Services University 
Professor (adjunct), Surgical Oncology, MD Anderson Cancer 
Center 
600 Navarro Street, Suite 500 
San Antonio, TX 78205 
Telephone: (210) 557-4291 
gpeoples@cancerinsight.com 
 
 
Investigator: 
Yun Wu, MD, PhD 
 
 
 
 
 
Associate Professor 
Department of Pathology  
1515 Holcombe Blvd.  
Unit 0085  
Houston, TX 77030 
 
 
 
 
 
Telephone: (713) 563-1847 
YunWu@mdanderson.org  
 


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Organization Name: 
 
 
Thomas Jefferson University 
Investigator:  
 
Adam C. Berger, M.D., FACS 
Chief, Section of Surgical Oncology 
Department of Surgery 
1100 Walnut Street, MOB, Suite 500 
Philadelphia, PA 19107 
T (215) 955-1622 
F (215) 923-8222 
Adam.Berger@jefferson.edu  
 
 
Organization: 
 
University of Texas MD Anderson Cancer Center 
Statistician: 
 
J. Jack Lee, Ph.D. 
 
 
1515 Holcombe Boulevard, Unit 447 
Houston, TX 77230 
 
 
Telephone: (713) 794-4158 
 
 
Fax: (713) 563-4242 
 
jjlee@mdanderson.org  
 
 
IND Sponsor:  
NCI/Division of Cancer Prevention  
 
IND#  
 
 
 
Agent(s)/Supplier: 
NeuVax TM (nelipepimut-S) supplied by SELLAS, Inc.; GM-CSF (rhuGM-CSF; 
Leukine® [sargramostim]) supplied by MRI Global 
 
 
 
 
 
NCI Contract # 
HHSN261201200034I 
 
Protocol Version Date: 10/24/2019 
 
Protocol Revision or   
Amendment #  
Version 16 
 


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SCHEMA 
 
VADIS Trial: Phase II trial of the Nelipepimut-S Peptide Vaccine in Women with DCIS of the 
Breast 
 
Pre- or post-menopausal women with DCIS on core biopsy 
 
Baseline Testing/Prestudy Evaluation (within 30 days prior to randomization) 
Screening visit informed consent, registration, screening for eligibility including HLA typing, cardiac evaluation 
 
Randomization 
Eligibility confirmation (the trial is limited to HLA-A2 positive participants), randomization 
 
 
 
 
 
 
 
Intervention: 
 
Vaccine 1 (day 0) 
Blood draw 
 
Vaccine 2 (day 14 +/- 3 days) 
 
Surgery (day 28 +/- 5 days) 
Blood draw 
 
Post-vaccinations follow up clinic visit (1 Month +/- 7days post-op; but prior to radiation, if radiation after 
surgery is advised) 
Blood draw; cardiac evaluation 
 
 
Final follow up clinic visit (3-6 Months after surgery) 
Blood draw 
 
 
Endpoints 
Primary:  Evaluate for nelipepimut-S-specific cytotoxic T lymphocyte (CTL; CD8+ T cell) response in vaccinated 
participants compared to participants receiving GM-CSF alone. 
Secondary: 
a) Toxicity profile and frequency of adverse events in women with DCIS of the breast receiving nelipepimut-S 
vaccine as compared to women receiving GM-CSF alone. 
b) Presence of DCIS at resection; 
c) Difference in HER2 expression in the biopsy and the surgical specimen excised post-vaccination; 
d) Histologic responses: 
a. 
Degree of lymphocyte infiltration as determined on H&E stained slides and immune infiltration as 
determined by multiplex immunofluorescence staining for markers including but not limited to CD3, CD4 
and CD8, 
e) Immune infiltrates in normal tissue maximally distant from the tumor (in mastectomy samples). 
NeuVax™ (nelipepimut-S+ GM-CSF);  
32 randomized / 27 evaluable
GM-CSF alone (Control) 
16 randomized / 13 evaluable


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TABLE OF CONTENTS 
 
COVER PAGE.............................................................................................................................................. 1 
SCHEMA ...................................................................................................................................................... 4 
1. 
OBJECTIVES .................................................................................................................................. 7 
1.1 
Primary Objectives .......................................................................................................................................... 7 
1.2 
Secondary Objectives ...................................................................................................................................... 7 
2. 
BACKGROUND ............................................................................................................................. 7 
2.1 
DCIS of the Breast .......................................................................................................................................... 7 
2.2 
NeuVax ........................................................................................................................................................... 8 
2.3 
Rationale ......................................................................................................................................................... 9 
3. 
SUMMARY OF STUDY PLAN ................................................................................................... 12 
4. 
PARTICIPANT SELECTION ....................................................................................................... 12 
4.1 
Inclusion Criteria ........................................................................................................................................... 12 
4.2 
Exclusion Criteria .......................................................................................................................................... 13 
4.3 
Inclusion of Women and Minorities .............................................................................................................. 14 
4.4 
Recruitment and Retention Plan .................................................................................................................... 15 
5. 
AGENT ADMINISTRATION ...................................................................................................... 15 
5.1 
Dose Regimen and Dose Groups: Vaccine dosage and preparation .............................................................. 15 
5.2 
NeuVax Inoculation Series Administration ................................................................................................... 15 
5.3 
Run-in Procedures ......................................................................................................................................... 16 
5.4 
Contraindications .......................................................................................................................................... 16 
5.5 
Concomitant Medications .............................................................................................................................. 16 
5.6 
Dose Modification ......................................................................................................................................... 16 
5.7 
Adherence/Compliance ................................................................................................................................. 18 
6. 
PHARMACEUTICAL INFORMATION ...................................................................................... 18 
6.1 
Study Agent (IND # TBA, IND Sponsor NCI DCP) ..................................................................................... 18 
6.2 
Reported Adverse Events and Potential Risks .............................................................................................. 19 
6.3  
Availability .................................................................................................................................................... 20 
6.4 
Agent Distribution ......................................................................................................................................... 20 
6.5  
Agent Accountability .................................................................................................................................... 21 
6.6 
Packaging and Labeling ................................................................................................................................ 21 
6.7 
Storage........................................................................................................................................................... 21 
6.8 
Registration/Randomization .......................................................................................................................... 21 
6.9 
Blinding and Unblinding Methods ................................................................................................................ 22 
6.10 Agent Destruction/Disposal .......................................................................................................................... 23 
7. 
CLINICAL EVALUATIONS AND PROCEDURES ................................................................... 24 
7.1 
Schedule of Events ........................................................................................................................................ 24 
7.2 
Baseline Testing/Prestudy Evaluation ........................................................................................................... 25 
7.3 
Evaluation During Study Intervention ........................................................................................................... 26 
7.4 
Evaluation at Completion of Study Intervention ........................................................................................... 27 
7.5 
Post-intervention Follow-up Period .............................................................................................................. 28 
7.6 
Methods for Clinical Procedures ................................................................................................................... 28 
8.  
CRITERIA FOR EVALUATION AND ENDPOINT DEFINITION ........................................... 28 
8.1 
Primary Endpoint .......................................................................................................................................... 28 
8.2 
Secondary Endpoints ..................................................................................................................................... 28 
8.3 
Off-Agent Criteria ......................................................................................................................................... 30 
8.4 
Off-Study Criteria ......................................................................................................................................... 30 
8.5 
Study Termination ......................................................................................................................................... 31 
9. 
CORRELATIVE/SPECIAL STUDIES ......................................................................................... 31 
9.1 
Rationale for Methodology Selection ............................................................................................................ 31 
9.2 
Comparable Methods .................................................................................................................................... 31 
10. 
SPECIMEN MANAGEMENT ...................................................................................................... 32 
10.1 Laboratories ................................................................................................................................................... 32 


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10.2 Collection and Handling Procedures ............................................................................................................. 32 
10.3 Shipping Instructions ..................................................................................................................................... 34 
10.4 Tissue Banking .............................................................................................................................................. 34 
11. 
REPORTING ADVERSE EVENTS ............................................................................................. 35 
11.1 Adverse Events .............................................................................................................................................. 35 
11.2 Serious Adverse Events ................................................................................................................................. 37 
12. 
STUDY MONITORING ............................................................................................................... 39 
12.1 Data Management ......................................................................................................................................... 39 
12.2 Case Report Forms ........................................................................................................................................ 39 
12.3 Source Documents ......................................................................................................................................... 39 
12.4 Data and Safety Monitoring Plan .................................................................................................................. 39 
12.5 Sponsor or FDA Monitoring ......................................................................................................................... 40 
12.6 Record Retention ........................................................................................................................................... 40 
12.7 Cooperative Research and Development Agreement (CRADA)/Clinical Trials Agreement (CTA) ............ 40 
13. 
STATISTICAL CONSIDERATIONS ........................................................................................... 41 
13.1 Study Design/Description .............................................................................................................................. 41 
13.2 Randomization/Stratification ......................................................................................................................... 42 
13.3 Accrual and Feasibility .................................................................................................................................. 42 
13.4 Primary Objective, Endpoint, Analysis Plan ................................................................................................. 42 
13.5 Secondary Objectives, Endpoints, Analysis Plans ........................................................................................ 42 
13.6 Reporting and Exclusions .............................................................................................................................. 43 
13.7 Evaluation of Toxicity ................................................................................................................................... 43 
13.8 Evaluation of Response ................................................................................................................................. 43 
13.9 Interim Analysis ............................................................................................................................................ 43 
13.10 Ancillary Studies ........................................................................................................................................... 44 
14. 
ETHICAL AND REGULATORY CONSIDERATIONS ............................................................. 44 
14.1 Form FDA 1572 ............................................................................................................................................ 44 
14.2 Other Required Documents ........................................................................................................................... 44 
14.3 Institutional Review Board Approval ............................................................................................................ 45 
14.4 Informed Consent .......................................................................................................................................... 45 
14.5 Submission of Regulatory Documents .......................................................................................................... 45 
14.6 Other .............................................................................................................................................................. 46 
15. 
FINANCING, EXPENSES, AND/OR INSURANCE ................................................................... 46 
REFERENCES ........................................................................................................................................... 47 
APPENDIX A ............................................................................................................................................. 50 
APPENDIX B ............................................................................................................................................. 51 
 


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1. 
OBJECTIVES 
 
1.1 
Primary Objectives  
1.1.1 
Evaluate for nelipepimut-S-specific cytotoxic T lymphocyte (CTL; CD8+ T cell) response 
in participants receiving NeuVax (nelipepimut-S plus GM-CSF) compared to participants 
receiving GM-CSF alone (Control). 
 
1.2 Secondary Objectives  
1.2.1 
Toxicity profile and frequency of adverse events in women with DCIS of the breast 
receiving nelipepimut-S vaccine as compared to women receiving GM-CSF alone; 
1.2.2 
Presence of DCIS at resection; 
1.2.3 
Difference in HER2 expression in the biopsy and the surgical specimen excised post-
vaccination; 
1.2.4 
Histologic responses: 
1.2.4.1 Degree of lymphocyte infiltration determined on H&E stained slides and immune 
infiltration as determined by multiplex immunofluorescence staining for markers 
including but not limited to CD3, CD4 and CD8;   
1.2.4.2 Immune infiltrates in normal tissue maximally distant from the tumor (in 
mastectomy samples). 
 
2. 
BACKGROUND 
 
2.1 
DCIS of the Breast 
 
Breast cancer accounts for approximately 41% of all cancers in women in the U.S [1].  It is further 
estimated that, in 2014 alone, new cases of cancer in U.S. women will include 232,670 diagnoses of 
invasive breast cancer and 51,933 diagnoses of ductal carcinoma in situ [2].  Current treatment guidelines 
for DCIS recommend local therapy to include either lumpectomy without lymph node surgery plus whole 
breast radiation therapy or total mastectomy with or without sentinel lymph node biopsy.  For patients 
that have ER+ DCIS and undergo breast conserving therapy with lumpectomy and radiation, tamoxifen 
may be considered for 5 years to reduce the risk of an ipsilateral breast recurrence. Despite high cure rates 
with this current standard of care for DCIS, there are still patients that recur and ultimately succumb to 
breast cancer.  A recent observational study reported on 108,196 women who had been diagnosed with 
DCIS from 1988 to 2011 and were captured in the Surveillance, Epidemiology, and End Results (SEER) 
program [3]. The mean age of diagnosis was 54 years of age.  For patients undergoing lumpectomy, the 
risk of an ipsilateral invasive recurrence at 10 years was 4.9% which was decreased to 2.5% if radiation 
was administered.  At 20 years, the breast cancer-specific mortality was 3.3% (95% CI, 3.0%-3.6%).  
Importantly, the risk of dying of breast cancer was increased after an ipsilateral invasive breast cancer 
recurrence (HR 18, 95% CI: 14-24, p<.001).  Interestingly, there were also patients (n=517) that died of 
breast cancer following a diagnosis of DCIS that did not experience a known in-breast invasive cancer 
recurrence.  Given these data, there is a need for more effective prevention strategies.  Our group has been 
investigating immune-based prevention strategies in breast cancer and this protocol represents one of 
these efforts specifically for patients with DCIS. 
 
Little is known regarding the endogenous immune response to DCIS or the potential role of targeting 
DCIS with immunotherapy.  In a study of 69 participants performed at the MD Anderson Cancer Center 
looking at the biologic and immunologic effects of pre-operative trastuzumab (monoclonal antibody 
targeting HER2), it was shown that a single dose of trastuzumab administered to participants with HER2+ 
DCIS does not result in histologic, antiproliferative or apopototic changes but did result in the ability to 
mount antibody-dependent cell mediated cytotoxicity in all participants [4].  Czerniecki et al. conducted a 
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histocompatibility class I and class II peptides. The trial enrolled participants with DCIS with HER2 
overexpression (>2+ intensity) in at least 10% of cells as determined using HerceptTest [5-7].  The trial 
enrolled 29 participants who were administered the vaccine as four weekly intranodal injections 
administered prior to surgery.  The vaccination strategy was shown to be feasible and safe.  In addition, 
the investigators demonstrated an antigen specific immune response with induction of IFNγ producing 
CD4+ and CD8+ T cells in the peripheral blood as well as in increased lymphocyte infiltrate into the 
surgically excised tumors versus the biopsy specimens.  Interestingly, in a small subset of participants, 
there was decreased HER2 expression in the surgically excised tumor compared to the pre-treatment 
biopsy in seven participants suggesting immunoediting of the HER2+ cells. 
 
Our group has also been investigating vaccination strategies in breast cancer.  Specifically, we have been 
investigating HER2-derived peptide vaccines.  Compared to DC vaccines which require the patients to 
undergo leukapheresis followed by an ex vivo processing of the monocyte precursors to generate the DC 
product, peptide vaccines represent a simple, “off the shelf” strategy that combines a peptide with an 
immunoadjuvant that can be administered as an intradermal inoculation.  One of the vaccines that we 
have studied is nelipepimut-S which is a major histocompatibility class (MHC) I vaccine that consists of 
the immunogenic peptide (nelipepimut-S) combined with the immunoadjuvant GM-CSF.  Previous data 
from clinical trials has shown that the nelipepimut-S vaccine is safe, with minimal toxicity, and effective 
in stimulating an antigen-specific immune response in metastatic [8-10] and early-stage invasive [11-14] 
breast cancer.  For patients with early stage cancer that have been rendered disease free with standard of 
care therapy, clinical trials have shown that the nelipepimut-S vaccine decreases the risk of recurrence 
[11-13]. Nelipepimut-S is currently being evaluated in a phase III registration trial (NCT01479244). In 
addition to its immunologic effects in breast cancer, the effect of nelipepimut-S treatment has been 
examined in the peripheral blood mononuclear cells (PBMCs) from healthy people.  Following 
stimulation with nelipepimut-S, the PBMCs from 50% of healthy donors exhibited  nelipepimut-S-
specific cytolytic activity (2 responded at priming, 3 responded after 2 additional restimulations [15].  In 
addition, the study suggested that nelipepimut-S-precursory cytotoxic T lymphocytes (CTLs) were not 
tolerized in the majority of donors [15]. 
 
Based upon these positive results, we developed this study testing the Nelipepimut-S vaccine in women 
with DCIS of the breast to determine whether antigen-specific immunity is induced and whether the 
induced immune response suppresses the growth of DCIS cells.  Completion of the current study will 
provide the necessary data to determine the next phase of study, specifically whether a subsequent, larger 
phase II will be conducted versus a phase III study. 
 
 
2.2 
NeuVax 
 
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 that can be recognized by T cells.  
Tumor-associated antigens 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 tumor-associated antigen.  Several peptides capable of inducing CTL have been described from 
the HER2 protein including 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. [16]).   
 
The vaccine consists of the nelipepimut-S peptide (1000 µg) mixed with 250 µg GM-CSF (significantly 
less than the GM-CSF dose of 250 µg/m2 administered daily either IV or SC for most clinical indications 
for GM-CSF).  This dose was determined to be the optimal dose in early phase clinical trials [17].  It has 




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symptoms and fatigue which are attributable to the GM-CSF.  There were no grade 4 or 5 toxicities.  All 
participants demonstrated in vitro immunologic responses and in vivo delayed type hypersensitivity 
(DTH) responses post-vaccination. With respect to in vitro immune responses, they were assessed using a 
dimer assay which enumerates the number of epitope-specific CTL (i.e. nelipepimut-S-CTL). A recurring 
pattern of clonal expansion was observed among vaccinated participants with an increasing percentage of 
CD8+ nelipepimut-S-CTL with successive vaccinations that peak during the vaccination series and then 
contract and plateau by the completion of the series.  The peak clonal expansion most commonly occurred 
after the third inoculation [12, 13].  At a median follow-up of 20 months, the recurrence rate seen in the 
vaccinated participants was 6% compared to 15% for participants in the observation group (p=0.04).  The 
level of nelipepimut-S-specific CTL generated in response to vaccination was assessed in HLA-A2+ 
participants enrolled on the study and 35% of participants were found to have a nelipepimut-S-CTL level 
above the mean with the recurrence rate among those participants being 3%.  For participants with a 
nelipepimut-S-CTL level below the mean, the recurrence rate was 14% [20].  
 
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 nelipepimut-S-specific CTL.  A voluntary booster program was initiated and 
vaccinated patients ≥ 6 months from completion of their primary vaccination series were eligible for 
participation.  Participants enrolled onto the trial after initiation of the booster program were consented 
prospectively to receive booster inoculations.  An initial report of the booster inoculations showed them to 
be safe and effective in stimulating nelipepimut-S-specific immunity in participants who had waning 
levels of nelipepimut-S -specific CTL six months following completion of their primary vaccination 
series [21].  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 [11].  Among the 187 participants (108 
HLA-A2/A3+ vaccinated; 79 HLA-A2/3- controls), the 60-month DFS rates were 90% for vaccinated 
participants versus 80% for controls (p=0.08), a 50% reduction in the relative risk of recurrence [11].  
Because the trials began as dose- and schedule-finding trials, not all participants received the dose that 
was eventually determined to be optimal (1000µg nelipepimut-S + 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 participants received a booster 
inoculation, the DFS rate in the 53 boosted participants was 96% versus 83% in vaccinated participants 
that did not receive a booster inoculation (p=.04). 
 
Taken together, these data suggested clinical benefit [11].  Nelipepimut-S was recently evaluated in a 
multicenter, multinational, prospective, randomized, double-blind, controlled phase III study 
(NCT01479244; PI:  Mittendorf) [22].  The trial randomized 758 lymph node-positive breast cancer 
participants with HER2 1+/2+ tumors in the adjuvant setting. Randomization was completed in April 
2015.  In June 2016, the Independent Data Monitoring Committee (IDMC) met to complete a planned 
safety and futility interim analysis that was triggered after 70 qualifying DFS events were reached.  At 
that time, the IDMC recommended that the trial be stopped due to futility.  The AEs observed in the 
Phase III study as of the date of the last data cutoff 27 June 2016 were consistent with the reactions 
observed in previous studies.  AEs reported for ≥ 10% of participants (includes both arm of the trial) in 
the pivotal study were limited to local injection site reactions, including induration (56%), swelling 
(46%), pain (40%), pruritus (55%), and edema (17%).  AEs reported for <20% of participants have 
included headache, fatigue, back pain and nausea.  AEs have been generally mild in severity, with no 
safety trends identified for Grade 3 or higher AEs or SAEs/deaths.  Two participants in the pivotal phase 
III study reported AEs related to study drug hypersensitivity including 1 patient with non-serious, Grade 2 
angioedema and 1 participant with a Grade 4 SAE of anaphylactic reaction, both of which were 
considered definitely related to study drug, resulted in discontinuation of treatment, and were noted to 
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as related to rhGM-CSF.  No other allergic- or anaphylactic-type reactions to E75 plus the rhGM-CSF 
adjuvant have been reported. 
 
Having demonstrated clinical efficacy in the adjuvant setting to prevent disease recurrence, the next 
logical step evaluating the nelipepimut-S peptide vaccine is to look at it in patients with DCIS of the 
breast.  DCIS of the breast represents premalignant disease and vaccinating in this setting is ideal for 
initial testing of the efficacy and safety of prophylactic vaccines.  Giving a vaccine to a patient with a 
premalignant lesion would boost preexisting immunity to one or more tumor antigens against which the 
patient has already generated an adaptive immune response.  In an analogous setting, Kimura et al. 
vaccinated participants with colonic adenomas and showed that vaccination was effective in inducing 
immune responses and immune memory in approximately 50% of participants [23]. This result led to Dr. 
Olivera Finn, a recognized expert in the field, to suggest that the next step in developing a preventive 
vaccine would be to evaluate vaccines known to generate an antigen-specific immune response in patients 
with premalignant disease [24].  
 
The current study will randomize participants to vaccine (NeuVax™; nelipepimut-S+GM-CSF) or GM-
CSF alone.  The use of GM-CSF alone as a control arm will allow us to investigate whether the 
nelipepimut-S -specific CTL response generated after inoculation is secondary to the vaccine 
(nelipepimut-S + GM-CSF), versus GM-CSF alone.  Because the local toxicity (erythema, pruritus) seen 
in our phase I/II trials is attributable to the GM-CSF, use of GM-CSF alone as a control arm will allow for 
participants to be blinded to their treatment allocation.  Similarly, the use of GM-CSF alone as a control 
arm will allow us to investigate the effects of vaccination with the immunizing peptide (nelipepimut-S) 
versus GM-CSF for the protocols secondary endpoints.  The participants will receive two inoculations 
(either vaccine or GM-CSF alone) prior to surgery.  In the completed phase I/II trials evaluating 
NeuVax™, we have seen no interactions between vaccination and endocrine therapy.  For the phase III 
trial evaluating NeuVax™, evaluations regarding interactions between vaccination and endocrine therapy 
are ongoing.  That phase III trial is allowing for concomitant administration of the vaccine with endocrine 
therapy; therefore, for the current trial, for participants recommended to take endocrine therapy, the 
timing of beginning endocrine therapy will be at the discretion of the participant’s treating oncologist and 
will not impact timing of vaccination.  As part of the recent Phase III study the FDA required cardiac 
monitoring every 3 months during vaccination then every 6 months until 36 months and annually 
thereafter. Following a meeting in June 2015, the IDMC recommended routine cardiac monitoring by 
echocardiogram (ECHO) or multiple-gated acquisition (MUGA) scans could be reduced and that such a 
reduction was justified and consistent with the pre-specified Cardiac Toxicity Monitoring Stopping Rules 
defined in the study protocol. The reduced monitoring included a baseline assessment, 6 months, 12 
months, 24 month, 36 months, and end of the study.    Based on the safety data obtained from the phase 
III study, cardiac monitoring at baseline and at the post vaccinations follow-up visit will be conducted to 
ensure participant safety. 
 
Data from our phase I/II trials evaluating the nelipepimut-S vaccine show a correlation between clinical 
efficacy and in vitro immune responses (specifically, enumeration of nelipepimut-S-specific CTL) [20] 
hence we will be looking at immunologic responses as the primary endpoint in this trial.  The ability of 
the generated immune response to traffic to the tumor will be investigated as a secondary endpoint, 
specifically, we will be determining the degree of lymphocyte infiltration into the tumor in the resected 
surgical specimen.  In order to determine the functional impact of the lymphocytes infiltrating the tumor, 
we will evaluate HER2 expression (i.e., will evaluate HER2 expression in the pre-vaccination biopsy and 
the post-vaccination surgical specimen to determine if the antigen-specific immune response can 
eliminate the HER2+ clones in the tumor).  
 


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Completion of this study will determine the safety and immunologic efficacy of vaccination with NeuVax 
in patients with DCIS thereby providing necessary data to support the conduct of a larger study in DCIS 
as well as to build the foundation for a breast cancer prevention trial. 
 
 
3. 
SUMMARY OF STUDY PLAN 
 
A maximum of 48 participants will be accrued and randomized 2:1 to NeuVax or GM-CSF alone groups.  
Accounting for 10% attrition rate while on study and for approx. 5% non-evaluable sample rate, we 
expect to have 40 evaluable participants, 27 in vaccine and 13 in GM-CSF alone group, that have all 
baseline, post-vaccination (pre-surgery), and post-surgery measures of nelipepimut-S-specific CTL. 
Assuming a screening rate of approximately 3-4 participants per month and an accrual rate of 
approximately 1-2 participants per month, we expect the study to complete accrual within 30 months (2.5 
years). 
 
Women whose diagnostic biopsies show DCIS will be invited to participate in this trial.  The tissue 
obtained from this biopsy will be used for the pre-treatment biomarker analysis.  Participants with HER2 
0, 1+, 2+ and 3+ will be eligible.  Participants with confirmed DCIS will be HLA-typed. Because 
nelipepimut-S is an HLA-restricted peptide, the trial is limited to patients who are HLA-A2 positive.   
 
HLA-A2+ participants who meet all other eligibility criteria will be randomized to receive NeuVax™ 
(1000 µg of nelipepimut-S and 250 µg of GM-CSF) or GM-CSF alone (Control).  Participants 
randomized to the nelipepimut-S + GM-CSF arm will receive two vaccinations of nelipepimut-S (1000 
μg) and GM-CSF (250 μg) administered intradermally two weeks apart prior to surgery. Participants 
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.  Participants will be blinded as to whether 
they are receiving nelipepimut-S + GM-CSF or GM-CSF alone (Cotnrol).  One post-vaccination clinic 
visit will occur 1 month (+/- 7 days) after surgery and before initiation of radiation, if post-operative 
radiation is advised; blood for biomarkers will be collected at this visit.  The final follow up visit will 
occur 3-6 months post-surgery. 
 
The following measurements will be taken to meet study objectives.  Blood will be drawn for HLA typing 
at baseline.   Blood for immunologic assays will be drawn at the time of the 1st vaccination, surgery, at the 
post-vaccination follow up visit and at the final follow up visit.  Research tissue will be obtained from the 
surgical specimen. 
 
Each participant will be on study for up to 8 months from randomization to the final follow up visit. 
 
 
4. 
PARTICIPANT SELECTION 
 
4.1 
Inclusion Criteria 
 
4.1.1 
Participants must be female ≥18 years of age, pre- or post-menopausal.  The minimum age of 18 
was chosen as breast cancer is extremely rare in women less than 18 years.   Because no dosing or 
adverse event data are currently available on the use of NeuVax in participants <18 years of age, 
children are excluded from this study but will be eligible for future pediatric trials, if applicable. 
 
4.1.2 
Participants must have a diagnosis of DCIS made by core needle biopsy 
 
4.1.3 
Participants must be HLA-A2 positive. 
 


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4.1.4 
ECOG performance status must be 0 or 1 (Karnofsky ≥ 60%; see Appendix A). 
 
4.1.5 
Clinical chemistry less than 2x normal upper limit of normal range; adequate kidney and liver 
function as measured by creatinine, bilirubin, and liver enzymes (see below):  
• 
Platelets 
 
 
 
≥100,000/mm3 
• 
Hemoglobin  
 
≥10 g/dL 
• 
Blood Urea Nitrogen 
<2 x ULN 
• 
Alkaline Phosphatase 
<2 x ULN 
• 
Lactate Dehydrogenase  
<2 x ULN 
• 
Creatinine  
 
<2 x ULN 
• 
Bilirubin 
 
 
<2 x ULN 
• 
AST (SGOT)/ALT (SGPT) <2 x ULN 
(ULN = upper limit of normal as defined by the participating institutions laboratory). 
 
4.1.6 
A normal ejection fraction, as defined by the participant’s institution (see footnote 1 below). 
 
4.1.7 
Willingness to comply with all study interventions and follow-up procedures.  
 
4.1.8 
The ability to understand and willingness to sign a written informed consent document. 
 
1  Only limited ECHOs will be used as cardiac evaluation.  No other tests are allowed. ECHO is to be done only in 
HLA-A2 positive participants. If ECHO has been done within 30 days prior to randomization and results showing a 
normal ejection fraction have been obtained prior to randomization, an additional ECHO is not needed at Baseline. 
 
 
4.2 
Exclusion Criteria 
 
4.2.1 
Invasive breast cancer.  Areas of microinvasion or suspicious for microinvasion on the core 
biopsy is allowed. 
 
4.2.2 
History of prior breast cancer treated within the past two years.  Patients completing all breast 
cancer-specific treatment over two years prior to the current diagnosis are eligible. 
 
4.2.3 
History of prior ductal carcinoma in situ (DCIS) treated within the past two years.  Patients 
completing all treatment for a previous diagnosis of DCIS over two years prior to the current 
diagnosis are eligible.  
 
4.2.4 
Prior lobular carcinoma in situ (LCIS) is allowed. 
 
4.2.5 
Pregnant, unwilling to use adequate contraception during study treatment duration or 
breastfeeding (see footnote 1 below). 
 
4.2.6 
Any autoimmune disease or other medical condition that, in the opinion of the investigator, 
would compromise the subject's safety. 
 
4.2.7 
Immune deficiency diseases such as immunoglobulin deficiency or immunosuppressive therapy 
that might interfere with appropriate immune response. 
 
4.2.8 
Known history of or known active infection with human immunodeficiency virus (HIV), hepatitis 
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4.2.9 
Patients on chronic steroid therapy or other immunosuppressive therapy except for topical or 
inhaled steroids known to have low systemic absorption. 
 
4.2.10 Patients with a known hypersensitivity to GM-CSF, yeast-derived products, or any 
component of the GM-CSF product (e.g., mannitol). 
 
4.2.11 Concurrent treatment with other investigational agent. 
 
4.2.12 History of non-breast malignancy within 5 years prior to randomization, except curatively 
treated superficial bladder cancer, carcinoma in situ of the cervix (Stage 0 - 1), and basal cell 
or squamous cell carcinoma of the skin. 
 
4.2.13 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to NeuVax. 
 
4.2.14. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situations that would limit compliance with study requirements. 
 
4.2.15. No recent or planned immunotherapy. 
 
1  The effects of NeuVax on the developing human fetus are unknown. For this reason and because NeuVax may be 
teratogenic, pregnant women will be excluded. All heterosexually active women who may become pregnant must 
agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry 
and for the duration of study participation OR be post-menopausal defined as any one of the following 1) prior 
hysterectomy, 2) absence of menstrual period for 1 year in the absence of prior chemotherapy or 3) absence of 
menstrual period for 2 years in women with a prior history of chemotherapy exposure who were pre-menopausal 
prior to chemotherapy.  Should a woman become pregnant or suspect she is pregnant while participating in this 
study, she should inform her study physician immediately.  
 
 
4.3 
Inclusion of Women and Minorities 
 
Participants will be adult women of all races and ethnic groups who are at least 18 years old.  Breast 
cancer is extremely rare in men and men have limited amounts of normal breast tissue; therefore, male 
participants will not be recruited to this trial. Children will not be recruited to the trial because breast 
cancer is not relevant to the child population.  
 
Our goal is to ensure that the study is available to women of all races and ethnic groups.  To ensure 
enrollment of minorities we will work with the minority community and social network groups available 
at each accruing site within the US, such as Black Women's Health Imperative, Sisters Network, Army of 
Women, Cancer Forward, Breast Health Collaborative, Asian Cancer Council (Indian American Cancer 
Network, Filipino Cancer Network, Light and Salt) and Susan G. Komen Breast Cancer Foundation.   
 
Although minority groups will be targeted in recruitment, the HLA-A2 haplotype is less common in 
minorities than in Caucasians.  In our previous studies close to 77% of HLA-A2 positive participants 
were Caucasian.  This gives us grounds to expect that although we will approach all racial groups, the 
majority of participants who will test HLA-A2 positive during eligibility verification will be Caucasian.  
 
 


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4.4 
Recruitment and Retention Plan 
 
Participants will be recruited for enrollment on this trial from the primary clinics of the Investigators 
and/or surgical collaborators. Efforts will be made to enroll women from a diversity of ethnic and socio-
economic backgrounds.  There will be no payments made to participants or physicians for participation in 
this study. Prior to enrollment on the study, the physician will discuss the study protocol in detail with the 
patient, including possible toxicities. The informed consent document will be reviewed by the physician 
with the patient. To facilitate accrual, information about the trial will be advertised, for example, on the 
websites of participating institutions.  
 
Please refer to the study-specific Recruitment and Retention Plan for more details.  
 
 
5. 
AGENT ADMINISTRATION 
 
Vaccine and GM-CSF alone inoculations will be administered in the clinic.   
 
5.1 
Dose Regimen and Dose Groups: Vaccine dosage and preparation 
 
• 
Agents: The NeuVax vaccine (nelipepimut-S; as 1.5 mg/mL nelipepimut-S acetate solution) will 
be supplied to the NCI Repository by SELLAS. GM-CSF (as 250 μg lyophilized Leukine) will be 
supplied by the NCI Repository.  
• 
Administration: Using aseptic technique, 0.8 mL of nelipepimut-S acetate solution (1136 μg 
nelipepimut-S peptide) is added to the re-constituted Leukine. Taking into account vial losses, 
this procedure will yield a total dose of approximately 1010 μg of nelipepimut-S peptide in a 
volume of 1.6 mL.  Divide 1.6 mL into 4 equally divided 0.4 mL syringes for intradermal 
injection. The detailed guidance for mixing nelipepimut-S peptide mixed with lyophilized 
Leukine® (sargramostim, GM-CSF) or Control (sterile water for injection) with Leukine is 
presented in Appendix B. 
• 
Duration:  Participants randomized to the nelipepimut-S/GM-CSF arm will receive two vaccinations of 
nelipepimut-S (1000 μg) and GM-CSF (250 μg) administered intradermally two weeks apart prior to 
surgery.  Participants 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.  
 
5.2 
NeuVax Inoculation Series Administration 
 
Subjects will receive 2 vaccinations each consisting of 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. 
 
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 
administered in the same lymph node draining area (same leg). Vaccinations will be made in the same 
thigh each time (Vaccines #1 through 2 ) to optimize the immune response by using the same draining 
lymph node each time.  
 
Participants randomized to the nelipepimut-S/GM-CSF arm will receive two vaccinations of nelipepimut-
S (1000 μg) and GM-CSF (250 μg) administered intradermally two weeks apart prior to surgery. 
Participants 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. 
 


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A healthcare provider approved at each institution to deliver inoculations will administer the inoculations 
sterilely in the clinical facility at each study site. Post inoculation vital signs will be assessed at 30 +/- 5 
minutes after inoculation by the research nurse / or coordinator.  For female participants with childbearing 
potential, a urine pregnancy test will be performed before each inoculation. If this test is positive at any 
time, the participants will be discontinued from the study. 
 
 
5.3 
Run-in Procedures  
 
This trial will not include a run-in period. 
 
 
5.4 
Contraindications 
 
Drug interactions between NeuVax and other agents have not been studied.  
 
Leukine is contraindicated: 
1. in patients with excessive leukemic myeloid blasts in the bone marrow or peripheral blood (≥ 
10%); 
2. in patients with known hypersensitivity to GM-CSF, yeast-derived products or any component of 
the product; 
3. for concomitant use with chemotherapy and radiotherapy. 
Due to the potential sensitivity of rapidly dividing hematopoietic progenitor cells, Leukine should not be 
administered simultaneously with cytotoxic chemotherapy or radiotherapy or within 24 hours preceding 
or following chemotherapy or radiotherapy. In one controlled study, participants with small cell lung 
cancer received Leukine and concurrent thoracic radiotherapy and chemotherapy or the identical 
radiotherapy and chemotherapy without Leukine. The participants randomized to Leukine had 
significantly higher incidence of adverse events, including higher mortality and a higher incidence of 
grade 3 and 4 infections and grade 3 and 4 thrombocytopenia. Refer to the rhGM-CSF Leukine® 
[sargramostim] package insert for information on drug interactions with rhGM-CSF Leukine® 
[sargramostim]. 
 
 
5.5 
Concomitant Medications 
 
All medications (prescription and over-the-counter), vitamin and mineral supplements, and/or herbs taken 
by the participant will be documented on the concomitant medication CRF and will include: 1) start and 
stop date, dose and route of administration, and indication.  
 
Participants experiencing a local or systemic reaction to vaccination may be advised to take Tylenol.  For 
significant pruritis at the injection site, participants may use an over the counter oral anti-histamine.  
Steroids are prohibited unless, in the determination of the Investigator, the participant is experiencing 
systemic hypersensitivity or an allergic reaction. 
 
 
5.6 
Dose Modification 
 
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 or greater 
than or equal to grade 2 hypersensitivity reactions [26].  For a description of how local reaction will be 


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measured, refer to section 7.3.1.  To avoid higher grade toxicities, for participants meeting any of these 
criteria, we will dose-reduce the GM-CSF component of the vaccine as outlined below.  Site PIs cannot 
make the decision to dose-reduce without consulting with Dr. Mittendorf.  The decision to dose-reduce 
the GM-CSF will be made by the Protocol Principal Investigator, Dr. Mittendorf, who will communicate 
the need for dose-reduction to the individual site PI and site study research nurse / or coordinator who will 
be generating the orders for the pharmacy for preparation of vaccine.  Adverse events that do not respond 
to either dose modification or clinical management of the reaction will result in taking the participant off 
agent but followed for study endpoints (refer to Section 8.3: Off-Agent Criteria).   
 
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 participant or require medical or surgical 
intervention to prevent an outcome listed above.   
 
Participants experiencing Grade 4 toxicities of any attribution will be taken off agent and followed (refer 
to Section 8.3: Off-Agent Criteria). Any participant with Grade 3 systemic toxicities possibly, probably or 
definitely attributable to study drug will be taken off agent and followed, except for participants with skin 
toxicities.  Any participant experiencing Grade 3 skin toxicities will continue to receive inoculations as 
long as their skin toxicity has resolved prior to the next scheduled inoculation.  Participants with Grade 3 
toxicities not related or unlikely related to study agent will continue on study.  
 
Dose Reductions of Vaccine.  Toxicities observed with the vaccine are attributable to the GM-CSF.  
Although the toxicity profile of GM-CSF is the same for a 250 μg dose as for a 125 μg dose, the extent of 
the toxicity will vary between participants.  Therefore, the provision for dose reductions of the GM-CSF 
component of the vaccine is a safety issue for an individual participant.    For participants experiencing 
robust local reactions (>100mm x 100mm); grade 3 local reactions; or greater than or equal to grade 2 
hypersensitivity reactions the dose of GM-CSF will be reduced for the subsequent inoculation as outlined 
in Table 1 below.  This dose reduction will be done to decrease the likelihood that the participant will 
experience the same degree of reaction. Management of hypersensitivity reactions will be at the discretion 
of the Investigator or an appropriately qualified member of the study team designated by the Investigator. 
 
Dose Reduction of GM-CSF.  
Table 1. Dose Reduction Using Reconstituted Lyophilized Leukine 
 
Amount of reconstituted 
Leukine® 
solution 
removed prior to addition 
of E75 acetate solution 
Amount of WFI diluent 
added 
back 
to 
E75 
acetate solution 
Dose of rhGM-CSF 
No 
dose 
reduction 
0 mL 
0 mL 
250 µg 
1st 
dose 
reduction 
0.5 mL 
0.5 mL 
125 µg 
 
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 primary vaccination series.  


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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), over the counter antihistamines may be used before or after subsequent 
inoculations at the discretion of the Investigator or an appropriately qualified member of the study team 
designated by the Investigator.  If urticaria persists with the use of over the counter anti-histamines, then 
prescription anti-histamines will be prescribed.  If urticarial persists with the use of prescription anti-
histamines, the participant will be give oral steroids (Medrol Dosepak).  If the urticarial continues to 
persist then the participant will be given intravenous steroids.  Use of intravenous steroids has been 
required in <1% of participants on prior trials evaluating this vaccine. If intravenous steroids or 
hospitalization are required, or in the opinion of the Principal Investigator, further inoculations will be 
discontinued, the participant will be taken off agent but followed for study endpoints (refer to Section 8.3: 
Off-Agent Criteria). 
 
 
5.7 
Adherence/Compliance 
 
• 
We plan to enroll 40 evaluable participants.  Adjusting for the 5% non-evaluable sample rate and 
10% attrition rate, our goal is to randomize a total of 48 participants to yield at least 40 evaluable 
participants.   
• 
Compliance is defined as 100% adherence to the vaccination schedule. 
• 
For the primary endpoint of change in the number of nelipepimut-S-CTL for an individual 
participants between the pre-vaccination timepoint and the 1 month (+/- 7 days) after surgery 
timepoint, “evaluable” will be defined as completing all pre-surgical vaccinations and the visit at 
1 month (+/- 7 days) after surgery.  
• 
For the safety endpoint, participants who receive at least one vaccination will be included in the 
safety evaluation.  
• 
For all secondary endpoints, all non-compliant participants will be analyzed according to the 
intent-to-treat principle.  All levels of compliance will be included in the full evaluation of 
endpoints if paired biopsies are available.   
 
 
6. 
PHARMACEUTICAL INFORMATION 
 
6.1 
Study Agent (IND # 
, IND Sponsor NCI DCP)   
 
Physical description of agents: 
NeuVaxTM appearance: Clear, colorless solution, essentially free from visible particulates; 
GM-CSF (Leukine®, sargramostim) appearance: The liquid Leukine presentation is formulated as a 
sterile, preserved, injectable solution in a vial. Lyophilized Leukine is a sterile, white, preservative-free 
powder that requires reconstitution with 1 mL Sterile Water for Injection.  
 
Formulation to be used in this study and excipients: 
The vaccine NeuVax TM, also known as nelipepimut-S, is a nine amino acid peptide (KIFGSLAFL) 
derived from the HER2 protein that is overexpressed in many breast cancer patients (reviewed in [16]). 
Vaccines containing the nelipepimut-S peptide mixed with an immunoadjuvant have proven to be 
efficacious in early phase (phase I/II clinical trials) conducted in the adjuvant setting to prevent 
recurrence of breast cancer [11]. 
 
The NeuVax vaccine (nelipepimut-S; as 1.5 mg/mL nelipepimut-S acetate solution) will be supplied by 
SELLAS. GM-CSF (as 250 μg lyophilized Leukine) will be supplied by NCI Repository, MRI Global.  


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GM-CSF is a potent cytokine. 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 1.4 x 106 IU/mL. See GM-CSF (Leukine® (sargramostim)) package insert and labeling for 
additional details. 
 
The nelipepimut-S 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, LLC, Albuquerque, NM as a 1.5 mg/mL solution in 1 mL sterile water for injection 
(SWFI) 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 1.42 mg/mL nelipepimut-S peptide. The control 
is SWFI.   
 
Prior to administration, 1,136 μg (0.8 mL) of the nelipepimut-S acetate or control is mixed by the 
pharmacist or pharmacy technician under the supervision of the pharmacist with 1 mL of reconstituted 
lyophilized Leukine® GM-CSF (reconstituted as 250 μg/mL in solution). Immediately after mixing, the 
study drug is withdrawn into four 1 mL syringes and within 2 hours of mixing, a total of 1.6 mL is 
administered as 4 intradermal injections to deliver a total of 1,000 μg of nelipepimut-S peptide vaccine 
(1,010 μg of nelipepimut-S peptide). The control vial contains 1 mL SWFI. The detailed guidance for 
mixing nelipepimut-S (or control) solution from lyophilized Leukine® 250 μg and test drug (nelipepimut-
S solution or control) is stated in Appendix B. 
 
6.2 
Reported Adverse Events and Potential Risks 
 
Phase I trials addressing the immunogenicity and toxicity of escalating  nelipepimut-S peptide doses 
mixed with GM-CSF as adjuvant were conducted in patients with metastatic breast and ovarian cancer.22 
Of the 14 subjects enrolled, a majority experienced mild pain at the injection site. Low-grade fever, 
nausea, fatigue, myalgia, itching at the injection site, and back and abdominal pain were also noted. One 
subject reported grade 2 chills, headache and ulceration at the injection site. No grade 3 AEs were 
reported. The side effects observed were not dose-dependent.   
 
Two concurrent Phase II trials in both node-positive and node-negative breast cancer patients disease-free 
after standard treatment were recently completed [13, 27] (CCRE08140797). These trials were designed 
to address the ability of the vaccine to prevent disease recurrence. Subjects were observed for an hour 
after injection for any signs of hypersensitivity, and asked to return 48‒72 hours later to monitor 
local/systemic reactions to injection. In a total of 187 subjects, local reactions included grade 1 erythema 
and induration, pain and soreness at the injection site (81% of vaccinated subjects) and grade 2 pruritus 
(19%). Systemic reactions included grade 1 headache and fatigue (71%); grade 2 pain, flu like symptoms, 
and fatigue (14%); and grade 3 angioedema of tongue and bone pain (2%). No grade 4 or 5 events were 
observed. Because the inoculations are very well tolerated with few immediate adverse events, only post 
inoculation vital signs will be assessed at 30 +/- 5 minutes after inoculation by the research nurse or 
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The same vaccine (nelipepimut-S+GM-CSF) was evaluated in prostate cancer patients expressing varying 
levels of HER2/neu protein [25].  Of the 40 enrolled subjects, grade 1 local reactions were observed in all; 
systemic reactions were observed in 38.1% (grade 1) and 9.5% (grade 2). 
 
These effects are likely attributable to GM-CSF. In studies utilizing this adjuvant, patients sometimes 
complained of mild to moderate flu-like symptoms (fever, chills, achiness, and fatigue) for 1‒2 days after 
vaccination.  In subsequent studies evaluating the AE37 vaccine (AE37 + GM-CSF), the toxicity profile 
was similar in patients randomized to receive the vaccine and those randomized to receive GM-CSF only 
(unpublished data). 
 
Nelipepimut-S was recently evaluated in a multicenter, multinational, prospective, randomized, double-
blind, controlled phase III study (NCT01479244; PI:  Mittendorf)  The trial randomized 758 lymph node-
positive breast cancer patients with HER2 1+/2+ tumors in the adjuvant setting. Randomization was 
completed in April 2015.  In June 2016, the Independent Data Monitoring Committee (IDMC) met to 
complete a planned safety and futility interim analysis that was triggered after 70 qualifying DFS events 
were reached.  At that time, the IDMC recommended that the trial be stopped due to futility.  The AEs 
observed in the Phase III study as of the date of the last data cutoff (16 Jan 2015) were consistent with the 
reactions observed in previous studies.  AEs reported for ≥ 10% of patients in the pivotal study have been 
limited to local injection site reactions, including induration (56%), swelling (46%), pain (40%), pruritus 
(55%), and edema (17%).  AEs reported for <20% of patients have included headache, fatigue, back pain 
and nausea.  AEs have been generally mild in severity, with no safety trends identified for Grade 3 or 
higher AEs or SAEs/deaths.  Two patients in the pivotal phase III study reported AEs related to study 
drug hypersensitivity including 1 patient with non-serious, Grade 2 angioedema and 1 patient with a 
Grade 4 SAE of anaphylactic reaction, both of which were considered definitely related to study drug, 
resulted in discontinuation of treatment, and were noted to have resolved.  In addition, one patient in the 
early phase trials reported Grade 3 angioedema assessed as related to rhGM-CSF.  No other allergic- or 
anaphylactic-type reactions to E75 plus the rhGM-CSF adjuvant have been reported. 
 
 
 
6.3  
Availability 
 
NeuVax TM is provided to the NCI under a Clinical Trials Agreement (CTA) between SELLAS and the 
DCP, NCI (see §12.7). 
 
 
6.4 
Agent Distribution 
 
Agents will only be released by NCI, DCP after documentation of IRB approval of the DCP-approved 
protocol and consent is provided to DCP and the collection of all Essential Documents is complete (see 
DCP website for description of Essential Documents).  
 
NCI, DCP-supplied agents may be requested by the Investigator (or their authorized designees) at each 
Organization. DCP guidelines require that the agent be shipped directly to the institution or site where the 
agent will be prepared and administered. DCP does not permit the transfer of agents between institutions 
(unless prior approval from DCP is obtained). DCP does not automatically ship agents; the site must 
make a request. Agents are requested by completing the DCP Clinical Drug Request form (NIH-986) (to 
include complete shipping contact information) and faxing or mailing the form to the DCP agent 
repository contractor: 
 


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John Cookinham 
MRIGlobal 
DCP Repository 
1222 Ozark Street 
North Kansas City, MO 64116 
Phone: (816) 360-3805 
FAX:  (816) 753-5359 
Emergency Telephone: (816) 360-3800 
 
 
6.5  
Agent Accountability 
 
The Investigator, or a responsible party designated by the Investigator, must maintain a careful record of 
the inventory and disposition of all agents received from DCP using the NCI Drug Accountability Record 
Form (DARF) or an institutionally-approved accountability system. The Investigator is required to 
maintain adequate records of receipt, dispensing and final disposition of study agent.  Include on receipt 
record from whom the agent was received and to whom study agent was shipped, date, quantity and batch 
or lot number. On dispensing record, note quantities and dates study agent was dispensed and 
administered to each participant.  Drs. Mittendorf or Garber, Bedrosian and Berger or their 
representatives will be responsible for study agent accountability for participants at Dana-Farber Cancer 
Center, UT MD Anderson Cancer Center, and Thomas Jefferson University, respectively. 
 
 
6.6 
Packaging and Labeling 
 
NeuVax TM will be provided by SELLAS and will be distributed to the clinical sites by MRI Global.  GM-
CSF (as 250 μg lyophilized Leukine) will be supplied to the clinical sites by NCI Repository, MRI Global. 
 
 
 
6.7 
Storage 
 
For detailed storage instructions of the components of the vaccine (nelipepimut-S acetate and GM-CSF) 
please follow the instructions in the Investigator’s Brochure.  The components of the active study drug 
and the control will be stored separately in the research pharmacy and reconstituted when needed.  The 
components will be stored as per appropriate storage temperature designated on clinical carton label.  All 
study drug components (nelipepimut-S acetate and GM-CSF) will be stored under refrigerated conditions 
(5°C ± 3°C).   
 
6.8 
Registration/Randomization 
 
Screening and Registration into the DMI Database:  
Once informed consent has been signed, participants will be registered into the DMI database.  The DMI 
database will assign a participant’s PID upon completion of the registration process. 
 
Randomization:  
Participants will be assigned a randomization number once the following has been accomplished: 
eligibility has been verified at the site level, eligibility has been confirmed by the site PI, and eligibility 
CRF has been entered into the DMI web application.  The randomization number will be generated by the 
database and assigned to the participant. Refer to Section 13.2 for details of randomization.  
 


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Screening/Registration/Randomization into site-specific databases: 
The DMI is the database of record for the study. Registration and randomization should occur per the 
procedures outlined above. If the site staff need to enter study data into site-specific electronic databases 
per their institutional requirements, they should do so in accordance with their institutional policies and 
procedures. 
 
Appropriate CRFs must be completed for any participant who signs an informed consent. If a consented 
participant is a screen failure and deemed ineligible, the following CRFs must be completed: 1) the 
Registration CRF; 2) the Randomization CRF with the eligibility box checked “no”, 3) the Inclusion and 
Exclusion CRFs showing why the participant is ineligible, 4) the Off-Study CRF, 5) the Adverse Event 
CRF, 6) the Concomitant Medication CRF and 7) the Verification CRF. If no Adverse Event and/or 
Concomitant Medications were assessed by the time the participant is deemed ineligible, the “NONE” 
box will be checked to complete both CRFs.  All participants who sign an informed consent must 
formally go off study. All participant registration information will be entered into DMI. If a participant 
experiences a serious Adverse Event during the screening process, a Serious Adverse Event (SAE) form 
must be completed. 
 
6.9 Blinding and Unblinding Methods 
 
6.9.1 
Participants will be blinded to NeuVax TM or GM-CSF alone arm assignment. 
6.9.2 
The Statistician, Site Study Pharmacist and other investigators will not be blinded to NeuVax TM 
or GM-CSF alone arm assignment. 
6.9.3 
Participants randomized to the nelipepimut-S/GM-CSF arm will receive two vaccinations of 
nelipepimut-S (1000 μg) and GM-CSF (250 μg) consisting of 4 intradermal inoculations two 
weeks apart prior to surgery. Participants 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.   
6.9.4 
At the end of the study participants will be unblinded to their assignment.  
6.9.5 
Study agents may be unblinded during the course of the study if deemed medically necessary in 
the event of a serious adverse event after discussion with the NCI medical monitor. Unblinding 
will be conducted as follows: 
1) The Site PI contacts the Protocol Principal Investigator (Elizabeth Mittendorf, MD, 
PhD) and requests the participant’s treatment status be unblinded. 
2) The Protocol Principal Investigator (Elizabeth Mittendorf, MD, PhD) contacts the 
NCI DCP Medical Monitor (Margaret (Malgorzata) Wojtowicz, MD) and requests 
the participant’s treatment status be unblinded.  The Protocol Principal Investigator 
then conveys the Medical Monitor’s decision to the Site PI.  The Site PI then 
proceeds with unblinding as written out below.  
3) If the NCI Medical Monitor cannot be reached and the participant requires 
emergency care, the Protocol Principal Investigator (Dr. Mittendorf) may authorize 
the site PI to break the blind. 
4) If the Site PI is unable to reach the Protocol Principal Investigator and the participant 
requires emergency care, then the Site PI must proceed with unblinding as written out 
below.  
5) The Site PI officially unblinds the participant and takes the participant off-study.  
6) The date and reason for breaking the blind must be submitted by the Site PI to the 
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7) It is the responsibility of the Protocol Principal Investigator to report the date and 
reason for breaking the blind to the NCI Medical Monitor, Margaret (Malgorzata) 
Wojtowicz, MD as soon as possible after receiving this information from the Site PI. 
 
NCI Medical Monitor: 
Margaret (Malgorzata) Wojtowicz, MD 
NCI/Division of Cancer Prevention 
Room 5E104 
9609 Medical Center Drive 
MSC 9781 
Bethesda, MD 20892 
Phone: (240) 276-7012; Fax: (240) 276-7848 
wojtowim@mail.nih.gov 
 
8) The date and reason for breaking the blind must be submitted by the Protocol 
Principal Investigator to the MD Anderson Consortium Principal Investigator, Powel 
H. Brown, MD, PhD, or designee as soon as possible via email to 
phbrown@mdanderson.org. 
9) The date and reason for breaking the blind will be reported by Dr. Brown or designee 
to the MD Anderson DSMB as soon as possible. 
 
 
6.10 
Agent Destruction/Disposal 
 
At the completion of the study, all unused study agent will be returned to NCI, DCP Repository according 
to the DCP “Guidelines for AGENT RETURNS” and using the DCP form “Return Drug List”.  
 
The guidelines and the form are available on the DCP website. 
 




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6 May be performed up to 5 days prior to surgery relative to the visit target date. 
7 May be assessed the day prior to surgery in clinic or on the phone. 
 
 
7.2 
Baseline Testing/Prestudy Evaluation (Within 30 days prior to randomization) 
Baseline Testing/Prestudy Evaluation interventions will be conducted within 30 days of 
randomization, may require multiple visits and will consist of the following procedures:  
• 
Informed consent must be obtained prior to starting any further study procedures. 
• 
Registration: Once informed consent has been signed, participants will be registered into the 
DMI database.  The DMI database will assign a participant’s PID upon completion of the 
registration process. Participants will also be registered into site-specific registry databases as 
applicable.  
• 
Baseline medical history, to include a review of breast cancer history and previous medical 
history, previous surgery (e.g., lumpectomy or mastectomy), chemotherapy and radiation 
therapy history, reproductive history, family history, demographic information, including age 
and race. 
• 
Diagnosis of ductal carcinoma in situ by core needle biopsy; must be confirmed by review of 
the pathology report. 
• 
Participants with confirmed DCIS will be HLA-typed. Because nelipepimut-S is an HLA-
restricted peptide, the trial is limited to patients who are HLA-A2 positive (approximately 
45% of the US population). HLA typing will be done locally by each site. Approximately 6-8 
mL of blood will be drawn for HLA-typing (or as directed by the site HLA typing facility). 
• 
Use of concomitant medications will be reviewed. 
• 
A physical examination will be done that will include vital signs (temperature, blood 
pressure, heart rate, respiratory rate).  
• 
Height, weight.  
• 
Pre-study routine laboratory evaluations will include the following: 
o Platelets 
o Hemoglobin 
o Blood Urea Nitrogen 
o Alkaline Phosphatase 
o Lactate Dehydrogenase 
o Creatinine 
o Bilirubin 
o AST (SGOT)/ALT (SGPT) 
• 
In women of child bearing potential a pregnancy test must be done within 30 days prior to 
Randomization. The pregnancy test will be repeated prior to each vaccination. Results must 
be known before vaccination.  Either a serum or urine pregnancy test may be performed. If a 
urine pregnancy test is performed, the test must be done in the clinic. 
o If the pregnancy test result is positive at Baseline (any time before randomization), 
the participant is a Screen Failure. 
o If the pregnancy test result is positive prior to any Vaccination, do not vaccinate, take 
the participant off-study, complete the Off-Study Case Report Form (CRF), follow 
the pregnancy to term and complete the Outcome of Pregnancy Case Report Form 
(CRF).  
• 
Cardiac evaluation: limited ECHOs to determine the ejection fraction.  A normal ejection 
fraction, as defined by the participant’s institution is required for randomization.  Only 
limited ECHOs will be used as cardiac evaluation.  No other tests are allowed.  ECHO is to 
be done only in HLA-A2 positive participants (i.e., participants with confirmed DCIS will be 
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randomization and results showing a normal ejection fraction have been obtained prior to 
randomization, an additional ECHO is not needed at Baseline. 
• 
Confirm eligibility: At the completion of the screening period eligibility must be confirmed. 
Only after eligibility is confirmed, can eligible participants be randomized.  
 
Randomization  
• 
Once registration is complete, eligibility has been confirmed and eligibility CRF is entered 
into the web application, participants will be assigned a randomization number pre-generated 
by the database. Participants will be randomized to receive either NeuVax™ (nelipepimut-S; 
1000 µg and 250 µg of GM-CSF) or GM-CSF alone.  Participants will be blinded as to 
whether they are receiving nelipepimut-S/GM-CSF or GM-CSF alone.  
• 
An H&E stained slide and 10 unstained slides (or 11 unstained slides) from the initial breast 
biopsy should be sent to Dr. Al-Atrash’s laboratory at the address provided in Section 10.3. 
The biopsy slides should be sent within 6 weeks of randomization. 
• 
Randomization and Vaccination #1 may occur on the same day.  
 
 
7.3 
Evaluation During Study Intervention 
 
7.3.1 Vaccination #1 (Day 0 +/-3 days): 
 
Pre-Vaccination: 
• 
Pregnancy test prior to vaccination. Negative pregnancy result must be confirmed prior to 
vaccination:  
o If the pregnancy test result is positive prior to any Vaccination, do not vaccinate, take the 
participant off-study, complete the Off-Study Case Report Form (CRF), follow the 
pregnancy to term and complete the Outcome of Pregnancy Case Report Form (CRF). 
• 
Blood for immunologic assays. 
• 
One set of vital signs prior to inoculation. 
• 
Adverse Events. 
• 
Concomitant medications.  
• 
Symptom assessment.  
 
Vaccination 
• 
Agent administration: inoculation. 
• 
Post inoculation vital signs will be assessed at 30 +/- 5 minutes after inoculation. 
 
Post-Vaccination (48-72 hrs after inoculation): 
• 
Telephone contact with research nurse /or coordinator or nurse visit during the initial 48-72 
hrs after inoculation: Participants will either return to their study site or be contacted by 
phone for questioning regarding any systemic toxicity during the initial 48-72 hrs after 
inoculation.  If they return to their study site, the local reaction at the inoculation sites will be 
examined and measured.  For participants who do not return to their study site, a tool will be 
given, and instructions provided to measure the local reaction.  The NCI CTCAE version 4.03 
graded toxicity scale will be utilized to assess local and systemic toxicity. 
• 
Local reaction (for participants who return to the study site after inoculation): 
o Participants 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.   
• 
Local reaction (for participants who do not return to the study site after inoculation): 


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o For participants that do not return to their study site, a self-assessment tool will be 
given, and instructions provided to measure the local reaction.  This self-assessment 
tool is currently being utilized in the phase III trial evaluating NeuVax as well as two 
ongoing phase II trials evaluating NeuVax in combination with trastuzumab.  The 
tool has been well received by the participants who appreciate its ease of use (the tool 
consists of a transparent piece of lightweight plastic with concentric circles depicted 
on it to allow for measurement) and the fact that it reduces the need to return to the 
study site for injection site assessment.  To date, at MD Anderson, we have had 
100% compliance with the use of the tool. 
 
 
7.3.2 Vaccination #2 (Day 14 +/-3 days); 
• 
Pregnancy test prior to vaccination. Negative pregnancy result must be confirmed prior to 
vaccination:  
o If the pregnancy test result is positive prior to any Vaccination, do not vaccinate, take the 
participant off-study, complete the Off-Study Case Report Form (CRF), follow the 
pregnancy to term and complete the Outcome of Pregnancy Case Report Form (CRF). 
• 
One set of vital signs prior to inoculation. 
• 
Post inoculation vital signs will be assessed at 30 +/- 5 minutes after inoculation. 
• 
Adverse Events. 
• 
Concomitant medications.  
• 
Symptom assessment.  
• 
Local reaction (during the initial 48-72 hrs after inoculation).  
• 
Telephone contact with research nurse /or coordinator or nurse visit during the initial 48-72 
hrs after inoculation. 
 
7.3.3 Day of Surgery (Day 28 +/-5 days): 
• 
Blood for immunologic assays will be drawn prior to the surgical procedure. 
• 
Blood will be drawn for laboratory evaluations prior to surgery. Pre-surgery laboratory 
evaluations will include the following: 
o Platelets 
o Hemoglobin 
o Alkaline Phosphatase 
o Lactate Dehydrogenase 
o Bilirubin 
o AST (SGOT)/ALT (SGPT) 
• 
Research tissue for tissue biomarkers (from the surgical specimen). For patients undergoing 
mastectomy, a representative sample of normal tissue maximally distant from the tumor will 
be obtained to assess for immune infiltrates.  
• 
Adverse Events. 
• 
Concomitant medications.  
• 
Symptom assessment.  
 
 
7.4 
Evaluation at Completion of Study Intervention 
 
7.4.1 Post Vaccinations Follow-Up Visit (Day +30 post op +/-7 days): the post vaccinations follow-up 
visit will be conducted on Day +30 (+/- 7 days) post op and prior to initiating radiation, if post-op 
radiation is advised.   
The following will be assessed: 


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• 
Adverse Events. May be assessed on the phone.  
• 
Concomitant medications. May be assessed on the phone. 
• 
Symptom assessment. May be assessed on the phone. 
• 
Blood for immunologic assays. 
Cardiac evaluation: limited ECHOs to determine the ejection fraction.  Decreases of > 10% in the ejection 
fraction from baseline will be reported as an SAE and the participant experiencing the SAE will be 
managed according to the best clinical practice. Only limited ECHOs will be used as cardiac evaluation.  
No other tests are allowed.   
 
7.5 
Post-intervention Follow-up Period 
 
The Final Follow-Up Visit (3-6 Months post op): the Final Follow-Up Visit will be conducted 3-6 
Months post-surgery.   
The following will be assessed: 
• 
Physical exam. 
• 
Vital signs.  
• 
Adverse Events. 
• 
Concomitant medications.  
• 
Symptom assessment.  
• 
Blood for immunologic assays. 
 
7.6 
Methods for Clinical Procedures 
 
7.6.1  Local reaction assessment.  The local reaction will be measured by either the research nurse /or 
coordinator (if the participant returns to clinic) or by the participant using the local reaction assessment 
tool, 48-72 hours after inoculation.  The local reaction will be measured and reported in two dimensions. 
 
8.  
CRITERIA FOR EVALUATION AND ENDPOINT DEFINITION 
 
8.1 
Primary Endpoint 
 
Primary Endpoint: Enumeration of nelipepimut-S-specific CTL 
The primary endpoint of the trial is to evaluate for nelipepimut-S-specific CTL response in vaccinated 
participants compared to participants receiving GM-CSF alone.  The primary biomarker therefore is 
nelipepimut-S-specific CTL, which will be detected using a dextramer assay, which is a flow cytometry 
based assay.  The number of nelipepimut-S-CTL will be determined in all participants, regardless of 
randomization to vaccine or GM-CSF alone, at the following time points:  pre-vaccination, at the time of 
surgery (after two preoperative vaccinations), 1 month (+/- 7 days) after surgery, and 3-6 months at the 
time of the final study visit.  The change in the number of nelipepimut-S-CTL for an individual 
participant between the pre-vaccination timepoint and the 1 month (+/- 7 days) after surgery timepoint 
will be used as the primary endpoint measure for this study.  Planned subset analyses will be performed 
based on the extent of HER2 expression in the biopsy specimen (HER2 3+ vs HER2 0, 1+ or 2+), 
hormone receptor status, and menopausal status. 
 
8.2 
Secondary Endpoints 
 
Secondary endpoints include: 
 
8.2.1 
Toxicity profile and frequency of adverse events in women with DCIS of the breast receiving 
nelipepimut-S vaccine as compared to women receiving GM-CSF alone. 


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8.2.2 
Presence of DCIS at resection. The histologic data will be presented in tabular form to examine 
whether the vaccine treatment is associated with the absence of DCIS, induction of necrosis, 
reduction in histologic grade, or a reduction in the size of DCIS.   These data will be descriptive 
only.  The data will be obtained from the standard surgical pathology report generated as part of 
the patient’s clinical care. Formal comparisons of these parameters would likely require a larger 
sample size to see a statistically significant difference in presence of DCIS, induction of necrosis, 
or reduction in size of DCIS. 
 
8.2.3 
Difference in HER2 expression in the biopsy and the surgical specimen excised post-vaccination. 
HER2 scoring will be determined according to the American Society of Clinical 
Oncology/College of American Pathologists clinical guidelines [7].  Positive cases are those with 
circumferential membrane staining that is complete, intense, and within >10% of tumor cells 
(Score 3+).  Negative cases are defined as those with no observable staining, or membrane 
staining that is incomplete and is faint/barely perceptible and within less than or equal to 10% of 
tumor cells (Score 0) or incomplete membrane staining that is faint/barely perceptible and within 
>10% of tumor cells (Score 1+).  Equivocal or indeterminate cases are those with circumferential 
membrane staining that is incomplete and or weak/moderate and within >10% of tumor cells or 
complete and circumferential membrane staining that is intense and within less than or equal to 
10% of tumor cells (Score 2+).  Pre-vaccination and post-vaccination specimens will be 
compared. 
 
8.2.4 
Degree of lymphocyte infiltration.  We will define intra-tumoral TIL as those within the basement 
membrane.  Stromal TIL will be defined as those in the periductal/lobular stroma including the 
intralobular stromal infiltrate.  Cells in the interlobular stromal inflammatory infiltrate will be 
excluded.  All mononuclear cells will be scored but polymorphonuclear leukocytes will be 
excluded.  Intra-tumoral and stromal TILs will be scored as a continuous variable and the 
percentage of in the surgical specimen will be compared to that in the pre-vaccination diagnostic 
biopsy. 
 
8.2.5 
 
We will utilize tissue-based cyclic immunofluorescence (t-CyCIF), a novel, highly multiplexed 
imaging modality that allows for the imaging of formalin-fixed, paraffin embedded (FFPE) tissue 
sections at subcellular resolution across 20-60 distinct antigen channels.4-6  Antibody panels that 
will be used include but may not be limited to an already optimized immune panel (Table 2). 
 
Table 2. Optimized t-CyCIF immune panel. 
 
ImageJ an Matlab computer software will be used to analyze single cells and yield high-
dimensional data analysis including tSNE analysis, dot plot analysis, heatmap analysis and scatter 
plots.  The total number of prioritized immune cell types (Table 3) per mm2 of tumor tissue and 


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will be asked to return to the clinic site prior to going off study for an early termination visit according to 
the Schedule of Events.    
 
 
8.5 
Study Termination 
 
NCI, DCP as the study sponsor has the right to discontinue the study at any time.  
 
 
9. 
CORRELATIVE/SPECIAL STUDIES 
 
9.1 
Rationale for Methodology Selection 
 
9.1.1 
Enumeration of nelipepimut-S-specific CTL 
 
Rationale.  MHC class I vaccines such as nelipepimut-S+GM-CSF are designed to stimulate clonal 
expansion of antigen-specific CTL.  In a subset of 86 patients from our phase I/II trials investigating 
nelipepimut-S+GM-CSF, we used a dimer assay to quantify nelipepimut-S-specific CTL and found that 
only 1 of 30 (3%) patients with -specific CTL levels above the mean recurred compared to 8 of 56 (14%) 
with levels below the mean [21].  This suggests that robust in vitro immunologic responses may identify 
participants in whom vaccination will be efficacious.  The primary objective of this trial is to evaluate the 
nelipepimut-S-specific CTL response in participants receiving NeuVax versus control participants 
receiving GM-CSF alone. 
 
 
9.1.2 
Histologic Responses 
 
Rationale.  Because this trial will administer vaccines before surgical therapy, the effects of 
immunization will be assessed on residual tumor at the time of surgical resection.  A previous trial 
investigating a HER2-targeting dendritic cell vaccine in participants with DCIS showed a decrease in the 
number of HER2-expressing tumor cells in residual DCIS lesions suggesting that vaccination had 
stimulated an immune response capable of eliminating the HER2+ clones within the tumor.  Dendritic 
cell vaccines are limited by their complexity which requires leukapharesis to obtain an individual 
participant’s blood from which dendritic cells can be isolated, matured, and stimulated with the antigen of 
interest at a central processing facility.  Our peptide vaccine strategy is also capable of stimulating an 
antigen-specific immune response but has the obvious advantage of being much simpler as it is an “off 
the shelf” therapy.  In order to determine the intratumoral immune response stimulated in response to 
vaccination, we will evaluate for: 1) HER2 expression, and 2) the degree of lymphocyte infiltration 
(determined on H&E stained slides as well as determined by multiplex immunofluorescence),  . 
 
 
9.2 
Comparable Methods 
 
The methodology described above are those previously used (see details above), and the resulting data 
will be able to be compared to existing data. 
 
 


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10. 
SPECIMEN MANAGEMENT  
 
10.1 
Laboratories 
 
We plan to evaluate biomarkers in both PBMC and tissue specimens.  For our primary biomarker, 
nelipepimut-S-specific CTL, we will use a dextramer assay, which is a flow cytometry based assay, to 
enumerate the number of nelipepimut-S-specific CTL generated in response to vaccination with the 
nelipepimut-S peptide.  Phase I/II studies of NeuVax (= nelipepimut-S + GM-CSF) showed a correlation 
between nelipepimut-S-specific CTL and disease free survival [20].  Our tissue based assays will be 
performed in a CLIA certified laboratory (HER2 [32] and lymphocyte infiltration [assessed on 
hematoxylin & eosin stained slides] [33] and multiplex immunofluorescence will be analyzed in the 
laboratory of our pathology collaborator using established protocols .   
 
10.1.1 PBMC and serum samples 
 
Dextramer Assay: 
to enumerate the number of nelipepimut-S-
specific CTL  
Gheath Al-Atrash, DO, PhD 
The University of Texas MD Anderson Cancer 
Center 
 
10.1.2 Tissue samples 
 
Histologic analysis (presence or absence of 
DCIS, invasive breast cancer, grade of tumor) 
 
Breast Tumor Immunology Lab 
Dana Farber Cancer Institute 
HER2 
 
Breast Tumor Immunology Lab 
Dana Farber Cancer Institute 
Multiplex Immunofluorescence 
 
Laboratory of Systems Pharmacology 
Harvard Medical School 
PI: Peter Sorger, PhD 
Immune infiltration in normal tissue maximally 
distant from the tumor (in mastectomy 
samples) 
Undetermined* 
MD Anderson Cancer Center 
*  In the mastectomy samples we will perform core needle biopsies of the normal tissue, 
maximally distant from the tumor (ex vivo after the mastectomy if performed) and store for future 
studies of immune infiltrates. 
 
 
10.2 Collection and Handling Procedures 
 
Random, blinded, unique ID numbers will be assigned to specimens at each time point and will be 
distributed with the PID numbers.  All specimens will be labeled with the unique specimen number 
(XXXX), date and time specimen collected and protocol # MDA2014-04-02, at a minimum.  Laboratories 
receiving the specimens will be blinded from distinguishing participant information and visit point but 
this information will be kept available in a password protected electronic log at each site to indicate which 
specimen number belongs to which participant and at what visit.  The master log of specimen IDs will be 
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Multiple blood draws will be required for immunologic assessments in this trial. Approximately 70 mL 
will be drawn at each time point when blood is drawn for immunologic assessments.   Participant blood 
samples labeled as described above will be sent from study sites via overnight delivery to the laboratory 
of Gheath Al-Atrash, DO, PhD at MD Anderson where immunologic response assays will be performed.  
Assays will be run within 30 days of receipt of specimen.  Blood not used to perform the dextramer assay 
will be frozen and stored in the laboratory of Gheath Al-Atrash, DO, PhD under the unique protocol 
number and identifier for up to five years for additional immunologic studies related to this protocol.  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 or/ 
coordinator at any time. Additionally, any stored blood may also be utilized to assess new generations of 
vaccines.      
 
For blood draws performed for immunologic assessment, 10 mL are collected into a BD Vacutainer Rapid 
Serum tube (BD, Franklin Lakes, NJ) which contain a clot activator and silicone coated interior.  After 
centrifugation, serum will be collected, aliquoted in 1 mL vials and frozen for batch analysis. The samples 
will undergo a single freeze-thaw.  The remaining 60 mL 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.  
Cells will be counted, aliquoted at 5x106 cells in 1 mL vials and either used to perform the indicated 
immunologic assays or frozen. 
 
10.2.1 Materials and Methods for nelipepimut-S-CTL Determination 
PBMC will be isolated by ficoll gradient and stained with aqua live/dead stain (Invitrogen) and 
the following antibodies:  CD8 APC-H7 (BD Biosciences), CD3 PE Cy7 (BD Biosciences), 
nelipepimut-S -APC-conjugated dextramer (Immudex), and the following pacific blue conjugated 
lineage antibodies:  CD4 (BD Biosciences), CD14 (BD Biosciences), CD16 (BD Biosciences), 
and CD19 (Biolegend).  Cells will be analyzed on a LSR Fortessa Analyzer (BD Biosciences) and 
the number of nelipepimut-S-specific CTL will be determined.   
 
 
10.2.2 Immunohistochemistry (IHC) 
We will utilize standard IHC techniques to measure expression of specific biomarkers in tissue 
samples.  Tissue samples will be required from both the initial breast biopsy and the subsequent 
surgical excisional procedure.  An H&E stained slide and 10 unstained slides (or 11 unstained 
slides) from the initial breast biopsy and an H&E and 10 unstained slides (or 11 unstained slides) 
from the subsequent surgical excisional procedure should be sent to the Breast Tumor 
Immunology Lab  at the address provided in Section 10.3 below. The biopsy slides should be sent 
within 6 weeks of randomization.  The slides from the surgical procedure should be sent within 6 
weeks of the surgery.  All slides can be sent at room temperature. The study pathologist will 
review all of the H&E slides as well as subsequent slides prepared for biomarker assessment and 
mark the relevant areas for evaluation.  Standard antibody recognition techniques will be used to 
assess expression of specific biomarkers of interest listed below.  Both positive and negative 
controls will be included in each batch to ensure the functionality of the antibody.  We plan to 
assess 
the 
following 
biomarkers: 
 
HER2 
and 
lymphocyte 
infiltration. 
Multiplex 


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immunofluorescence will be analyzed in the laboratory of our collaborator using established 
protocols.  
 
In the mastectomy samples only we will perform core needle biopsies of the normal tissue, 
maximally distant from the tumor (ex vivo after the mastectomy if performed) and store for future 
studies of immune infiltrates.  These core needle biopsy samples should be formalin fixed and 
paraffin embedded.  The block or 10 unstained slides should be sent to  the Breast Tumor 
Immunology Lab with the surgical samples at the address provided in Section 10.3. 
 
 
10.3 
Shipping Instructions 
 
Tissue samples will be shipped to: 
 
Breast Tumor Immunology Lab (BTIL)  
Elizabeth Mittendorf, MD, Ph.D.  
Jennifer Guerriero, Ph.D. 
Dana-Farber Cancer Institute 
450 Brookline Avenue, Smith Building, Room 930 
Boston, MA 02215 
 
Blood samples will be shipped to:  
 
Gheath Alatrash, DO PhD  
Department of Stem Cell Transplantation and Cellular Therapy 
c/o Na Qiao, PhD 
The University of Texas MD Anderson Cancer Center  
7435 Fannin St 
Unit 0900 
Houston, TX  77030 
Telephone: (713) 563-3337 
Fax: 713-794-5720 
 
Notify the following via email: 
emittendorf@bwh.harvard.edu 
ibedrosian@mdanderson.org 
nqiao@mdanderson.org 
galatras@mdanderson.org 
 
All samples will be shipped in compliance with the International Air Transport Association (IATA) 
Dangerous Goods Regulations. 
 
 
10.4 
Tissue Banking 
 
Biologic specimens collected during the conduct of each clinical trial that are not used during the course 
of the study will be considered deliverables under the contract and thus the property of the NCI. At study 
completion, NCI reserves the option to either retain or relinquish ownership of the unused biologic 
specimens. If NCI retains ownership of specimens, the Contractor shall collect, verify and transfer the 
requested biologic specimens from the site to a NCI-specified repository or laboratory at NCI’s expense. 


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11. 
REPORTING ADVERSE EVENTS  
 
DEFINITION: AE means any untoward medical occurrence associated with the use of a drug in humans, 
whether or not considered drug related. An AE can therefore be any unfavorable and unintended sign), 
symptom, or disease temporally associated with participation in a study, whether or not related to that 
participation. This includes all deaths that occur while a participant is on a study. 
 
Please note that all abnormal clinical laboratory values that are determined to be of clinical significance 
based on a physician’s assessment are to be reported as AEs. Those labs determined to be of no clinical 
significance or of unknown clinical significance (per the physician’s assessment) should not be reported 
as AEs. Any lab value of unknown clinical significance should continue to be investigated/followed-up 
further for a final determination, if possible. 
 
An abnormal lab value should be deemed clinically significant if either of the following conditions is met: 
• 
The abnormality suggests a disease and/or organ toxicity that is new or has worsened from 
baseline. 
• 
The abnormality is of a degree that requires additional active management, e.g., change of dose, 
discontinuation of the drug, close observation, more frequent follow-up assessments, or further 
diagnostic investigation. 
Therefore, a clinically significant lab value is one that indicates a new disease process, an exacerbation or 
worsening of an existing condition, or requires further action(s) to be taken.  
 
A list of AEs that have occurred or might occur (Reported Adverse Events and Potential Risks) can be 
found in §6.2, Pharmaceutical Information, as well as the Investigator Brochure or package insert.  
 
 
11.1 
Adverse Events 
 
11.1.1 Reportable AEs 
 
11.1.1.1 
All AEs that occur after the informed consent is signed and baseline assessments are 
completed (including run-in) through the day preceding the day of surgery must be recorded on the AE 
CRFs (paper and/or electronic) whether or not related to study agent.  
 
11.1.1.2 
AEs not related to surgery that occur from the day of surgery through the post 
vaccination follow-up visit (30 days±7 days post-op) must be recorded on the AE CRF (paper and/or 
electronic) whether or not related to study agent. 
 
11.1.1.3 
All SAEs, including all hospitalizations, during the study period will be reported as per 
DCP SAE reporting procedures, with the following exception: 
 
Hospitalization for planned surgery will not be reported. However, if this hospitalization 
event lasts longer than the usual period at the institution (as determined by the Site 
Principal Investigator or Sponsor), it will be reportable as an SAE. Adverse events (AEs) 
relevant to the prolongation of this hospitalization will also be collected and reported on 
AE CRFs. 
 
11.1.2 AE Data Elements: 
 
The following data elements are required for adverse event reporting. 
• 
AE verbatim term 


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• 
System Organ Class (SOC) 
• 
Common Terminology Criteria for Adverse Events v4.0 (CTCAE) AE term 
• 
Event onset date and event ended date 
• 
Severity grade 
• 
Attribution to study agent (relatedness) 
• 
Whether or not the event was reported as a serious adverse event (SAE) 
• 
Whether or not the subject dropped due to the event 
• 
Outcome of the event 
 
11.1.3  Severity of AEs 
 
 
 
11.1.3.1 
Identify the AE using the NCI Common Terminology Criteria for Adverse Events 
(CTCAE) version 4.0. The CTCAE provides descriptive terminology and a grading scale for each adverse 
event 
listed. 
A 
copy 
of 
the 
CTCAE 
can 
be 
found 
at 
http://ctep.cancer.gov/protocolDevelopment/electronic applications/ctc.htm 
 
AEs will be assessed according to the CTCAE grade associated with the AE term. AEs that do not have a 
corresponding CTCAE term will be assessed according to the general guidelines for grading used in the 
CTCAE v4.0. as stated below.  
 
 
CTCAE v4.0 general severity guidelines:  
 
Grade 
Severity 
Description  
1 
Mild 
Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated. 
2 
Moderate 
Moderate; minimal, local or noninvasive intervention indicated; 
limiting age-appropriate instrumental activities of daily living 
(ADL)*. 
3 
Severe 
Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; 
disabling; limiting self-care ADL**. 
4 
Life-threatening 
Life-threatening consequences; urgent intervention indicated. 
5 
Fatal 
Death related to AE. 
   
ADL 
 
*Instrumental ADL refers to preparing meals, shopping for groceries or clothes, using the telephone, 
managing money, etc. 
 
**Self-care ADL refers to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 
 
11.1.4 Assessment of relationship of AE to treatment 
 
 
The possibility that the adverse event is related to study agent will be classified as one of the following: 
not related, unlikely, possible, probable, definite. 
  
11.1.5 Follow-up of AEs 
 


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All AEs, including lab abnormalities that in the opinion of the investigator are clinically significant, will 
be followed according to good medical practices and documented as such. 
 
Unresolved AEs with a “possibly”, “probably” or “definitely” attribution to study agent at the post 
vaccination follow-up visit will be managed according to good medical practices and documented as such 
with a minimum of a telephone call to follow-up on resolution at 20-30 days after the post vaccination 
follow-up visit. 
 
 
11.2 
Serious Adverse Events  
 
11.2.1 DEFINITION: Fed. Reg. 75, Sept. 29, 2010 defines SAEs as those events, occurring at any dose, 
which meet any of the following criteria: 
• 
Results in death 
• 
Is life threatening (Note: the term life-threatening refers to an event in which the participant was 
at risk of death at the time of the event; it does not refer to an event which hypothetically might 
have caused death if it were more severe). 
• 
Requires inpatient hospitalization or prolongation of existing hospitalization 
• 
Results in persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• 
Is a congenital abnormality/birth defect 
• 
Important medical events that may not result in death, be life-threatening or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the patient or subject and may require medical or surgical intervention to prevent 
one of the outcomes listed. 
 
11.2.2 Reporting SAEs to DCP 
 
11.2.2.1 The Lead Organization and all Participating Organizations will report SAEs on the 
DCP SAE form found at  
https://prevention.cancer.gov/clinical-trials/clinical-trials-management/protocol-
information-office/pio-instructions-and-tools/2012-consortia. 
11.2.2.2 Reporting within 24 hours of knowledge of the event.  
11.2.2.2(A) Report to the NCI DCP Medical Monitor within 24 hours: 
Contact the DCP Medical Monitor by email or phone within 24 hours of knowledge of 
the event.   
 
Margaret (Malgorzata) Wojtowicz, MD 
NCI/Division of Cancer Prevention 
Room 5E104 
9609 Medical Center Drive 
MSC 9781 
Bethesda, MD 20892 
Phone: (240) 276-7012 
wojtowim@mail.nih.gov  
 
 
Include the following information when emailing / calling the Medical Monitor: 
• 
Date and time of the SAE 


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• 
Date and time of the SAE report 
• 
Name of reporter 
• 
Call back phone number 
• 
Affiliation/Institution conducting the study 
• 
DCP protocol number 
• 
Title of protocol 
• 
Description of the SAE, including attribution to drug and expectedness 
 
 
11.2.2.2(B) Report to the Consortium Lead Organization (CLO) (Dr. Powel Brown or 
designee) within 24 hours of knowledge of the event: 
The same information reported to the DCP Medical Monitor should be provided to the 
Consortium PI, Dr. Powel Brown or the CLO Coordinator (Saba Abutaseh) via email, 
phone or fax within 24 hours of knowledge of the event. The contact details are provided 
on the cover page of this protocol.  
 
 
11.2.2.3 Reporting within 48 hours of knowledge of the event: 
11.2.2.3 (A) Email the written SAE reports to the DCP Medical Monitor within 48 hours 
of learning of the event using the paper SAE form. The SAE forms should be obtained at 
https://prevention.cancer.gov/sites/default/files/uploads/clinical_trial/SAE-Form_05-02-
2016.pdf. 
 
11.2.2.3 (B) The written SAE reports will also be sent to DCP’s Regulatory Contractor, 
CCS Associates, at safety@ccsainc.com. 
 
11.2.2.3 (C) The written SAE report will also be emailed to the CLO PI (Dr. Powel 
Brown), or faxed to (713) 792-4003.  
 
It is the responsibility of the CLO to inform the Protocol Principal Investigator upon 
receipt of the report from the organization experiencing the event.  
 
11.2.2.4 The DCP Medical Monitor and regulatory staff will determine which SAEs require FDA 
submission. 
 
11.2.2.5 The Lead Organization and all Participating Organizations will comply with applicable 
regulatory requirements related to reporting SAEs to the IRB/IEC.  
 
11.2.2.6 Follow-up of SAE 
Site staff should send follow-up reports as requested when additional information is available.  
Additional information should be entered on the DCP SAE form in the appropriate format.  
Follow-up information should be sent to DCP as soon as available. SAEs related to the study 
agent will be followed until resolved. 
 
 


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12. 
STUDY MONITORING 
 
12.1 
Data Management 
 
This study will report clinical data using the Data Management Initiative (DMI) web-based application 
managed by the Consortium Biostatistics and Data Management Core.  Data Management Initiative 
(DMI) infrastructure has been developed in the Division of Quantitative Sciences (DQS), MD Anderson 
Cancer Center.  This infrastructure supplies integrated database and software services for web-based data 
collection, randomized treatment assignment, reporting, query, data download, and data quality 
management.  The DMI will be the database of record for the protocol and subject to NCI and FDA audit.  
All DMI users will be trained to use the DMI system and will comply with the instructions in the 
protocol-specific “DMI User Manual” as well as applicable regulatory requirements such as 21 CFR; Part 
11.  Data management procedures for this protocol will adhere to the Data Management Plan (DMP) on 
file at the DCP for contract HHSN261201200034I.  
 
 
12.2 
Case Report Forms 
 
Participant data will be collected using protocol-specific case report forms (CRF) developed from the 
standard set of DCP Chemoprevention CRF Templates and utilizing NCI-approved Common Data 
Elements (CDEs). The approved CRFs will be used to create the electronic CRF (e-CRF) screens in the 
DMI application. Site staff will enter data into the e-CRF. Amended CRFs will be submitted to the DCP 
Protocol Information Office for review and approval.  Approved changes will be programmed into the 
DMI database by the Consortium Biostatistics and Data Management Core. 
 
12.3 
Source Documents 
 
Source documentation will include only those documents containing original forms of data, including 
clinic charts, shadow files, hospital charts, and physician notes.  Data recorded directly on the CRFs 
designated as source documents (i.e., no prior written or electronic record of data) will be considered 
source data.  All other data recorded on the CRFs will not be considered source documentation. 
 
 
12.4 
Data and Safety Monitoring Plan 
 
The Data and Safety Monitoring Plan for the MD Anderson Consortium is on file at the DCP.  This study 
will be monitored by the MDACC Data and Safety Monitoring Board (DSMB), the data and safety 
monitoring board of record for this study.   The DSMB reports to the President, or his designee, as the on-
campus representative of The University of Texas Board of Regents. It oversees the data and patient 
safety issues for randomized clinical trials that originate at MD Anderson; that are coordinated or 
analyzed by MD Anderson and are not being monitored by any other DSMB; or have been designated as 
requiring DSMB monitoring at the request of the IRB, the CRC, or institution. The primary objectives of 
the DSMB are to ensure that patients' rights pertaining to participation in a research study are protected, 
and that patients' interests are prioritized over the interests of the scientific investigation. Responsibilities 
include: 
(a) Review interim analyses of outcome data (prepared by the study statistician or other 
responsible person at the time points defined in the study) approved by the IRB and 
additional time points as determined by the DSMB, and to recommend, if necessary, whether 
the study needs to be changed or terminated based on these analyses; 
(b) Determine whether, and to whom, outcome results should be released prior to the reporting of 
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(c) Review interim toxicity data and efficacy of treatment; 
(d) Review major research modifications proposed by the investigator or appropriate study 
committee prior to implementation (e.g., termination, dropping an arm based on toxicity 
results from the study or results of other studies, increasing target sample size). 
 
Refer to the Data and Safety Monitoring Plan for the MD Anderson Consortium on file at the DCP for 
further details. 
 
 
12.5 
Sponsor or FDA Monitoring 
 
The NCI, DCP (or their designee), pharmaceutical collaborator (or their designee), or FDA may 
monitor/audit various aspects of the study. These monitors will be given access to facilities, databases, 
supplies and records to review and verify data pertinent to the study.  
 
 
12.6 
Record Retention 
 
Clinical records for all participants, including CRFs, all source documentation (containing evidence to 
study eligibility, history and physical findings, laboratory data, results of consultations, etc.), as well as 
IRB records and other regulatory documentation will be retained by the Investigator in a secure storage 
facility in compliance with Health Insurance Portability and Accountability Act (HIPAA), Office of 
Human Research Protections (OHRP), Food and Drug Administration (FDA) regulations and guidances, 
and NCI/DCP requirements, unless the standard at the site is more stringent. The records for all studies 
performed under an IND will be maintained, at a minimum, for two years after the approval of a New 
Drug Application (NDA). For NCI/DCP, records will be retained for at least three years after the 
completion of the research. NCI will be notified prior to the planned destruction of any materials. The 
records should be accessible for inspection and copying by authorized persons of the Food and Drug 
Administration. If the study is done outside of the United States, applicable regulatory requirements for 
the specific country participating in the study also apply. 
 
 
12.7 
Cooperative Research and Development Agreement (CRADA)/Clinical Trials Agreement 
(CTA) 
 
The agent(s) supplied by DCP, NCI, used in this protocol, is/are provided to the NCI under a 
Collaborative Agreement (CRADA, CTA, MTRA) between SELLAS (hereinafter referred to as 
Collaborator(s)) 
and 
the 
NCI 
Division 
of 
Cancer 
Prevention. 
Therefore, 
the 
following 
obligations/guidelines, in addition to the provisions in the “Intellectual Property Option to Collaborator(s) 
contained within the terms of award, apply to the use of Agent(s) in this study: 
 
12.7.1 The Research Material will only be used for research purposes by Recipient, its contractors, 
and agents and Investigator for the Research Project described in Attachment A, under suitable 
containment conditions. This Research Material will not be used for any other purpose including but 
not limited to commercial purposes for screening, production or sale, for which a commercialization 
license may be required. DCP and its Investigator shall not use the Research Material in assays for 
the identification of potential commercial products. At the end of the Research Project, Recipient and 
its Investigator will be responsible for ensuring the return of any unused Research Material to Sponsor, 
unless otherwise directed by Sponsor.  Collaborator(s) data for Agent(s) are confidential and 
proprietary to Collaborator(s) and shall be maintained as such by the investigators. The protocol 
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not be shared or distributed without the permission of the NCI. If an individual participating on the 
study or participant’s family member requests a copy of this protocol, the individual should sign a 
confidentiality agreement. A suitable model agreement can be downloaded from the DCP website. 
 
12.7.2 
Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial by 
any other Collaborator solely to the extent necessary to allow said other Collaborator to develop, 
obtain regulatory approval, or commercialize its own investigational agent. 
 
12.7.3 
Any Collaborator having the right to use the Multi-Party Data from these trials must agree 
in writing prior to the commencement of the trials that it will use the Multi-Party Data solely for 
development, regulatory approval, and commercialization of its own investigational agent. 
 
12.7.4 
Clinical Trial Data and Results and Raw Data developed under a collaborative agreement will 
be made available exclusively to Collaborator(s), the NCI, and the FDA, as appropriate. All data 
made available will comply with HIPAA regulations. 
 
12.7.5 
When a Collaborator wishes to initiate a data request, the request should first be sent to the 
NCI, who will then notify the appropriate investigators of Collaborator's wish to contact them. 
 
12.7.6 
Any abstract or manuscript reporting the results of this clinical trial must be provided to DCP 
for immediate delivery to Collaborator(s) for advisory review and comment prior to submission for 
publication. Collaborator(s) will have 30 days (or as specified in the CTA) from the date of 
receipt for review. Press releases and other media presentations must also be forwarded to DCP 
prior to release. Each Party agrees to provide proposed press releases that reference or rely upon the 
work under this MTRA to the other party for review and comment at least seven business (7) days 
prior to publication. Copies of any manuscript, abstract, and/or press release/ media presentation 
should be sent to the Protocol Information Office at NCI DCP PIO@mail.nih.gov. 
 
The Protocol Information Office will forward manuscripts to the DCP Project Officer for distribution 
to the Collaborator(s). No publication, manuscript or other form of public disclosure shall contain 
any of Collaborator’s confidential/proprietary information. 
 
 
13. 
STATISTICAL CONSIDERATIONS 
 
13.1 
Study Design/Description 
 
This is a Phase II, randomized, single-blind study with 2 intervention arms.  A total of 108 DCIS 
participants will be consented and screened for eligibility and, with a conservative estimate, 48 (45%) 
participants are expected to be HLA-A2 positive.  These 48 participants will be randomized 2:1 to 
vaccine and GM-CSF alone groups.  Accounting for 10% attrition rate while on study and for approx.  
5% non-evaluable sample rate, we expect to have 40 evaluable participants, 27 in vaccine and 13 in GM-
CSF alone group, that have all baseline, post-vaccination (pre-surgery), and post-surgery measures of 
nelipepimut-S-specific CTL.  With 27 and 13 in vaccine and GM-CSF alone groups, respectively, we will 
have 82% power to detect an effect size of 1 on the change of nelipepimut-S-specific CTL (“1 month (+/- 
7 days) after completion of the vaccination series timepoint” minus pre-1st vaccine) between the two 
groups, corresponding to change of 0.05% ± 0.05% in vaccine group and 0 ± 0.05% in control group, 
using two-sided t-test with a significance level of 0.05. 
 
The decision rule for declaring the agent promising will be if we detect an effect size of 1 on the change 
of nelipepimut-S-specific CTL, and if there are not greater than 10% grade III AEs attributed to the active 


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study drug or IV AEs of any attribution.  Completion of the current study will provide the necessary data 
to determine the next phase of study, specifically whether a subsequent, larger phase II will be conducted 
versus a phase III study. 
 
 
13.2 
Randomization/Stratification  
 
Participants will be assigned a randomization number once the following has been accomplished: 
eligibility has been verified at the site level, eligibility has been confirmed by the site PI, and eligibility 
CRF has been entered into the DMI web application.  The randomization number will be generated by the 
database and assigned to the participant. 
 
A total of 48 eligible participants will be randomized 2:1 to vaccine and GM-CSF alone groups.  
Accounting for approx. 10% attrition rate and approx. 5% non-evaluable sample rate, we expect to 
achieve 40 evaluable participants, 27 in vaccination and 13 in GM-CSF alone group.  We will stratify the 
randomization by site using random permutated block design with the block size of 6. 
 
 
13.3 
Accrual and Feasibility 
 
We plan to consent and screen 108 potentially eligible subjects in the study over 2.5 years at four clinical 
centers.  Our goal is to randomize 48 participants.  The randomized participants will receive the 
intervention for approx. 4 weeks; and will be followed up for an additional approx. 3-6 months period.  
They will be followed closely to monitor the toxicity and drug compliance. 
 
13.4 
Primary Objective, Endpoint, Analysis Plan 
 
The primary objective of this study is to evaluate the effect of the nelipepimut-S vaccine on nelipepimut-
S-specific CTL measured in blood in DCIS participants that are HLA-A2 positive. We will use a 
dextramer assay, which is a flow cytometry based assay, to enumerate the number of nelipepimut-S-
specific CTL generated in response to vaccination with the nelipepimut-S peptide.  The number of 
nelipepimut-S-CTL will be determined in participants at the following time points:  pre-vaccination, at 
the time of surgery (after two preoperative vaccinations), 1 month (+/- 7 days) after surgery, and 3-6 
months after surgery at the time of the final study visit.  The change in the number of nelipepimut-S-CTL 
for an individual participant between the pre-vaccination timepoint and the 1 month (+/- 7 days) after 
surgery timepoint will be used as the primary endpoint measure for this study. 
 
At the end of the study, the change of nelipepimut-S-specific CTL at the 1 month (+/- 7 days) after 
completion of the vaccination series timepoint from baseline will be estimated for each group using mean, 
standard deviation, median, minimum and maximum.  Two-sample t-test or Wilcoxon rank sum test, 
whichever appropriate, will be used to compare the change between the two groups.  Nelipepimut-S-
specific CTL will also be measured repeatedly through 3 months after surgery.  Repeated measures 
analysis including mixed effects model will be performed to analyze the effect of treatment on 
nelipepimut-S-specific CTL change over time [35].  We will evaluate the results in the whole participant 
population as well as the participants with or without radiation treatment separately, when appropriate 
 
 
13.5 
Secondary Objectives, Endpoints, Analysis Plans 
The secondary objectives of the study include evaluation of the toxicity profile of nelipepimut-S vaccine 
and to compare the toxicity profile between the two groups.  Additional secondary objectives include 
presence of DCIS at resection, difference in HER2 expression in the biopsy and the surgical specimen 


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excised post-vaccination and histologic responses (degree of lymphocyte infiltration determined on H&E 
stained slides as well as immune infiltration as determined by multiplex immunofluorescence staining for 
markers including but not limited to CD3, CD4 and CD8).  Comparisons of intraperson changes in the 
vaccination versus GM-CSF alone arms will be made. 
 
Adverse events by grade and relationship will be summarized by tabulation for each group.  Correlation 
among the biomarkers described above at baseline in each specimen and between different specimens will 
be assessed.  The association among various continuous and discrete biomarkers or treatment groups will 
be assessed by the exploratory data analysis using scatter plot matrix, box plots, BLiP plot [10] and trellis 
plot, etc., and may be tested by t-test/ANOVA/Wilcoxon rank sum test/Kruskal-Wallis test, when 
appropriate. Correlation between continuous biomarkers will be examined by Pearson or Spearman rank 
correlation coefficients. The association between discrete biomarkers will be tested by chi-square or 
Fisher’s exact test.  Paired t-test/Wilcoxon rank sum test and McNemar’s test may be used to test the 
change of a single continuous biomarker and discrete biomarker, respectively, over time within each 
treatment group.  Repeated measures analysis including mixed effects model will be performed to analyze 
the effect of treatment on biomarkers change over time [35]. 
 
 
13.6 
Reporting and Exclusions 
 
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 participant.   
 
 
13.7 
Evaluation of Toxicity 
 
All participants will be evaluable for toxicity from the time of their first vaccination.  The toxicity profile 
in terms of type, attribute, and grade of toxicities will be described in each of the treatment arms.  
 
 
13.8 
Evaluation of Response 
 
All randomized and treated participants are assessed for response to intervention including participants 
with protocol deviations.  For the primary endpoint of change in the number of nelipepimut-S-CTL for an 
individual participant between the pre-vaccination timepoint and the 1 month (+/- 7 days) after surgery 
timepoint, “evaluable” will be defined as completing all pre-surgical vaccinations and all post-surgical 
vaccinations up to the 1 month (+/- 7 days) timepoint.   However, the response outcome for both the 
evaluable and the non-evaluable participants will be computed and compared.  For the safety endpoint, 
participants who receive at least one vaccination will be included in the safety evaluation.  For the 
biomarker (biologic efficacy) endpoints, all non-compliant participants will be analyzed according to the 
intent-to-treat principle.  All levels of compliance will be included in the full evaluation of endpoints if 
paired biopsies are available. 
 
The primary endpoint is defined as the change in the number of E75-CTL for an individual participant 
between the pre-vaccination timepoint and the 1 month (+/- 7 days) after surgery.  
 
 
13.9 
Interim Analysis 
 
No formal statistical interim analysis and no early stopping rules have been planned for the study primary 
and secondary endpoints.   However, we plan to evaluate HER2 expression of the baseline biopsy 


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specimens of the first 12 participants to ensure that accrual is balanced and the trial enrolls women with 
DCIS on core biopsy who are HER2 0, 1+, 2+ or 3+ approximately equally.   If, on the other hand, we 
determine that the trials enrolls disproportionately more participants with a particular level of HER2 
expression, we will re-design the trial to include HER2 testing at baseline and stratification based on 
HER2 expression.  
 
 
13.10 Ancillary Studies 
There are no plans for ancillary studies.  
 
 
14. 
ETHICAL AND REGULATORY CONSIDERATIONS 
 
14.1 
Form FDA 1572 
 
Prior to initiating this study, the Protocol Lead Investigator at the Lead or Participating Organization(s) 
will provide a signed Form FDA 1572 stating that the study will be conducted in compliance with 
regulations for clinical investigations and listing the investigators, at each site that will participate in the 
protocol. All personnel directly involved in the performance of procedures required by the protocol and 
the collection of data should be listed on Form FDA 1572. 
 
 
14.2 
Other Required Documents 
 
14.2.1 Signed and dated current (within two years) CV or biosketch for all study personnel listed on the 
Form FDA 1572 and Delegation of Tasks form for the Lead Organization and all Participating 
Organizations.  
 
14.2.2 Current medical licenses (where applicable) for all study personnel listed on Form FDA 1572 and 
Delegation of Tasks form for the Lead Organization and all Participating Organizations. 
 
14.2.3 Lab certification (e.g., CLIA, CAP) and lab normal ranges for all labs listed on Form FDA 1572 
for the Lead Organization and all Participating Organizations. 
 
14.2.4 Documentation of training in “Protection of Human Research Subjects” for all study personnel 
listed on the FDA Form 1572 and Delegation of Tasks form for the Lead Organization and all 
Participating Organizations. 
 
14.2.5 Documentation of training in “Good Clinical Practice” for all study personnel listed on the FDA 
Form 1572 and Delegation of Tasks form for the Lead Organization and all Participating Organizations. 
 
14.2.6 Documentation of Federalwide Assurance (FWA) number for the Lead Organization and all 
Participating Organizations. 
 
14.2.7 Signed Investigator’s Brochure/Package Insert acknowledgement form. 
 
14.2.8 Delegation of Tasks form for the Lead Organization and all Participating Organizations signed by 
the Principal Investigator and all study personnel listed on the form for each site. 
 
14.2.9 Signed and dated NCI, DCP Financial Disclosure Form for all study personnel listed on Form 
FDA 1572 for the Lead Organization and all Participating Organizations. 


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14.3 
Institutional Review Board Approval 
 
Prior to initiating the study and receiving agent, the Investigators at the Lead Organization and the 
Participating Organization(s) must obtain written approval to conduct the study from the appropriate IRB. 
Should changes to the study become necessary, protocol amendments will be submitted to the DCP PIO 
according to DCP Amendment Guidelines. The DCP-approved amended protocol must be approved by 
the IRB prior to implementation 
 
14.4 
Informed Consent 
 
All potential study participants will be given a copy of the IRB-approved Informed Consent to review. 
The investigator will explain all aspects of the study in lay language and answer all questions regarding 
the study. If the participant decides to participate in the study, he/she will be asked to sign and date the 
Informed Consent document. The study agent(s) will not be released to a participant who has not signed 
the Informed Consent document. Subjects who refuse to participate or who withdraw from the study will 
be treated without prejudice. 
 
Participants must be provided the option to allow the use of blood samples, other body fluids, and tissues 
obtained during testing, operative procedures, or other standard medical practices for further research 
purposes. If applicable, statement of this option may be included within the informed consent document 
or may be provided as an addendum to the consent. A Model Consent Form for Use of Tissue for 
Research is available through a link in the DCP website. 
 
Prior to study initiation, the informed consent document must be reviewed and approved by NCI, DCP, 
the CLO, and the IRB at each participating organization at which the protocol will be implemented. Any 
subsequent changes to the informed consent must be approved by NCI, DCP, the CLO’s IRB, and then 
submitted to each organization’s IRB for approval prior to initiation. 
 
 
14.5 
Submission of Regulatory Documents 
 
All regulatory documents are collected by the CLO and reviewed for completeness and accuracy. Once 
the CLO has received complete and accurate documents from a participating organization, the CLO will 
forward the regulatory documents to the DCP Regulatory Contractor: 
 
 
Paper Document/CD-ROM Submissions: 
 
Regulatory Affairs Department 
 
CCS Associates 
 
1923 Landings Drive 
 
Mountain View, CA 94043 
 
Phone: 650-691-4400 
 
Fax: 650-691-4410 
 
 
E-mail Submissions: 
 
regulatory@ccsainc.com 
 
Regulatory documents that do not require an original signature may be sent electronically to the CLO for 
review, which will then be electronically forwarded to the DCP Regulatory Contractor. 
 


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14.6 
Other 
 
This trial will be conducted in compliance with the protocol, Good Clinical Practice (GCP), and the 
applicable regulatory requirements. 
 
 
15. 
FINANCING, EXPENSES, AND/OR INSURANCE 
 
Participants will not be responsible for the costs of this study. Study agent will be provided at no cost to 
the subject.  If, as a result of participation in this study, an individual experiences injury from known or 
unknown risks of the research procedures as described in the informed consent, immediate medical care 
and treatment, including hospitalization, if necessary, will be available.  No monetary compensation is 
available for the costs of medical treatment for an injury, thus, the participant will be responsible for the 
costs of such medical treatment, either directly or through their medical insurance and/or other forms of 
medical coverage. 
   
 


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APPENDIX A 
 
Performance Status Criteria 
 
 
ECOG Performance Status Scale 
 
 
Karnofsky Performance Scale 
Grade Descriptions 
Percent 
Description 
0 
Normal activity. Fully active, able to 
carry on all pre-disease performance 
without restriction. 
100 
Normal, no complaints, no evidence of 
disease. 
90 
Able to carry on normal activity; minor 
signs or symptoms of disease. 
1 
Symptoms, but ambulatory. 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). 
80 
Normal activity with effort; some signs 
or symptoms of disease. 
70 
Cares for self, unable to carry on 
normal activity or to do active work. 
2 
In bed <50% of the time. Ambulatory and 
capable of all self-care, but unable to 
carry out any work activities. Up and 
about more than 50% of waking hours. 
60 
Requires occasional assistance, but is 
able to care for most of his/her needs. 
50 
Requires considerable assistance and 
frequent medical care. 
3 
In bed >50% of the time. Capable of only 
limited self-care, confined to bed or chair 
more than 50% of waking hours. 
40 
Disabled, requires special care and 
assistance. 
30 
Severely 
disabled, 
hospitalization 
indicated. Death not imminent. 
4 
100% bedridden. Completely disabled. 
Cannot carry on any self-care. Totally 
confined to bed or chair. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
10 
Moribund, fatal processes progressing 
rapidly. 
5 
Dead. 
0 
Dead. 
 
 
 
 
 


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Appendix B 
 
 
Guidance for mixing NeuVax (or control) solution  
 
Dosing Summary:   
The study treatment is 1000 μg of nelipepimut-S peptide mixed with 250 μg of lyophilized Leukine® 
(sargramostim, GM-CSF) or control (sterile water for injection [SWFI]) mixed with Leukine, administered in four 
separate 0.4 mL intradermal injections.  
 
The nelipepimut-S peptide is provided as 1.5 mg/mL of nelipepimut-S acetate (contains a 1.42 mg/mL solution of 
the nelipepimut-S peptide). Using aseptic technique, 0.8 mL of nelipepimut-S acetate solution (1136 μg 
nelipepimut-S peptide) is added to the re-constituted Leukine. Taking into account vial losses, this procedure will 
yield a total dose of approximately 1010 μg of nelipepimut-S peptide in a volume of 1.6 mL.  
 
Stability:   
Once prepared, administer the nelipepimut-S peptide/control and lyophilized Leukine mixture within 2 hours.   
 
Materials:  
 
1. 
Test Drug kit with Subject ID number. This kit contains a single 1.0 mL vial of nelipepimut-S acetate 
solution (1.5 mg/mL)    
2.  
Lyophilized Leukine kit with Subject ID number. This kit contains a single vial of lyophilized Leukine 
(250 μg). 
3.  
Sterile Water for Injection (SWFI) 
4.  
Ancillary Supplies: 7-1.0 mL TB syringes, 5-3/8 inch 26G needles, 4-2 inch 21G needles, 2-3 mL luer 
lock syringes.  
 
NeuVax Dose Preparation:  
 
NOTE: DO NOT INVERT ANY STUDY DRUG VIALS DURING PREPARATION 
 
1. 
Remove cap from vial of sterile water for injection (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.  
• 
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 (nelipepimut-S acetate, 1.5 mg/mL) 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.  


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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. [Note: Using the same needle from step 
#9 or changing the needle to a new one is allowed.] 
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 2 hours of mixing.  
 
Control Dose Preparation:  
 
NOTE: DO NOT INVERT ANY STUDY DRUG VIALS DURING PREPARATION 
 
1. 
Remove cap from vial of sterile water for injection (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.  
• 
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. 
Using the same vial of SWFI from step#1, attach a 2 inch 21G needle to a 1.0 mL TB syringe and draw 
up 0.8 mL from the vial of SWFI, slowly inject an additional 0.8 mL of SWFI into the reconstituted 
Leukine vial.  
6. 
Keeping the vial upright, slowly roll the Leukine/Control vial between your hands. Allow any foaming to 
dissipate before continuing. 
7. 
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/Control 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.  
8. 
Using the needle and syringe containing the Leukine/Control from step #9, top-fill four 1.0 mL TB 
syringes with 0.4 mL of the Leukine/Control in each syringe. [Note: Using the same needle from step #7 
or changing the needle to a new one is allowed.] 
9. 
Attach a new sterile 3/8 inch 26G needle to each syringe containing 0.4 mL of Leukine/Control.  
Appropriately label each syringe and dispense syringes for intradermal administration of study drug.  The 
syringes must be used within 2 hours of mixing.  
 
NeuVax Dose Preparation for 1st Dose Reduction 
 
NOTE: DO NOT INVERT ANY STUDY DRUG VIALS DURING PREPARATION 


MDA2014-04-02 
Protocol Version 16, dated 10/24/2019 
DCP Protocol Template Version 8.3 
–53– 
05/21/2013 
 
 
1. 
Remove cap from vial of sterile water for injection (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.  
• 
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. 
Using the needle and syringe from step #4, draw up 0.5 mL from the vial of reconstituted Leukine. 
• 
Dispose of the needle and syringe containing 0.5 mL of reconstituted Leukine. 
6. 
Remove cap from vial of Test Drug (nelipepimut-S acetate, 1.5 mg/mL) and wipe septum with alcohol 
prep pad.  
7. 
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.   
8. 
Using the needle and syringe from step #7, slowly inject the 0.8 mL of Test Drug into the reconstituted 
Leukine vial.  
10. 
Using the same vial of SWFI from step#1, using the needle and syringe from step #8, draw up 0.5 mL 
from the vial of SWFI. 
• 
Insert the needle into the Leukine/Test Drug vial. 
• 
Angle the needle to the side of the Leukine/Test Drug vial and slowly expel 0.5 mL SWFI into the 
Leukine/Test Drug. 
• 
Keeping the vial upright, slowly roll the Leukine/Test Drug vial between your hands. Allow any 
foaming to dissipate before continuing. 
11. 
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.  
12. 
Using the needle and syringe containing the Leukine/Test Drug from step #11, top-fill four 1.0 mL TB 
syringes with 0.4 mL of the Leukine/Test Drug in each syringe. [Note: Using the same needle from step 
#11 or changing the needle to a new one is allowed.] 
13. 
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 2 hours of mixing. 
 
Control Dose Preparation for 1st Dose Reduction 
 
NOTE: DO NOT INVERT ANY STUDY DRUG VIALS DURING PREPARATION 
 
1. 
Remove cap from vial of sterile water for injection (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.  


MDA2014-04-02 
Protocol Version 16, dated 10/24/2019 
DCP Protocol Template Version 8.3 
–54– 
05/21/2013 
 
• 
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. 
Using the needle and syringe from step #4, draw up 0.5 mL from the vial of reconstituted Leukine. 
• 
Dispose of the needle and syringe containing 0.5 mL of reconstituted Leukine.  
6. 
Using the same vial of SWFI from step#1, attach a 2 inch 21G needle to a 1.0 mL TB syringe and draw 
up 0.8 mL from the vial of SWFI, slowly inject an additional 0.8 mL of SWFI into the reconstituted 
Leukine vial. 
8. 
Using the same vial of SWFI from step#1, Using the needle and syringe from step #6, draw up 0.5 mL 
from the vial of SWFI. 
• 
Insert the needle into the Leukine/Control vial. 
• 
Angle the needle to the side of the Leukine/Control vial and slowly expel 0.5 mL SWFI into the 
Leukine/Test Drug. 
• 
Keeping the vial upright, slowly roll the Leukine/Control 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/Control 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/Control from step #9, top-fill four 1.0 mL TB 
syringes with 0.4 mL of the Leukine/Control in each syringe. [Note: Using the same needle from step #9 
or changing the needle to a new one is allowed.] 
11. 
Attach a new sterile 3/8 inch 26G needle to each syringe containing 0.4 mL of Leukine/Control.  
Appropriately label each syringe and dispense syringes for intradermal administration of study 
drug.  The syringes must be used within 2 hours of mixing.