Clinical trial NCT02425306
Conditions: Melanoma
Keywords: peptide vaccine adjuvant polyICLC cyclophosphamide Montanide ISA-51
Sponsor: Craig L Slingluff, Jr
Start date: 2015-05-12; Completion date: 2018-01-08
Enrollment: 48 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study evaluates whether it is safe to administer a peptide vaccine in combination with different adjuvants. Adjuvants are substances that may boost immune responses vaccines. In this study, the adjuvants are Montanide ISA-51, polyICLC and cyclophosphamide. This study will also evaluate the effects of the combination of the peptide vaccine and the adjuvants on the immune system. The investigators will monitor these effects by performing tests in the laboratory on participants' blood, a lymph node, and tissue from the sites of vaccination.

Detailed description:

Arms: arm 1: Part 1: 200 mcg of 6MHP emulsified in Montanide ISA-51 adjuvant will be administered on 1, 8, 15, 36, 57 and 78. arm 2: Part 1: 200 mcg of 6MHP emulsified in Montanide ISA-51 adjuvant will be administered on 1, 8, 15, 36, 57 and 78. Cyclophosphamide (50 mg) will be taken orally once a day for 7 days followed by a 7 day rest period. This will be repeated for 5 cycles. Cycles will begin on the following days:

* Day -6 (Cycle 1)
* Day 8 (Cycle 2)
* Day 22 (Cycle 3)
* Day 36 (Cycle 4)
* Day 50 (Cycle 5) arm 3: Part 1: 200 mcg of 6MHP plus 1 mg of polyICLC emulsified in Montanide ISA-51 adjuvant will be administered on 1, 8, 15, 36, 57 and 78. arm 4: Parts 1 and 2: 200 mcg of 6MHP plus 1 mg of polyICLC emulsified in Montanide ISA-51 adjuvant will be administered on 1, 8, 15, 36, 57 and 78. Cyclophosphamide (50 mg) will be taken orally once a day for 7 days followed by a 7 day rest period. This will be repeated for 5 cycles. Cycles will begin on the following days:

* Day -6 (Cycle 1)
* Day 8 (Cycle 2)
* Day 22 (Cycle 3)
* Day 36 (Cycle 4)
* Day 50 (Cycle 5)
Interventions: intervention 1: 6MHP intervention 2: Montanide ISA-51 intervention 3: polyICLC intervention 4: Cyclophosphamide
Intervention descriptions: intervention 1: 6 melanoma helper vaccine comprised of 6 class II MHC-restricted helper peptides intervention 2: Montanide ISA-51 (Incomplete Freund's Adjuvant), local adjuvant intervention 3: polyICLC, local adjuvant intervention 4: Cyclophosphamide, systemic adjuvant

Protocol documents:
Prot_SAP_000.pdf: 
Protocol Mel63/IRB# 17860/IND#10825 
Version Date:  03-13-17 
1 
 
 
 
 
 
 
 
UNIVERSITY OF VIRGINIA 
HUMAN IMMUNE THERAPY CENTER 
 
UVA-MEL-63 
A TRIAL TO EVALUATE THE IMMUNOGENICITY AND SAFETY OF A 
MELANOMA HELPER PEPTIDE VACCINE PLUS NOVEL ADJUVANT 
COMBINATIONS  
 
Cyclophosphamide, Hiltonol, And Melanoma Peptides (CHAMP) 
 
 
 
 
 
 
 
 
 
 
 
 
ATTACH TO THE FRONT OF EVERY COPY OF THIS PROTOCOL 
 
Confidentiality Statement 
The information contained in this document is the property of the University of Virginia Human 
Immune Therapy Center.  Distributing, copying, or disclosing information contained in this 
document requires prior authorization from the Human Immune Therapy Center, except that 
this document may be disclosed to the appropriate institutional review boards and 
representatives of the U.S. Food and Drug Administration.   


Protocol Mel63/IRB# 17860/IND#10825 
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Signature Page 
 
Sponsor-Investigator 
 
______________________  
_______________________  
_______________ 
Name  
 
 
 
Signature 
 
 
 
Date 
 
 
SPONSOR-INVESTIGATOR’S AGREEMENT 
I confirm that I have read this protocol and I agree to conduct the study as outlined herein.  I agree to 
conduct the study in accordance with the ethical principles that have their origin in the Declaration of 
Helsinki and that are consistent with Good Clinical Practices, as outlined in ICH E6, and the applicable 
laws and regulations.  
 
 
 


Protocol Mel63/IRB# 17860/IND#10825 
Version Date:  03-13-17 
3 
 
UNIVERSITY OF VIRGINIA 
HUMAN IMMUNE THERAPY CENTER 
Mel-63 
A TRIAL TO EVALUATE THE IMMUNOGENICITY AND SAFETY OF A MELANOMA HELPER 
PEPTIDE VACCINE PLUS NOVEL ADJUVANT COMBINATIONS 
 
Sponsor 
University of Virginia Human Immune Therapy Center 
Director, Craig L. Slingluff, Jr., MD 
1340 Jefferson Park Ave., Jordan Hall, Room 1352 
Charlottesville, VA 22908 
Principal Investigator 
Director, Human Immune 
Therapy Center 
Craig L. Slingluff, Jr. 
434-924-1730 
cls8h@virginia.edu 
Co-Investigators 
University of Virginia 
William Grosh, MD 
wwg9u@virginia.edu 
 
Elizabeth Gaughan, MD 
emg5x@virginia.edu 
 
Philip Smith, MD 
pws7v@virginia.edu 
 
James Patterson, MD 
jwp9e@virginia.edu 
 
Paul Levine, MD 
pal@virginia.edu 
 
David Shonka, MD 
dcs5z@virginia.edu 
 
Carmel Nail, FNP-C 
cjn2r@virginia.edu 
 
Kathleen Haden, ANP-C 
km3s@virginia.edu 
 
Kimberly Bullock, PhD 
kb9d@virginia.edu 
 
Mark Jameson, MD 
mjj4e@virginia.edu 
 
Lynn Dengel, MD 
ltd5b@virginia.edu 
 
Katherine Fedder, MD 
klf2e@virginia.edu 
 
Jennifer Eccles, PA-C 
jme5j@virginia.edu 
 
Biostatisticians: 
University of Virginia 
Gina R. Petroni, PhD 
grp4c@virginia.edu 
 
Nolan Wages, PhD 
naw7n@virginia.edu 
 
Mark Smolkin, MS 
mes6r@virginia.edu 
 
Clinical Research 
Coordinators and Data 
Management 
University of Virginia 
Emily Allred, PhD 
eh4m@virginia.edu 
 
Jessica Zareno, MS 
jhz4f@hscmail.mcc.virginia.edu 
 
Alexandra Carney, MPH 
alc6a@virginia.edu 
Annika Shuali, MS 
ah2ze@virginia.edu 
 
Sarah Kelley 
slk6vj@virginia.edu 
 
Sarah Lewis 
set5v@virginia.edu 
Authors: 
 
Craig L. Slingluff, Jr., MD 
Kimberly Bullock, PhD 
Gina R. Petroni, PhD 


Protocol Mel63/IRB# 17860/IND#10825 
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PROTOCOL SYNOPSIS ....................................................................................................................... 7 
LIST OF ABBREVIATIONS ................................................................................................................. 11 
1.0 
BACKGROUND AND SCIENTIFIC RATIONALE ..................................................................... 13 
1.1 
INTRODUCTION ............................................................................................................................ 13 
1.2 
STUDY RATIONALE ....................................................................................................................... 13 
1.3 
6MHP ......................................................................................................................................... 14 
1.4 
MONTANIDE ISA-51 ..................................................................................................................... 15 
1.5 
6MHP ADMINISTERED IN MONTANIDE ISA-51 .............................................................................. 16 
1.6 
POLYICLC ................................................................................................................................... 21 
1.7 
METRONOMIC CYCLOPHOSPHAMIDE ............................................................................................ 22 
1.8 
DOSING ....................................................................................................................................... 23 
1.9 
SUMMARY .................................................................................................................................... 23 
2.0 
OBJECTIVES ........................................................................................................................... 24 
2.1 
PRIMARY OBJECTIVES AND ENDPOINTS ....................................................................................... 24 
2.2 
SECONDARY OBJECTIVES AND ENDPOINTS .................................................................................. 24 
2.3 
EXPLORATORY OBJECTIVES AND ENDPOINTS ............................................................................... 24 
3.0 
PARTICIPANT SELECTION CRITERIA ................................................................................... 25 
3.1 
INCLUSION CRITERIA .................................................................................................................... 25 
3.2 
EXCLUSION CRITERIA .................................................................................................................. 27 
3.3 
REGISTRATION AND RANDOMIZATION ........................................................................................... 28 
4.0 
STUDY DRUG .......................................................................................................................... 28 
4.1 
PEPTIDE SYNTHESIS .................................................................................................................... 28 
4.2 
STORAGE OF INDIVIDUAL PEPTIDES ............................................................................................. 29 
4.3 
RECONSTITUTION AND VIALING OF THE VACCINE .......................................................................... 29 
4.4 
VACCINE STORAGE ...................................................................................................................... 29 
4.5 
LOT TESTING ............................................................................................................................... 29 
4.6 
STABILITY TESTING ...................................................................................................................... 29 
4.7 
LABELING .................................................................................................................................... 29 
4.8 
MONTANIDE ISA-51 ..................................................................................................................... 29 
4.9 
STUDY DRUG ACCOUNTABILITY ................................................................................................... 30 
5.0 
POLYICLC (HILTONOL) .......................................................................................................... 30 
5.1 
PACKAGING AND LABELING .......................................................................................................... 30 
5.2 
STORAGE .................................................................................................................................... 30 
5.3 
SUPPLY ....................................................................................................................................... 30 
5.4 
STUDY DRUG ACCOUNTABILITY ................................................................................................... 30 
6.0 
CYCLOPHOSPHAMIDE ........................................................................................................... 30 
6.1 
STORAGE .................................................................................................................................... 30 
6.2 
SUPPLY ....................................................................................................................................... 30 
7.0 
TREATMENT PLAN ................................................................................................................. 30 
7.1 
MANAGEMENT OF PARTICIPANTS ................................................................................................. 30 
7.2 
ADMINISTRATION OF 6MHP, MONTANIDE ISA-51, AND POLYICLC ................................................ 31 
7.3 
ADMINISTRATION OF CYCLOPHOSPHAMIDE ................................................................................... 31 


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7.4 
DOSE MODIFICATIONS ................................................................................................................. 32 
7.5 
DOSE DELAYS ............................................................................................................................. 32 
7.6 
DISCONTINUATION OF THERAPY ................................................................................................... 35 
7.7 
REPLACEMENT OF STUDY PARTICIPANTS ..................................................................................... 35 
7.8 
CONCOMITANT MEDICATIONS ...................................................................................................... 35 
7.9 
PERMITTED MEDICATIONS OR TREATMENTS ................................................................................. 36 
7.10 
TREATMENT COMPLIANCE ............................................................................................................ 36 
7.11 
BIOPSIES ..................................................................................................................................... 36 
8.0 
CLINICAL AND LABORATORY EVALUATIONS .................................................................... 37 
8.1 
PHYSICAL EXAMS AND EVALUATIONS ........................................................................................... 37 
8.2 
PATHOLOGY REVIEW ................................................................................................................... 37 
8.3 
PERFORMANCE STATUS ............................................................................................................... 37 
8.4 
CLINICAL LABS ............................................................................................................................. 37 
8.5 
TOXICITY ASSESSMENTS ............................................................................................................. 37 
8.6 
RESEARCH BLOOD SAMPLES ....................................................................................................... 38 
8.7 
VACCINE SITE BIOPSIES............................................................................................................... 38 
8.8 
SENTINEL IMMUNIZED NODE BIOPSY ............................................................................................ 38 
8.9 
TUMOR BIOPSIES ......................................................................................................................... 39 
8.10 
ASSESSMENTS ............................................................................................................................. 41 
8.11 
STUDY CALENDAR ....................................................................................................................... 42 
9.0 
STATISTICAL CONSIDERATIONS .......................................................................................... 42 
9.1 
OVERVIEW ................................................................................................................................... 42 
9.2 
STUDY DESIGN ............................................................................................................................ 42 
9.3 
ACCRUAL ALLOCATION FOR DETERMINATION OF THE RECOMMENDED OPTIMAL COMBINATION 
(PART 1 ONLY) ........................................................................................................................................ 43 
9.4 
STATISTICAL PROPERTIES ........................................................................................................... 45 
9.5 
SAMPLE SIZE AND ACCRUAL ........................................................................................................ 47 
9.6 
STOPPING RULES (PART 1 ONLY) ................................................................................................. 48 
9.7 
DATA ANALYSIS PLANS ................................................................................................................ 48 
10.0 
ADVERSE EVENT DATA COLLECTION AND MONITORING ................................................. 49 
10.1 
DEFINITIONS ................................................................................................................................ 49 
10.2 
ATTRIBUTION ASSESSMENT ......................................................................................................... 51 
10.3 
DATA COLLECTION ....................................................................................................................... 52 
10.4 
RISKS AND SAFETY ...................................................................................................................... 52 
10.5 
ADVERSE EVENT CLASSIFICATIONS ............................................................................................. 55 
10.6 
REPORTING ADVERSE EVENTS .................................................................................................... 57 
10.7 
ADVERSE EVENT REVIEW AND MONITORING ................................................................................ 59 
10.8 
RECORDING LABORATORY VALUES .............................................................................................. 60 
10.9 
DOSE-LIMITING TOXICITIES .......................................................................................................... 61 
10.10 MANAGEMENT OF TOXICITY ......................................................................................................... 61 
10.11 DATA COLLECTION ....................................................................................................................... 62 
10.12 MONITORING PLAN ...................................................................................................................... 62 
11.0 
STUDY CONDUCT AND ETHICAL CONSIDERATIONS ......................................................... 62 
11.1 
UVA INSTITUTIONAL REVIEW BOARD FOR HEALTH SCIENCES RESEARCH .................................... 62 
11.2 
CONSENT FORMS AND THE CONSENTING PROCESS ..................................................................... 63 
11.3 
MAINTENANCE OF STUDY DOCUMENTS ........................................................................................ 63 
12.0 
APPENDICES ........................................................................................................................... 64 
APPENDIX 1:  STUDY CALENDAR ................................................................................................... 65 


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APPENDIX 3:  ECOG PERFORMANCE STATUS .............................................................................. 69 
APPENDIX 4:  NEW YORK HEART ASSOCIATION DISEASE CLASSIFICATION ........................... 70 
APPENDIX 5:  RECIST 1.1 CRITERIA ................................................................................................ 71 
APPENDIX 6:  VACCINE LOT RELEASE AND STABILITY TESTING............................................... 72 
APPENDIX 7:  SUMMARY OF CHANGES ......................................................................................... 74 
REFERENCE LIST .............................................................................................................................. 77 
 
 
 


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Protocol Synopsis 
Title 
A trial to evaluate the immunogenicity and safety of a melanoma helper peptide vaccine plus novel 
adjuvant combinations (MEL63) 
Investigational Drugs 
 
6 melanoma helper peptides (6MHP)(Table 1) 
 
Montanide ISA-51 (incomplete Freund’s adjuvant)  
 
polyICLC  
 
cyclophosphamide  
 
 
 
 
 
 
 
 
 
 
FDA approved drugs for the intended indication 
N/A 
Indication 
Part 1:  High-risk resected melanoma: Stage IIB-IV, plus stage IIA with high risk features on Melanoma 
DecisionDx assay. 
Part 2:  Stage IIIB-IV melanoma with one or more tumor deposits accessible for biopsy and/or excision. 
Objectives and Endpoints 
Primary  
Part 1: 
1. Safety:  to determine whether it is safe to administer a multipeptide vaccine comprised of 6 
melanoma-associated class II MHC-restricted helper peptides (6MHP) plus Montanide ISA-51 
(Incomplete Freund’s adjuvant, IFA), or IFA + polyICLC (Hiltonol), with or without oral 
metronomic cyclophosphamide (mCy). This will be evaluated by adverse event assessments, 
including CTCAE 4.03 and subclassified by irAE categories. 
 
2. Immunogenicity: To determine which of the following treatments is most effective for inducing 
CD4+ T cell responses to 6MHP after 6MHP vaccination (assessed in PBMC and vaccine-
draining nodes) as measured initially by ELIspot assay: 
a. Montanide ISA-51 (IFA) 
b. IFA + systemic metronomic cyclophosphamide (mCy) 
c. IFA + polyICLC  
d. IFA + polyICLC + mCy 
Part 2: 
3. To obtain preliminary data on whether treatment with the recommended optimal combination 
(determined in Part 1) in patients with one or more tumor deposits accessible for biopsy or 
excision modifies the tumor microenvironment (increases infiltration of CD4+ and CD8+ T cells 
into tumor metastases). 
Table 1.  Peptides used in the 6 Melanoma Helper Peptide (6MHP) vaccine 
Amino Acid Sequence 
Epitope (source protein, residues) 
ref 
AQNILLSNAPLGPQFP 
Tyrosinase 56-70
 (alanine added#) 
(1) 
FLLHHAFVDSIFEQWLQRHRP 
Tyrosinase 386-406 
(2) 
RNGYRALMDKSLHVGTQCALTRR 
Melan-A/MART-151-73 
(3) 
TSYVKVLHHMVKISG 
MAGE-3 281-295 
(4) 
LLKYRAREPVTKAE 
MAGE-1,2,3,6 121-134 
(5) 
WNRQLYPEWTEAQRLD 
gp100 44-59 
(6,7) 


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Secondary  
4. To determine which of the following treatments is most effective for inducing CD8+ T cell 
responses to melanoma antigens (assessed in PBMC and vaccine-draining nodes) to 6MHP 
vaccination as measured initially by ELIspot assay: 
a. Montanide ISA-51 (IFA) 
b. IFA + systemic metronomic cyclophosphamide (mCy) 
c. IFA + polyICLC  
d. IFA + polyICLC + mCy  
Exploratory 
5. To assess whether the vaccine adjuvant most effective for inducing CD4+ T cell responses:  
a. induces Th1-dominant cytokine responses in the VSME and SIN. 
b. induces IgG antibodies to 6MHP. 
 
6. To obtain preliminary data about the vaccine-site microenvironment (VSME) induced by the 
vaccines administered in different adjuvants. This will include assessment of Th1/Th2/Th17 bias 
of cells in the VSME, T cell retention in the VSME, and induction of retention integrins. 
 
7. To assess whether immune responses to melanoma antigens will be associated with survival in 
each study cohort and overall. These include measures of: 
a. CD4+ T cell response to 6MHP. 
b. IgG antibody to 6MHP. 
c. CD8+ T cell response to a panel of melanoma proteins.  
 
8. To estimate clinical outcomes for participants in each study arm, and overall, including disease-
free survival (DFS) or progression-free survival (PFS), as appropriate, and overall survival. 
Design 
This is an open-label, single center phase I/II study  
 
Regimen 
Part 1:  All participants will receive the 6MHP vaccine.  Participants will be randomized to receive the 
vaccine with the following: 
a. Montanide ISA-51 (IFA) 
b. IFA + systemic metronomic cyclophosphamide (mCy) 
c. IFA + polyICLC  
d. IFA + polyICLC + mCy  
 
Part 2:  Participants will receive the 6MHP vaccine with the optimal adjuvant combination identified from 
Part 1 of the study. 
 
 
 


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Figure 1A:  Protocol Schema:  Part 1 
 
 
 
Figure 1B:  Protocol Schema:  Part 2 
Optimal adjuvant combination identified from Part 1 of the study. 
 
 
 
 
 
 
 
 


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Biopsies 
 
Part 1: 
Vaccine Site Biopsies 
Each participant enrolled in Part 1 will undergo a biopsy of a vaccine site at two time points (days 8 and 
22; one week after vaccines 1 and 3).  The biopsies will consist of three 4-mm punch biopsies of skin. 
 
Sentinel Immunized Node 
A lymph node draining the 3rd vaccine site (sentinel immunized node (SIN) will be biopsied on day 22. 
 
Tumor Biopsy 
Optional at the time of recurrence or later as clinically indicated. 
 
Part 2: 
Tumor Biopsies 
Each participant enrolled in Part 2 will undergo a tumor biopsy and/or tumor excision at two time points: 
 
Pre-vaccine:  Optimally, patients will have a tumor biopsy within 3 weeks of starting treatment.  
However, tumor tissue from a prior biopsy can serve as the pre-treatment sample provided: 1) 
there is no intervening treatment in between the pre-study biopsy and study treatment, and 2) 
formalin-fixed tumor tissue is available and adequate to provide at least 20 unstained slides with 
sufficient tumor for analysis.  
 
Day 22.  The day 22 biopsy will typically include complete resection in accord with clinical 
indications for disease control. 
 
Optional biopsies may be completed at the time of progression or later as clinically indicated. 
 
Population 
The main criteria for inclusion include: 
Part 1: 
 
Age 18 years or older.  
 
A diagnosis of melanoma at high risk of recurrence/metastasis after surgical or ablative therapy 
(stage IIB-IV).  Patients with stage IIA melanoma who are found to be high-risk based on the 
DecisionDx Melanoma test may also be eligible.   
Part 2: 
 
Age 18 years or older.  
 
A diagnosis of stage IIIB-IV melanoma with one or more tumor deposits accessible for biopsy 
and/or excision. 
 
Accrual Goal 
Maximum total target accrual should not exceed 74 patients for Parts 1 and 2 but is estimated to be 
approximately 65 patients. 
 
 
 


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List of Abbreviations 
 
Abbreviation 
Full text 
HCG 
Beta Human chorionic gonadotropin (pregnancy test) 
6MHP 
6 melanoma-derived class II MHC-restricted helper peptides 
12-MP 
12 melanoma-derived class I MHC-restricted peptides 
AE 
adverse event 
AGC 
absolute granulocyte count 
AJCC 
American Joint Committee on Cancer 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
APC 
antigen presenting cell 
AST 
aspartate aminotransferase 
BRAFi 
BRAF inhibitor 
BSA 
body surface area 
BUN 
urea nitrogen 
CC 
Cancer Center 
cc 
cubic centimeter 
CFR 
Code of Federal Regulations 
cm 
centimeter 
CO2 
carbon dioxide 
CRF 
case report form 
CT 
computed tomography 
CTA 
cancer-testis antigens 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTL 
cytotoxic T lymphocyte 
CTO 
Clinical Trials Office 
Cy 
cyclophosphamide 
DC 
dendritic cells 
dL 
deciliter 
DLT 
dose limiting toxicity 
DSMC 
Data and Safety Monitoring Committee 
DTH 
delayed type hypersensitivity 
ECOG 
Eastern Cooperative Oncology Group 
ELISA 
enzyme linked immunosorbent assay 
FACS 
fluorescence activated cell sorter 
FBS 
fetal bovine serum 
FDA 
Food and Drug Administration 
FITC 
Fluorescein isothiocyanate 
g 
gram 
GCRC 
General Clinical Research Center 
GM-CSF 
granulocyte-macrophage stimulating colony 
GMP 
good manufacturing practice 
Hgb 
hemoglobin 
HGBA1C 
hemoglobin a1c 
HITC 
Human Immune Therapy Center 
HIV 
human immunodeficiency virus 
HLA 
human leukocyte antigen 
HPLC 
high performance liquid chromatography 
IBW 
ideal body weight 
id 
intradermal 
IFN 
interferon 
IL-2 
interleukin-2 
IL-7 
interleukin-7 
IL-15 
interleukin-15 


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IML 
Immune Monitoring Laboratory 
In. 
inch 
IND 
investigational new drug 
ip 
intraperitoneal 
IRB 
Institutional Review Board 
IU 
international unit 
IV 
intravenous 
kg 
kilogram 
KLH 
keyhole limpet hemocyanin 
LC 
Langerhans cells 
LDH 
lactate dehydrogenase 
Lf 
flocculation value 
m 
meter 
mcCi 
microcurie 
mcg 
microgram 
mcl 
microliter 
mCy 
metronomic cyclophosphamide 
MDP 
melanocyte differentiation proteins 
mg 
milligram 
MHC 
major histocompatability complex 
mIU 
million international units 
ml 
milliliter 
mm 
millimeter 
MRI 
magnetic resonance imaging 
NBT/BCIP 
Nitro blue tetrazolium chloride/5-Bromo-4-Chloro-3-Indolyl Phosphate 
NCI 
National Cancer Institute 
NOS 
not otherwise specified 
NSAID 
non-steroidal anti-inflammatory drug 
OCS 
Office of Collaborative Studies 
PAP 
pulmonary alveolar proteinosis 
PBL 
peripheral blood lymphocytes 
PBS 
phosphate buffered saline 
PD-1 
Programmed-death 1 
PET 
positron emission tomography 
PHA 
phytohemagglutinin 
PI 
principal investigator 
PMA 
phorbol myristate acetate 
ppm 
parts per million 
PRC 
protocol review committee 
RPMI 
Roswell Park Memorial Institute 
sc 
subcutaneous 
SD 
standard deviation 
SIN 
sentinel immunized node 
TAA 
tumor associated antigens 
tet 
tetanus helper peptide 
TFA 
trifluoroacetic acid 
Th 
CD4+ helper T cells  
TIL 
tumor infiltrating lymphocytes 
TNF 
tumor necrosis factor 
TPF 
Tissue Procurement Facility 
ULN 
upper limits of normal 
USP 
United States Pharmacopeia 
UVA 
University of Virginia 
WBC 
white blood cell 
w/v 
weight to volume 
v/v 
Volume to volume 


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1.0 
BACKGROUND AND SCIENTIFIC RATIONALE 
1.1 
Introduction 
Cancer immunotherapy for solid tumors is coming of age, with FDA-approved 
immuno-therapeutics in prostate cancer, melanoma, and renal cell cancer.  Interleukin-2 
(IL-2) and the CTLA4 antibody ipilimumab are approved for melanoma; both induce 
durable clinical regressions. Recent data also show that blockade of PD-1/PD-L1 
induces durable clinical regressions of melanoma, renal cell cancer and non-small cell 
lung cancer (NSCLC) (8,9). Furthermore, antigen-specific adoptive T cell therapy 
induces clinical regressions that are durable in about 20% of treated patients(10). There 
is excitement about this growing armamentarium of systemic immunotherapeutics, 
whose effects are mediated predominantly by T lymphocytes.  Despite the effectiveness 
of those therapies, still about 70-80% of patients fail them, and go on to develop 
progressive disease. There is a need for new combination approaches that build on the 
demonstrated clinical value of immune therapy. 
Cancer vaccines inducing antigen-specific T cell responses are emerging as a 
component of combination immunotherapy.  In the past few years, a cancer vaccine has 
been approved for prostate cancer, based on two randomized trials showing improved 
survival (11), and a randomized prospective trial showed that adding a peptide vaccine 
to high-dose IL-2 significantly prolonged progression-free survival (PFS) when compared 
to IL-2 alone(12).  Thus, after several decades of development and optimization, there is 
now evidence that cancer vaccines may improve clinical outcomes, in particular in 
combination with other active therapy.  
We have developed a vaccine incorporating 6 intermediate-length peptides that 
induce CD4+ helper T cell (TH) responses (6 helper peptides, 6MHP), which has clinical 
activity in patients with advanced melanoma. The current proposal is to optimize the 
6MHP vaccine (Aim 1), by testing two local adjuvants and one systemic adjuvant. This 
clinical trial also will incorporate correlative studies of immune responses in blood, skin, 
and lymph nodes, to obtain a more complete understanding of the host: tumor 
relationship in the context of these vaccination approaches.  This study holds promise to 
optimize the immunogenicity of vaccines comprising class II MHC peptides with 
improved adjuvants, which may have relevance across a spectrum of cancers,  
1.2 
Study Rationale 
 Evidence for the Role of the Immune System in Protecting Against the 
1.2.1
Development of Solid Tumors 
 
There has long been evidence of immune responses to cancer, but evidence 
of impact on tumor progression has not been well demonstrated until recently.  
The most convincing evidence of the importance of immune surveillance in 
preventing the development of solid tumors is provided by recent work in murine 
models, in which 50-100% of knockout mice lacking STAT1 and/or IFN receptor 
function developed spontaneous solid tumors of various histologies within 12-15 
months, whereas normal mice never developed malignancies during the same 
time period (13).  These studies strongly support the role of cellular immune 
function in the control of cancer progression. 


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 The Role of CD4+ Helper T Lymphocytes in Anti-tumor Immune Responses 
1.2.2
Initially, the majority of cancer vaccines were designed to activate the CD8+ 
cytotoxic T cell arm of the host immune system. However, more recent 
approaches target the activation of CD4+ Th cells.  This is based in part on results 
from earlier studies which demonstrated depletion of CD4+ T-cells abrogates all 
or part of protective immune response to vaccines (14).  Furthermore, adoptive 
therapy with CD4+ T-cells has been shown to induce tumor protection in some 
model systems (15) (16).  Thus, the protective immunity induced by tumor cell 
vaccines appears to be mediated both by CD8+ T-cells and by CD4+ T-cells.   
 
Th cells can activate dendritic cells (DC) for heightened antigen presentation, 
causing the DC to secrete IL-2 and other cytokines that may help to direct the 
immune response. Furthermore, strong Th1 help produces the proper cytokine 
milieu which is critical to the induction of immune-mediated tumor destruction 
(17,18).  In addition, Th responses are believed to be involved in the 
establishment of memory responses.   
 Safety of Peptide-based Vaccines   
1.2.3
Peptide-based vaccines have been administered safely to humans in a 
number of clinical trials, with toxicity limited usually to local injection site reactions 
and transient grade I-II systemic reactions.  In our experience, other systemic 
toxicities are attributable to cytokines added as adjuvants, but the peptides 
themselves appear to be very well tolerated (19). In previous studies conducted 
by the UVA-HITC and by the Eastern Cooperative Oncology Group (ECOG), 
vaccines containing 6MHP have been administered to over 200 patients and 
have been well-tolerated(20-22).  In fact, injection site reactions have been lower 
with 6MHP than with Class I MHC associated peptides (23). 
1.3 
6MHP 
 Selection of Class II MHC-restricted Epitopes for Melanoma-reactive Th Cells 
1.3.1
In the current protocol we are including class II MHC-restricted peptides 
derived from melanoma proteins in an effort to generate melanoma-specific Th 
responses.  The melanoma specific class II MHC-restricted peptides (Table 1) 
are derived from melanocytic differentiation proteins (MDP) and cancer-testis 
antigens (CTA).  The peptides were originally reported to bind to HLA-DR1, -
DR4, -DR11, -DR13, and/or -DR15, and approximately 90% of the melanoma 
patient population will express at least one of those class II alleles.  Our prior 
work has demonstrated that these peptides, like other HLA-DR restricted 
peptides are also presented promiscuously on many other HLA-DR molecules 
(24); thus, we do not restrict enrollment based on HLA expression.  
 
The melanoma-associated class II MHC-restricted peptides in the 6MHP 
vaccine include 4 from MDPs (tyrosinase (2), gp100 (1), MelanA/MART-1 (1)), 
and 2 from CTAs (MAGE proteins). The first report of HLA-DR-restricted peptides 
recognized by T-cells on melanoma identified tyrosinase56-70 and tyrosinase448-462 
(1). Both peptides require high concentrations to induce T cell responses, but the 
former peptide has a higher binding affinity for HLA-DR4 than the latter; 
therefore, tyrosinase 56-70  (QNILLSNAPLGPQFP) was chosen for use in this 
study.  A DR15-restricted peptide, tyrosinase 386-406, was reported also to be an 
antigen for Th cells and was selected for use in this study (2).  Peptides 


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presented by HLA-DR4 from MART-1/Melan-A have been identified and we have 
chosen to include Melan-A/MART-151-73 in this trial.  The last MDP represented in 
the vaccine is gp100, from which a peptide at residues 44-59 has been identified. 
T-cells sensitized against this peptide can recognize melanoma cells, and this 
epitope has been demonstrated to be naturally processed and presented in the 
context of HLA-DR4 (6,7,25).  
   
The peptides from CTA to be included are from MAGE proteins.  The peptide 
MAGE-A3 281-295 can stimulate peptide reactive CTL in vitro and is strongly 
recognized by DR11-restricted MAGE-3 reactive CTL (4).  Another MAGE 
peptide is homologous with MAGE-1, 2, 3, and 6, and is restricted by DR13.  It 
represents MAGE 121-134 (5).   
 Immune Monitoring Assays for CD4+ T-cells    
1.3.2
An essential step in the development of effective cancer vaccines is 
identification of the immune response parameters that effectively measure 
relevant immunologic endpoints, such as immunogenicity. Ideally, these 
endpoints will be associated with clinical response. A number of immunologic 
assays have been evaluated over the years for their ability to serve as sensitive 
and reliable tools for immune monitoring purposes.   
One method for measuring epitope-specific helper T cell responses is the 
ELIspot assay, which was derived to evaluate functional antigen-specific 
responses by permitting direct counting of T-cells reacting to antigen by 
production of IFN or other cytokines (26) (27,28).  T-cells that are not anergized 
should secrete IFN after exposure to their cognate antigen, especially if they 
have a memory phenotype (29).  ELIspot assays can reproducibly detect 
functional T cell responses to defined antigens at levels below 0.01% (26,30), 
and they do not require prolonged in vitro culture prior to evaluation.      
 
We have also found measures of proliferation, by incorporation of tritiated 
thymidine, are effective for detection of helper responses, and that 
multiparameter flow cytometry enables detection of multifunctional T cells and 
differentiating CD4+ and CD8+ T cell responses.  For the present study, we 
propose to use flow cytometry as the primary assay, and ELIspot assays as a 
secondary assay.  Characterization of the helper T cell response may aid in 
detection of differences in the immunologic milieu when the vaccines are 
administered. This can be achieved by measuring cytokines secreted into the 
media by CD4+ T cells proliferating in response to antigen, using an ELISA assay 
(31) or flow cytometry, with calculation of the Th1/Th2/Th17 balance. 
1.4 
Montanide ISA-51  
 Montanide ISA-51 as a Vaccine Adjuvant 
1.4.1
Montanide ISA-51 adjuvant has been effective at inducing immune responses 
against murine viral antigens when administered with a synthetic peptide epitope 
(32,33) and is widely used as a vaccine adjuvant in veterinary practices.  The 
product consists of a mineral oil base similar to incomplete Freund's adjuvant 
(IFA).  However, the Arlacel A emulsifying agent of incomplete Freund's, which 
has caused reactions in the past, has been replaced with a purified manoside 
monooleate called "montanide", which appears to be safer.  The UVA HITC has 
sponsored studies where peptide-based vaccines in Montanide ISA-51 have 


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been safely administered to more than 600 participants.  Immunological 
responses against the immunizing peptides have been detected in most 
participants (20-22,34-36). 
Montanide ISA-51 may or may not be an optimal adjuvant; concerns have 
been raised about its usefulness as an adjuvant with a short peptide vaccine, 
attributed to changes from a prior formulation to the current one (34), but in our 
hands, we found that both formulations were very similar in their effectiveness as 
vaccine adjuvants with peptide vaccines (34).  Another concern is that 
vaccination with short (9-mer) peptides in IFA induces chronic inflammation at 
the site of vaccination, with retention of antigen-reactive T cells at the vaccine 
site, both in mice and humans (37,38). However, this does not appear to be the 
case with longer peptides (37). We believe, thus, that these 14-23 mer peptides 
in 6MHP are not likely susceptible to retention at the vaccine site; so that IFA is a 
good adjuvant to use.   
1.5 
6MHP Administered in Montanide ISA-51 
 Immunogenicity and Clinical Activity 
1.5.1
This protocol builds on 3 prior NIH-funded clinical trials performed at the 
University of Virginia and collaborating centers: 1) Mel41 (NCT00089219): Phase 
I/II first-in-humans trial of 6MHP vaccine in stage III/IV melanoma (supported by 
R21 
CA105777)(22); 
2) 
Mel44 
(NCT00118274): 
Multi-peptide 
Vaccine 
Administered with Cyclophosphamide for High-Risk Melanoma (supported by 
R01 CA118386)(21), and 3) ECOG trial E1602 (NCT00071981) testing multi-
peptide vaccines to stimulate CD8+ and CD4+ T cells in advanced melanoma 
(supported by R01 CA104362)(39).  Together these trials demonstrate the 
safety, immunogenicity, and clinical activity of the 6MHP vaccine.  
 
Table 2. Clinical response rates to 6MHP vaccines in 
patients with advanced melanoma in Mel41 and E1602 
trials (RECIST) 
Study 
N 
CR+
PR 
CR+PR
+SD 
RR 
DCR 
Mel41 
17 
2 
4 
12% 
24% 
E1602 Arm D 
42 
3 
15 
  7.1% 
36% 
E1602 Arm C 
32 
2 
8 
  6.3% 
25% 
All studies 
91 
7 
27 
  7.7% 
30% 
Mel41+Arm D 
59 
5 
19 
  8.5% 
32% 
 
The 6MHP vaccine incorporates a set of 6 intermediate-length peptides that 
are promiscuous in binding to Class II MHC molecules (Table 1); thus, they are 
presented by a wide range of MHC molecules, and can be applied to patients 
without pre-testing MHC expression(22). Vaccination with 6MHP has been 
immunogenic in 81% of patients with stage III-IV melanoma, when administered 
with incomplete Freund’s adjuvant (IFA) + GM-CSF as the vaccine adjuvant, and 
when measuring immune responses in vaccine-draining nodes (sentinel 
immunized nodes)(22). When immune responses are measured in peripheral 
blood, they are detected in 40-60% of patients (21,22,39).In patients with 
advanced melanoma, this vaccination strategy has been associated also with 
clinical regressions in 7-12% of patients and with stable disease for an additional 
12-29% (mean disease control rate, DCR, 30%, Table 2. In the Mel41 trial, 
durations of SD and clinical responses have ranged from 1 to 7 years (22).  


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We have found in a separate study that GM-CSF did not increase 
immunogenicity compared to IFA alone. Instead, we found that IFA alone is a 
better adjuvant than IFA+GMCSF for induction of CD4+ T cell responses to an 
intermediate length helper peptide (35).  Though GM-CSF continues to be used 
as a vaccine adjuvant in other settings, studies by us and  others (40) provide 
randomized prospective data on its use in humans in combination with other 
adjuvants - in both cases, the addition of GM-CSF was associated with lower 
immunogenicity, worse clinical outcome, or both (35,40).  Thus, in another trial, 
we tested patients with no clinical evidence of disease (resected stage IIB-IV), 
with the 6MHP vaccine in Montanide ISA-51, without GM-CSF (21): immune 
responses to 6MHP in the blood were detected in 48% of patients.  
We tested, in two trials (Mel44 and E1602), whether co-administration of the 
6MHP vaccine with a peptide vaccine that stimulates CD8+ T cells (12 MHC 
Class I restricted peptides, 12MP (36)) would induce greater CD8+ T cell 
reactivity and increased clinical responses than vaccination with 12MP alone.   In 
the Mel44 trial, patients were vaccinated with 12MP+6MHP (MELITAC 12.6) or 
12MP+tetanus helper peptide (MELITAC 12.1), with IFA alone as the adjuvant, in 
patients with resected stage 
IIB-IV melanoma and no 
measurable disease. That  
trial was designed to enroll 
160 eligible patients, plus 
allowances for 
ineligibility 
and 
over-enrollment. 
170 
patients were enrolled at 3 
centers (3/05–1/08). Three 
(1.8%) were ineligible on 
post 
review. 
Immune 
responses were evaluable 
for 161 (96%) of 167 eligible 
patients. T cell responses 
were measured by IFN 
ELIspot assay directly ex 
vivo, among CD8+ T cells for 
12MP, and among CD4+ T 
cells 
for 
tetanus 
helper 
peptide (arms A and B), and 
6MHP (arms C and D).  
The 
combination 
of 
6MHP with 12MP paradoxically reduced the circulating CD8+ T-cell response rate 
(Figure 2) (21). Responses to the tetanus helper peptide were detected in 91% of 
patients vaccinated with it, and the mixture of 6 melanoma helper peptides 
(6MHP) induced TH cell responses in 52% of those vaccinated with it (21). 
Patients on the Mel44 trial also were randomized to pre-treatment with one dose 
of cyclophosphamide (CY, 300 mg/m2), which had no effect on CD8+ or CD4+ T 
cell responses (Figure 2).  Clinical outcome was not altered by adding 6MHP or 
CY to 12MP. 
 
Figure 2. Mel44 trial. Immune response rates for 12MP, 6MHP, 
and tetanus peptide by ELISpot assay for each study arm, by 
evaluation of PBMC through day 50. P values for the response 
to 12MP are noted for four pairs of study arms. 12.1 [MELITAC 
12.1], 
vaccines 
with 
12MP 
plus 
tetanus 
peptide 
AQYIKANSKFIGITEL; 12.6 [MELITAC 12.6], vaccines with 
12MP plus 6MHP. 


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In the E1602 trial, patients with advanced measurable melanoma were 
vaccinated in a 4-arm randomized phase II design: Patients on arms A, B, C, and 
D were vaccinated with 12MP, 12MP+tetanus peptide, 12MP+6MHP, or 6MHP 
alone, respectively, and with IFA+GMCSF as vaccine adjuvant(39). Similar to the 
finding in the Mel44 trial, the combination of 12MP+6MHP did not induce greater 
CD8+ T cell responses or better clinical outcome.  However, T cell responses  
were observed in the peripheral blood in about 40% of patients vaccinated with 
6MHP (Figure 3), and objective clinical responses were observed in arms C and 
D: 
2 
partial 
responses (PR) in 
Arm C (6.3%) and 3 
in Arm 
D 
(7.1%) 
(Table 2).  There is 
additional 
evidence 
of clinical benefit, as 
the 1 year survival 
for Arms C and D 
exceed 
the 
95% 
confidence 
interval 
from a meta-analysis 
of 
outcomes 
from 
cooperative 
group 
trials 
(Figure 
4A). 
Importantly, 
there 
also 
is 
a 
very 
significant 
prolongation 
of 
survival for patients who had a helper T cell response to the 6MHP vaccines 
(Arms C+D, p = 0.005, Figure 4B). That difference persisted in a multivariate 
analysis (p = 0.038, HR 0.5; report in preparation).  The association between 
immune response to helper peptides was specific for the 6MHP, as no difference 
in survival was observed with immune response to the tetanus peptide for Arm B 
(not shown). 
 Summary of Preliminary Immunogenicity and Clinical Response Data with 6MHP 
1.5.2
Vaccines  
A consistent finding from these 3 trials is that 6MHP vaccines are 
immunogenic, with 40-50% immune response rate, when measured in blood, and 
81% when measured also in vaccine-draining nodes. Also, in the 2 trials that 
enrolled patients with measureable disease, there were durable clinical 
responses(22), and there was clinical activity with an 8%  
RECIST-defined response rate, and 30% disease control rate (Table 2), similar 
to those of ipilimumab (RR 11%, DCR 29%)(41) and durable clinical responses.  
Importantly, in E1602, there was a strong and specific association between 
immune response to the 6MHP and survival, support the clinical relevance of 
immune responses induced to the 6MHP vaccine. 
 
 
 
 
Figure 3.  E1602 trial. Immune responses to 12MP, 6MHP, and 
tetanus peptide.   A) The proportions of analyzable patients with 
CTL response to 12MP (blue bars), helper T cell response to 
tetanus peptide (yellow bars), and helper T cell response to 6MHP 
(maroon bars) are shown for each study arm.  


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 Toxicities Previously Reported for Participants Receiving the 6MHP Vaccine with 
1.5.3
in Montanide ISA-51 adjuvant, with or without GM-CSF 
 
There is no reason to expect direct toxicity of the melanoma peptides; they 
are not directly cytotoxic in vitro and have been used safely in prior studies.  On 
the other hand, because some of these peptides are identical or similar to a 
portion of a normal protein, risks of autoimmunity in humans are important to 
evaluate. There is no murine system adequately modeling the human immune 
response to these peptides. The most meaningful evaluation of this peptide 
vaccine mixture is in patients with melanoma. This trial will focus on participants 
with high risk resected melanoma (stage IIB/C, III, or IV melanoma without 
measurable disease; and stage IIA melanoma found to be high risk based on 
gene expression profiling).  These individuals face a high risk (> 35%) of 
premature death, and the anticipated risk of short-term or long-term toxicity of 
this vaccine preparation is minimal, while the vaccine may delay or decrease the 
risk of morbidity and mortality due to melanoma in these patients(20,22).   
 
Potential implications of autoimmunity against cells of melanocytic lineage 
are illustrated by reported cases of vitiligo occurring coincident with regressions 
of melanoma (43).  Most of these are limited, often occurring in skin surrounding 
the regressing melanoma, but occasionally occurring systemically. While 
pathogenesis of this phenomenon can only be hypothesized, it is reasonable to 
consider this a worst-case scenario. The loss of skin pigment and hair pigment 
can be striking in these cases, but is not a cause of morbidity or mortality. Of 
greater potential concern is the theoretical risk of damage to the retinal pigment 
epithelium; however, visual loss has not been observed with these peptide 
vaccines, in over 200 patients treated, despite observing vitiligo in up to 10% of 
A 
B 
 
 
 
 
Figure 4.   Overall survival on E1602.  (A) 1 year survival for each treatment arm and for the full study 
population is shown on the plot of 1 year OS for all melanoma trials as previously published by Korn et al(42), 
and (B) Overall survival by helper T cell response to 6MHP in analyzable patients on arms C and D of E1602 
trial (n = 64).   


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patients (20-22).  Depigmentation of the retinal pigment epithelium has been 
observed in a small number of patients vaccinated with dendritic cells pulsed with 
MDP-derived peptides; however, this change was asymptomatic and was not 
associated with loss of visual acuity (personal communication – Frank Haluska).  
A careful study of the retinal pigment epithelium using monobenzyl ether of 
hydroquinone to induce pigment cell destruction on a biochemical basis suggests 
the safety of pigment cell destruction and supports immunotherapy directed 
against MDP as a strategy for melanoma therapy (personal communication, JM 
Kirkwood).   
Table 3.  Reported toxicities for 6MHP vaccines in humans (n=207) 
Toxicity (based on max grade) 
Gr 1 
Gr2 
Gr3 
Gr4 
 
 
 
 
 
LOCAL, INJECTION SITE 
 
 
 
 
Injection site reaction 
18% 
56% 
2.4% 
 
Ulceration 
 
5% 
1.4% 
 
 
 
 
 
 
CONSTITUTIONAL 
 
 
 
 
Fatigue  
43% 
8% 
3.4% 
 
Headache 
27% 
1.4% 
0.5% 
 
Rigors, Chills 
24% 
1.4% 
-- 
 
Nausea 
23% 
2% 
0.5% 
 
Sweating 
19% 
1% 
 
 
Myalgias 
18% 
0.5% 
 
 
Arthralgias 
17% 
0.5% 
 
 
Fever 
16% 
2% 
 
 
Dizziness 
13% 
 
 
 
Anorexia 
13% 
3% 
 
 
Diarrhea 
12% 
2% 
 
 
Cough 
13% 
 
 
 
Allergic rhinitis 
11% 
 
 
 
Nasal/paranasal reactions 
11% 
 
 
 
Pain larynx/throat 
10% 
 
 
 
Flushing 
10% 
 
 
 
Pruritis 
9% 
0.5% 
 
 
Rash 
6% 
3.4% 
 
 
Dyspnea 
5% 
1% 
0.5% 
 
Vomiting 
5% 
1% 
0.5% 
 
Flu-like syndrome 
6% 
 
 
 
Mucositis 
6% 
 
 
 
Constipation 
5% 
 
 
 
Autoimmune reaction 
4% 
0.5% 
 
 
Wound, non-infectious 
4% 
 
 
 
Pain, other 
2% 
1% 
0.5% 
 
Abdominal pain 
1.4% 
 
0.5% 
 
Tinnitus 
 
0.5% 
0.5% 
 
Tumor pain 
0.5% 
 
0.5% 
 
Hearing (without monitoring program) 
 
 
0.5% 
 
 
 
 
 
 
CLINICAL LABORATORY 
 
 
 
 
Hyperglycemia (not fasting) 
22% 
1% 
 
 
Hemoglobin, low 
17% 
1% 
0.5% 
 
Hyperkalemia 
13% 
 
 
 
Lymphopenia 
9% 
2.9% 
1% 
 
Leukocytes 
8% 
1% 
 
 
Hyponatremia 
7% 
 
 
 
Increased creatinine 
6% 
0.5% 
 
 
Hypoglycemia 
6% 
 
 
 
AST, SGOT 
5% 
 
 
0.5% 


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ALT, SGPT 
4% 
 
1% 
 
Neutrophils 
3% 
2% 
 
 
Metabolic, Other 
3% 
0.5% 
0.5% 
 
Alk phos 
4% 
0.5% 
 
 
Bilirubin 
3% 
 
0.5% 
 
Included are events reported in 4% or more of participants or events with one or more grade 3-4 
treatment-related AEs.  *One patient had a grade 4 ALT elevation (<0.5%) 
 
The 6MHP vaccines have been administered to 207 humans in 3 clinical trials 
(UVA trials Mel41 and Mel44, and ECOG trial E1602; NCT00089219, 
NCT00118274, NCT00071981).  Treatment-related adverse events were graded 
using the NCI Common Terminology Criteria for Adverse Events v3.0. Toxicities 
experienced by at least 4% of participants are included in Table 3. The vast 
majority of these toxicities are grade 1; a subset list of expected grade 2 or 3 
toxicities is in Table 4. 
  
 
Table 4.  Expected toxicities for 6MHP vaccines  
Toxicity 
Grade 2 
Injection site reaction 
+ 
Ulceration 
+ 
Fatigue 
+ 
1.6 
polyICLC 
 Rationale for use of polyICLC as a vaccine adjuvant and with Montanide ISA-51 
1.6.1
Polyinosinic-Polycytidylic acid (PolyIC) is a double-stranded RNA (dsRNA) 
that acts as a TLR3 agonist. However, its short half-life limits its usefulness. To 
increase half-life and its practical use in clinical settings, it has been stabilized 
with polylysine and carboxymethylcellulose as polyICLC. Like polyIC, polyICLC is 
a TLR3 agonist.  TLR3 is expressed in the early endosome of myeloid DC; thus 
polyICLC preferentially activates myeloid dendritic cells, favoring a Th1 cytotoxic 
T-cell response (44) (45). PolyICLC also activates NK cells and induces cytolytic 
potential.(45) It has been administered in the emulsion with Montanide ISA-51 
plus long peptides, with increased immunogenicity over Montanide ISA-51 plus 
peptides alone; the combination was safe, with some significant local reactions 
but no DLTs (46). 
 Toxicities of vaccination with peptides in IFA + polyICLC 
1.6.2
We have performed a clinical trial of a peptide vaccine using 12 short 
melanoma peptides (12MP) with IFA plus polyICLC (Mel58, NCT01585350), and 
others have vaccinated with long peptides with IFA plus polyICLC (47).  Data 
from the Mel58 trial are not yet final, but vaccination with the combination of 
polyICLC and IFA has been well-tolerated, though inflammation at the vaccine 
sites has been prominent, and - as with IFA and short peptides alone – has 
occasionally been associated with skin ulceration at the vaccine site.  The 
published experience with long peptides in IFA + polyICLC is that there were 
injection site reactions and fatigue, and that of 11 patients, 1 developed grade 2 
panniculitis at the injection site, and 3 patients developed grade 2 injection site 
reactions.  The injection site reactions were not considered DLTs, but further 
vaccinations in some patients were held to prevent more severe toxicity.  Our 
own experience with 6 vaccines using polyICLC + IFA is that it has been 
tolerated well in most patients, though we had one patient who came off early 
due to grade 3 toxicity (unpublished preliminary data, Mel58 trial). We have used 


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a lower dose of polyICLC (0.5 ml, 1 mg) than the initial experience with NY-ESO1 
peptides (0.7ml). 
1.7 
Metronomic Cyclophosphamide 
 Rationale for use of metronomic cyclophosphamide 
1.7.1
Cyclophosphamide (CY) has been studied as a systemic adjuvant for cancer 
vaccines.  CY doses lower than those used for tumor lysis have been reported to 
augment immune responses in mice and humans (48-52) through several 
potential mechanisms (50,53-59), including decreasing regulatory T cells (60,61), 
and supporting dendritic cell maturation (62).  In preclinical studies, 
immunopotentiation has been reported with CY administered 1 to 7 days prior to 
vaccination (63-66).  Prior human trials of immunomodulatory properties of CY 
have tested dose from 75 to 1000 mg/m2, with variable results (57,58,67-70).  For 
melanoma patients, pre-treatment with 300 mg/m2 of CY was associated with 
augmented delayed type hypersensitivity (DTH) responses to an autologous 
melanoma cell vaccine in sequential non-randomized studies (68,71).  Prior 
human experience suggested that CY increased immunogenicity when 
administered 3 days prior to a cell-based vaccine, but those studies were non-
randomized and were limited by semi-quantitative immunologic endpoints 
(68,69,71).  Other human experience failed to identify changes in regulatory T 
cells with CY treatment, (72)  and one study identified negative effects of CY 
(200 mg/m2 or greater) pretreatment on cellular immune responses to a breast 
cancer cell vaccine, proposing that even lower doses may support 
immunogenicity (70).   The largest experience has been with a dose of 300 
mg/m2 prior to vaccination.  Thus, in a prior randomized prospective trial, we 
evaluated that dose, administered once, 5 days prior to the first vaccine, but 
found that it had no significant effect on circulating CD4+ or CD8+ T cell 
responses (21).   
Other doses or timing of CY pretreatment may have different effects than 
those observed in that study. In fact, a very different dosing scheme for use of 
cyclophosphamide has shown promise, where T cell responses to peptide 
vaccines in patients with ovarian cancer were about 10-fold higher in patients 
receiving a metronomic dosing of very low dose cyclophosphamide over a 10-
week period in addition to vaccine, compared to patients receiving vaccine alone 
(73).  That trial used 50 mg per day oral dosing, for one week, followed by 1 
week with no CY, and repeating that 2 week cycle for a total of 5 cycles (10 
weeks) coincident with the vaccines.  Metronomic scheduling of various drugs 
has had differential and beneficial effects in multiple settings, and has been 
justified in particular for cyclophosphamide (74).  A goal of the present study is to 
evaluate whether T cell responses to the helper peptide vaccines will be 
increased by combination with this regimen of very low dose CY (50 mg per day) 
in a metronomic schedule (daily for seven days, followed by no treatment for 7 
days, repeated x5, over 10 weeks). 
 Toxicities of metronomic cyclophosphamide  
1.7.2
Cyclophosphamide administered at 52 mg orally per day induced grade 3 
nausea in 2/24 patients (8%), with no grade 4 treatment related toxicity.  When 
given at 50-100 mg 3 weeks out of 4, there was neutropenia in 2/13 patients and 
lymphopenia in 5/13 patients (grade 3 – 4, combined).  In another study of 80 
patients treated with cyclophosphamide 50 mg/m2/day, grade 3 toxicities 


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included lymphopenia in 19 (<25%), anemia in 1, leukopenia in 1, and no grade 4 
toxicities.  Thus, this low dose appears to be safe (74). 
 
1.8 
Dosing 
 6 MHP 
1.8.1
The peptide vaccine is sequestered locally, and the immune response occurs 
primarily locally and in the draining lymph nodes.  Thus, the dose of the vaccine 
does not need to be scaled up proportionately to the size (by weight or body 
surface area) of the recipient, as might be done for a drug whose effect is related 
to its distribution in body fluid.  Because direct toxicity of the peptide is not 
expected, dose escalation is not as meaningful as it would be with a drug with a 
narrow therapeutic index.  
 polyICLC 
1.8.2
PolyICLC also is administered locally with the vaccine with the intention of 
supporting activation of the immune response at the site of vaccine and in the 
draining lymph nodes. It has been used safely in cancer patients, with 
intravenous doses up to 300 mcg/kg.(75)  We will administer 1 mg (0.5 mL) per 
vaccine, as used in other trials (e.g.: NCT01008527).  It will be incorporated in 
the emulsion with peptides and Montanide ISA-51. 
 Metronomic Cyclophosphamide 
1.8.3
Dosing will be 50 mg orally once per day of dosing. Patients will begin taking 
cyclophosphamide once a day (any time of day) 1 week prior to the first vaccine 
(day -6), then will not take it for seven days. This 14-day cycle will be repeated 5 
times for a total of 35 doses of 50 mg over 10 weeks. 
1.9 
Summary 
We have developed a vaccine incorporating 6 intermediate-length peptides that 
induce CD4+ helper T cell (TH) responses (6 helper peptides, 6MHP), which has clinical 
activity in patients with advanced melanoma. The current proposal is to optimize the 
6MHP vaccine, by testing 2 local adjuvants and one systemic adjuvant. Part 1 of this  
clinical trial is to test whether the immune response to 6MHP can be improved by 
combining polyICLC with IFA, and/or by co-administration of low-dose metronomic 
cyclophosphamide.  In Part 2 of this study, we will obtain preliminary data on whether 
vaccination with the optimal combination of 6MHP and adjuvants increases infiltration of 
CD4+ and CD8+ T cells into tumor. 
 
This clinical trial also will incorporate correlative studies of immune responses in 
blood, skin, lymph nodes, and tumor to obtain a more complete understanding of the 
host: tumor relationship in the context of these vaccination approaches.  This study 
holds promise to optimize the immunogenicity of vaccines comprising class II MHC 
peptides with improved adjuvants, which may have relevance across a spectrum of 
cancers.  
 
 


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2.0 
OBJECTIVES 
2.1 
Primary Objectives and Endpoints 
Part 1: 
Safety:  to determine whether it is safe to administer a multipeptide vaccine comprised of 
6 melanoma-associated class II MHC-restricted helper peptides (6MHP) plus Montanide 
ISA-51 (Incomplete Freund’s adjuvant, IFA), or IFA + polyICLC (Hiltonol), with or without 
oral metronomic cyclophosphamide (mCy). This will be evaluated by adverse event 
assessments, including CTCAE 4.03 and subclassified by irAE categories. 
Immunogenicity: To determine which of the following treatments is most effective for 
inducing CD4+ T cell responses to 6MHP after 6MHP vaccination (assessed in PBMC 
and vaccine-draining nodes) as measured initially by ELIspot assay: 
a. Montanide ISA-51 (IFA) 
b. IFA + systemic metronomic cyclophosphamide (mCy) 
c. IFA + polyICLC  
d. IFA + polyICLC + mCy 
Part 2: 
To obtain preliminary data on whether treatment with the recommended optimal 
combination (determined in Part 1) in patients with one or more tumor deposits 
accessible for biopsy or excision modifies the tumor microenvironment (increases 
infiltration of CD4+ and CD8+ T cells into tumor metastases). 
 
2.2 
Secondary Objectives and Endpoints 
To determine which of the following treatments is most effective for inducing CD8+ T cell 
responses to melanoma antigens (assessed in PBMC and vaccine-draining nodes) to 
6MHP vaccination as measured initially by ELIspot assay: 
a. Montanide ISA-51 (IFA) 
b. IFA + systemic metronomic cyclophosphamide (mCy) 
c. IFA + polyICLC  
d. IFA + polyICLC + mCy  
 
2.3 
Exploratory Objectives and Endpoints 
To assess whether the vaccine adjuvant most effective for inducing CD4+ T cell 
responses:  
a. induces Th1-dominant cytokine responses in the VSME and SIN. 
b. induces IgG antibodies to 6MHP. 
To obtain preliminary data about the vaccine-site microenvironment (VSME) induced by 
the vaccines administered in different adjuvants. This will include assessment of 
Th1/Th2/Th17 bias of cells in the VSME, T cell retention in the VSME, and induction of 
retention integrins. 
To assess whether immune responses to melanoma antigens will be associated with 
survival in each study cohort and overall. These include measures of: 
a. CD4+ T cell response to 6MHP. 
b. IgG antibody to 6MHP. 
c. CD8+ T cell response to a panel of melanoma proteins.  


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To estimate clinical outcomes for participants in each study arm, and overall, including 
disease-free survival (DFS) or progression-free survival (PFS), as appropriate, and overall 
survival. 
 
3.0 
PARTICIPANT SELECTION CRITERIA 
3.1 
Inclusion Criteria 
 Part 1 
3.1.1
Patients with stage IIB, IIC, III, or IV melanoma at original diagnosis or at 
restaging after recurrence, rendered clinically free of disease by surgery, other 
therapy, or spontaneous remission within 6 months prior to registration. Patients 
with small radiologic or clinical findings of an indeterminate nature may still be 
eligible. 
Patients with high-risk stage IIA melanoma (by DecisionDx Melanoma test, 
Castle Biosciences, Inc,;Friendswood, TX) also may be eligible.  These 
participants may have had cutaneous, uveal, mucosal primary melanoma, or an 
unknown primary melanoma.  Diagnosis of melanoma must be confirmed by 
cytological or histological examination. Staging of cutaneous melanoma will be 
based on version 7 AJCC staging system (Appendix 2).   
Part 2 
Patients with a diagnosis of stage IIIB-IV melanoma with one or more tumor 
deposits accessible for biopsy and/or excision.  These participants may have had 
cutaneous, uveal, mucosal primary melanoma, or an unknown primary 
melanoma.  Diagnosis of melanoma must be confirmed by cytological or 
histological examination. Staging of cutaneous melanoma will be based on 
version 7 AJCC staging system (Appendix 2). 
Patients must have adequate cutaneous,subcutaneous, soft tissue, or nodal 
metastases of melanoma readily accessible for biopsy to provide a minimum of 
0.1 cm3 of tissue per biopsy (approximately 0.58 cm by 0.58 cm x 0.58 cm or two 
2mm core biopsies) and up to about 0.3 cm3 of tissue per biopsy at each time 
point, depending on the individual’s tumor availability and specifics.  We will try to 
maximize the amount of tissue collected at each time-point, trying to collect up to 
about 0.3 cm3 of tissue per biopsy when it is available.   
Several scenarios may fulfill the tumor burden requirement.  For example, a 
patient may have one large lesion from which core biopsies can be taken for the 
first biopsy time point and then the entire lesion excised for the final tissue 
sample.  Alternatively, a patient may have two lesions, each ≥0.1 cm3, and these 
lesions would be excised sequentially as biopsies 1 and 2.  Other combinations 
are acceptable. 
Optimally, patients will have a tumor biopsy within 3 weeks of starting treatment.  
However, tumor tissue from a prior biopsy can serve as the pre-treatment sample 
provided: 1) there is no intervening treatment in between the pre-study biopsy 
and study treatment and 2) formalin-fixed tumor tissue is available and adequate 
to provide at least 20 unstained slides with sufficient tumor for analysis. 


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 Participants will be required to have radiological studies to rule out radiologically 
3.1.2
evident disease.  Required studies include: 
 
Chest CT scan, 
 
Abdominal and pelvic CT scan, and 
 
Head CT scan or MRI 
 
PET/CT fusion scan may replace scans of the chest, abdomen, and pelvis. 
 Participants who have had brain metastases will be eligible if all of the following 
3.1.3
are true: 
 
Each brain metastasis must have been completely removed by surgery or 
each unresected brain metastasis must have been treated with stereotactic 
radiosurgery.   
 
There has been no evident growth of any brain metastasis since the most 
recent treatment. 
 
No brain metastasis is > 2 cm in diameter at the time of registration. 
 The most recent surgical resections or gamma-knife therapy for malignant 
3.1.4
melanoma must have been completed ≥ 1 week and for Part 1 ≤ 6 months prior 
to registration.  
 ECOG performance status of 0 or 1 (Appendix 3) 
3.1.5
 Ability and willingness to give informed consent 
3.1.6
 Laboratory parameters as follows: 
3.1.7
 
ANC > 1000/mm3 
 
Platelets > 100,000/mm3 
 
Hgb > 9 g/dL 
 
HgbA1c ≤ 7.5%  
 
Hepatic: 
 
AST and ALT ≤ 2.5 x upper limits of normal (ULN) 
 
Bilirubin ≤ 2.5 x ULN (except in patients with Gilbert’s disease, where bilirubin 
to 4x ULN is allowed) 
 
Alkaline phosphatase ≤ 2.5 x ULN 
 
Renal 
 
Creatinine ≤ 1.5 x ULN 
 
Serology (within 6 months of study entry) 
 
HIV negative 
 
Hepatitis C negative (no evidence of active virus)  
 
 Blood is to be collected for HLA typing (Class I and Class II), which will be 
3.1.8
analyzed as part of the immunologic endpoints, but HLA type will not be an 
inclusion/exclusion criterion. 
 Age 18 years or older at registration. 
3.1.9
 


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 Part 1:  Participants must have at least two intact (undissected) axillary and/or 
3.1.10
inguinal lymph node basins. 
Part 2:  Participants must have at least one intact (undissected) axillary and/or 
inguinal lymph node basin. 
3.2 
Exclusion Criteria 
 Participants who have received the following medications or treatments at any 
3.2.1
time within 4 weeks of registration: 
 
Chemotherapy 
 
Interferon (e.g. Intron-A®) 
 
Radiation therapy (Stereotactic radiotherapy, such as gamma knife, can be 
used ≥ 1 week and ≤ 6 months prior to registration) 
 
Allergy desensitization injections 
 
High doses of systemic corticosteroids, with the following qualifications and 
exceptions: 
o In patients with adrenal or pituitary insufficiency replacement steroid 
doses are allowed; however, daily doses of 10 mg or more of 
prednisone (or equivalent) per day administered parenterally or orally 
are not allowed in patients with normal adrenal and pituitary function. 
o Inhaled steroids (e.g.: Advair®, Flovent®, Azmacort®) are permitted at 
low doses (less than 500 mcg fluticasone per day, or equivalent) 
(76,77).  
o Topical, nasal, and intra-articular corticosteroids are acceptable. 
 
Growth factors (e.g. Procrit®, Aranesp®, Neulasta®) 
 
Interleukins (e.g. Proleukin®) 
 
Any investigational medication 
 
Targeted therapies specific for mutated BRAF or for MEK 
 Participants who are currently receiving nitrosoureas or who have received this 
3.2.2
therapy within the preceding 6 weeks 
 Participants who are currently receiving a checkpoint molecule blockade therapy, 
3.2.3
or who have received this therapy within the preceding 12 weeks.   
 Participants with known or suspected allergies to any component of the vaccine. 
3.2.4
 Participants may not have been vaccinated previously with any of the synthetic 
3.2.5
peptides included in this protocol. Participants who have received vaccinations 
containing agents other than the synthetic peptides included in this protocol and 
have recurred during or after administration of the vaccine will be eligible to enroll 
12 weeks following their last vaccination. 
 Pregnancy. Female participants of childbearing potential must have a negative 
3.2.6
pregnancy test (urinary or serum beta-HCG) obtained within 2 weeks prior to 
registration. Males and females must agree, in the consent form, to use effective 
birth control methods during the course of vaccination. 
 Female participants must not be breastfeeding 
3.2.7


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 Participants in whom there is a medical contraindication or potential problem in 
3.2.8
complying with the requirements of the protocol in the opinion of the investigator. 
 Participants classified according to the New York Heart Association classification 
3.2.9
as having Class III or IV heart disease (Appendix 4). 
 Participants with uncontrolled diabetes, defined as having a HgbA1c > 7.5%. 
3.2.10
 Participants must not have had prior autoimmune disorders requiring cytotoxic or 
3.2.11
immunosuppressive therapy, or autoimmune disorders with visceral involvement.  
Participants with an active autoimmune disorder requiring these therapies are 
also excluded.  The following will not be exclusionary: 
 
The presence of laboratory evidence of autoimmune disease (e.g. positive 
ANA titer) without symptoms 
 
Clinical evidence of vitiligo 
 
Other forms of depigmenting illness 
 
Mild arthritis requiring NSAID medications 
 Participants with known addiction to alcohol or drugs who are actively taking 
3.2.12
those agents, or participants with recent (within 1 year) or ongoing illicit IV drug 
use. 
 Body weight < 110 pounds (without clothes) at registration, due to the amount 
3.2.13
and frequency with which blood will be drawn. 
3.3 
Registration and Randomization 
All participants must sign the consent form prior to determination of eligibility for this 
study.  All participants who meet the inclusion/exclusion criteria may be registered.  
Registration will occur following verification of eligibility by the treating physician. 
Participants should receive their first study treatment within 3 weeks of registration. 
Part 1: 
Treatment allocation will be discussed with participants during the process of informed 
consent and will occur after registration.  Treatment allocation will be based upon an 
adaptive design until a safety bound has been triggered or target accrual has been met. 
Arm allocation slots are generated by the study statisticians.  
 
Part 2: 
All participants will be assigned to the optimal treatment determined in Part 1. 
 
4.0 
STUDY DRUG 
4.1 
Peptide Synthesis 
The vaccine drug product 6MHP to be administered consists of 6 peptides.  All peptides 
were synthesized directly from amino acids by Multiple Peptide Systems (now 
Polypeptide Group, San Diego, CA) under GMP conditions.  Recombinant vectors in 
bacteria or viruses were not used.  The synthetic peptides were purified by HPLC.  The 
identity of the synthetic peptides has been confirmed by verifying their mass and amino 
acid sequences by mass spectrometry.  Details of the synthesis, certificates of analysis, 
and technical summaries are included in the IND application.  


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4.2 
Storage of Individual Peptides 
Each bulk peptide was supplied to the HITC as lyophilized powder without excipients 
and stored at a temperature ≤ -70°C and protected from light. 
4.3 
Reconstitution and Vialing of the Vaccine 
Lyophilized peptides were reconstituted, mixed and vialed under GMP conditions by 
Clinalfa (Merck Biosciences AG, Laufelfingen, Switzerland).  Lyophilized peptides were 
supplied to the HITC as individual use vials.  Lot release testing of the final vialed 
peptide has also been completed by Clinalfa in accord with FDA guidelines. Details of 
the vialing are included in the IND application. 
4.4 
Vaccine Storage   
The vials of lyophilized peptide are stored by the HITC at a temperature ≤ -70°C and 
protected from light.  Once thawed, the vial(s) must be used for preparation of the 
vaccine within 24 hours. 
4.5 
Lot Testing   
Each lot of peptide vaccine is evaluated as required by the FDA for identity, sterility, 
general safety, purity, and pyrogenicity.  The details of these tests are outlined in the 
IND application. 
4.6 
Stability testing    
The peptide vaccine will undergo stability testing as described in Appendix 6.  
4.7 
Labeling   
Each vial of lyophilized peptide is labeled with the following information: 
 
Short name of the product 
 
Product number 
 
Proper name of the product 
 
Name and address of the vialing facility 
 
Lot number 
 
Date of manufacture (the date of vialing the reconstituted peptides) 
 
Serial number 
 
Quantity of each peptide per vial 
 
Vial contains no preservative, store at ≤ -70°C 
 
“Caution:  New Drug – Limited by US Federal law to investigational use” 
 
4.8 
Montanide ISA-51  
Montanide ISA-51 is available from Seppic, Inc. (Fairfield, NJ).  A drug master file for 
Montanide ISA-51 is filed with the FDA and is cross-referenced in the IND application. 
Class II MHC-restricted melanoma peptides (6-MHP; 200 mcg) in aqueous solution are 
mixed 1/1 with Montanide ISA-51 to form water-in-oil emulsions (see vaccine mixing 
sheets for complete mixing instructions).  


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4.9 
Study Drug Accountability 
The investigational drug will be stored in accord with directions specified in Section 4 of 
the protocol in a secure area with the UVA-HITC laboratories.  Study drug accountability 
is maintained using the InvestMed database. 
5.0 
polyICLC (Hiltonol) 
5.1 
Packaging and Labeling 
This is provided for used as an adjuvant as a clinical grade reagent in single-use vials 
containing 1 mL of a 2 mg/mL solution. We will administer 1 mg (0.5 mL) per vaccine, 
as used in other trials (e.g.: NCT01008527).   
5.2 
Storage 
The polyICLC is stored at 2-8oC. 
5.3 
Supply 
Hiltonol is provided by the Ludwig Institute for Cancer Research and its Cancer Vaccine 
Consortium at no charge; as they have purchased a lot of it from Dr. Andres Salazar 
from Oncovir, Inc. (Washington, D.C). 
5.4 
Study Drug Accountability 
Study drug will be kept in the University of Virginia Investigational Pharmacy or Human 
Immune Therapy Center.   
Study drug accountability will be maintained by the University of Virginia Investigational 
Pharmacy or Human Immune Therapy Center. 
6.0 
CYCLOPHOSPHAMIDE 
6.1 
Storage 
Cyclophosphamide can be stored at room temperature. 
6.2 
Supply 
Cyclophosphamide is available commercially and is an FDA approved chemotherapy 
agent for other indications. Generic formulations are available and are acceptable.  For 
this study, 35 doses of 50 mg tablets or capsules of cyclophosphamide (or combination 
of tablets or capsules adding up to 50 mg) will be administered. 
7.0 
TREATMENT PLAN 
7.1 
Management of Participants 
This study will be conducted on an outpatient basis, with participants scheduled to be 
evaluated as needed for clinical care, and as specified in the study calendar (Appendix 
1) through 8 months (or more often if needed for testing or medical reasons).  
Participants will be off treatment follow-up at the end of 8 months, or when another 
therapy is initiated, whichever occurs first.  Once off treatment follow-up, participants will 
be followed yearly for progression-free survival and overall survival. 


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7.2 
Administration of 6MHP, Montanide ISA-51, and polyICLC 
 Overview 
7.2.1
The peptides will be prepared by combining the 6MHP mixture of 6 peptides, 
which are provided as 300 mcg (0.3mg) of each peptide per vial with Montanide 
ISA-51 or polyICLC and Montanide ISA-51. More information is provided below, 
for each study arm.  Details of making emulsions with IFA (Montanide ISA-51) 
are provided in the vaccine mixing sheets.   
All subjects will receive vaccines on days 1, 8, 15, 36, 57 and 78.  Each vaccine 
will be administered subcutaneously (50%) and intradermally (50%) at one skin 
location. The same skin location will be used for vaccines 1-3.  If the vaccine site 
has severe inflammation or ulceration after 1-2 vaccines, the next vaccine may 
be placed near the original site.  For vaccines 4-6 (after excision of the sentinel 
immunized node), a different site will be used and may be rotated among 
extremities or administered in adjacent skin areas on the same extremity. 
 Dose Calculations 
7.2.2
Study Arms A, B (IFA):  At each designated time-point, 200 mcg each of the 6 
peptides (Table 1) will be emulsified in Montanide ISA-51 adjuvant (2 ml total) 
and administered. 
Study Arms C, D (IFA + polyICLC):  At each designated time-point, 200 mcg 
each of the 6 peptides (Table 1) plus 1 mg of polyICLC will be emulsified in 
Montanide ISA-51 adjuvant (2 ml total) and administered. 
 Pre-medications 
7.2.3
None required. 
 Preparation of Study Drug 
7.2.4
Directions on how to prepare the investigational drug will be provided in vaccine 
mixing sheets.  Prepared vaccines will be stored in a plastic syringe and 
delivered to the clinicians in a plastic bag.  This bag with the syringe will be 
stored at room temperature until the vaccine is administered.  Ideally, the vaccine 
should be administered 1-2 hours after mixing.  If the vaccine is not administered 
within 4 hours after mixing, it should be discarded. 
 Post-Vaccine Observation 
7.2.5
All participants will be closely observed for adverse events for at least 20 minutes 
following each vaccination.  Any time thereafter, participants should report any 
adverse events to the research coordinator or research clinician. 
7.3 
Administration of Cyclophosphamide 
 Overview 
7.3.1
Study Arms B and D:  Cyclophosphamide will be provided in 50 mg tablets or 
capsules (or combination of tablets or capsules adding up to 50 mg), which will 
be taken orally once a day for 7 days followed by a 7 day rest period.  This will be 
repeated for 5 cycles.  Cyclophosphamide may be taken before or after a 
vaccine, on days when both vaccine and cyclophosphamide are administered.   


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Patients will be provided with a daily patient diary on which to document the days 
on which they took a dose of cyclophosphamide.  Cycles will begin on the 
following days:  
 
Day -6 (Cycle 1) 
 
Day 8 (Cycle 2) 
 
Day 22 (Cycle 3) 
 
Day 36 (Cycle 4) 
 
Day 50 (Cycle 5) 
 Dose Calculations 
7.3.2
At each designated time-point, 50 mg of cyclophosphamide will be taken orally. 
 Pre-medications 
7.3.3
None required. 
7.4 
Dose Modifications 
 6MHP, Montanide ISA-51, and polyICLC 
7.4.1
There will be no dose modifications of vaccine components. 
 Cyclophosphamide 
7.4.2
There will be no dose modifications of cyclophosphamide. 
7.5 
Dose Delays 
 Dose Delays Due to Toxicity 
7.5.1
 
In circumstances where assessment of an AE is limited, such as by 
intercurrent illness, or when laboratory studies are required to assess for 
other causes of toxicity, the vaccine schedule may be interrupted for up to 21 
days.  Delay of one vaccine administration by up to 21 days will not be 
considered a protocol violation if due to an AE, regardless of attribution.  If 
more than one vaccine is delayed by 21 days due to an AE, regardless of 
attribution, vaccine treatment should be discontinued.   
 
 
Dose delays for cyclophosphamide  
 
Toxicity of cyclophosphamide is expected to be very mild. However, if a 
patient does not tolerate cyclophosphamide or has an expected grade 2- 3 
toxicity attributable to this drug, the mCy may be held until toxicities resolve to 
grade 1 or lower.  If a grade 3 toxicity that is not expected occurs, the mCy 
should be discontinued. Reasons for discontinuation should be noted.  If mCy 
is resumed, it should resume as close to the original schedule as possible 
and not be extended beyond week 10. 
If there is a change in the schedule of other protocol interventions for toxicity 
or reasons other than toxicity (Table 5, section 7.5), the mCy should either: 
o begin at the time of the other interventions of the protocol, and 
continue for 7 sequential calendar days, or 


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o may continue as originally scheduled.  This latter choice is preferable 
if the shift in schedule of other interventions is temporary. 
For example, a temporary shift may occur when visits through day 15 are on 
a Wednesday, but  the visit scheduled day 22 is moved to a Monday or 
Tuesday (1-2 days earlier)  to perform the SIN biopsy or to avoid a holiday, 
followed by resuming with subsequent treatment visits on Wednesdays.   In 
that case, starting the mCy on a Wednesday each week is preferred. 
Missed doses of cyclophosphamide should not be repeated, but will be 
recorded.  
 Delayed Visits for Reasons Other Than Toxicity 
7.5.2
A schedule for return visits should be established at the first visit.  If a participant 
misses a treatment, the missed treatment will be administered as soon as 
possible, so that subsequent treatments will be given in the appropriate intervals.  
Treatment may be continued for an additional time period, if needed.  
Participants who are treated outside of the established schedule should return to 
the original schedule as soon as possible  
Table 5 defines what constitutes a delayed visit, whether the participant should 
continue to be treated, and whether a protocol violation should be reported and 
recorded.  The range of days is counted from the original scheduled date.   
Table 5.  Delayed Visit for Reasons other than Toxicity 
Treatment Period 
Range of Days 
Participant Treatment 
Protocol 
Deviation 
Pre-study Biopsy(Not 
applicable to those 
patients who had a prior 
biopsy and have left-
over tissue available 
 
 
 
Day -6 
Day -27 through 
Day -6  
Pre-study biopsy 
No 
 
-28 or less 
Pre-study biopsy 
Yes 
 
-5 or greater 
Pre-study biopsy 
Yes 
Initiate mCy* 
 
 
 
Day -6 
 2 days 
Treatment/Labs/mCy 
No 
 
 3 to 7 days 
Treatment/Labs/mCy 
Yes 
 
 8 or more 
days 
Labs 
Yes 
Vaccine 1 
 
 
 
Day 1 
 2 days 
Vaccine/Labs 
No 
        
 3 to 7 days 
Vaccine/Labs 
Yes 
 
 8 or more 
days 
Labs 
Yes 
Vaccine 2/Biopsy*/start 1 week mCy* 
 
 
Day 8 
 2 days 
Vaccine/Labs/Biopsy/mCy 
No 
        
 3 to 7 days 
Vaccine/Labs/Biopsy/mCy 
Yes 
 
 8 or more 
days 
Labs 
Yes 
 
 
 
 
Vaccine 3* 
 
 
 
Day 15 
 2 days 
Vaccine/Labs 
No 


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Table 5.  Delayed Visit for Reasons other than Toxicity 
Treatment Period 
Range of Days 
Participant Treatment 
Protocol 
Deviation 
        
 3 to 7 days 
Vaccine/Labs 
Yes 
 
 8 or more 
days 
Labs 
Yes 
 
 
 
Assessment/Biopsy/start 1 wk mCy* 
 
 
Day 22 
 2 days 
Biopsy/Labs/mCy 
No 
 
 3 to 7 days 
Biopsy/Labs/mCy 
Yes 
 
 8 or more 
days 
Labs 
Yes 
 
 
 
 
Vaccine 4*/start 1 wk mCy* 
 
 
Day 36 
 7 days 
Vaccine/Labs/mCy 
No 
 
 8 to 14 days 
Vaccine/Labs/mCy 
Yes 
 
 15 or more 
days 
Labs 
Yes 
 
 
 
 
Day 50/start 1 wk mCy* 
 2 days 
Vaccine/Labs/Biopsy/mCy 
No 
        
 3 to 7 days 
Vaccine/Labs/Biopsy/mCy 
Yes 
 
 8 or more 
days 
Labs 
Yes 
 
 
 
 
Vaccines 5-6 
 
 
 
Days 57, 78 
 7 days 
Vaccine/Labs 
No 
 
 8 to 14 days 
Vaccine/Labs 
Yes 
 
 15 or more 
days 
Labs 
Yes 
 
 
 
 
Assessment 
 
 
 
Week 12 (day 85) 
 7 days 
Labs/Scans 
No 
 
 8 to 14 days 
Labs/Scans 
Yes 
 
 15 or more 
days 
Labs 
Yes 
Follow-up 
 
 
 
Week 18 (day 127) 
 7 days 
Labs 
No 
 
 8 to 30 days 
Labs 
Yes 
 
 
 
 
Week 26 (day 180) 
 7 days 
Scans/Labs 
No 
 
 8 to 30 days 
Scans/Labs 
Yes 
 
 
 
 
Week 32 (day 225) 
 7 days 
Labs 
No 
 
 8 to 30 days 
Labs 
Yes 
* 
mCy = metronomic cyclophosphamide.  This will be administered orally for seven 
sequential calendar days (eg days -6 through day 0, then days 8-14, 22-28, 36-
44, 50-56. 
**    A participant will be taken off protocol treatment if more than one vaccination is 
delayed [ 3 to 7 days] during the treatment period. 
 
 


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7.6 
Discontinuation of Therapy 
Protocol treatment will be discontinued for any of the following reasons: 
 
Any dose-limiting toxicity as defined in section 10.9. 
 
Disease progression requiring other therapy (e.g. surgery under general 
anesthesia, radiation, chemotherapy, or steroid therapy).  The appearance of 
small metastases or recurrent tumor deposits will not be a basis for discontinuing 
the vaccinations.  Biopsy to determine the nature of new lesions, biopsies 
completed as part of this study, or minor surgical procedures to excise a new 
lesion, will not be a basis for discontinuing vaccinations.  Even surgery under 
general anesthesia will be acceptable for biopsies done pre-vaccine or day 22, 
and will not be a basis for discontinuing protocol treatment.  
 
Initiation of cytotoxic chemotherapy (other than metronomic cyclophosphamide), 
radiation therapy, surgery for resection of disease, steroid therapy, or other 
immunosuppressive therapy. 
 
Any other potential adverse reaction deemed sufficiently serious to warrant 
discontinuation of therapy by the Principal Investigator or one of the Associate 
Investigators. 
 
Noncompliance with the requirements of the study. 
 
Therapy may be discontinued at the participant's request. 
 
Therapy may be discontinued at the discretion of an Investigator.   
 
Pregnancy.  Pregnant participants will continue to be followed for the duration of 
the pregnancy. 
 
Participants who discontinue treatment will be followed according to the follow-up 
schedule, unless a participant has withdrawn consent.   
7.7 
Replacement of Study Participants 
A participant who is enrolled but who does not receive study drug or any of the study 
related procedures may be replaced.  Every attempt will be made to evaluate any data 
from these participants for endpoint assessment.   
7.8 
Concomitant Medications 
Medications taken in the month prior to registration will be recorded on the baseline case 
report form.  This includes prescription medications, over-the-counter medications, 
injected medications, biological products, blood products, imported drugs, or street 
drugs.  Participants should be maintained on drugs that they were taking prior to entry 
unless a change in regimen is medically indicated. 
 
 
The following are non-permitted medications or treatments  
 
Cytotoxic chemotherapy 
 
Interferon therapy (e.g. Intron-A®) 
 
Radiation therapy 
 
Nitrosoureas 
 
Allergy desensitization injections 
 
Corticosteroids, as detailed in section 3.2.1. 
 
Growth factors (e.g. Procrit®, Aranesp®, Neulasta®) 
 
Interleukins (e.g. Proleukin®) 
 
Antibodies to PD-1 or other immune checkpoint blockade therapies (e.g. Keytruda®) 


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 
Other investigational medications 
 
Street drugs 
7.9 
Permitted Medications or Treatments 
 
Nonsteroidal anti-inflammatory agents 
 
Anti-histamines (e.g. Claritin®, Allegra®) 
 
Topical corticosteroids or steroids 
 
Short-term therapy for acute conditions not specifically related to melanoma 
 
Chronic medications except those listed in section 7.8 
 
Influenza vaccines are permitted, but should be administered at least 2 weeks prior 
to or at least 2 weeks after a study vaccine. 
7.10 
Treatment Compliance 
Treatment compliance may be evaluated through drug accountability assessments and 
through the evaluation of subject medical records and CRF documents. 
7.11 
Biopsies 
Part 1 
 
Vaccine Site Biopsies 
Each participant will undergo a biopsy of a vaccine site at two time points (days 8 and 
22; one week after vaccines 1 and 3).  The biopsies will consist of three 4-mm punch 
biopsies of skin. 
 
Sentinel Immunized Node 
A lymph node draining the 3rd vaccine site (sentinel immunized node (SIN) will be 
biopsied on day 22. 
 
Tumor Biopsy 
Optional at the time of recurrence or later as clinically indicated. 
 
Part 2 
 
Tumor Site Biopsies 
Each participant enrolled in Part 2 will undergo a tumor biopsy and/or tumor excision at 
two time points: 
 
Pre-vaccine:  Optimally, patients will have a tumor biopsy within 3 weeks 
of starting treatment.  However, tumor tissue from a prior biopsy can 
serve as the pre-treatment sample provided: 1) there is no intervening 
treatment in between the pre-study biopsy and study treatment and 2) 
formalin-fixed tumor tissue is available and adequate to provide at least 
20 unstained slides with sufficient tumor for analysis. 
 
Day 22.  The day 22 biopsy will typically include complete resection in 
accord with clinical indications for disease control. 
 
Optional biopsies may be completed at the time of progression or later as clinically 
indicated. 
 


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8.0 
CLINICAL AND LABORATORY EVALUATIONS 
The following evaluations will be performed on an outpatient basis.  Please refer to the study 
calendar Appendix 1 for scheduling. 
8.1 
Physical Exams and Evaluations 
 
Medical History 
 
Complete Physical Exam (includes vital signs, weight, performance status, 
medication review, neurologic function-general) 
 
Assessment of skin and nodal basins for evidence of disease recurrence or 
metastasis 
 
Assessment of skin for vitiligo   
 
Assessment of hair and eye color  
 
Visual acuity (Snellen chart)  (baseline only) 
 
Color vision exam (Ishihara eye chart)  (baseline only)  
 
Assessment of baseline symptoms (baseline only) 
 
Designation of vaccination sites (at screening only) 
Evidence suggests nodes proximal to a tumor site may be relatively 
immunosuppressed; therefore, the vaccination sites will be selected to be distant 
from the sites of tumor whenever possible.  In general, participants will be vaccinated 
in upper arm or thigh sites with intact draining nodes.  Vaccines will be administered 
at the designated site(s). 
8.2 
Pathology Review 
 
Review of pathology at the University of Virginia 
8.3 
Performance Status 
 
ECOG performance status criteria will be used in the evaluations (Appendix 3) 
8.4 
Clinical Labs 
 
CBC with differential, including automated lymphocyte count (0.3 ml) 
 
Comprehensive chemistry panel to include sodium, potassium, creatinine, glucose, 
calcium total bilirubin, ASL, ALT, and alkaline phosphatase.  NOTE:  fasting blood 
sugars, when required, may be evaluated 4 hours or more after last eating. (0.9 ml) 
 
Urinalysis 
 
β-HCG for women of childbearing potential (2 ml) 
 
HgB-A1C (3 ml) 
 
HIV screening (antibody screen); reflexive testing to determine whether active 
disease is present. (3 ml) 
 
Hepatitis C Virus screening (antibody screen);  reflexive testing to determine whether 
active disease is present (combined with HIV) 
 
ANA and Rf 
 
8.5 
Toxicity Assessments 
 
Assessment of adverse events.  The NCI Common Terminology Criteria for Adverse 
Events (CTCAE) version 4.03 will be used for the characterization and grading of 
adverse events. 
 
Toxicity diaries will be distributed to participants and reviewed by study personnel. 
 


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8.6 
Research Blood Samples 
Blood should be obtained prior to the vaccine injection if a vaccine is scheduled to be 
administered.  Results of research blood tests are not required prior to administering the 
vaccine on that date.   
The following blood samples for research will be collected and processed by the UVA 
Biorepository and Tissue Research Facility (BTRF). 
o 80 cc -120 cc blood collected in heparinized green top tubes for lymphocytes.  
o 20 cc blood collected in red top tubes for serum 
 
8.7 
Vaccine Site Biopsies 
 Sampling 
8.7.1
Each participant will undergo biopsy of a vaccine site at two time points (days 8 
and 22; one week after vaccines 1 and 3).  The biopsies will consist of three 4-
mm punch biopsies of skin. The biopsies on day 8 will be performed on skin at 
the site of vaccine #1.  Vaccines 2 and 3 are to be administered immediately 
adjacent to the site of vaccine #1, so that they are likely to drain to the same 
lymph node(s).  Vaccine 3 is to be administered to the same site as vaccine 
#2.  The biopsies on day 22 will be of the skin at the sites of vaccines 2 and 3. 
 Procedure 
8.7.2
Three 4-mm punch biopsies will be obtained under local anesthesia.  As each is 
removed, it will be passed off to staff of the BTRF, who will place one specimen 
in liquid nitrogen, one in formalin, and one in RNA-later. 
 Evaluations 
8.7.3
The skin of the vaccine sites may be evaluated for immune activation and cellular 
infiltrates using multiple assays, including, but not limited to: 
a) Formalin-fixed paraffin-embedded specimen:  Immunohistochemistry for 
infiltration 
by 
immune 
cells 
(CD8, 
CD4, 
dendritic 
cells), 
for 
Th1/Th2/Th17/Treg bias (T-bet, GATA3, RORγt, FoxP3). 
b) Quick frozen tissue:   Luminex or other protein assays for cytokines and 
chemokines 
c) RNA-later:  Gene expression profiling of vaccine sites for immune 
signatures. 
8.8 
Sentinel Immunized Node Biopsy 
 Procedure 
8.8.1
The node (sentinel immunized node, SIN) will be identified by radiocolloid 
(usually technetium 99 sulfur colloid) injection, with or without lymphoscintigraphy 
imaging, and with use of a handheld gamma probe during the procedure. This 
will be performed under local anesthesia in the clinic, in conjunction with the 
vaccine site biopsy, by a qualified surgeon.   
Lymphatic mapping will be initiated, usually in the nuclear medicine suite, after 
intradermal injection with radiocolloid (typically technetium 99-sulfur colloid).  The 
node excision will be performed under local anesthesia (usually lidocaine HCl 1-
2%, with or without epinephrine 1:100,000 injection, with or without 8.4% sodium 
bicarbonate), in the outpatient clinic or comparable procedure room, using sterile 
technique.  A handheld gamma probe will be used. 


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When possible, the node will be sectioned into 5 sections:  a central section (10-
20% of the node), leaving two adjacent sections of about 40% each.  These latter 
two sections will be bisected.  They will be allocated into various preservation 
conditions:1 central section will be fixed in formalin, then paraffin-embedded  
a. (for histology/immunohistology) 
b)  1 section will be placed in RNA-later. (for RNA/RT PCR) 
c)  1 section will be quick-frozen (for immunohistology/protein studies) 
d) 2 sections (40%) will be processed for single cell suspension by  
mechanical disaggregation, then enzymatic digestion (collagenase, 
hyaluronidase, 
DNAase). 
 
The 
resulting 
suspensions 
will 
be 
cryopreserved in FBS and DMSO (for cellular immune function and flow 
cytometry).   
e) If there is additional tissue, it may be processed for additional 
immunologic or angiogenic studies. 
 
The incisions will be sutured closed. 
Toxicities related to the biopsies will be recorded. 
8.9 
Tumor Biopsies 
Tumor biopsies will be completed in subjects enrolled in part 2 of the study.  Biopsy sites 
may be in nodes, skin, soft tissue, liver, or other sites that can be accessed by needle 
biopsy, incisional or excisional biopsy.  Biopsies may be completed with or without 
image guidance.   
Size Requirements 
A critical component of this protocol is the histologic and cytologic evaluation of changes 
in immune effectors and the tumor microenvironment after vaccination and systemic 
therapy.  A minimum of 0.16 cm3 but ideally 0.3 cm3 or more of tumor tissue will be 
needed for each biopsy time point as described in the inclusion criteria. Biopsies may be 
taken from a single lesion or multiple lesions at each of the time points depending on the 
size of each lesion.  If taken from multiple lesions, those lesions should be similar.  For 
example, three non-ulcerated skin metastases would be considered similar; one 
bleeding small bowel metastasis would not be considered similar to a subcutaneous 
nodule). 
Sampling 
The biopsies will vary based on the clinical scenario and may include six core biopsies, 
an incisional biopsy or an excisional biopsy. 
Procedure 
When appropriate (and we anticipate the majority of cases) the biopsies will be 
performed under local anesthesia (typically lidocaine HCl 1% and epinephrine 1:100,000 
injection + or - 8.4% sodium bicarbonate), in the outpatient clinic or comparable 
procedure room, using sterile technique.  In cases when clinical standard of care 
requires a larger procedure the biopsies may be performed in the operating room under 
standard technique. 
To minimize errors in analysis due to sampling error and specimen heterogeneity, each 
study biopsy specimen will be divided into several components and randomly allocated 
into various preservation conditions. Ideally, tissue will be divided into the following 
preservation conditions, using core needle biopsies (19 mm long and 2 mm diameter; 


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about 80 mm3), or incisional or excisional biopsies with at least the same minimum 
tissue volume: 
It is most critical to obtain the following: 
 
Formalin: 1 core biopsy or similar tissue volume (about 80 mm3 or greater) will be 
fixed in formalin, then paraffin-embedded (for histology/immunohistology) 
 
Quick-frozen: 2 core biopsies or similar tissue volume (each about 80 mm3 or 
greater) quick-frozen processed for protein studies, histology, or nucleic acid studies. 
If only one core can be obtained, this portion should be provided as two specimens 
(eg cut the core biopsy specimen in half). 
When sufficient tissue is available, the following should also be obtained: 
 
RNA-later: 1 core biopsy or similar tissue volume (about 80 mm3 or greater) will be 
placed in RNA-later (for RNA/RT PCR) 
 
Viable cell suspension:  2 core biopsies or similar tissue volume (total about 160 
mm3 or greater) will be processed for single-cell suspension by mechanical 
disaggregation, then enzymatic digestion (collagenase, hyaluronidase, DNAase).  
The resulting suspensions will be cryopreserved in FBS serum and DMSO (for 
cellular immune function and flow cytometry). 
 
If there is additional tissue, it may be processed for additional immunologic studies. 
The incisions will be sutured closed.  Toxicities related to the biopsies will be recorded.   
Based on our experience in prior clinical trials (Mel48 (NCT00705640), Mel51 
(NCT00977145 ), and Mel53 (NCT01264731)), it is adequate, for each specimen, to 
have at least the equivalent of a core biopsy specimen that is 19 mm long and 2 mm in 
diameter (about 80 mm3), or a cubic specimen 5 mm in width. Thus, for 1 FFPE and 1-2 
QF specimens, we need about 160-240 mm3 (2-3 cores, or 1 lesion about 7-10 mm in 
diameter).  For those with larger specimens (>1 cm diameter), some will also be saved 
as viably cryopreserved single cell suspensions.  
A 5 micron section of each tumor specimen will be stained by H&E and reviewed to 
assess the extent and quality of viable tumor. For FFPE specimens, only those with at 
least 4 mm2 viable tumor (on cross-section) will be considered evaluable for histologic 
and immunohistologic studies. FFPE tissue will be evaluated for immunotype and for 
immune cell infiltrates. For QF specimens, those with at least 70% tumor will be 
considered evaluable.  
If during the study, participants develop metastases or recurrences, or progress, these 
may be removed, and following receipt by pathology, may be evaluated by the study 
research team.   
Tissue samples may be screened for antigen expression or protein profiles using tests 
such as Western blots, immunohistochemistry, PCR, flow cytometry or gene chip 
analysis.  Tumor escape mechanisms may also be evaluated.  Specimens will be used 
in immunological assays to assess T cell infiltration, T cell function or antibody response.  
Assays generally used for this type of testing include, but are not limited to, 
immunohistochemistry, flow cytometric analyses, T cell receptor sequencing, ELIspot 
assays, ELISAs, chromium-release assays, proliferation assays and intracellular 
cytokine staining.  Specimens may be used to study the immunologic aspects of the 
tumor microenvironment or as targets or controls in laboratory assays.  Specimens may 
be used to establish cell lines for long-term studies. 


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This tissue may also be compared to lesions resected prior to enrollment, which will be 
requested from the pathology department of each institution as paraffin-embedded 
tissue samples, and these tissues may be banked for use in future studies.  If 
participants are removed from the study or progress during or after follow-up, tissue may 
be collected for use as part of this study, as described above, or banked for use in future 
studies. 
 
8.10 
Assessments 
 Anti-tumor Activity 
8.10.1
Anti-tumor activity will be assessed by the following: 
Tumor Imaging 
Tumor imaging may include CT/PET-CT scans and/or MRI.  These will complement 
physical exam and other imaging as required, but the primary measures of clinical 
response will be based on CT/PET-CT and/or MRI. For each participant, the same 
method of assessment will be used to evaluate tumor burden at baseline and throughout 
the course of the study. 
Tumor Measurements 
RECIST 1.1 Criteria will be used to evaluate tumor burden.  These are summarized in 
Appendix 5. 
 Immunologic Assessments  
8.10.2
Assessments of T cell function may include, but are not limited to the following:  
 
ELIspot assays:  measure of primary response 
Analysis of T cell responses by IFNγ ELIspot assay, comparing pre-
vaccine to post-vaccine.  The magnitude of T cell response will be defined 
by the ratio of responding cells at the time tested over background 
reactivity (and corrected for any pre-existing response.  A 5-fold reactivity 
over background at two or more time points by day 85 will be considered 
a positive response.  Flow cytometry may be performed to assess T cell 
responses as well, measuring multifunctional T cell responses. 
 
ELISAs 
 
Chromium-release assays  
 
Proliferation assays  
 
Intracellular cytokine staining  
 
T cell receptor sequencing. 
 
Cytokine bead array 
 
Flow cytometry and/or CY-TOF mass cytometry 
 
HLA typing 
 
Characterization of cellular populations may include, but are not limited to the following:  
 
Immunohistochemistry 
 
Gene expression analysis 
 
Flow cytometry and/or CY-TOF mass cytometry 
 
ELISAs 


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 
Western-blot analysis 
 
Intracellular cytokine staining 
 
Cytokine bead array 
 
8.11 
Study Calendar  
See Appendix 1. 
9.0 
STATISTICAL CONSIDERATIONS 
9.1 
Overview 
This is an early phase study evaluating the safety and immunogenicity of a vaccine 
comprised of a mixture of 6 synthetic melanoma helper peptides (6MHP) administered 
with one of 2 local adjuvant combinations (IFA or IFA + polyICLC), alone or with 
systemic low-dose cyclophosphamide (mCy).  The trial is designed to find the range of 
optimal treatment combinations, defined by a combination with early and durable 
immunologic response and an acceptable level of toxicity. An adaptive design will be 
used to guide accrual decisions with toxicity assessments and the potential for a durable 
immune response characterizing the primary decision measures. 
An additional study objective is to obtain preliminary data on whether treatment with 
the recommended optimal combination in patients with one or more tumor deposits 
accessible for biopsy or excision modifies the tumor microenvironment as quantified as a 
significant increase in infiltration of CD4+ or CD8+ T cells into tumor metastases.  
9.2 
Study Design 
Part 1: 
The primary objective is to determine the range of optimal combinations (one or 
more), among the set defined in Table 6, and then to expand accrual within the 
acceptable range to determine which overall treatment strategy is best. An optimal 
combination is a combination that is estimated to have an acceptable toxicity profile as 
measured by dose limiting toxicities (DLT) and a high rate of early and durable immune 
response (dRsp) as measured by CD4+ T cell response to 6MHP during the time period 
of vaccine administration.  
Table 6:  Design Definitions 
Zone 
Arm/Combination 
6MHP+ 
1 
A 
IFA 
2 
B 
IFA + mCy 
2 
C 
IFA + PolyICLC 
3 
D 
IFA + PolyICLC + mCy 
 
A DLT is defined in section 10.9. An early dRsp is defined as at least a 5-fold 
increase in immune response to the 6MHP peptide as measured by CD4+ T cells (see 
section 8.10.2) over two consecutive  time periods during vaccination (days 0 to 85). As 
data accumulates each patient will be classified as experiencing a DLT (yes/no) and 
experiencing a dRsp (yes/no). Although DLTs are not anticipated they will be used to 
guide accrual decisions and protect against the unanticipated. Treatment-related grade 3 
or higher adverse event (AE) data from our prior studies will be used to gauge DLT 
rates.  Patients on Mel 44 that received the 6MHP + IFA vaccine (arms C & D), patients 


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on Mel 55 that received AS-15 and patients on Mel 58 that received IFA + PolyICLC 
experienced grade 3 or higher treatment-related AEs in 3.5%, 0% and 16% of patients, 
respectively.  Using these results the DLT tolerance level was chosen to be 25% (i.e., 
any optimal combination that we are satisfied has an estimated DLT probability ≤25% to 
be considered “acceptable” in terms of safety). Data from the 6MHP arms of Mel 44 
resulted in potential durable immune responses in 18% (90% CI(11, 26%)) of patients 
and provides the baseline to evaluate durable immune response.   
Part 2: 
To obtain preliminary data on the effects of the optimal treatment to modify the tumor 
microenvironment as quantified as a significant increase in infiltration of CD4+ or CD8+ T 
cells into tumor metastases.  
9.3 
Accrual Allocation for Determination of the Recommended Optimal Combination 
(Part 1 only) 
Accrual to arms will occur in two stages. The initial stage will accrue eligible patients 
in cohorts of two on each arm, until a patient experiences a DLT. The second stage will 
allocate eligible patients based upon a continual reassessment method (CRM) for 
combinations of agents (78,79). The minimum follow-up period for escalation between 
Zones is 3 weeks after the initial vaccine.  
 First Stage Patient Allocation 
9.3.1
The escalation plan for the 1st-stage is based on grouping treatment 
combinations into “zones.” With this design patients can be accrued and 
assigned to other open arms within a zone but escalation will not occur outside 
the zone until the minimum follow-up period is observed for the first patient 
accrued to an arm. Initial allocation within a zone will be based upon random 
allocation (1:1) between the possible arms. Escalation to a higher zone occurs 
only when all arms in the lower zone have been tried, and no DLT has been 
observed. Patient allocation to subsequent arms within the new Zone will follow 
the same accrual strategy. This allocation strategy is followed for accrual to 
increasing zones until a patient experiences a DLT or a stopping rule is triggered 
(Section 9.6).  Once a DLT has been observed, the 2nd-Stage using CRM 
modeling begins. 
 Second Stage Patient Allocation 
9.3.2
The 2nd-stage will allocate eligible patients based upon a CRM that accounts 
for both toxicity and immune response in combinations of agents. Toxicity 
assessment is based upon the occurrence of DLT’s and immune response 
assessment is based on achievement of dRsp. The modeling stage uses (a) a 
selected set of possible orderings for both the DLT and dRsp probabilities and (b) 
a working model for both the DLT and dRsp probabilities under each ordering. 
 
 
 
Table 7: Possible orderings of DLT probabilities 
Order 
Orderings 
Working models** 


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A 
B 
C 
D 
1 
A-B-C-D 
0.01 
0.05 
0.11 
0.17 
2 
A-C-B-D 
0.01 
0.11 
0.05 
0.17 
**Working models were chosen according to the algorithm of Lee and Cheung, 2009 (80) 
 
The orderings of immune response are formulated under two different 
assumptions (1) the probabilities are increasing with increasing zone, or (2) the 
probabilities increase initially and then plateau after a certain zone. 
Table 8: Possible orderings of dRsp probabilities 
Under the assumption of increasing dRsp probabilities 
Order 
Orderings 
Working models** 
A 
B 
C 
D 
1 
A-B-C-D 
0.25 
0.40 
0.55 
0.70 
2 
A-C-B-D 
0.25 
0.55 
0.40 
0.70 
3 
A-B-D-C 
0.25 
0.40 
0.70 
0.55 
4 
A-C-D-B 
0.25 
0.70 
0.40 
0.55 
5 
A-D-B-C 
0.25 
0.55 
0.70 
0.40 
6 
A-D-C-B 
0.25 
0.70 
0.55 
0.40 
Under the assumption of plateau dRsp probabilities 
Order 
Orderings 
Working models** 
A 
B 
C 
D 
7 
A-B-C-D 
0.45 
0.55 
0.70 
0.70 
8 
A-B-C-D 
0.55 
0.70 
0.70 
0.70 
9 
A-B-C-D 
0.70 
0.70 
0.70 
0.70 
10 
A-C-B-D 
0.45 
0.70 
0.55 
0.70 
11 
A-D-B-C 
0.45 
0.70 
0.70 
0.55 
**Working models were chosen according to the algorithm of Lee and Cheung, 2009(80). 
 
The 2nd-stage allocation scheme begins once a DLT is observed regardless 
of which Zone it occurs in. Within each ordering, the CRM is fit for both toxicity 
and immune response, using the working model and the accumulated data. The 
2nd-stage will accrue eligible patients in cohorts of one and use a CRM model fit 
to estimate DLT probabilities and immune response probabilities at each arm 
combination. For each order, 𝑚= 1, 2, in Table 7, the DLT probabilities for each 
arm combination 𝑖= 1, ⋯, 4, are modeled via a one-parameter power model, 
𝑃𝑟⁡(DLT⁡at⁡combination⁡𝑖) ≈𝑝𝑚𝑖
𝜃𝑚, where the 𝑝𝑚𝑖 are the working model values for 
order 𝑚 given in Table 8. After accrual of each patient into the trial, the 
parameter 𝜃𝑚 is estimated for each ordering by maximum likelihood estimation 
where the likelihood is given by ∏
(𝑝𝑚𝑖
𝜃𝑚)
𝑦𝑖(1 −𝑝𝑚𝑖
𝜃𝑚)
𝑛𝑖−𝑦𝑖
4
𝑖=1
where 𝑦𝑖= the 
number of DLTs and 𝑛𝑖=the number of treated patients in arm combination 𝑖. The 
order with the largest likelihood is chosen and, within this ordering, DLT 
probability estimates are updated for each combination. If there is a tie between 
the likelihood values of two or more orderings, then the selected order is 
randomly chosen from among the tied orderings.  
These DLT probabilities will be used to define a set of “acceptable” 
combinations. For arm combinations B-D, a one-sided 80% confidence interval is 
calculated using the estimated DLT probability for that arm, based on confidence 
interval estimation for CRM models (81). If the lower bound of this confidence 
interval exceeds the maximum toxicity tolerance of 25%, then this arm is deemed 


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too toxic and excluded from the acceptable set of combinations. If arm A is 
excluded from the acceptable set then no arm is considered acceptable and the 
trial is stopped for safety, therefore, for arm A the level of confidence is set at 
95% instead of 80%. The toxicity tolerance of 25% was chosen based on the 
expectedness of adverse events. Note this process is performed for each new 
accrual.  For each dRsp working model, ℎ= 1, ⋯, 11, in Table 8, the dRsp 
probabilities 
are 
modeled 
via 
a 
one-parameter 
power 
model 
P r(dRsp⁡at⁡combination⁡i) ≈𝑞ℎ𝑖
exp⁡(𝛽ℎ), where the 𝑞ℎ𝑖 are the working model values 
for order ℎ given in Table 8. After accrual of each patient into the trial, the 
parameter 𝛽ℎ is estimated for each working model by maximum likelihood 
estimation where the likelihood is given by ∏
(𝑞ℎ𝑖
exp⁡(𝛽ℎ))
𝑧𝑖(1 −𝑞ℎ𝑖
exp⁡(𝛽ℎ))
𝑛𝑖−𝑧𝑖
4
𝑖=1
where 
𝑧𝑖= the number of immune responses and 𝑛𝑖=the number of treated patients in 
combination 𝑖. Again, the model with the largest likelihood is chosen and, within 
this working model, dRsp probability estimates are updated for each 
combination. If there is a tie between the likelihood values of two or more 
models, then the selected model is randomly chosen from among the tied 
models. 
 Accrual Deviations 
9.3.3
If the minimum follow-up period is not satisfied at the time a new patient is 
ready to be put on-study, then the patient may be accrued to any arm, by random 
allocation, which has accrued at least one patient and is in the set of possible 
optimal combinations.  Data from these patients will be used in the modeling 
stage as their data becomes available for DLT and dRsp determination. 
 Arm Recommendation 
9.3.4
Once the set of “acceptable” combinations is determined, the recommended 
combination will be based upon how many patients have been entered into the 
study to that point. For the first third of the trial (i.e. 1/3 the maximum sample 
size), the combination recommendation is based on randomization using a 
weighted allocation scheme. The recommended combination for the next entered 
patient is chosen at random from the “acceptable” combinations with each 
acceptable combination weighted by its estimated immune response probability. 
That is, acceptable combinations with higher estimated dRsp probabilities have a 
higher chance of being randomly chosen as the next recommended combination. 
For the latter third of the trial (i.e. final 2/3 of maximum sample size), the 
recommended combination for the next entered patient is defined as the 
“acceptable” combination with the highest estimated dRsp probability. After each 
patient, a new recommended combination is obtained, and the next entered 
patient is allocated to the recommended combination. The trial will stop once 
sufficient information about the optimal dose range has been obtained, according 
to the stopping rules outlined in Section 9.6.  
9.4 
Statistical Properties 
Simulation results were run (R-package, pocrm) to display the performance of the 
design characteristics, see Table 9. For each scenario, 1000 simulated trials were run. 
Displayed in the table in each scenario for each arm is the true DLT probability, the true 
dRsp response rate, the percentage of trials in which the arm was recommended as the 
optimal combination, and the percentage of patients treated. Displayed in the last four 
columns is the average and selected percentiles for the trial size at study closure, the 


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percentage of times in the simulations that the trial closed due to safety concerns, the 
percentage of simulated patients that had a DLT, and the percentage of simulated 
patients that had a dRsp.  The following scenarios were chosen to display the operating 
characteristics with the optimal combination(s) indicated in bold type in the table. 
Scenario 1: All true DLT probabilities are safe (i.e. less toxic than 25%) and the 
highest Zone has the combination with the highest dRsp rate.  
Scenario 2: All true DLT probabilities are safe (i.e. less toxic than 25%) and the dRsp 
probabilities begin to plateau at arm C in Zone 2.  
Scenario 3: Two combinations (C and D) have true DLT probabilities more toxic than 
25% and combination B has the highest dRsp rate among safe 
combinations. 
Scenario 4: All true DLT probabilities are safe (i.e. less toxic than 25%) and equal 
dRsp for combination B and D.  
Scenario 5: Two combinations (B and D) have true DLT probabilities more toxic than 
25% and combination C has the highest dRsp rate among safe 
combinations.  
Scenario 6: All combinations are too toxic (i.e. more toxic than 25%).  
Table 9: Design performance 
Maximum sample size set at 70, stop when the optimal arm has accrued 30 patients 
Scenario: 
True probabilities (DLT, dRsp) 
% optimal regimen recommended 
% patient allocation 
Avg size, 
percentiles 
% 
stop 
% 
DLT 
% 
dRsp 
Zone: 
1 
2 
3 
 
 
 
 
     Regimen: 
A 
B 
C 
D 
 
1: 
 
(0.02, 0.19) 
0.05 
0.15 
(0.07, 0.30) 
0.09 
0.17 
(0.05, 0.40) 
0.11 
0.18 
(0.17, 0.70) 
0.75 
0.50 
48, 
25th = 44 
50th = 46 
75th = 49 
90th = 58 
95th = 64 
0.000 
0.107 
0.501 
 
2: 
 
(0.01, 0.35) 
0.15 
0.20 
(0.03, 0.45) 
0.24 
0.25 
(0.05, 0.60) 
0.44 
0.34 
(0.10, 0.60) 
0.17 
0.21 
50, 
25th = 45 
50th = 47 
75th = 54 
90th = 63 
95th = 67 
0.002 
0.046 
0.513 
 
3: 
 
(0.14, 0.19) 
0.28 
0.31 
(0.20, 0.40) 
0.62 
0.43 
(0.44, 0.50) 
0.07 
0.20 
(0.44, 0.70) 
0.02 
0.06 
49, 
25th = 42 
50th = 48 
75th = 56 
90th = 65 
95th = 70 
0.020 
0.242 
0.373 
 
4: 
 
(0.05, 0.15) 
0.13 
0.20 
(0.05, 0.40) 
0.67 
0.46 
(0.17, 0.20) 
0.04 
0.14 
(0.17, 0.40) 
0.17 
0.20 
48, 
25th = 44 
50th = 46 
75th = 50 
90th = 59 
95th = 64 
0.003 
0.090 
0.332 
 
5: 
 
(0.05, 0.20) 
0.10 
0.21 
(0.40, 0.40) 
0.12 
0.22 
(0.20, 0.50) 
0.75 
0.47 
(0.45, 0.70) 
0.03 
0.10 
52, 
25th = 45 
50th = 50 
75th = 60 
90th = 69 
95th = 70 
0.005 
0.237 
0.438 
 
6: 
 
(0.60, 0.20) 
0.00 
0.92 
(0.70, 0.40) 
0.00 
0.04 
(0.80, 0.50) 
0.00 
0.04 
(0.90, 0.70) 
0.00 
0.01 
8, 
25th = 2 
50th = 7 
75th = 12 
90th = 17 
95th = 21 
0.997 
0.620 
0.215 
 


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9.5 
Sample Size and Accrual 
 Optimal Combination (Part 1): 
9.5.1
Target sample size for the optimal combination is based upon acquiring 
sufficient information to assess the objective of estimating dRsp rates, assuming 
at least one optimal combination has been found. Based upon results from the 
Mel44 clinical trial (NCT00118274), 30 eligible patients treated at the optimal 
combination will provide adequate data to assess dRsp. The target of thirty 
patients was chosen based on having sufficient information to determine if the 
optimal arm shows an increase dRsp rate compared to the baseline rate 
observed in the 6MHP arms of Mel 44 of 18% (90% CI(11, 26%)).  If at least 
13/30 (43% 90% CI(28, 60%)) patients on the optimal arm experience a dRsp the 
results will be considered promising since the lower limit of the confidence 
interval exceeds the upper limit from the Mel 44 estimated rate. Total study 
sample size is estimated from the simulations, but in reality is an outcome 
determined by the stopping rules in Section 9.6. We set the maximum total 
sample size to 70 eligible patients; however, as indicated in the simulation results 
the maximum average trial size over all scenarios is 52 patients. Adjusting for a 
5% drop-out/ineligibility rate, maximum total target accrual to part 1 should not 
exceed 74 patients but based upon the simulations is estimated to be 
approximately 50 patients. 
 Assessment of the tumor microenvironment (Part 2): 
9.5.2
Published data from the Mel51 clinical trial  provides preliminary estimates of 
variability and baseline change in infiltration of CD4+ or CD8+ T cells into tumor 
metastases (82).  Shown in the following table is the alternative mean change 
and effect size that could be detected with accrual of 10 to 16 eligible participants 
based upon assessment of mean change estimated from Mel51 data in 9 
participants.  The alternative was estimated assuming a one-side 10% level non-
parametric test with 90% power. Based upon these results, a target sample of 14 
eligible participants treated at the recommended combination would provide for 
determination of a moderate to large effect size, ES=0.737, a feasible and 
meaningful result. Adjusting for a 5% drop-out/ineligibility rate, maximum total 
target accrual to part 2 should not exceed 15 participants. 
Infiltrate 
measure in 
tumor 
N 
Null 
Mean 
Change 
Alternative 
Mean 
Change 
StD 
Effect 
Size 
CD4 
10 
208.0 
1291.1 
1199.0 
0.903 
 
12 
208.0 
1176.1 
1199.0 
0.807 
 
14 
208.0 
1091.7 
1199.0 
0.737 
 
16 
208.0 
1026.3 
1199.0 
0.682 
 
 
 
 
 
 
CD8 
10 
993.0 
3060.7 
2289.0 
0.903 
 
12 
993.0 
2841.3 
2289.0 
0.807 
 
14 
993.0 
2680.1 
2289.0 
0.737 
 
16 
993.0 
2555.1 
2289.0 
0.682 
 
 


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9.6 
Stopping Rules (Part 1 only) 
Accrual to the study will be halted and trigger a safety review by the study 
investigators and DSMC to determine if the study should be modified, or permanently 
closed to further accrual according to the following: 
 
Accrual will be halted for safety if the first four entered patients in Zone 1 
experience a DLT on all arms in Zone 1 in the 1st-Stage.  
 
If at any point in the 2nd-Stage, the set of acceptable combinations is empty, the 
trial will stop for safety.  
 
Otherwise, accrual to part 1 of the study will end if the recommendation is to 
assign the next patient to a combination that already has 30 patients treated at 
that combination.  
9.7 
Data Analysis Plans 
All patients who are placed on-study will be included in the final report. 
 Safety: 
9.7.1
All patients who receive any protocol treatment will be monitored for adverse 
events. Adverse events will be described and coded based upon the NCI CTCAE 
v4.03. A DLT is defined as any unexpected adverse event that is possibly, 
probably or definitely related to treatment and satisfies the criteria in Section 
10.9.  Occurrence of DLTs will guide escalation and stopping decisions.  At study 
conclusion frequency, proportion and severity of adverse events, and DLTs by 
arm will be tabulated.  
 Efficacy: 
9.7.2
All eligible patients who receive any protocol treatment will be evaluated for 
immunologic and clinical endpoints. 
a. Immunologic (For all patients unless otherwise specified) 
CD4+ T cell responses to 6MHP after 6MHP vaccination as assessed in 
the PBMC over the treatment course.  Those data will be used to define the 
optimal treatment combination in Part 1 and to describe response overall for 
both parts of the study.  Prolonged duration of immunologic response as 
measured over the follow-up period will be used to determine whether 
response persists beyond the treatment period.  Point estimates assessing 
fold increase at each time point will be described and if applicable, repeated 
measure models will be used to describe the pattern of change over time.  
Other study endpoints that will be summarized by arm for Part 1 only include 
CD4+ T cell responses to 6MHP assessed in the vaccine-draining nodes and 
CD8+ T cell responses to melanoma antigens assessed in vaccine-draining 
nodes. CD8+ T cell responses to melanoma antigens will also be assessed in 
PBMC in Parts 1 and 2. 
For Part 1, any arm that was in the range of optimal combinations, we will 
describe whether the vaccine adjuvant contained in the range of optimal 
combinations induces Th1-dominant cytokine responses in the VSME and 
SIN, and induces IgG antibodies to 6MHP.  Other preliminary data about the 
vaccine-site microenvironment (VSME) induced by the vaccines administered 
in different adjuvants will be summarized and include assessment of 


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Th1/Th2/Th17 bias of cells in the VSME, T cell retention in the VSME, and 
induction of retention integrins.   
b. Clinical: 
Patients will be assessed for disease-free survival (DFS) or progression-
free survival (PFS), as appropriate, and overall survival (OS). DFS is defined 
as the time from the date of start of treatment to the date of melanoma 
recurrence or metastasis, or date of death, whichever occurs first.  Patients 
who have neither recurred/progressed nor died will be censored on the date 
of last evaluable tumor assessment. PFS is defined as the time from the date 
of start of treatment to the date of melanoma progression or death, whichever 
occurs first.  Patients who have neither progressed nor died will be censored 
on the date of last evaluable tumor assessment. OS is defined as the time 
from the date of start of treatment to the date of death from any cause. 
Patients who do not experience an event (death) will be censored at date of 
last follow-up/contact. DFS, PFS and OS distributions will be estimated by the 
product limit method of Kaplan and Meier. 
c. Tumor microenvironment (Part 2 only): 
A one-sample sign rank test will be used to assess change (post-pre) in 
tumor infiltrate measures. Specifically, the tumor biopsy pre vaccine (timing 
as detailed in section 3.1.1) and, tumor biopsy or excision at week 3 (1 week 
after 3rd vaccine) will be used to determine whether T cells induced by 
vaccination infiltrate tumor deposits and whether CD8+ T cell infiltration 
increases with treatment.  
 Study Conclusion (Part 1 only) 
9.7.3
For Part 1, if more than one combination is contained within the range of 
optimal combinations, then immune response during the follow-up period, days 
85 through 180, may be used to define which patients have prolonged durable 
responses with the arm with the highest rate defining the better combination. 
Other secondary endpoints may be used to differentiate which combination is the 
most worthy of further study.   For Part 2, similar assessments will be performed 
as for Part 1 except that sentinel immunized node and vaccine site biopsies will 
not be available for Part 2, but assessments of the tumor microenvironment will 
be performed for Part 2, as detailed above. 
 
 
10.0 
ADVERSE EVENT DATA COLLECTION AND MONITORING 
10.1 
Definitions 
 Adverse event (AE) – Any unfavorable and unintended sign (including an 
10.1.1
abnormal laboratory finding), symptom, or disease temporally associated with the 
use of a medical treatment or procedure regardless of whether it is considered 
related to the medical treatment or procedure (attribution of unrelated, unlikely, 
possible, probable, or definite).  Medical conditions or diseases present before 
starting the investigational drug will be considered as treatment-related AEs if 
they worsen after starting study treatment. 


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 Unexpected AE – Any adverse event not listed in section 10.4.3. 
10.1.2
 Serious AE – Any adverse drug experience occurring at any dose that results in 
10.1.3
any of the following outcomes:  
 
death;  
 
a life-threatening adverse drug experience;  
 
inpatient hospitalization, or prolongation of existing hospitalization (as 
defined below in this section);  
 
a 
persistent 
or 
significant 
disability/incapacity; 
or 
a 
congenital 
anomaly/birth defect.   
 
Important medical events that may not result in death, be life threatening, 
or require hospitalization may be considered a serious adverse drug 
experience when, based upon medical judgment, they may jeopardize the 
patient or participant and may require medical or surgical intervention to 
prevent one of the outcomes listed in this definition.  
 
Hospitalization for expedited AE reporting purposes is defined as an 
inpatient hospital stay equal to or greater than 24 hours.  Hospitalization is 
used as an indicator of the seriousness of the adverse event and should 
be reserved for situations where the adverse event truly fits this definition 
and not for hospitalizations associated with less serious events.  For 
example, the following are not considered serious adverse events: 
o a hospital visit where a patient is admitted for observation or minor 
treatment (e.g. hydration) and released in less than 24 hours  
o hospitalization for pharmacokinetic sampling  
o admission to hospice  
o hospitalizations planned before entry into the clinical study 
o hospitalization for elective treatments  
o hospitalizations to work up Grade 1 adverse events 
 Unanticipated problem - An unanticipated problem is any event/experience that 
10.1.4
meets ALL 3 criteria below: 
 
Is unexpected in terms of nature, severity or frequency given the research 
procedures that are described in the protocol-related documents AND in 
the characteristics of the participant population being studied. 
 
Is related or possibly related to participation in research.  This means that 
there is a reasonable possibility that the incident may have been caused 
by the procedures involved in the research study.  
 
The incident suggests that the research placed the participant or others at 
greater risk of harm than was previously known or recognized OR results 
in actual harm to the participant or others. 
 
 Protocol Violation-  A protocol violation is defined as any change, deviation, or 
10.1.5
departure from the study design or procedures of a research project that is NOT 
approved by the institution’s IRB prior to its initiation or implementation, OR 
deviation from  standard operating procedures, Good Clinical Practices (GCPs), 
federal, state or local regulations. Protocol violations may or may not be under 
the control of the study team or UVa staff. These protocol violations may be 
major or minor violations. 
 Suspected Adverse Reaction (as defined in 21 CFR 312.32 (a))- Any adverse 
10.1.6
event for which there is a reasonable possibility that the drug caused the adverse 
event. 


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10.2 
Attribution Assessment 
 Attribution – The determination of whether an adverse event is related to a 
10.2.1
medical treatment or procedure.  The attribution groups are: 
Definite – Applies to those adverse events which, the investigator feels are 
incontrovertibly related to study drug.  An adverse event may be assigned an 
attribution of definitely related if or when (must have all of the following): 
 It follows a reasonable temporal sequence from administration of the test 
drug. 
 It could not be reasonably explained by the known characteristics of the 
participant’s clinical state, environmental or toxic factors, or other modes of 
therapy administered to the participant. 
 It disappears or decreases on cessation or reduction in dose with re-
exposure to drug.  (Note: This is not to be constructed as requiring re-
exposure of the participant; however, the group of definitely related can 
only be used when a recurrence is observed.) 
 It follows a known pattern of response to the test drug. 
 
Probable – Applies to those adverse events for which, after careful consideration 
at the time they are evaluated, are felt with a high degree of certainty to be 
related to the test drug.  An adverse event may be considered probably related if 
or when (must have three of the following): 
 It follows a reasonable temporal sequence from administration of the test 
drug. 
 It could not be reasonably explained by the known characteristics of the 
participant’s clinical state, environmental or toxic factors, or other modes of 
therapy administered to the participant. 
 It disappears or decreases on cessation or reduction in dose.  There are 
important exceptions when an adverse event does not disappear upon 
discontinuation of the drug, yet drug-relatedness clearly exists (e.g. bone 
marrow depression, fixed drug eruptions, tardive dyskinesia). 
 It follows a known pattern of response to the test drug. 
 
Possible – Applies to those adverse events for which, after careful consideration 
at the time they are evaluated, a connection with the test drug administration 
appears unlikely but cannot be ruled out with certainty.  An adverse event may 
be considered possibly related if or when (must have two of the following): 
 It follows a reasonable temporal sequence from administration of the test 
drug. 
 It could not readily have been produced by the participant’s clinical state, 
environmental or toxic factors, or other modes of therapy administered to 
the participant. 
 It follows a known pattern of response to the test drug. 
 
Unlikely – Applies to those adverse events for which, after careful consideration 
at the time they are evaluated, are judged to be unrelated to the test drug.  An 


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adverse event may be considered unlikely if or when (must have two of the 
following): 
 It does not follow a reasonable temporal sequence from administration of 
the test drug. 
 It could readily have been produced by the participant’s clinical state, 
environmental or toxic factors, or other modes of therapy administered to 
the participant. 
 It does not follow a known pattern of response to the test drug. 
 It does not reappear or worsen when the drug is re-administered. 
 
Unrelated – Applies to those adverse events, which after careful consideration, 
are clearly and incontrovertibly due to extraneous causes (disease, environment, 
etc.). 
10.3 
Data collection  
Data will be collected using a centralized electronic case report form called ON-line 
Clinical Oncology Research Environment = Oncore. 
10.4 
Risks and Safety 
 Adverse Event Descriptions and Grading Scales 
10.4.1
The NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.03 will be used for the characterization and grading of adverse events. 
 Time Span for Reporting Adverse Events 
10.4.2
Reporting of AEs will begin when the participant is administered the study drug or 
has a study related biopsy. Events occurring through 30 days after administration 
of the last dose of 6MHP or mCy, regardless of attribution, will be reported. AEs 
should be followed to resolution or stabilization.  If an AE worsens and becomes 
an SAE, it should be reported as serious per the guidelines specified for SAE 
reporting. 
AEs that are possibly, probably, or definitely related to any of the study drugs will 
be recorded until the participant completes treatment follow-up.  If, during 
treatment follow-up, the participant receives an alternative anti-cancer treatment, 
participants will be off treatment follow-up and will be followed yearly for disease 
progression and survival. 
 Agent-Specific Expected Adverse Events List: 
10.4.3
 
 
Any AE not in this list will be considered an unexpected AE. 
 
 
Table 10: Toxicities related to 6MHP in prior trials 
Toxicity (based on max grade) 
Gr 1 
Gr2 
Gr3 
Gr4 
 
 
 
 
 
LOCAL, INJECTION SITE 
 
 
 
 
Injection site reaction 
18% 
56% 
2.4% 
 
Ulceration 
 
5% 
1.4% 
 
 
 
 
 
 
CONSTITUTIONAL 
 
 
 
 
Fatigue  
43% 
8% 
3.4% 
 
Headache 
27% 
1.4% 
0.5% 
 


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Toxicity (based on max grade) 
Gr 1 
Gr2 
Gr3 
Gr4 
Rigors, Chills 
24% 
1.4% 
-- 
 
Nausea 
23% 
2% 
0.5% 
 
Sweating 
19% 
1% 
 
 
Myalgias 
18% 
0.5% 
 
 
Arthralgias 
17% 
0.5% 
 
 
Fever 
16% 
2% 
 
 
Dizziness 
13% 
 
 
 
Anorexia 
13% 
3% 
 
 
Diarrhea 
12% 
2% 
 
 
Cough 
13% 
 
 
 
Allergic rhinitis 
11% 
 
 
 
Nasal/paranasal reactions 
11% 
 
 
 
Pain larynx/throat 
10% 
 
 
 
Flushing 
10% 
 
 
 
Pruritis 
9% 
0.5% 
 
 
Rash 
6% 
3.4% 
 
 
Dyspnea 
5% 
1% 
0.5% 
 
Vomiting 
5% 
1% 
0.5% 
 
Flu-like syndrome 
6% 
 
 
 
Mucositis 
6% 
 
 
 
Constipation 
5% 
 
 
 
Autoimmune reaction 
4% 
0.5% 
 
 
Wound, non-infectious 
4% 
 
 
 
Pain, other 
2% 
1% 
0.5% 
 
Abdominal pain 
1.4% 
 
0.5% 
 
Tinnitus 
 
0.5% 
0.5% 
 
Tumor pain 
0.5% 
 
0.5% 
 
Hearing (without monitoring program) 
 
 
0.5% 
 
 
 
 
 
 
CLINICAL LABORATORY 
 
 
 
 
Hyperglycemia (not fasting) 
22% 
1% 
 
 
Hemoglobin, low 
17% 
1% 
0.5% 
 
Hyperkalemia 
13% 
 
 
 
Lymphopenia 
9% 
2.9% 
1% 
 
Leukocytes 
8% 
1% 
 
 
Hyponatremia 
7% 
 
 
 
Increased creatinine 
6% 
0.5% 
 
 
Hypoglycemia 
6% 
 
 
 
AST, SGOT 
5% 
 
 
0.5% 
ALT, SGPT 
4% 
 
1% 
 
Neutrophils 
3% 
2% 
 
 
Metabolic, Other 
3% 
0.5% 
0.5% 
 
Alk phos 
4% 
0.5% 
 
 
 
The following toxicities are those greater than grade 1 that are to be considered 
expected from the standpoint of defining DLTs.  They are selected because they 
occur in at least 4% of patients and are no greater than grade 2. 
Table 11: Expected 
Toxicities for 6MHP 
vaccines 
Grade 2 
Injection site reaction 
+ 
Ulceration 
+ 
Fatigue 
+ 
 
 
 
 
 


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Expected toxicities related to polyICLC 
The following expected toxicities for polyICLC are based on the data from 45 
patients treated with 20 mcg/kg 3x/week.  This would be about 1.4 mg per dose 
and 4.2 mg per week (much higher than we will use). 
 
 
Table 12:  Toxicity Data for PolyICLC 
Category 
Toxicity 
Grade 
Comment 
Nervous system disorders 
 
Headache 
Grade 2 
1 of 22 (<5%) 
Tremors 
Grade 3 
2 of 45 (4%) poss related 
Musculoskeletal and 
connective tissue disorders 
Muscle weakness 
Grade 2 
1 of 45 (<2%) experienced at 
Grade 3 
Respiratory, thoracic and 
mediastinal disorders 
 
Dyspnea 
Grade 2 
1 of 45 (<2%) experienced at 
Grade 3 
Hypoxia 
Grade 2 
1 of 45 (<2%) experienced at 
Grade 3 
Metabolism and nutrition 
disorders 
Hypernatremia  
Grade 2 
1 of 45 (<2%) experienced at 
Grade 3 
Investigations 
 
Elevated 
transaminases (GPT) 
Grade 3 
4 of 45 (9%) 3 cases possibly 
related; one case probably 
related.  Typically transient. 
Elevated Alkaline 
phosphatase 
Grade 3 
7% of patients, in IB 
Leukocytopenia 
Grade 3 
2 of 45 (<4%) in published 
work; 20% in IB 
Thrombocytopenia 
Grade 3 
14% of patients in IB 
Neutropenia 
Grade 3 
10% of patients in IB 
Blood/lymph disorders 
Anemia 
Grade 3 
13-31% of patients in IB 
General disorders and 
administration site conditions 
 
Vaccine site reaction 
Grade 2 
1 of 22 (<5%) 
Fever 
Grade 3 
14% of patients in RCC trial 
Chills 
Grade 3 
10% of patients in RCC trial 
Fatigue 
Grade 3 
10% of patients in RCC trial 
 
Thus, this higher dosage of polyICLC induced grade 3 toxicities in a subset of 
patients. However, in a trial of a peptide vaccine administered in an emulsion 
with polyICLC and Montanide ISA-51, there were no grade 3 toxicities.  For that 
cohort of 11 patients, the following grade 2 toxicities were reported: injection site 
reaction (27%), panniculitis (1/11 = 9%)(46).  In that trial they administered 1.4 
mg of polyICLC with each dose, every 3 weeks x 5 (total 7 mg over 12 weeks). 
We will administer 1 mg of polyICLC with each dose x 6 (total 6 mg over 11 
weeks).  Thus, we expect toxicities from polyICLC in this trial to be similar to 
those in that peptide vaccine trial.  The following toxicities are those greater than 
grade 1 that we consider expected from the standpoint of defining DLTs.   Grade 
3 toxicities are not expected.  Panniculitis is not listed because we have not 
observed it in our ongoing experience with polyICLC (data not yet summarized 
for ongoing trials). 
Table 13: Expected Toxicity for 
polyICLC 
Grade 2 
Injection site reaction 
+ 
 
 


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Expected toxicities related to mCy.   
We anticipate that a low dose of metronomic cyclophosphamide will not carry the 
same level of risk as a dose that is intended to be used as a chemotherapy 
medication.  Toxicities that have been associated with low-dose administration of 
cyclophosphamide  are summarized in section 1.7.2.  At doses and schedules 
slightly higher than those planned for the present  study, a minority of patients 
(but >4%) experienced grade 3 lymphopenia, neutropenia, and nausea.  
Because the current proposal will use somewhat lower doses, grade 3 toxicities 
may not occur. However, if they do occur, cyclophosphamide will be held until the 
resolve to grade 1 or lower, and cyclophosphamide may be resumed at that time.  
Expected 
Grade 
2 
and 
Grade3 
toxicities 
for 
low-dose 
metronomic 
cyclophosphamide are listed in Table 14. 
Table 14:  Expected toxicities related to mCy 
 
Grade 2 
Grade 3 
Fatigue 
+ 
 
Nausea 
+ 
 
Thrombocytopenia 
+ 
 
Anemia 
+ 
 
Lymphocyte count decreased 
 
+ 
White blood cell decreased 
 
+ 
Neutrophil count decreased 
 
+ 
 
 
 
Expected toxicities from vaccine site and node biopsies. 
 
 
 
Below is a list of expected AEs related to vaccine site and SIN biopsies: 
 
 
 
Vaccine Site Biopsies 
 Bleeding 
 Bruising 
 Pain 
 Very low risk of infection (less than 2%) 
 Delayed wound healing  
 Scarring 
 
 
  
SIN Biopsies 
 Bleeding 
 Bruising 
 Pain 
 Infection 
 Delayed wound healing  
 Scarring 
 Very low risk of lymphedema (less than 2%) 
 Numbness 
 
10.5 
Adverse Event Classifications  
Adverse events (AEs) are classified into sections, specified in the CTCAE v4.03.  For 
specific classifications pertaining to the protocol, we specify the following: 


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Hematologic/Metabolic- Any AE coded under one of the following CTCAE v4.03 
categories should be reported under the Hematologic/Metabolic adverse event 
classification: 
 
Table 15:   Hematologic/Metabolic Classifications 
Section 
AE 
Blood and lymphatic 
Anemia 
Leukocytosis 
Investigations 
ALL EXCEPT:  
Carbon monoxide diffusing capacity decreased 
Ejection fraction decreased 
Forced expiratory volume decreased 
Vital capacity abnormal 
Weight gain 
Weight loss 
Metabolism and nutrition 
disorders 
ALL EXCEPT: 
Alcohol intolerance 
Anorexia 
Dehydration 
Glucose intolerance 
Iron overload 
Obesity 
Tumor lysis syndrome 
 
Non-hematologic/Non-Metabolic- Any AE not reported under hematologic/metabolic, 
ocular, or allergic/autoimmune, should be reported under the non-hematologic/non-
metabolic adverse event classification. 
Ocular – Any AE coded under one of the following CTCAE v4.03 Adverse Event Terms 
should be reported under the Ocular adverse event classification: 
1) A single treatment-related experience of the following adverse events will be 
classified as a DLT: 
 
Eye Disorders: Night blindness (nyctalopia) 
 
Eye Disorders: Papilledema 
 
Eye Disordersl: Retinopathy 
 
Participants will be referred for an ophthalmologic exam if any of these ocular 
adverse events occur. 
2) A prolonged treatment-related experience (e.g., lasting > 5 days) of the 
following non-severe adverse events will be classified as a DLT: 
 Eye Disorders: Blurred vision 
 Eye Disorders: Flashing lights 
 Eye Disorders: Floaters 
 
Participants will be referred for an ophthalmologic exam if any of these ocular 
adverse events occur. 
Allergic/Autoimmune – Only AEs coded as Immune System Disorder: Allergic reaction, 
autoimmune disorder, or anaphylaxis should be reported under the Allergic/Autoimmune 


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adverse event classification. Other AEs coded under Immune System Disorder should 
be reported under Non-hematologic/Non-metabolic adverse event classification. 
10.6 
Reporting Adverse Events 
 Process for Reporting AEs: 
10.6.1
 
Dose-limiting toxicities 
DLTs will be entered into Oncore within 5 calendar days of the study team 
learning of the event. DLT’s that are deemed serious and unexpected will be 
submitted to the IRB per institutional guidelines (see below). 
 
 
 
Other AEs 
AEs must be recorded into the University of Virginia Cancer Center OnCore 
database per the following guidelines Table 16. 
 
 Table 16:  AE reporting 
  
 Pregnant-Partner Outcomes 
10.6.2
If a male has been exposed to the investigational agent prior to or around the 
time of conception, this will not be considered an SAE.  The HITC will ask 
permission of the pregnant partner to be followed until term. 
Pregnancy 
If a female has been exposed to the investigational agent prior to or around the 
time of conception, this will not be considered an SAE.  The HITC will follow the 
pregnancy until term. 
 
 High Risk Studies  
Reporting requirements for AEs that occur within 30 days of the last dose of protocol specified treatment 
 
Grade 1 
Grade 2 
Grade 3 
Grade  
4 & 5 
Expected 
and 
unexpected 
 
Expected 
 
Unexpected 
Expected 
 
Unexpected 
Expected 
and 
Unexpected 
Without 
hospitalization 
With 
hospitalization 
Without 
hospitalization 
With 
hospitalization 
Unrelated 
Unlikely 
OnCore 
30 daysa 
OnCore 
30 days 
OnCore 
30 days 
OnCore 
30 days 
OnCore 
15 days 
OnCore 
30 days 
OnCore 
15 days 
OnCore 
7 days 
Possible 
Probable 
Definite 
OnCore 
30 daysa 
OnCore 
30 days 
OnCore 
15 days 
OnCore 
30 days 
OnCore 
15 days 
OnCore 
7 days 
OnCore 
7 days 
OnCore 
(24-hrs)* 
7 days 
 
*Enter into OnCore database within 24 hours if unexpected and definitely related to protocol specified treatment 
Hospitalization defined as an inpatient hospital stay or prolongation of a hospital stay equal to or greater than 24 hours 
a Grade 1 unexpected or expected hematologic/metabolic events will be recorded in the Cancer Center Database; however, 
regardless of attribution, these events do not have to be reported. 
 
 


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 IRB Reporting Requirements 
10.6.3
The University of Virginia is responsible for reporting to the UVA IRB-HSR per 
the following guidelines: 
 
Table 17:  UVA IRB-HSR reporting 
Type of Event 
To whom will it 
be reported: 
Time Frame for 
Reporting 
How reported? 
Any internal event resulting 
in death that is deemed 
DEFINITELY related to 
(caused by) study 
participation 
(Note:  An internal event is 
one that occurs in a subject 
enrolled in a UVa protocol.) 
IRB-HSR 
Within 24 hours 
IRB Online and phone call 
 
www.irb.virginia.edu/ 
 
Internal, Serious, 
Unexpected adverse event. 
 
IRB-HSR 
Within 7 calendar 
days from the time 
the study team 
received knowledge 
of the event. 
 
Timeline includes 
submission of signed 
hardcopy of AE form. 
IRB Online 
 
www.irb.virginia.edu/ 
 
Unanticipated Problems 
that are not adverse events 
or protocol violations  
This would include a Data 
Breach.   
IRB-HSR 
 
 
Within 7 calendar 
days from the time 
the study team 
received knowledge 
of the event.  
Unanticipated Problem report 
form.  
 
http://www.virginia.edu/vprgs/irb/
HSR_docs/Forms/Reporting_Re
quirements-
Unanticipated_Problems.doc ) 
 
  
Protocol Violations (The 
IRB-HSR only requires that 
MAJOR violation be 
reported, unless otherwise 
required by your sponsor, if 
applicable.) 
 
Or  
 
Enrollment Exceptions 
IRB-HSR 
 
 
Within 7 calendar 
days from the time 
the study team 
received knowledge 
of the event.  
 
Protocol Violation and Enrollment 
Exception Reporting Form 
 
 
http://www.virginia.edu/vprgs/irb/
hsr_forms.html 
 
 
Data Breach  
The UVa 
Corporate 
Compliance and 
Privacy Office 
and 
 
ITC:  if breach 
involves  
electronic data-  
 
 
UVa Police if 
breach includes 
such things as 
stolen 
computers.  
As soon as possible 
and no later than 24 
hours from the time 
the incident is 
identified. 
 
As soon as possible 
and no later than 24 
hours from the time 
the incident is 
identified. 
 
 
IMMEDIATELY.  
 
 
UVa Corporate Compliance and 
Privacy Office- Phone 924-9741 
 
 
 
 
ITC:  Information Security 
Incident Reporting procedure,  
http://www.itc.virginia.edu/securit
y/reporting.html 
 
 
Phone- (434) 924-7166 
 


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 Additional Reporting Requirements for the Sponsor (UVA) 
10.6.4
Reporting to the FDA 
 Serious and unexpected suspected adverse reactions will be reported to the 
FDA no later than 15 calendar days after the sponsor determines that the 
requirements for an IND safety report have been met. The FDA will be 
notified using an FDA Form 3500a. 
 Unexpected fatal or life-threatening suspected adverse reactions will be 
reported to the FDA no later than 7 calendar days after the Sponsor receives 
the initial information of the event. The FDA will be notified using an FDA 
Form 3500a. 
 Other adverse event information will be sent to the FDA in the IND annual 
report. 
 
Reporting to the Ludwig Institute for Cancer Research (LICR)-pertains to arms 
receiving polyICLC  
 
All Serious Adverse Events (SAEs), for which a relationship with polyICLC 
cannot be ruled out. These SAEs should be reported within 15 calendar 
days of their onset (7 calendar days for life-threatening or fatal SAEs). 
 
 Reporting of Participant Withdrawals/Dropouts Prior to Study Completion 
10.6.5
Participants who withdraw consent and those dropping out of the study 
secondary to an AE will be reported to the UVA IRB yearly on the IRB 
continuation form.   
10.7 
Adverse Event Review and Monitoring 
 Capturing Adverse Events 
10.7.1
In addition to clinic notes, adverse events will be initially captured using study-
specific tools and participant toxicity diaries.  
Each participant will be evaluated by a licensed clinician.  The following will be 
performed as designated in the protocol: routine disease-directed physical exam 
including performance status and blood collection for clinical labs.   
Participants should keep a daily diary of toxicities until the next protocol clinic 
visit.  The diaries will be reviewed by a research clinician prior to the next 
scheduled infusion or vaccine, if one is scheduled.  During clinic visits, 
participants will also be asked about subjective symptoms including headache, 
malaise, fatigue, dyspnea, nausea, rash, diarrhea, abdominal discomfort, 
peripheral nerve pain, visual changes, appetite, tremors, night sweats, and ability 
to concentrate.  Additional toxicities will be captured from laboratory tests.  For 
each AE (with the exception of Grade 1 hematologic/metabolic events), date of 
onset, duration, grade, and attribution will be noted in the participant’s study 
chart, on study documents, or in the clinic note, and will be entered into the UVA 
Cancer Center database.   


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After administration of each 6MHP vaccine, participants will be observed for AEs 
for at least 20 minutes. Vital signs will be collected at the end of the observation 
period.  Follow-up phone calls will be made per the judgment of the research 
clinicians with regard to individual participant need.  Participants will be instructed 
on how to reach their provider should they have any questions and/or problems 
during the study. 
 
In the event of an AE, appropriate action will be taken to ensure adequate care 
for the participant.  If the participant is still on protocol, treatment delay or 
withdrawal from the protocol will be considered according to the protocol 
guidelines. 
 Review of Adverse Events by the Study Team 
10.7.2
Individual AEs will be reviewed by the treating physician, principal investigator, 
and the clinical research coordinator(s) (CRC).  Other staff on the research team 
may also review AEs. 
 
SAEs will be reviewed about once per month by the PI and Sponsor during the 
UVA Melanoma Team Meeting.  This meeting will occur at least 20 times in a 
calendar year.  Those present at the meeting may include the sponsor/overall 
study PI, sub-investigators, protocol development staff, biostatisticians, research 
nurses, research coordinators, laboratory specialists, and laboratory research 
managers.  These meetings also include the review of individual participants to 
assess whether they are protocol candidates, whether AEs warrant 
discontinuation, and whether existing protocols should be continued or closed. 
10.8 
Recording Laboratory Values 
The following laboratory values will be recorded in the UVA Cancer Center database, 
graded using the CTCAE v4.03 (if a grading category exists), and reported as described 
in section 10.6: 
1. Alk Phosphatase 
2. ALT (SGPT) 
3. ANA 
4. AST (SGOT) 
5. Bilirubin, total  
6. Creatinine 
7. Eosinophil #  
8. Hepatitis C serology or virus measures 
9. beta-HCG 
10. Hgb 
11. HIV 
12. HLA type 
13. Potassium 
14. RF 
15. Urinalysis 
16. WBC 
 


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Any abnormal laboratory values captured which are not included in the above list, 
but are considered to be pertinent positive clinical signs/symptoms, and 
laboratory results obtained as part of routine care of patients will be recorded in 
the UVA Cancer Center database and reported as described in Section 10.6.  If 
there is any doubt on the part of study personnel concerning what constitutes a 
pertinent positive finding, the PI and sponsor will be consulted. 
10.9 
Dose-limiting Toxicities 
The study will be monitored continuously for treatment-related adverse events. 
A DLT is defined as any unexpected adverse event that is possibly, probably or 
definitely related to treatment and meets the following criteria: 
 
≥ Grade 3, with the exception of grade 3 injection site reaction with ulceration ≤ 2 
cm 
 
≥ Grade 1 ocular adverse events as defined below 
 
≥ Grade 2 allergic/autoimmune reactions as defined below 
 
Ocular – Any AE coded under one of the following CTCAE v4.03 Adverse Event Terms 
should be reported under the Ocular adverse event classification: 
1) A single treatment-related experience of the following adverse events will be 
classified as a DLT: 
 
Eye Disorders: Night blindness (nyctalopia) 
 
Eye Disorders: Papilledema 
 
Eye Disordersl: Retinopathy 
 
Participants will be referred for an ophthalmologic exam if any of these ocular 
adverse events occur. 
2) A prolonged treatment-related experience (e.g., lasting > 5 days) of the following 
non-severe adverse events will be classified as a DLT: 
 
Eye Disorders: Blurred vision 
 
Eye Disorders: Flashing lights 
 
Eye Disorders: Floaters 
 
Participants will be referred for an ophthalmologic exam if any of these ocular 
adverse events occur. 
Allergic/Autoimmune – Only AEs coded as Immune System Disorder: Allergic reaction, 
autoimmune 
disorder, 
or 
anaphylaxis 
should 
be 
reported 
under 
the 
Allergic/Autoimmune adverse event classification.  Other AEs coded under Immune 
System Disorder should be reported under  
Non-hematologic/Non-metabolic 
adverse event classification. 
10.10 Management of Toxicity 
The study will be monitored continuously for treatment-related adverse events.  
Expected treatment-related toxicities of 6MHP combined with IFA and/or polyICLC, with 


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or without mCy, will be managed in accord with section 7.5.1, which allows for dose 
delays, but not dose reductions.  
10.11 Data Collection 
 
Endpoint Data 
10.11.1
 
Endpoint data will be collected using HITC IML data forms, participant-
specific binders, and the HITC laboratory database. 
 
The HITC laboratory database, which has password-restricted access, is 
stored on the UVA Health System Computing Services secured server. 
10.12 Monitoring Plan 
 
The University of Virginia Cancer Center Data and Safety Monitoring 
10.12.1
Committee (CC DSMC) will provide oversight of the conduct of this study.  The 
CC DSMC will report to the UVA Protocol Review Committee (PRC). 
 
The UVA CC DSMC will review the following: 
10.12.2
 
All adverse events 
 
Audit results 
 
Application of study designed stopping/decision rules 
 
Whether the study accrual pattern warrants continuation/action 
 
Protocol violations 
 
 
The UVA CC DSMC will meet every month for aggregate review of data.  
10.12.3
Tracking reports of the meetings are available to the PI for review.  Issues of 
immediate concern by the DSMC are brought to the attention of the sponsor 
(and if appropriate to the PRC and IRB) and a formal response from the 
sponsor is requested.  Per the UVA Cancer Center NIH approved institutional 
plan, this study will be audited approximately every 6 months.  The audit may 
include direct access to source data/documents. 
11.0 
STUDY CONDUCT AND ETHICAL CONSIDERATIONS 
This study will be conducted in accordance with ICH Good Clinical Practice (GCP) Guidelines 
and in accord with the ethical principles that originated in the Declaration of Helsinki. In addition, 
all local laws and regulations will apply.  The PI will ensure that staff are trained and carry out the 
study in accord with the protocol specifications. 
11.1 
UVA Institutional Review Board for Health Sciences Research  
The UVA Institutional Review Board for Health Sciences Research (UVA IRB-HSR) will 
approve all aspects of this study, including the clinical trial protocol, informed consent 
documents, and patient materials.  Modifications to the protocol or consent form will be 
reviewed and approved by the UVA IRB-HSR prior to implementation, except when 
necessary to eliminate apparent immediate hazards to the study participants. The study 
will undergo continuing IRB review based on the level of risk as assessed by the IRB.  
This review will take place no less than annually. Reporting to the UVA IRB-HSR will 
occur as specified in Section 9.6. 


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11.2 
Consent Forms and the Consenting Process 
Consent forms will be written in accord with 21 CFR 50 and will be reviewed and 
approved by the UVA IRB-HSR prior to use. Participants will be given a consent form to 
review and a member of the study team will be available to answer any questions.   
Informed consent will be obtained from each participant prior to conducting any study-
specific procedures or administering study drug. 
11.3 
Maintenance of Study Documents 
Signed consent forms and other research records will be retained in a confidential 
manner. Study records will be kept for at least 6 years after completion of the study.  


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12.0 
APPENDICES 
Appendix 1:  Study Calendar 
Appendix 2:  AJCC Staging System 
Appendix 3:  ECOG Performance Status 
Appendix 4:  New York Heart Association Disease Classification 
Appendix 5:  RECIST 1.1 Criteria 
Appendix 6:  Vaccine Lot Release and Stability Testing 
Appendix 7:  Summary of Changes 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix 1:  Study Calendar 
Studies & Tests 
Pre 
Active Treatment  
Follow-up 
Day 
-6 
1 
8 
15 
22 
36 
50h 
57 
78 
85 
127 
183 
225 
Week 
-1 
0 
1 
2 
3 
5 
7h 
8 
11 
12 
18 
26 
32 
Informed consent  
Xa 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pathology review  
Xa 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential  
Xb,e 
Xf 
X 
 
X 
X 
X 
X 
X 
 
X 
 
 
 
Comprehensive chemistry  
Xb 
Xf 
X 
 
X 
X 
X 
X 
X 
 
X 
 
 
 
HGBA1C  
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis  
Xb 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
-HCG 
Xc 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV / Hepatitis C  
Xd 
 
 
 
 
 
 
 
 
 
 
 
 
 
CT chest/abdomen/pelvis or PET-CT  
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
CXR, or other imaging as indicated. 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
Head MRI / CT  
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
History & physical  
Xb 
Xf 
X 
 X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
Medication review 
Xb 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
Toxicity assessment (or baseline) 
 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
Designation of potential vaccination sites 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment of skin and nodal basins for evidence of disease 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment of skin for vitiligo 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
X 
X 
Assessment of hair and eye color 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
X 
X 
Visual acuity exam/ color vision 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
120cc green top tubes   
 
Xg 
 
 
 
 
 
 
 
 
 
 
 
 
80cc green top tubes 
 
  
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
20cc red top tubes  
 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
Anti-nuclear antibody / Rf factor 
 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
Vaccination 
 
 
X 
X 
X 
 
X 
 
X 
X 
 
 
 
 
Skin biopsy at vaccine site 
 
 
 
Xi 
 
Xi 
 
 
 
 
 
 
 
 
Biopsy of sentinel immunized node 
 
 
 
 
 
Xi 
 
 
 
 
 
 
 
 
Tumor Biopsyk 
 
Xj,l 
 
 
 
Xj 
 
 
 
 
 
 
 
 
Participant diary reviewed and/or distributed 
 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
 
Oral cyclophosphamide 50 mg daily x 7 days (Arms B and D only) 
 
X 
 
X 
 
X 
X 
X 
 
 
 
 
 
 


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a Any point prior to registration 
b Pre-study within 6 weeks of registration 
c Within 2 weeks of registration (for childbearing women) 
d Within 6 months of registration 
eTo include fasting glucose 
f History & physical, comprehensive chemistry, and CBC with differential scheduled for Day -6 are not required if  prestudy assessments were within 10 calendar days of 
day -6. 
gBlood for HLA typing is included in the research bloods. 
hThe day 50 study visit will only be required for participants on arms B & D. 
i Part 1 only. 
j Part 2 only. 
kOptional biopsies may occur at the time of recurrence or later as clinically indicated for subjects enrolled in either Part 1 or Part 2 of the study. 
lOptimally, biopsies  will occur within 3 weeks of starting study treatment; however, archival tissue may be used in the pre-study analyses. 
 
 
 
 
 
 
 
 
 


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Appendix 2:  AJCC Staging System 
  
Melanoma TNM Classification 
T Classification 
Thickness 
Ulceration Status 
T1 
≤ 1.0 mm 
a:  without ulceration or mitoses 
b:  with ulceration or mitoses  1 
T2 
1.01 – 2.0 mm 
a:  without ulceration 
b:  with ulceration 
T3 
2.01 – 4.0 mm 
a:  without ulceration 
b:  with ulceration 
T4 
> 4.0 mm 
a:  without ulceration 
b:  with ulceration 
 
 
 
N Classification 
# of Metastatic Nodes 
Nodal Metastatic Mass 
N1 
1 node 
a:  micrometastasis* 
b:  macrometastasis† 
N2 
2 – 3  nodes 
a:  micrometastasis* 
b:  macrometastasis† 
c: in transit met(s)/satellite(s) 
without metastatic nodes 
N3 
4 or more metastatic nodes, 
or matted nodes, or  
in transit met(s)/satellites(s) 
with metastatic node(s) 
 
 
 
 
M Classification 
Site 
Serum Lactate Dehydrogenase 
M1a 
Distant skin, subcutaneous 
or nodal mets 
Normal 
M1b 
Lung metastases 
Normal 
M1c 
All other visceral metastases 
Normal 
Any distant metastatsis 
Elevated 
* 
Micrometastases are diagnosed after sentinel or elective lymphadenectomy. 
† 
Macrometastases are defined as clinically detectable nodal metastases confirmed by therapeutic 
lymphadenectomy or when nodal metastasis exhibits gross extracapsular extension. 
 
 
 
 


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Stage Groupings for Cutaneous Melanoma 
 
Clinical Staging 
Pathologic Staging 
 
T 
N 
M 
T 
N 
M 
0 
Tis 
N0 
M0 
Tis 
N0 
M0 
IA 
T1a 
N0 
M0 
T1a 
N0 
M0 
IB 
T1b 
T2a 
N0 
N0 
M0 
M0 
T1b 
T2a 
N0 
N0 
M0 
M0 
IIA 
T2b 
T3a 
N0 
N0 
M0 
M0 
T2b 
T3a 
N0 
N0 
M0 
M0 
IIB 
T3b 
T4a 
N0 
N0 
M0 
M0 
T3b 
T4a 
N0 
N0 
M0 
M0 
IIC 
T4b 
N0 
M0 
T4b 
N0 
M0 
III‡ 
Any T 
N1-3 
M0 
 
IIIA 
 
T1-4a 
T1-4a 
N1a 
N2a 
M0 
M0 
IIIB 
 
T1-4b 
T1-4b 
T1-4a 
T1-4a 
T1-4a/b 
N1a 
N2a 
N1b 
N2b 
N2c 
M0 
M0 
M0 
M0 
M0 
IIIC 
 
T1-4b 
T1-4b 
Any T 
N1b 
N2b 
N3 
M0 
M0 
M0 
IV 
Any T 
Any N 
Any M1 
Any T 
Any N 
Any M1 
* 
Clinical staging includes microstaging of the primary melanoma and clinical/radiologic evaluation for 
metastases.  By convention, it should be used after complete excision of the primary melanoma with clinical 
assessment for regional and distant metastases. 
† 
Pathologic staging includes microstaging of the primary melanoma and pathologic information about the 
regional lymph nodes after partial or complete lymphadenectomy.  Pathology stage 0 or stage 1A patients are 
the exception; they do not require pathologic evaluation of their lymph nodes. 
‡ 
There are no stage III subgroups for clinical staging. 
 
 
Staging for Mucosal Melanomas 
This system is based on the staging of cutaneous melanomas. 
 
Stage IIB: Clinically localized primary melanoma > 4mm thick 
Stage III: Lymph node metastases 
Stage IV:   Distant metastases 
 
 
 
 
 
 
 
 
 


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Appendix 3:  ECOG Performance Status 
 
 
ECOG PERFORMANCE STATUS* 
Grade ECOG 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light 
or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all selfcare but unable to carry out any work activities. Up and 
about more than 50% of waking hours 
3 
Capable of only limited selfcare, confined to bed or chair more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any selfcare. Totally confined to bed or chair 
5 
Dead 
* As published in Am. J. Clin. Oncol.: 
Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., McFadden, E.T., Carbone, P.P.: 
Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol 5:649-
655, 1982. 
 
 


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Appendix 4:  New York Heart Association Disease Classification 
 
Functional Capacity 
Objective Assessment 
Class I.  Patients with cardiac disease but 
without resulting limitation of physical activity. 
Ordinary physical activity does not cause undue 
fatigue, palpitation, dyspnea, or anginal pain. 
No objective evidence of cardiovascular 
disease. 
Class II.  Patients with cardiac disease resulting 
in slight limitation of physical activity. They are 
comfortable at rest. Ordinary physical activity 
results in fatigue, palpitation, dyspnea, or 
anginal pain. 
Objective evidence of minimal cardiovascular 
disease 
Class III.  Patients with cardiac disease 
resulting in marked limitation of physical activity. 
They are comfortable at rest. Less than ordinary 
activity causes fatigue, palpitation, dyspnea, or 
anginal pain. 
Objective evidence of moderately severe 
cardiovascular disease. 
Class IV.  Patients with cardiac disease 
resulting in inability to carry on any physical 
activity without discomfort. Symptoms of heart 
failure or the anginal syndrome may be present 
even at rest. If any physical activity is 
undertaken, discomfort is increased. 
Objective evidence of severe cardiovascular 
disease. 
* The Criteria Committee of the New York Heart Association. Nomenclature and Criteria for Diagnosis of Diseases of the 
Heart and Great Vessels. 9th ed. Boston, Mass: Little, Brown & Co; 1994:253-256 
 
 
 


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Appendix 5:  RECIST 1.1 Criteria  
 
Please refer to the following publication for evaluation of clinical response by RECIST 1.1. 
 
E.A. Eisenhauer et al.  New response evaluation criteria in solid tumours: Revised RECIST guideline 
(version 1.1).  European Journal of Cancer, 2009, 45: 228-247. 
 
PMID:  19097774 
 
 


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Appendix 6:  Vaccine Lot Release and Stability Testing 
A. Preparation of the synthetic melanoma and tetanus peptides 
All peptides were synthesized under GMP conditions by Multiple Peptide Systems (San Diego, CA).   
 
Peptide preparation and vialing were performed under GMP conditions by Clinalfa (Merck 
Biosciences AG, Laufelfingen, Switzerland).  Documentation relating to the procedures used to 
prepare and vial the peptides were included in the Chemistry and Manufacturing Section of prior 
IND application applications (10825 and 12191).   
 
B. Quality Assurance Testing 
Prepared peptides were subjected to the following tests: 
1. Identity.  Identity was confirmed by structural studies.  The individual peptides were tested for 
identity by mass spectrometry (to define molecular mass and amino acid sequence) and HPLC 
(to confirm purity) in a GMP laboratory (Polypeptide Group).  
2. Purity.  Purity was assessed before and after vialing the peptide mixtures.  Before vialing the 
peptide mixtures, each synthetic peptide was evaluated for the presence of a single dominant 
species by high pressure liquid chromatography (HPLC) in a GMP laboratory (Polypeptide 
Group).  Purity of each peptide component exceeded 90%. Variants of the original peptide may 
have included incomplete products of synthesis, minor degradation products due to oxidation of 
methionine residues, and dimerization of cysteine-containing peptides.  After vialing the peptide 
mixture, purity was reconfirmed by HPLC in a GMP laboratory (Clinalfa). 
3. Trifluoroacetic acid (TFA).  The amount of total fluorine in each peptide preparation was less 
than 0.5% or 5000 ppm as determined by Multiple Peptide Systems. 
4. Potency.  Peptides are synthesized under GMP conditions and the net peptide content 
calculated for each.  The amounts of each peptide (mcg quantities) added to the vaccine vials 
are calculated based on the net peptide content of the original stock of lyophilized peptides. 
5. Pyrogenicity.  Pyrogenicity testing was conducted by Clinalfa in accordance with USP 
guidelines. 
6. General Safety.  General safety testing was conducted by Clinalfa in accordance with USP 
guidelines. 
7. Sterility.  Sterility testing was conducted by Clinalfa in accordance with USP guidelines. 
8. Stability.  The peptide preparations were assayed for stability at months 3, 6, 12, 24, and 36 and 
were shown to be stable. The peptides will continue to be assessed yearly for stability while 
subjects are on active treatment.  The following analyses will be performed to confirm stability. 
a. HPLC:  HPLC will be performed to confirm purity.  An optical comparison to previous HPLC 
data will be performed.  Ideally, the purity of each peptide component will exceed 90% 
(94%-98%).  Variants of the original peptide may include incomplete products of synthesis, 
minor degradation products due to oxidation of methionine residues, and dimerization of 
cysteine-containing peptides.  Such minor variants will be tolerated as long as the peptide 
represents at least 75% of the intended peptide species.  Because measures of peptide 
quantity are subject to variability, a peptide lot will be rejected only if two sequential 
measures fail to meet the criterion stated above 


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b. Sterility.  One vial of peptide will be submitted to the Clinical Microbiology Laboratory at the 
University of Virginia or Microbiology Research Associates, Inc. (Acton, MA) for sterility 
testing.   
 
 


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Appendix 7:  Summary of Changes 
12-04-14 
1) Table of Contents:  Updated 
2) Section 1.3.2:  editorial correction 
3) Section 1.8.2:  editorial correction 
4) Section 6.2:  The language regarding the supply of mCy has been reworded to 
eliminate the requirement to receive mCy by prescription. 
5) Section 9.5:  editorial correction 
6) Section 10.4.3:  Separated out the risks and side effects for the vaccine site biopsy 
and the SIN biopsy.  Added numbness as a possible side effects for the SIN 
biopsy. 
 
 
02-18-15 
1) Updated IRB # in header and removed PRC# 
2) Section 1.1:  changed “three” to “two” in reference to the number of local adjuvants. 
3) Added investigator’s statement. 
4) Formatting adjusted throughout study document 
5) Table of Contents-updated 
6) Section 4.6:  reference to Appendix 6 replaces “vaccine manual” 
7) Section 4.8:  “mixing sheets” replace “manual” 
8) Section 7.2.1:  “mixing sheets” replaces “manual” 
9) Section 7.2.4:  “vaccine mixing sheets” replace “laboratory manual” 
10) Section 10.6.4:  AE reporting requirements for reporting to the LICR were added. 
11) Appendix 6:  Lot release and stability testing of the vaccine were added. 
12) Summary of Changes is now Appendix 7. 
03-17-15 
1) Updated investigator list 
2) Updated investigator’s statement 
3) Formatting and editing changes made throughout the study document 
4) Updated Table of contents 
5) Section 7.2.1:  Additional text added to clarify the contents of the vaccine. 
6) Section 7.3.1:  Added text to clarify that cyclophosphamide may be taken before or 
after a vaccine, on days when both vaccine and cyclophosphamide are 
administered. 
7) Section 8.4:  Added reflexive testing for HCV and HIV screening, if needed to be 
consistent with screening criteria.  Also added ANA and Rf testing in the text to be 
consistent with the X-page (Appendix 1) 
8) Section 10.4.3 and Table 14:  expected toxicities for cyclophosphamide were 
updated. 
9) Section 10.10:  removed text related to Cy dose reductions. Dose reductions in Cy 
will not be permitted per protocol. 
10) Appendix 1:  added urinalysis testing at days 22 and 57. 
11) Appendix 1:  added CBC and comp chem at days 15, 36, and 50. 
04-17-15 
1) Section 6.2 and Section 7.3.1:  added capsules as an option for cyclophosphamide 
2) Section 7.2.2: Volume of vaccine to be administered has been changed from 1 ml 
to 2 ml. 
3) Editorial changes made throughout document. 
4) Updated investigator’s list. 
5) Updated Table of Contents 
07-16-15 
1) Updated investigator list: removed Geoffrey Weiss, Christopher Blackwell, and 
Connor Poland. 
2) Updated Table of Contents. 
3) Section 3.1.1: Clarified in inclusion criteria that subjects should be clinically free of 
disease, as originally intended and stated in the protocol synopsis indication.  
Included the statement indicating that patients with small radiologic or clinical 
findings of an indeterminate nature may be eligible.   
4) Section 3.2.12: Removed exclusion criteria regarding other cancer diagnosis.  . 
5) Section 3.3: Removed statement indicating randomization would be discussed “no 
sooner than 7 days prior to the start of treatment” and indicated that subjects 
should receive study treatment within 3 weeks of registration.  


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6) Section 10.4: Clarified risk of infection from vaccine site biopsy is less than 2%.  
7) Appendix 1: Updated the study calendar to indicate day 50 visit would only be 
Yes.required for subjects on arms B and D. 
10-26-16 
1) Updated investigator list 
2) Updated table of contents 
3) Updated Investigator’s Agreement 
4) Protocol Synopsis— 
 
Indication:  Added details to describe Part 2 of the study including the addition 
of a new patient population-Stage IIIB-IV melanoma with one or more tumor 
deposits accessible for biopsy and/or excision. 
 
Objectives and Endpoints:  Added objective 3 to address Part 2 of the study. 
 
Regimen:  Specified the study regimen for Part 2.  The study regimen includes 
the 6MHP vaccine with the optimal adjuvant combination identified from Part 1 
of the study. 
 
Schema:  Added Figure 1B (Part 2). 
 
Biopsies:  Clarified that vaccine site biopsies and SIN biopsies will be 
performed in  Part 1 of the study and that tumor biopsies in Part 1 are optional 
at the time of recurrence or later as clinically indicated.  Added a section to 
describe the tumor biopsies that are required in Part 2.  Additional, optional 
tumor biopsies may also be completed in Part 2 at the time of progression or 
later as clinically indicated. 
 
Population:  Added a summary of the patient population that will be accrued to 
Part 2. 
 
Increased the approximate accrual goal from 50 to 65 patients. 
5) Section 1.9:  Added text to the summary section to clarify the purpose of the 
addition of Part 2 of the study.  Added text to specify that immune responses will 
be evaluated at the tumor site. 
6) Section 2.1:  Revised to include a study objective for Part 2. 
7) Section 3.1.1:  Revised to include  inclusion criteria to describe the patient 
population that will be accrued to Part 2 of the study. 
8) Section 3.1.4:  Revised to specify that the most recent surgical resections or 
gamma-knife therapy for malignant melanoma must have been completed ≥ 1 
week and for Part 1 ≤ 6 months prior to registration. 
9) Section 3.1.5:  removed “all participants must have”  
10) Section 3.1.10:  Revised entry criteria to specify that participants in part 1 must 
have at least 2 intact (undissected) axillary and/or inguinal lymph node basins and 
that participants in part 2 need to have at least 1 intact (undissected) axillary and/or 
inguinal lymph node basin. 
11) Section 3.2.10:  corrected to specify that participants with uncontrolled diabetes are 
defined as having a HgbA1c > 7.5% 
12) Section 3.3:  Revised to distinguish the treatment allocation procedures for Parts 1 
and 2.  Moved text related to the timing of the first study treatment (within 3 weeks 
of registration) to the first paragraph of this section. 
13) Table 5:  Added a treatment window for the pre-study biopsies.  
14) Section 7.6:  Revised protocol to clarify the conditions whereby biopsies are not a 
basis for discontinuing therapy. 
15) Section 7.11:  Revised to clarify that vaccine site biopsies and SIN biopsies will be 
performed in  Part 1 of the study and that tumor biopsies in Part 1 are optional at 
the time of recurrence or later as clinically indicated.  Added a section to describe 
the tumor biopsies that are required in Part 2.  Additional, optional tumor biopsies 
may also be completed in Part 2 at the time of progression or later as clinically 
indicated. 
16) Section 8.9:  Added this section to describe the size requirements, sampling, and 
procedures for the tumor biopsies. 
17) Sections 9.1 and 9.2:  Revised the statistical overview and study design sections to 
provide a description of the study objective for Part 2. 
18) Section 9.3:  Revised to specify that the accrual allocation is used for the 
determination of the recommended optimal combination in Part 1 only.  


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19) Section 9.5.1:  Revised to specify that the optimal combination in this section is for 
Part 1. 
20) Section 9.5.2:  Added this section to describe the sample size calculation for Part 
2. 
21) Section 9.6:  Revised to specify that the stopping rules in this section pertain to 
Part 1 only. 
22) Section 9.7.2: Revised to distinguish and describe the efficacy endpoints for Parts1 
and 2. 
23) Section 9.7.3:  Revised to include study conclusions for Part 2. 
24) Appendix 1:  Study Calendar-revised to include tumor biopsies.  Revised to specify 
that vaccine site biopsies and SIN biopsies are only required for Part 1.  
25) References:  Reference #82 was added.. 
 
03-13-17 
1) Section 10.9:  corrected the DLT definition to specify that grade 3 injection site 
reactions with ulceration ≤ 2 cm are not DLTs.   
2) Updated TOC 
 
 
 
 


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Reference List 
 
 
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