Clinical trial NCT02385669
Conditions: Melanoma
Keywords: vaccine peptide ipilimumab Yervoy Montanide ISA-51
Sponsor: Craig L Slingluff, Jr
Start date: 2015-04-01; Completion date: 2019-09-18
Enrollment: 3 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This study evaluates whether it is safe to administer a peptide vaccine with ipilimumab. This study will also evaluate the effects of the combination of the peptide vaccine and ipilimumab on the immune system. The investigators will monitor these effects by performing tests in the laboratory on participants' blood, a lymph node, and tumor samples.

Detailed description:

Arms: arm 1: The vaccine drug product, 6MHP, consists of 6 class II MHC-restricted peptides derived from melanoma proteins. Each vaccine consists of 200 mcg of each of the six peptides. An aqueous solution of vaccine is mixed 1/1 with Montanide ISA-51 to form water-in-oil emulsions. Vaccines are administered days 1, 8, 15, 43, 64, and 85. All peptide vaccines are administered intradermally and subcutaneously.

Ipilimumab will be administered in accord with the official prescribing information: 3 mg/kg intravenously once every 3 weeks, for 4 doses. Ipilimumab will be administered on days 1, 22, 43, and 64.
Interventions: intervention 1: Ipilimumab intervention 2: 6MHP
Intervention descriptions: intervention 1: Checkpoint blockade inhibitor intervention 2: 6 melanoma helper vaccine comprised of 6 class II MHC-restricted helper peptides

Protocol documents:
Prot_SAP_000.pdf: 
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1 
 
 
 
 
 
 
 
 
 
 
 
 UNIVERSITY OF VIRGINIA 
HUMAN IMMUNE THERAPY CENTER 
 
 
MEL 62 
 
 
A PHASE I/II TRIAL TO EVALUATE THE SAFETY AND 
IMMUNOGENICITY OF A HELPER PEPTIDE VACCINE PLUS CTLA-4 
BLOCKADE IN MELANOMA PATIENTS 
 
 
Abbreviated title:  6PAC (6 helper Peptides plus Anti-CTLA-4) 
 
 
 
 
 
 
 
 
 
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.   


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Signature Page 
 
 
 
Sponsor/Sponsor’s Representative 
 
 
 
 
______________________   _______________________   _______________ 
Name              Signature            Date 
 
 
 
 
 
 
Investigator 
 
 
 
 
_______________________  _________________________  ________________ 
Name             Signature            Date 
 
 
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.  


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UNIVERSITY OF VIRGINIA 
HUMAN IMMUNE THERAPY CENTER 
MEL 62 
A PHASE I/II TRIAL TO EVALUATE THE SAFETY AND IMMUNOGENICITY OF A 
HELPER PEPTIDE VACCINE PLUS CTLA-4 BLOCKADE IN MELANOMA PATIENTS 
 
Sponsor 
University of Virginia Human Immune Therapy Center 
Principal Investigator 
Director, Human Immune 
Therapy Center 
Elizabeth Gaughan, MD 
University of Virginia 
434-924-7678 
emg5x@virginia.edu 
Co-Investigators 
Craig L. Slingluff, Jr. 
434-924-1730 
cls8h@virginia.edu 
 
William Grosh, MD 
434-924-1904 
wwg9u@virginia.edu 
 
Lynn Dengel, MD 
ltd5b@virginia.edu 
 
 
Varinder Kaur, MD 
vk4q@virginia.edu 
 
Carmel Nail, FNP-C 
434-982-4042 
cjn2r@virginia.edu 
 
Kathleen Haden, ANP-C 
434-924-1730 
km3s@virginia.edu 
 
Kimberly Bullock, PhD 
434-924-0180 
kb9d@virginia.edu 
 
Jennifer Eccles, MPAS, PA-C 
434-243-0066 
jme5j@virginia.edu 
Biostatisticians: 
Gina R. Petroni, PhD 
434-924-8363 
grp4c@virginia.edu 
Mark Smolkin, MS 
434-982-1032 
mes6r@virginia.edu 
Clinical Research 
Coordinators and Data 
Management 
 
Emily Allred, PhD 
434-982-1902 
eh4m@virginia.edu 
 
Jessica Zareno, MS 
University of Virginia 
434-982-1901 
jhz4f@virginia.edu 
 
Sarah Lewis, BA 
University of Virginia 
434-924-8709 
set5v@virginia.edu   
Alex Carney, MPH 
434-982-6714 
alc6a@virginia.edu 
 
Annika Shuali, MS 
University of Virginia 
434-243-7242 
ah2ze@virginia.edu 
 
  
Authors: 
 
Craig L. Slingluff, Jr., MD        Gina R. Petroni, PhD 
Kimberly Bullock, PhD 
 
 


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Table of Contents 
Protocol Synopsis .............................................................................................................. 7 
1.0 BACKGROUND AND SCIENTIFIC RATIONALE .........................................................13 
 Introduction ..................................................................................................................... 13 
 Study Rationale ................................................................................................................ 13 
 6MHP Vaccine .................................................................................................................. 14 
 Montanide ISA-51 ............................................................................................................. 15 
 6MHP Administered in Montanide ISA-51 ......................................................................... 16 
 Checkpoint blockade with antibody to CTLA-4 ................................................................... 21 
 Dosing .............................................................................................................................. 24 
 Summary .......................................................................................................................... 24 
2.0 OBJECTIVES ...............................................................................................................25 
 Primary objectives and endpoints ..................................................................................... 25 
 Secondary objectives and endpoints ................................................................................. 25 
 Exploratory objectives and endpoints ................................................................................ 25 
3.0 PARTICIPANT SELECTION CRITERIA .......................................................................25 
 Inclusion Criteria ............................................................................................................... 25 
 Exclusion Criteria .............................................................................................................. 27 
 Registration and Randomization ....................................................................................... 29 
4.0 STUDY DRUG (6MHP and MONTANIDE ISA-51) ........................................................30 
 Peptide Synthesis ............................................................................................................. 30 
 Storage of Individual Peptides ........................................................................................... 30 
 Reconstitution and Vialing of the Vaccine .......................................................................... 30 
 Vaccine Storage ................................................................................................................ 30 
 Lot Testing ........................................................................................................................ 30 
 Stability testing................................................................................................................. 30 
 Labeling ............................................................................................................................ 30 
 Montanide ISA-51 ............................................................................................................. 31 
 Study Drug Accountability ................................................................................................. 31 
5.0 IPILIMUMAB .................................................................................................................31 
 Packaging and Labeling ..................................................................................................... 31 
 Storage ............................................................................................................................. 31 
6.0 TREATMENT PLAN .....................................................................................................31 


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 Management of Participants ............................................................................................. 31 
 Administration of 6MHP ................................................................................................... 31 
 Administration of Ipilimumab ........................................................................................... 32 
 Dose Modifications ........................................................................................................... 32 
 Dose Delays ...................................................................................................................... 33 
 Discontinuation of Therapy ............................................................................................... 34 
 Replacement of Study Participants .................................................................................... 35 
 Concomitant Medications ................................................................................................. 35 
 Permitted Medications or Treatments ............................................................................... 36 
 Biopsies .......................................................................................................................... 36 
7.0 CLINICAL AND LABORATORY EVALUATIONS .........................................................36 
 Physical Exams and Evaluations ........................................................................................ 37 
 Pathology Review ............................................................................................................. 37 
 Performance Status .......................................................................................................... 37 
 Clinical Labs ...................................................................................................................... 37 
 Toxicity Assessments ........................................................................................................ 37 
 Research Blood Samples ................................................................................................... 38 
 Tumor Biopsies ................................................................................................................. 38 
 Sentinel Immunized Node Biopsy ...................................................................................... 40 
 Assessments ..................................................................................................................... 40 
 Study Calendar ............................................................................................................... 41 
8.0 STATISTICAL CONSIDERATIONS ..............................................................................41 
 Design .............................................................................................................................. 41 
 Evaluation of Sample Populations and Criteria .................................................................. 41 
 Sample Size and Accrual .................................................................................................... 43 
 Cohort and post entry classification .................................................................................. 43 
 Safety Monitoring ............................................................................................................. 43 
 Analyses ........................................................................................................................... 44 
9.0 ADVERSE EVENT DATA COLLECTION AND MONITORING .....................................45 
 Definitions ........................................................................................................................ 45 
 Attribution Assessment .................................................................................................... 46 
 Data collection ................................................................................................................. 48 
 Risks and Safety ................................................................................................................ 48 


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 Adverse Event Classifications ............................................................................................ 49 
 Reporting Adverse Events ................................................................................................. 50 
 Adverse Event Review and Monitoring .............................................................................. 52 
 Recording Laboratory Values............................................................................................. 53 
 Dose-limiting Toxicities (DLT) ............................................................................................ 54 
 Management of Toxicity ................................................................................................. 54 
 Data Collection ............................................................................................................... 54 
 Monitoring Plan .............................................................................................................. 54 
10.0 STUDY CONDUCT AND ETHICAL CONSIDERATIONS ...........................................55 
 UVA Institutional Review Board for Health Sciences Research .......................................... 55 
 Consent Forms and the Consenting Process ..................................................................... 55 
 Maintenance of Study Documents ................................................................................... 55 
11.0 APPENDICES .............................................................................................................56 
Appendix 1:  Study Calendar ............................................................................................57 
Appendix 2: AJCC Staging System .................................................................................59 
Appendix 3:  ECOG Performance Status .........................................................................61 
Appendix 4:  New York Heart Association Disease Classification ................................62 
Appendix 5:  RECIST 1.1 Criteria .....................................................................................63 
Appendix 6:  NCI Common Terminology Criteria for Adverse Events v4 ......................64 
Appendix 7:  Vaccine Lot Release and Stability Testing ................................................65 
Appendix 8:  Summary of Changes .................................................................................67 
Reference List ...................................................................................................................70 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Protocol Synopsis 
 
Title 
A Phase I/II trial to evaluate the safety and immunogenicity of a helper peptide vaccine plus 
CTLA-4 blockade in melanoma patients (Mel62; 6PAC)   
 
Investigational Drugs 
 6MHP:  a mixture of 6 synthetic melanoma-derived class II MHC-restricted helper 
peptides 
 Montanide ISA-51 
 
# An alanine residue was added to the N-terminus to prevent cyclization 
 
FDA-approved agent: Ipilmumab 3 mg/kg IV every 3 weeks for four doses 
 
Objectives and endpoints 
Primary: 
Safety: To determine whether the combination of 6MHP vaccine plus ipilimumab is safe 
as evaluated by adverse event assessments, including CTCAE 4.03 and subclassified 
by irAE categories. 
Immunogenicity: To estimate in the blood and in the sentinel immunized node the CD4+ 
T cell response rate to 6MHP peptides in patients treated with 6MHP vaccine plus 
ipilimumab. 
Secondary: 
Induction of epitope-spreading: To estimate the proportion of patients in whom the 
combination of 6MHP vaccine plus ipilimumab induces epitope-spreading for CD8+ T 
cells in the blood and in the sentinel immunized node that are reactive to a panel of 
defined melanoma antigens. 
 
T cell infiltration of the tumor microenvironment (for cohorts 1 and 2):  To determine 
whether the combination of 6MHP vaccine plus ipilimumab increases infiltration of CD8+ 
and/or CD4+FoxP3neg T cells into melanoma metastases in at least 50% of patients with 
tumor evaluable pre- and post-treatment. 
Exploratory: 
Induction of antibody responses:  To estimate the production of serum IgG reactive to 
6MHP in patients treated with 6MHP vaccines plus ipilimumab. 
Table 1.  Peptides used in the 6 Melanoma Helper Peptide (6MHP) vaccine 
Sequence 
Epitope (source protein, 
residues) 
Reference 
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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Clinical outcome (for cohorts 2 and 3): To obtain preliminary data on risk of recurrence 
and time to recurrence after resection of tumor to no evidence of disease and 
administration of the combination of 6MHP vaccine plus ipilimumab. 
 
Associations of immune response and clinical outcome.  To obtain preliminary estimates 
of the associations between T cell and antibody responses to melanoma antigens and 
survival and with disease control.  
 
Clinical outcome (for cohort 1 only):  To obtain preliminary estimates of clinical 
outcomes, including overall survival, progression-free survival, and clinical response by 
RECIST 1.1, following administration of the combination of 6MHP vaccine plus 
ipilimumab.  
 
 
Design 
This is an open-label, phase I/II study.   
 
Treatment Cohorts: 
   
Cohort 1: Unresectable stage III/IV advanced melanoma: 
 
Cohort 2: Neoadjuvant therapy for patients with any of the following clinical presentations, 
when disease is amenable to complete surgical resection to render the patient clinically 
free of disease: 
a. Primary melanoma with clinically evident lymph node involvement 
b. Primary melanoma with clinically evident in transit disease, with or without 
clinically evident lymph node involvement. 
c. In transit recurrent disease amenable to complete excision. 
d. Soft tissue or visceral metastatic lesion, amenable to complete surgical excision 
to render patient free of evident disease 
Cohort 3: Adjuvant therapy, to start within 12 weeks of surgery, for: 
a. Stage IIA melanoma after complete excision and with class 2 DecisionDx 
b. Stage IIB-IV, NED 
 
Treatment Regimen: 
All patients will receive 1) the 6MHP vaccine administered in Montanide ISA-51 adjuvant 
(incomplete Freud’s adjuvant) and 2) ipilimumab, an anti-CTLA-4 blocking antibody.  
Ipilimumab will be administered at 3 mg/kg IV infusion on days 1, 22, 43, and 64. 
Vaccines will be administered days 1, 8, 15, 43, 64 and 85. On days when both 
ipilimumab and vaccine are administered, the vaccine may be administered before or after 
ipilimumab, but preferably before. All peptide vaccines will be administered intradermally 
and subcutaneously. Blood and biopsy samples of a vaccine-draining node and tumor will 
be analyzed for immunologic changes. Details are provided in the protocol schema 
(Figure 1, Figure 2, Figure 3) and in the study calendar (x-page; Appendix 1). 
 
 
 
 


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Figure 1: Protocol Schema Cohort 1 
 
Figure 2: Protocol Schema Cohort 2 
 
 
 
Figure 3: Protocol Schema Cohort 3 
 
 
 


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Biopsies 
Tumor 
For participants with metastases safely accessible for biopsy, biopsies will be obtained at 
baseline and day 22. Such biopsies must be obtained from at least 14 patients in this 
study. Additional optional biopsies may be performed throughout the study or during follow-
up (e.g. at the time of disease progression).   
 
Sentinel Immunized Node 
A lymph node draining the 3rd vaccine site (sentinel immunized node (SIN)) will be biopsied 
on day 22. 
 
Population:  The main criteria for inclusion include: 
A. Age 18 or older 
B. Participants with 
a. Cohort 1 (Advanced Patients): Unresectable stage III or IV melanoma 
with clinical or radiographic evidence of disease.   
b. Cohort 2  (Neoadjuvant therapy): primary melanoma with clinically 
apparent lymph node or in transit/satellite lesions with or without lymph 
node involvement or recurrence amenable to complete resection to no 
evidence of disease 
c. Cohort 3 (Adjuvant therapy): Stage IIA with class 2 DecisionDx Score, or 
Stage IIB-IV melanoma resected to no evidence of disease. These 
patients may have had cutaneous, uveal, mucosal or unknown primary 
melanoma.  
C. Patients may have had cutaneous, uveal, mucosal or unknown primary 
melanoma 
D. Participants must be eligible to be treated with ipilimumab based on clinician 
judgement 
E. ECOG performance status of 0 or 1 
 
Accrual Goal: 
To evaluate study endpoints target accrual is estimated at 24 eligible participants, of 
whom at least 14 must have biopsiable disease. Based upon prior studies, a 10% 
dropout/ineligibility/lost to follow-up among study participants is possible; therefore 
maximum accrual to the study is set at 27 participants. The annual accrual rate is 
projected to be approximately 16 participants; thus, accrual to the study should be 
completed in less than two years.  
 
 
 


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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 
AJCC 
American Joint Committee on Cancer 
ALT 
alanine aminotransferase 
ANA 
Anti-nuclear antibodies 
ANC 
absolute neutrophil count 
APC 
antigen presenting cell 
AST 
aspartate aminotransferase 
BRAFi 
BRAF inhibitor 
CC 
Cancer Center 
cc 
cubic centimeter 
CFR 
Code of Federal Regulations 
cm 
centimeter 
CR 
complete response 
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 
DCR 
disease control rate 
dL 
deciliter 
DLT 
dose limiting toxicity 
DSMC 
Data and Safety Monitoring Committee 
ECOG 
Eastern Cooperative Oncology Group 
ELISA 
enzyme linked immunosorbent assay 
FACS 
fluorescence activated cell sorter 
FBS 
fetal bovine serum 
FDA 
Food and Drug Administration 
g 
Gram 
GM-CSF 
granulocyte-macrophage stimulating colony 
GMP 
good manufacturing practice 
Hgb 
hemoglobin 
HGBA1C 
hemoglobin a1c 
HCV 
hepatitis C virus 
HITC 
Human Immune Therapy Center 
HIV 
human immunodeficiency virus 
HLA 
human leukocyte antigen 
HPLC 
high performance liquid chromatography 
id 
intradermal 
IFN 
interferon 
IL-2 
interleukin-2 
IML 
Immune Monitoring Laboratory 
IND 
investigational new drug 
ip 
intraperitoneal 
IRB 
Institutional Review Board 
IU 
international unit 
IV 
intravenous 
kg 
kilogram 
LDH 
lactate dehydrogenase 
m 
meter 
mcg 
microgram 


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mcl 
microliter 
MDP 
melanocyte differentiation proteins 
mg 
milligram 
MHC 
major histocompatability complex 
ml 
milliliter 
mm 
millimeter 
MRI 
magnetic resonance imaging 
NCI 
National Cancer Institute 
NED 
no evidence of disease 
NSAID 
non-steroidal anti-inflammatory drug 
PBL 
peripheral blood lymphocytes 
PD-1 
Programmed-death 1 
PET 
positron emission tomography 
PI 
principal investigator 
PR 
partial response 
PRC 
protocol review committee 
Rf 
Rheumatoid factor 
SD 
stable disease 
SQ 
subcutaneous 
SIN 
sentinel immunized node 
Th 
CD4+ helper T cells  
TIL 
tumor infiltrating lymphocytes 
TPF 
Tissue Procurement Facility 
ULN 
upper limits of normal 
USP 
United States Pharmacopeia 
UVA 
University of Virginia 
WBC 
white blood cell 
 


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1.0 BACKGROUND AND SCIENTIFIC RATIONALE 
 
 Introduction 
Cancer immunotherapy for solid tumors is coming of age, with FDA-approved 
immuno-therapeutics in prostate cancer, melanoma, non-small cell lung cancer and 
renal cell cancer.  Interleukin-2 (IL-2), the CTLA-4 blocking antibody (ipilimumab), 
and PD-1 blocking antibodies (pembrolizumab and nivolumab) are approved for 
melanoma; all induce durable clinical regressions. In 2015, the combination of 
ipilimumab and nivolumab therapy for advanced melanoma was approved based 
on high response rates and improvement in progression free survival over 
ipilimumab alone (8).  Other antibodies that block PD-1/PD-L1 also induce durable 
clinical regressions of melanoma, renal cell cancer, and non-small cell lung cancer 
(NSCLC) (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.  However, despite 
the effectiveness of available immunotherapeutic approaches, these therapies still 
fail in about 70-80% of patients. Thus, 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 3 years, a cancer vaccine 
has been approved for prostate cancer, based on two randomized trials showing 
improved survival (11).  Also, a randomized prospective trial in melanoma met its 
primary endpoint, showing 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), and which 
has clinical activity in patients with advanced melanoma (13). Goals of the current 
protocol are to obtain preliminary data on whether the combination of the 6MHP 
vaccine with immune checkpoint blockade is safe and improves immunologic and 
clinical outcomes compared to prior experience with each single agent alone. The 
proposed 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 peptide and immune checkpoint 
blockade combination therapies. The proposed study holds promise: 
 To extend the life of patients with advanced melanoma,  
 To reduce the risk of recurrence of resected melanoma, and   
 To obtain preliminary data on whether checkpoint blockade increases 
helper T cell responses to vaccine and clinical response rate 
 Study Rationale 
Evidence for the Role of the Immune System in Protecting Against the 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.  
Convincing evidence of the importance of immune surveillance in preventing the 
development of solid tumors is provided by work in murine models. In knockout 


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mice lacking STAT1 and/or IFN receptor function,  50-100% developed 
spontaneous solid tumors (benign and malignant) of various histologies within 12-
15 months, whereas normal mice never developed malignancies during the same 
time period (14).  These studies strongly support the role of cellular immune 
function in the control of cancer progression. 
 
The Role of CD4+ Helper T Lymphocytes in Anti-tumor Immune Responses.   
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 (15).  Furthermore, adoptive therapy with 
CD4+ T-cells has been shown to induce tumor protection in some model systems 
(16,17).  Thus, the protective immunity induced by tumor cell vaccines appears to 
be mediated both by CD8+ T-cells and by CD4+ T-cells.   
Activated Th cells express CD40L and can activate dendritic cells (DC) through 
ligation of CD40, for heightened antigen presentation, causing the DC to secrete IL-
12 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 (17-19).  In addition, Th responses are 
believed to be involved in the establishment of memory responses.  
 
 6MHP Vaccine 
Selection of Class II MHC-restricted Epitopes for Melanoma-reactive Th Cells 
In the current protocol we are including class II MHC-restricted peptides derived 
from shared 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 (20); 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 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 (6,7,21).    
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 


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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    
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 developed to evaluate functional antigen-specific responses by 
permitting direct counting of T-cells reacting to antigen by production of IFN or  
other cytokines (22-24).  T-cells that are not anergized should secrete IFN after 
exposure to their cognate antigen, especially if they have a memory phenotype (25).  
ELIspot assays can reproducibly detect functional T cell responses to defined 
antigens at levels below 0.01% (22,26), 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+ 
from 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 (27)or by flow cytometry, with 
calculation of the Th1/Th2/Th17 balance. 
 
  Montanide ISA-51 
Montanide ISA-51 as a Vaccine Adjuvant 
Montanide ISA-51 adjuvant has been effective at inducing immune responses 
against murine viral antigens when administered with a synthetic peptide epitope 
(28,29) 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 been 
safely administered to more than 600 participants.  Immunological responses 
against the immunizing peptides have been detected in most participants (13,30-
34). 
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 (35,36). However, this does not appear to be the case with 
longer peptides (35). We believe, thus, that these 14-23 mer peptides in 6MHP are 


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not likely susceptible to retention at the vaccine site; so that IFA is a good adjuvant 
to use.   
 
 6MHP Administered in Montanide ISA-51 
Immunologic and Clinical Results from Studies in Advanced Melanoma:  Mel41 and 
E1602 
In two prior advanced melanoma studies, one sponsored by the UVA-HITC 
(Mel41) and one sponsored by the Eastern Cooperative Oncology Group (E1602), 
the 6MHP vaccine was administered with the adjuvant Montanide ISA-51 plus GM-
CSF (13,30).  In the Mel41 trial, 81% of patients developed T cell responses to 
6MHP (57% as detected in the blood; 78% in a vaccine-draining node) (13), and in 
the E1602 trial, helper T cell responses to 6MHP were detected in 40% of patients 
in the blood (30).   
This vaccination strategy was 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 (13). 
 
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% 
 
Incorporation of GM-CSF with Peptide-Based Vaccines:  Results From Prior 
Clinical Studies, Mel43 and Mel44. 
In a separate peptide-based clinical study for patients with stage IIB-IV resected 
melanoma (Mel43), we demonstrated that GM-CSF did not increase 
immunogenicity of a peptide-based vaccine compared to IFA alone. Instead, we 
found that IFA alone was a better adjuvant than IFA+GM-CSF for induction of 
CD4+ T cell responses to an intermediate length helper peptide (33). Though GM-
CSF continues to be used as a vaccine adjuvant in other settings, studies by us 
and others (37) 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 (33,37).  
Thus, as a follow-up to the Mel43 study, we evaluated in another trial (Mel44), the 
immunogenicity of the 6MHP vaccine in Montanide ISA-51, without GM-CSF (32).  
Mel44 included patients with no clinical evidence of disease (resected stage IIB-
IV), and immune responses to 6MHP in the blood were detected in 48% of 
patients by direct ELISpot assay (32).       
 
Co-administration of the 6MHP vaccine with a peptide vaccine that stimulates 
CD8+ T cells:  Results from Mel44 and E1602 
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) (31) 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 


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helper peptide (MELITAC 12.1), with IFA alone as the adjuvant, in patients with 
resected stage IIB-IV melanoma. 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) (32). Responses to the tetanus helper peptide were 
detected in 91% of patients vaccinated with this peptide, and the 6MHP induced 
TH cell responses in 52% of vaccinated participants (32). 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).  Early 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. 
 
The E1602 trial was a 4-arm, randomized phase II study for patients with 
advanced measurable melanoma.  Study participants randomized to arms A, B, 
C, and D were vaccinated with 12MP, 12MP+tetanus peptide, 12MP+6MHP, or 
6MHP alone, respectively, and with IFA+GM-CSF as a vaccine adjuvant (30). 
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; (30)).  The 
association of survival and 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 here; (30)). 


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Figure 3.  E1602 trial. Immune responses to 12MP, 6MHP, and 
tetanus peptide.   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.  
 
 
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. (39), 
and (B) Overall survival by helper T cell response to 6MHP in analyzable patients on arms C and D of E1602 
trial (n = 64). 
 
Summary of preliminary immunogenicity data with 6-MHP vaccines 
A consistent finding from these 3 trials is that 6MHP vaccines are immunogenic, 
with 40-50% immune response rate, when measured in peripheral blood, and 
81% when measured in vaccine-draining nodes. Also, in the two trials that 
enrolled patients with measureable disease, there were durable clinical responses 
(13), 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%) (38) and durable clinical responses.  Importantly, in the E1602 trial, 
there was a strong and specific association between immune response to the 
6MHP and survival, supporting the clinical relevance of immune responses 
induced to the 6MHP vaccine.  Thus, the current proposal will test the 6MHP 


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vaccine approach in combination therapy of melanoma based on immunogenicity, 
safety, and association between immune response and survival. 
 
Safety of Peptide-based Vaccines 
Peptide-based vaccines have been administered safely to humans in many 
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 (40). In previous studies conducted by the UVA-
HITC and by the Eastern Cooperative Oncology Group (ECOG), vaccines 
containing 6MHP have been administered to about 200 patients and have been 
well-tolerated (13,30,32).  In fact, injection site reactions have been lower with 
6MHP than with Class I MHC associated peptides (41). 
The peptides used in peptide-based vaccines are identical or similar to a portion 
of a normal protein.  Thus, risks of autoimmunity in humans are important to 
evaluate. There is no murine system that adequately models the human immune 
response to these peptides. The most meaningful evaluation of this peptide 
vaccine mixture is in patients with melanoma. This trial will include participants 
with advanced melanoma (stage IIIB/C, or IV melanoma with measurable 
disease).  These individuals face a high risk (> 70-90%) 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 (13,30).    
The potential implications of autoimmunity against cells of melanocytic lineage 
are illustrated by reported cases of vitiligo occurring coincident with regressions of 
melanoma (42). 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 
patients (13,30,32).  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). 
 
Toxicities Previously Reported for Participants Receiving the 6-MHP Vaccine 
Administered in Montanide ISA-51 adjuvant, with or without GM-CSF. 
In Mel41, Mel44, and E1602 trials combined, vaccines containing 6MHP have 
been administered to about 207 patients and have been well-tolerated (13,30,32).  
In fact, injection site reactions have been lower with 6MHP than with Class I MHC 
associated peptides (41).  Toxicities were graded using the NCI Common 
Terminology Criteria for Adverse Events v3.0.  Treatment-related adverse events 


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experienced by at least 4% of participants and treatment-related toxicities with 
grade 3 or higher severity in any patient are listed in Table 3. 
 
Table 3.  Reported toxicities for 6MHP vaccines in humans (n=207) 
Toxicity (based on max grade) 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
 
 
 
 
 
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% 
ALT, SGPT 
4% 
 
1% 
 
Neutrophils 
3% 
2% 
 
 
Metabolic, Other 
3% 
0.5% 
0.5% 
 


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Alk phos 
4% 
0.5% 
 
 
Bilirubin 
3% 
 
0.5% 
 
 
 
Treatment related adverse events of a given grade are considered expected 
toxicities if they have been observed in 4% of patients at that grade or higher.  
These are listed in Table 4 for 6MHP.   
 
Table 4.  Expected toxicities for 6MHP vaccines  
Toxicity 
Grade 2 
Grade 3 
Injection site reaction 
+ 
+a 
Ulceration 
+ 
 
Fatigue 
+ 
 
a Note: Injection site reaction with ulceration <2 cm is expected.  This is 
consistent with the CTCAE v4.0 coding for ulceration (Grade 2).  Other grade 3 
injection site reactions are not expected 
 
 
 Checkpoint blockade with antibody to CTLA-4 
Checkpoint blockade  
Cancer immunotherapy for solid tumors is coming of age, especially with 
checkpoint blockade providing durable clinical benefit in about 30-50% of patients. 
The anti-CTLA-4 blocking antibody, ipilimumab, is approved for use in melanoma 
and has been shown to induce durable clinical regressions and to prolong survival 
in patients with stage IV disease (38,43).  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) (9,44).    Despite the effectiveness of 
checkpoint blockade therapies, about 50-70% of patients go on to develop 
progressive disease following treatment. Thus, there is a need for new combination 
approaches that build on the demonstrated clinical value of immune therapy.   
Ipilimumab at 3 mg/kg is approved by the FDA for the treatment of patients with 
advanced melanoma (Stage IV and unresectable stage IIIB/C).  A pooled analysis 
of the survival data for ipilimumab in the advanced setting demonstrated a 22% 
three year survival rate for all patients and a plateau in the survival curve identified 
around three years after treatment (45).  This agent is now widely used in 
combination with nivolumab for the management of advanced melanoma and as 
single agent therapy in the salvage setting.  
In 2015, based on the results of the EORTC 18072, high dose ipilimumab (10 
mg/kg) was FDA-approved in the adjuvant setting.  Patients eligible for this trial 
included those with stage IIIA melanoma with at least one metastasis measuring >1 
mm in greatest dimension in the involved node, and stage IIIB/IIIC were included if 
there were no in-transit metastases (46).  The treatment was administered every 
three weeks for four doses followed by every three months for up to three years.  
Survival analysis from this trial revealed an 11% improvement in overall survival 
with the use of ipilimumab versus placebo (47).  Fifty-four percent (54%) of patients 
treated with ipilimumab experienced a grade 3 or 4 adverse event including 41.6% 
with an immune-related adverse event.  There were also five deaths in the 
ipilimumab group attributed to immune-related toxicity.  
In the metastatic setting, Ipilimumab at 10 mg/kg has a higher response rate than 
3 mg/kg (11.1% vs 4.2%, p = 0.0015), but whether this benefit extends to the 
adjuvant setting is not known (48).  Preliminary results of a phase III study of 


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advanced melanoma patients randomized to 10 mg/kg versus 3 mg/kg ipilimumab 
were presented at the 2016 ESMO conference, demonstrating a statistically-
significant improvement in 3 year overall survival with the 10 mg/kg dose versus the 
lower dose (49).  Association of higher rates of toxicity with the 10 mg/kg dose of 
ipilimumab has been consistently identified across studies. At this time, a trial 
randomizing patients to Ipilimumab 3 mg/kg, Ipilimumab 10 mg/kg, and standard 
Interferon (ECOG 1609) in the adjuvant setting has completed accrual but results 
are not expected until 2018.  The potential of serious and life-threateining toxicity of 
ipilimumab 10 mg/kg administered in the adjuvant setting has limited enthusiasm for 
this treatment.  At this time, the adjuvant therapy options for most melanoma 
patients remains compromised by toxicity and low efficacy, and many patients opt 
for surveillance.   
In 2010, Hodi et al reported the results of a phase III trial combining ipilimumab 
with the gp100 vaccine which incorporates HLA-A*0201-restricted peptides 
designed to stimulate CD8+ cells (38).  There were three treatment groups including 
ipilimumab alone, ipilimumab with gp100 vaccine and gp100 vaccine alone.  The 
overall survival for the ipilimumab alone arm was 10.0 months versus 10.1 months 
for the ipilimumab with gp100 versus 6.4 months for the gp100 vaccine alone.  
There was no difference in overall survival between the two ipilimumab arms and 
there was no major differences in toxicity profile with the addition of the vaccine. 
Despite some promise for vaccines incorporating short peptides designed to 
stimulate CD8  T cells, overall clinical impact of these vaccines has been 
disappointing (12,31,50).  In contrast, we have found that vaccination with a mixture 
of longer peptides (14-23 amino acids) designed to induce helper T cell responses 
(6MHP) has induced durable clinical activity in a subset of patients  (30,51) and very 
encouraging overall survival for stage IV melanoma patients (13,30,52). This 
vaccine induces Th1-dominant helper T cells, which is expected to synergize with 
the effects of CTLA-4 blockade with ipilimumab, since ipilimumab’s antitumor effects 
are mediated in large part through effects on CD4 T cells (53). Thus, the present 
protocol explores combination therapy with 6MHP vaccine plus ipilimumab in 
several relevant clinical settings. 
In our study, we plan to include a broader patient population than the EORTC 
18072 study with a wider risk profile.  To balance the outcomes for our patients, we 
aim to augment the benefit of ipilimumab at 3 mg/kg with the addition of the 6MHP 
vaccine while limiting the risk to these patients with the lower dose of anti-CTLA-4 
antibody and the low toxicity profile of this vaccine.  The primary endpoints of our 
study are the safety and immunogenicity of this combination.  Our goal is to utilize 
this study to provide the foundation for future, more definitive investigations with this 
combination therapy. 
 
          Toxicities Previously Reported for Participants Receiving ipillimumab.  
Ipilimumab is approved by the FDA for the treatment of patients with advanced 
melanoma (Stage IV and unresectable stage IIIB/C).  Adverse events have been 
reported, and include a range of toxicities, including some that appear to be 
immune-mediated and some of which have been life-threatening or fatal.  The 
package insert lists the most common toxicities as fatigue, diarrhea, itching and 
rash.  It also lists the potential serious toxicities as colitis, hepatitis, toxic epidermal 
necrolysis, neuritis, hypophysitis, and inflammation of the eyes. Toxicity data 
reported for 380 patients treated with ipilimumab plus a gp100 peptide vaccine and 
131 treated with ipilimumab alone (total 511 participants) are summarized below in 
Table 5. 


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Table 5.  Expected toxicities in 511 patients with ipilimumab 3 mg/kg IV, 
with (n=380) or without (n=131) vaccine (adapted from (38)) 
 
 
Treatment related adverse events of a given grade are considered expected 
toxicities if they have been observed in 4% of patients at that grade or higher.  
These are listed in Table 6 for ipilimumab.   
 
 
 
 
 
 
 
Table 6. Expected toxicities for IV ipilimumab 3 mg/kg x 4 
Toxicity 
All grades 
Grade 3 
Grade 4 
ADVERSE EVENTS IN > 15% OF PARTICIPANTS 
Diarrhea 
189 (37%) 
23 ( 5%) 
1 (<1%) 
Nausea 
175 (34%) 
8 ( 2%) 
1 (<1%) 
Constipation 
108 (21%) 
6 ( 1%) 
. 
Vomiting 
106 (21%) 
9 ( 2%) 
1 (<1%) 
Abdominal pain 
87 (17%) 
8 ( 2%) 
. 
Fatigue 
179 (35%) 
28 ( 5%) 
. 
Anorexia 
123 (24%) 
7 ( 1%) 
1 (<1%) 
Fever 
94 (18%) 
2 (<1%) 
. 
Headache 
84 (16%) 
7 ( 1%) 
. 
Cough 
76 (15%) 
1 (<1%) 
. 
Dyspnea 
65 (13%) 
16 ( 3%) 
3 (<1%) 
Anemia 
56 (11%) 
15 ( 3%) 
. 
IMMUNE RELATED ADVERSE EVENTS 
Pruritis 
99 (19%) 
1 (<1%) 
. 
Rash 
92 (18%) 
6 ( 1%) 
. 
Vitiligo: (skin hypopigmentation) 
17 (  3%) 
. 
. 
Diarrhea 
151 (30%) 
20 ( 4%) 
. 
Colitis: 
(GI disorder or autoimmune disorder) 
30 (  6%) 
18 ( 4%) 
1 (<1%) 
Hypothyroid 
8 (  2%) 
1 (<1%) 
. 
Hypopituitarism:   
increased serum thyrotopin and decreased 
serum corticotropin  
(autoimmune disorder) 
6 (  1%) 
3 (<1%) 
1 (<1%) 
Hypophysitis:    
increased serum thyrotopin and decreased 
serum corticotropin  
(autoimmune disorder) 
4 (<1%) 
4 (<1%) 
. 
Adrenal insufficiency 
5 (  1%) 
2 (<1%) 
. 
Increased serum thyrotropin 
3 (<1%) 
. 
. 
Decreased serum corticotropin 
2 (<1%) 
. 
1 (<1%) 
Increased ALT  
(alanine aminotransferase increased) 
5 (  1%) 
2 (<1%) 
. 
Increased AST  
(aspartate aminotransferase increased) 
5 (  1%) 
1 (<1%) 
. 
Hepatitis (autoimmune disorder) 
3 (<1%) 
1 (<1%) 
 
Other 
18 (  4%) 
7 ( 1%) 
1 (<1%) 


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This list includes toxicities observed in >4% of participants. 
 
 Dosing 
6 MHP 
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.  
 
Ipilimumab 
Dosing for the ipilimumab is based on the current FDA approved dose and 
schedule.    
 
 Summary 
We have developed a vaccine incorporating 6 intermediate-length peptides that 
induce CD4+ helper T cell (TH) responses (6MHP), and have previously shown that 
the vaccine has clinical activity in patients with advanced melanoma. The current 
proposal is to obtain preliminary data on whether combination of the 6MHP vaccine 
with CTLA-4 blockade will be safe and will result in a CD4+ T cell response to 
6MHP peptides.  We intend to use this combination in three clinical cohorts: 
advanced disease, neoadjuvant therapy and adjuvant therapy.  This trial will 
provide preliminary information on the disease control rate of the combination in the 
advanced setting as well as the recurrence risk and time to recurrence in the 
neoadjuvant and adjuvant settings. The trial incorporates correlative studies of 
immune responses in the blood, sentinel immunized lymph node, and tumor to 
obtain a more complete understanding of the host:tumor relationship in the context 
of these combination therapies.  
 
Toxicity 
Grades 1 - 2 
Grade 3 
Diarrhea 
+ 
+ 
Nausea 
+ 
. 
Constipation 
+ 
. 
Vomiting 
+ 
. 
Abdominal pain 
+ 
. 
Fatigue 
+ 
+ 
Anorexia 
+ 
. 
Fever 
+ 
. 
Headache 
+ 
. 
Cough 
+ 
. 
Dyspnea 
 
+ 
. 
Anemia 
+ 
. 
Pruritis 
+ 
. 
Rash 
+ 
. 
Colitis  
(GI disorder or autoimmune disorder) 
+ 
+ 
Increased transaminases 
(alanine aminotransferase increased) 
(aspartate aminotransferase increased) 
+ 
. 


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2.0 OBJECTIVES  
 
 Primary objectives and endpoints 
Safety  
 
To determine whether the combination of 6MHP vaccine plus ipilimumab is 
safe as evaluated by adverse event assessments, including CTCAE 4.03 and 
subclassified by irAE categories (9). 
 
Immunogenicity 
 
To estimate in the blood and in the sentinel immunized node the CD4+ T cell 
response rate to 6MHP peptides in patients treated with 6MHP vaccine plus 
ipilimumab  
 
 Secondary objectives and endpoints 
Induction of epitope-spreading 
 
 To estimate the proportion of patients in whom the combination of 6MHP 
vaccine plus ipilimumab induces epitope-spreading CD8+ T cells in the blood 
and in the sentinel immunized node that are reactive to a panel of defined 
melanoma antigens. 
 
 Exploratory objectives and endpoints 
        Induction of antibody responses 
 
To estimate the production of serum IgG reactive to 6MHP in patients treated 
with 6MHP vaccines plus ipilimumab  
 
             Clinical outcome 
 
For cohort 1: To obtain preliminary estimates of clinical outcomes including 
overall survival, progression-free survival, and clinical response by irRC 
criteria following administration of the combination of 6MHP vaccine plus 
ipilimumab.  
 
 
For cohorts 2 and 3: To obtain preliminary data on risk of recurrence and time 
to recurrence after resection of tumor to no evidence of disease and 
administration of the combination of 6MHP vaccine plus ipilimumab. 
 
         Associations of immune response and clinical outcome 
 
To obtain preliminary estimates of the associations between T cell and 
antibody responses to melanoma antigens and survival and with disease 
control. 
 
T cell infiltration of the tumor microenvironment   
 
To determine whether the combination of 6MHP vaccine plus ipilimumab 
increases infiltration of CD8+ and/or CD4+FoxP3neg T cells into melanoma 
metastases in at least 50% of patients with tumor evaluable pre- and post- 
treatment. 
 
 
3.0 PARTICIPANT SELECTION CRITERIA 
 Inclusion Criteria  
 
 Participants with stage IIA (with class 2 DecisionDx Score) through IV 


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melanoma in cohorts defined below.  These participants may have had 
cutaneous, uveal, mucosal primary melanoma, or an unknown primary 
melanoma.  The diagnosis of melanoma must be confirmed by cytological 
or histological examination. Staging of cutaneous melanoma will be 
based on the revised AJCC staging system (Appendix 2) (54). 
Participants must be eligible to be treated with ipilimumab based on 
clinician judgment within standard of care. 
 
Cohort 1 (Advanced Patients): unresectable stage III or IV melanoma that 
have clinical or radiographic evidence of disease.   
 
Cohort 2  (Neoadjuvant therapy): primary melanoma with clinically 
apparent lymph node or in transit/satellite lesions with or without lymph 
node involvement, in transit recurrence or metastatic recurrence 
amenable to complete resection to no evidence of disease 
 
Cohort 3 (Adjuvant therapy): Stage IIA (with class 2 DecisionDx Score), 
IIB-IV melanoma resected to no evidence of disease.  
 
  Participants will be required to have radiological studies to define 
radiologically 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 melanoma available for biopsy pre-treatment and 
on day 22 must consent to having those biopsies.   
Melanoma lesions may be in nodes, skin, soft tissue, liver, or other sites 
that can be accessed by core needle biopsy, or incisional or excisional 
biopsy, with or without image guidance.   
The lesion(s) must be large enough to enable biopsy of at least 0.1 cm3 of 
tumor tissue (ideally 0.3 cm3 or more) in 5 core biopsies (ideally 14-16 
gauge, but 18 gauge is acceptable) or incisional/excisional biopsies at 
both time points. Biopsies may be taken from a single lesion or multiple 
lesions at each of the time points depending on the size of each lesion.  
Different lesions may be sampled at each time point. It is acceptable to 
perform a biopsy pretreatment, and then to perform an excision at day 22, 
even under general anesthesia if needed. 
The lesions to be biopsied must be specified at study enrollment and not 
included as target lesions for RECIST calculations.  There must be 
measurable disease in addition to the lesion(s) to be biopsied for cohort 
1. 
Up to 15 participants whose metastases are not available for biopsy may 
be enrolled in the first stage of enrollment, and up to 17 participants 
whose metastases are not available for biopsy may be enrolled in the 
second stage of enrollment. 
 


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  Participants who have had brain metastases will be eligible if all of the 
following are true: 
 
3.1.4.1 Each brain metastasis must have been treated by surgical removal,  
stereotactic radiosurgery or managed to complete resolution with 
immunotherapy.  Patients with brain lesions managed by 
immunotherapy without excision or radiosurgery are included provided 
that the brain metastases have completely resolved after systemic 
therapy and there are no neurologic symptoms or need for systemic 
therapy to control CNS-disease related symptoms.  
3.1.4.2 There has been no evident growth of any brain metastasis since the 
most recent treatment. If a patient has been managed by 
immunotherapy alone, the prior lesions must be completely resolved. 
3.1.4.3 No brain metastasis is > 2 cm in diameter at the time of registration 
3.1.4.4 The most recent surgical resections or gamma-knife therapy for 
malignant melanoma must have been completed ≥ 1 week prior to 
registration. 
 
  ECOG performance status of 0 or 1 (Appendix 3) (55) 
  Ability and willingness to give informed consent 
  Laboratory parameters as follows: 
3.1.7.1 ANC  1000/mm3 
3.1.7.2 Platelets  100,000/mm3 
3.1.7.3 Hgb  9 g/dL 
3.1.7.4 HgB-A1C ≤ 7.5%  
3.1.7.5 AST and ALT ≤ 2.5 x upper limits of normal (ULN) 
3.1.7.6 Bilirubin ≤ 1.5 x ULN (except in patients with Gilbert’s disease, where 
bilirubin to 4x ULN is allowed).   
3.1.7.7 Alkaline phosphatase ≤ 2.5 x ULN 
3.1.7.8 Creatinine ≤ 1.5 x ULN 
 
  Age 18 years or older at registration. 
  Participants must have at least two intact (undissected) axillary and/or 
inguinal lymph node basins 
 
 Exclusion Criteria  
  Participants who have received the following medications or treatments 
at any 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 prior to registration) 
 
Allergy desensitization injections 
 
High doses of systemic corticosteroids, with the following 
qualifications and exceptions: 
o   Daily doses of 10 mg predisone (or equivalent) per day 
administered parenterally or orally are not allowed in patients 


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with normal adrenal and pituitary function. 
o   In patients with adrenal or pituitary insufficiency replacement 
steroid doses are allowed. 
o   Inhaled steroids (e.g.: Advair®, Flovent®, Azmacort®) are 
permitted at low doses (less than 500 mcg fluticasone per 
day, or equivalent) (56,57).  
o  Topical and nasal 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 
 
  HIV positivity or evidence of active Hepatitis C virus (testing to be done 
within 6 months of study entry). 
  Participants who are currently receiving nitrosoureas or who have 
received this therapy within the preceding 6 weeks 
  Participants who are currently receiving a checkpoint molecule blockade 
therapy, or who have received this therapy within the preceding 6 weeks,  
with the following exception: 
3.2.4.1 Participants who have received a PD-1 blocking antibody (eg: 
pembrolizumab or nivolumab) may be enrolled 3 weeks after receiving 
the last dose of that antibody. 
3.2.4.2 Participants who have been treated previously with a CTLA-4 blocking 
antibody either as monotherapy or as part of combination CTLA-4/PD-
1 blockade will be ineligible if CTLA-4 therapy: 
1)  was discontinued early for toxicity, or  
2)  did not induce stable disease or objective clinical response (by 
RECIST or irRC criteria) lasting 6 months or more.   
 
Note:  Patients may be eligible if they have experienced progression 
after a period of stable disease (6 months or more) or an objective 
clinical response (by irRC or RECIST) (6 months or more) induced by 
CTLA-4 blockade or combination CTLA-4/PD-1 blockade 
 
Note:  Similar guidelines will apply for tremelimumab or other CTLA-4 
blocking antibodies. 
  Participants with known or suspected allergies to any component of the 
vaccine. 
  Participants may not have been vaccinated previously with any of the 
synthetic 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 pregnancy test (urinary or serum beta-HCG) obtained within 2 


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weeks prior to registration. Males and females must agree, in the consent 
form, to use effective birth control methods during the course of 
vaccination.  This is consistent with existing standards of practice for 
vaccine and chemotherapy protocols. 
  Female participants must not be breastfeeding 
  Participants in whom there is a medical contraindication or potential 
problem in complying with the requirements of the protocol in the opinion 
of the investigator. 
  Participants classified according to the New York Heart Association 
classification as having Class III or IV heart disease (Appendix 4). 
  Participants with uncontrolled diabetes, defined as having a HgB-A1C  
7.5%. 
  Participants must not have had prior autoimmune disorders requiring 
cytotoxic or 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 
 
A history of immune-related adverse events with immune therapy, if 
they have resolved completely. 
 
  Participants who have another cancer diagnosis, except that the 
following  diagnoses will be allowed: 
 
squamous cell cancer of the skin without known metastasis 
 
basal cell cancer of the skin without known metastasis 
 
carcinoma in situ of the breast (DCIS or LCIS) 
 
carcinoma in situ of the cervix 
 
any cancer without distant metastasis that has been treated 
successfully, without evidence of recurrence or metastasis for over 2 
years 
 
 Participants with known addiction to alcohol or drugs who are actively 
taking those agents, or participants with recent (within 1 year) or ongoing 
illicit IV drug use. 
 
 Body weight < 110 pounds at registration, due to the amount and   
frequency with which blood will be drawn. 
 
 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. 


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Participants should receive their first study treatment within 2 weeks of registration.  
 
No randomization is required; all participants will receive the same vaccine 
regimen.   
 
4.0 STUDY DRUG (6MHP and MONTANIDE ISA-51) 
  
 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.  
 
 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. 
 
 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. 
 
 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. 
 
 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. 
 
 Stability testing    
The peptide vaccine will undergo stability testing as described in Appendix 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) 


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 
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” 
 
 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.  
   
 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 IPILIMUMAB 
 Packaging and Labeling 
Ipilimumab (YERVOY®) is an FDA approved drug for usefor the treatment of 
patients with unresectable or metastatic melanoma.  The study drug is 
manufactured by Bristol Myers-Squibb and is packaged and labeled in accord with 
the package insert.  It is provided as a sterile aqueous liquid at 5 mg/ml in single-
use vials with either of two volumes (10 ml or 40 ml). 
 
 Storage 
Ipilimumab is stored refrigerated at 2°C– 8°C (36°F–46°F).  Vials are to be 
protected from light and should not be frozen 
 
6.0 TREATMENT PLAN 
 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 24 months (or more often if needed for testing or medical 
reasons).  Participants will be off treatment follow-up at the end of 24 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. 
 
 Administration of 6MHP 
 Overview 
The vaccine regimen will be the same for all subjects.  Days 1, 8, 15, 43, 64, 
85:  200 mcg each of the 6 peptides (Table 1), emulsified in Montanide ISA-
51 adjuvant (2 ml total will be administered).  Each of vaccines 1-3 will be 
administered subcutaneously (50%) and intradermally (50%) at one skin site. 
If the site of vaccines 1 or 2 has severe inflammation or ulceration, the next 
vaccine may be placed near the original site.  Vaccines 4-6 will be 
administered at a different site than vaccines 1-3, but vaccines 4-6 will all be 


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administered at one site.  If the site of vaccines 4 or 5 has severe 
inflammation or ulceration after 1-2 vaccines, the next vaccine may be placed 
near the site designated for vaccines 4-6.   
     
On days when both ipilimumab and vaccine are administered, the vaccine 
may be administered before or after ipilimumab, but preferably before.   
 
  Dose Calculations 
At each designated time-point, 200 mcg each of the 6 peptides (Table 1), 
emulsified in Montanide ISA-51 adjuvant (2 ml total) will be administered.  
  
  Pre-Medications 
        None required. 
 
  Preparation of Study Drug  
Directions on how to prepare the investigational drug will be provided.  The 
prepared peptide vaccines, containing 2 ml of peptide emulsion, 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 within 1-2 hours 
after mixing.  If the vaccine is not administered within 4 hours after mixing, it 
should be discarded. 
 
  Post-Vaccination Observation 
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. 
 
 Administration of Ipilimumab 
 
 Overview 
Ipilimumab will be administered in accord with the official prescribing 
information: 3 mg/kg intravenously once every 3 weeks, for 4 doses.  
 
  Pre-Medications 
None required. 
 
 Dose Modifications 
 
  6MHP 
There will be no dose modifications of the vaccine components 
 
 
  Ipilimumab 
In the event of treatment related toxicity, the dose of ipilimumab should be 
modified in accord with current practice guidelines.  Recommendations are 
also provided in the package insert.  Immune-related adverse events should 
be managed per standard clinical practice and in accord with  the package 


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insert for YERVOY® (ipilimumab) and the Immune-mediated Adverse 
Reaction Management Guide, available from Bristol-Myers Squibb. 
(https://www.hcp.yervoy.com/pages/rems.aspx) 
 
 Dose Delays  
Protocol treatment includes both the 6MHP vaccine and ipilimumab.  Either or 
both may be delayed for any of the following reasons: 
 
  Dose Delays Due to Toxicity 
 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, 
though the other systemic therapy may be continued. 
 Dose delays for ipilimumab will follow standard clinical guidelines. 
 
  Delayed Visits for Reasons Other Than Toxicity 
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  
 
The table below (Table 7) 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 7.  Delayed Visit for Reasons other than Toxicity  
Treatment Period 
Range of Days 
Participant Treatment 
Protocol  
Deviation 
Vaccine 1*/Ipilimumab 1 
 
 
 
Day 1 
 2 days 
Vaccine, Labs, Ipilimumab 
No 
        
 3 to 7 days 
Vaccine, Labs, Ipilimumab 
Yes 
 
 8 or more days 
Labs 
Yes 
Vaccines 2-3* 
 
 
 
Days 8, 15 
 2 days 
Vaccine/Labs 
No 
        
 3 to 7 days 
Vaccine/Labs 
Yes 
 
 8 or more days 
Labs 
Yes 
Biopsy/Ipilimumab 2 
 
 
 
Day 22 
 2 days 
Labs, Biopsies, Ipilimumab 
No 
 
 3 to 7 days 
Labs, Biopsies, Ipilimumab 
Yes 
 
 8 or more days 
Labs 
Yes 
 
 
 
 
Vaccine 4*/Ipilimumab 3 
 
 
 


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Days 43 
 2 days 
Vaccine, Labs, Ipilimumab 
No 
 
 3 to 7 days 
Vaccine, Labs, Ipilimumab 
Yes 
 
 8 or more days 
Labs 
Yes 
 
 
 
 
Assessment 
 
 
 
Day 57 
 7 days 
Scans** 
No 
 
 8 to 14 days 
Scans 
Yes 
 
 15 or more days 
Scans 
Yes 
Vaccine 5*/Ipilimumab 4 
 
 
 
Day 64 
 7 days 
Vaccine, Labs, Ipilimumab 
No 
 
 8 to 14 days 
Vaccine, Labs, Ipilimumab 
Yes 
 
 15 or more days 
Labs 
Yes 
 
 
 
 
Vaccine 6* 
 
 
 
Day 85 
 7 days 
Vaccine, Labs, Scans** 
No 
 
 8 to 14 days 
Vaccine, Labs, Scans** 
Yes 
 
 15 or more days 
Labs/Scans 
Yes 
 
 
 
 
Assessment 
 
 
 
Day 92 
 7 days 
Labs 
No 
 
 8 to 14 days 
Labs 
Yes 
 
 15 or more days 
Labs 
Yes 
 
 
 
 
 
 
 
 
Follow-up 
 
 
 
FUv1 and FUv2 
 
 14 days 
Labs/Scans** 
No 
 15 or more days 
Labs/Scans 
Yes 
 
 
 
 
Follow-up 
 
 
 
FUv3, FUv4, FUV5 
 28 days 
Labs/Scans** 
No 
 
 29 or more days 
Labs/Scans 
Yes 
 
 *  A participant will be taken off protocol treatment if more than one vaccination is delayed 
[ 3 to 7 days] during the treatment period. 
**  CT scans on days 57, 85, FUv1, FUv2, FUv3, FUv4, FUv5 should also be completed within the 
specified time limits.  The scan at day 57 is to measure response, with confirmatory scans at day 85 
only if needed.  Followup scans beyond day 85 may be performed earlier or more frequently than 
specified in the protocol if indicated as part of clinical care.  If earlier or more frequent scans have 
been performed for clinical care, it is not a violation to omit scans on followup dates if they have been 
performed within 3 months prior.  
 
 Discontinuation of Therapy 
Protocol treatment includes both the 6MHP vaccine and ipilimumab.  Either or 
both may be discontinued for any of the following reasons: 
 
Protocol treatment will be discontinued for a participant for any of the following 
reasons: 
 
 Any dose-limiting toxicity (DLT), as defined in Section 9 warrants 
discontinuation of the agent to which the DLT is attributed. If a DLT 
attributed to one intervention is not believed to be related to the other 
treatment, only the one to which the DLT is attributed must be 
discontinued. However, management of toxicities attributed to ipilimumab 


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should follow current standard practice guidelines, including use of 
steroids when indicated.    
 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, or minor surgical procedures to excise a new lesion, will not be a 
basis for discontinuing vaccinations.  Also, surgery to perform biopsy of 
tumor at day 22, in accord with the protocol, will not be a basis for 
discontinuing therapy, whether done under local anesthesia or general 
anesthesia. 
 Initiation of cytotoxic chemotherapy, radiation therapy, or targeted therapy 
for melanoma or other cancer. 
 Initiation of non-permitted medication, or other systemic 
immunosuppressive therapy, except that steroid or immunosuppressive 
therapy for management of toxicities of ipilimumab is allowed without 
discontinuation of vaccines. 
 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. 
 Treatment delays as specified in section 6.5.1. 
 
 
Safety follow-up Visits 
 In the event of an AE, appropriate action will be taken to ensure adequate care for 
the patient.  If the patient is still on protocol, treatment delay or withdrawal from the 
protocol will be considered according to the protocol guidelines (Section 6.6). 
 
Subjects who discontinue receiving investigational product will begin study-specific 
follow-up as outlined in Appendix 1.  Exceptions for the timing of the follow-up visits 
include the following: 
 Safety follow-up visits may occur prior to the designated time frames 
for subjects who will begin another cancer therapy and/or for 
subjects who withdraw consent at the time of discontinuation. 
 
 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.   
 
 Concomitant Medications 
Medications taken within 30 days  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. 


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 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®) 
 Other investigational medications 
 Street drugs 
 
 Permitted Medications or Treatments  
 
Nonsteroidal anti-inflammatory agents 
 Anti-histamines (e.g. Claritin®, Allegra®) 
 Topical corticosteroids or steroids for the reasons cited in section 3.2.1 
above. 
 Short-term therapy for acute conditions not specifically related to melanoma 
 Chronic medications except those listed in section 6.8 
 Influenza vaccines are permitted, but should be administered at least 2 
weeks prior to or at least 2 weeks after a study vaccine. 
 
 Biopsies 
For participants with biopsy accessible disease, biopsies (incisional, core or 
excisional biopsies) of cutaneous, subcutaneous, soft tissue or visceral 
metastatic melanoma will be obtained: 
 
at baseline: may be collected previously according to specifications 
below, during screening or once enrolled, prior to treatment on day 1.  
 
on day 22: 1 week after the third vaccine.  
 
Biopsies collected previously as part of standard treatment or other protocols 
may be used as the baseline biopsy provided they meet all of the following: 
a. Sufficient tissue is available 
b. Tissue was collected within 3 months of Day 1 
c. There were no intervening treatments in between the biopsy and the first 
protocol treatment (dietary supplements are permitted) 
Cohort 2 only: oncologic surgery to render patient free of disease on day 22 will 
provide tissue for day 22 biopsy.   
 
Additional optional biopsies may be performed during active treatment or during 
follow-up, e.g. for new lesions.  
 
 
7.0 CLINICAL AND LABORATORY EVALUATIONS  
 


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The following evaluations will be performed on an outpatient basis.  Please refer to the study 
calendar (Appendix 1) for scheduling. 
 
 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  
 Designation of vaccination sites (at screening only) 
 Visual acuity (Snellen chart)  (baseline only) 
 Color vision exam (Ishihara eye chart)  (baseline only)  
 Assessment of baseline symptoms (baseline only) 
 
 Pathology Review 
 Review of pathology at the University of Virginia 
 
 Performance Status 
 ECOG performance status criteria will be used in the evaluations (Appendix 
3).  
 
 Clinical Labs 
 
 CBC with differential, including automated lymphocyte count (0.3 ml) 
 Comprehensive chemistry panel to include sodium, potassium, creatinine, 
glucose, calcium total bilirubin, AST, ALT, and alkaline phosphatase.  NOTE:  
fasting blood sugars, when required, may be evaluated 4 hours or more after 
last eating. (0.9 ml) 
 Lactate dehydrogenase (LDH) (0.3 ml) 
 Urinalysis 
 β-HCG for women of childbearing potential (2 ml) 
 Cortisol (3 ml; combined with TSH and Free T4) 
 TSH 
 Free T4 
 HgB-A1C (3 ml) 
 HIV screening (antibody screen); if the antibody screen is positive, follow-up 
testing by RNA analysis may be completed to determine whether active 
disease is present. (3 ml) 
 HCV screening (antibody screen); if the antibody screen is positive, follow-up 
testing by RNA analysis may be completed to determine whether active 
disease is present. (combined with HIV) 
 Anti-nuclear antibody and rheumatoid factor (4 ml) 
 
 Toxicity Assessments 
 


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 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. 
 
 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 
 
 Tumor Biopsies  
Tumor biopsies will be completed in participants with adequate and accessible 
metastatic tumor in addition to measurable disease. 
 
 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 0.1 cm3 and up to about 0.3 
cm3 of 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. 
 
 Sampling 
The biopsies will vary based on the clinical scenario and may include five 
core biopsies, an incisional biopsy or an excisional biopsy as outlined in the 
inclusion criteria. 
 
  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 
when possible: 
 
a. 20% (1/5 of single biopsy or 1 core biopsy) will be fixed in formalin, then 
paraffin-embedded (for histology/immunohistology) 
b. 20% (1/5 of single biopsy or 1 core biopsy) will be processed for protein 
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c. 40% (2/5 of single biopsy or 2 core biopsies) 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). 
d. 20% (1/5 of single biopsy or 1 core biopsy) will be placed in RNA-later 
(for RNA/RT PCR) 
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. 
 
 Evaluations 
a. 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.   
b. Tumor escape mechanisms may also be evaluated.   
c. Specimens will be used in immunological assays to assess T cell 
function or antibody response.  Assays generally used for this type of 
testing include, but are not limited to, ELIspot assays, ELISAs, 
chromium-release assays, proliferation assays, intracellular cytokine 
staining, and T cell receptor sequencing.   
d. Specimens may be used to study the immunologic aspects of the 
tumor microenvironment or as targets or controls in laboratory assays.  
e. Specimens may be used to establish cell lines for long-term studies. 
f. 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.   
g. HLA typing (Class I and Class II) will be analyzed as part of the 
immunologic endpoints. (8 ml) 
 
 Optional Tumor Biopsies (at the time of progression, after study completion 
and withdrawal) 
If during the study, participants develop superficial metastatic deposits 
accessible to biopsy/excision with minimal morbidity, an optional biopsy may 
be collected for research purposes. 
 
If tumor samples are collected when a Mel 62 participant is no longer 
participating in the Mel 62 study and the participant consents to allow their 
tissue to be collected under the IRB #10598 tissue banking study to be 
analyzed for Mel 62, the tissue may be analyzed as part of the Mel 62 
analysis. 
 
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 Tumor Collected for Clinical Care 
If during the study, participants develop metastases or recurrences, or 
progress, these may be removed as part of their clinical care, and following 
receipt by pathology, may be evaluated by the study research team. 
 
 Sentinel Immunized Node Biopsy 
 
 Procedure 
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. 
 
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: 
a. 1 central section will be fixed in formalin, then paraffin-embedded (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).   
 
 Assessments 
 Anti-tumor Activity 
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 Measurments 
 


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RECIST 1.1 Criteria will be used to evaluate tumor burden.  These are 
summarized in Appendix 5 (58) 
. 
 Immunologic Assessments  
Assessments of T cell function may include, but are not limited to the 
following: 
 
ELIspot assays 
 
ELISAs 
 
Chromium-release assays  
 
Proliferation assays  
 
Intracellular cytokine staining  
 
T cell receptor sequencing. 
 
Cytokine bead array 
 
Flow cytometry 
 
HLA typing 
 
Characterization of cellular populations may include, but are not limited to the 
following: 
 
Immunohistochemistry 
 
Gene expression analysis 
 
Flow cytometry 
 
ELISAs 
 
Western-blot analysis 
 
Intracellular cytokine staining 
 
Cytokine bead array 
 
 Study Calendar  
(See Appendix 1) 
 
8.0 STATISTICAL CONSIDERATIONS 
 Design 
This is an early phase trial to evaluate whether the combination of the 6MHP 
vaccine with CTLA-4 blockade ipilimumab will be safe and will result in a CD4+ T 
cell response to 6MHP peptides.  The combination will be assessed in three clinical 
cohorts: advanced disease, neoadjuvant therapy and adjuvant therapy.  The trial 
should provide preliminary information on the disease control rate of the 
combination in the advanced setting as well as the recurrence risk and time to 
recurrence in the neoadjuvant and adjuvant settings. The primary goals are to 
assess safety and to determine if magnitude of immune response to the 6MHP 
vaccine can be improved by the addition of anti-CTLA-4.  
 
 Evaluation of Sample Populations and Criteria  
 Safety 
All participants receiving any protocol treatment will be evaluated for safety.  
 
The 6MHP vaccine has been safe, with lower toxicities than with class I 
peptides, in particular with lower autoimmune or hyperimmune toxicities. 
Ipilimumab causes toxicities related to unmasking autoimmunity, some of 
which can be severe, but are managed well with steroids. We do not 


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anticipate increased autoimmune toxicities by combining 6MHP vaccine with 
ipilimumab, but relatively high rates of hypophysitis have been reported by 
combining ipilimumab with each of two other vaccines. Thus, this trial will be 
designed with stopping rules for toxicities beyond those expected with 
ipilimumab alone. Formal safety bounds (Section 8.5) based upon monitoring 
dose limiting toxicities (DLT) will guide decisions about early stopping due to 
potential safety concerns. If a stopping bound is crossed then accrual to the 
study will be suspended until the study PI, co-investigators and the DSMC 
can review the data, and determine if the study should continue, be amended 
or be closed to further accrual. The proposed safety bound will be applied 
overall and within cohort. 
 
 Immunogenicity 
All eligible participants receiving any protocol treatment will be evaluated for 
the following immune response parameters. 
i. Primary: 
Circulating CD4+ T cell response to 6MHP peptides where the primary 
endpoint is the proportion of participants with at least two consecutive 
10-fold over baseline responses to the pool of 6MHP peptides. 
CD4+ T cell response to 6MHP peptides in the SIN. 
ii. Secondary: 
Evaluated in the blood and in the SIN 
Induction of epitope-spreading for CD8+ T cells reactive to a panel of 
defined melanoma antigens. 
Evaluated in the tumor microenvironment (In the subset of participants 
for which tumor biopsies are obtained pre- and post- treatment) 
T cell infiltration of CD8+ and/or CD4+FoxP3neg T cells.  
iii. Exploratory: 
Induction of antibody responses  
IgG antibody responses to 6MHP peptides. 
 
 Clinical efficacy 
All participants receiving any protocol treatment will be evaluated for clinical 
response to treatment as defined by the following measures.  
i. Exploratory: 
Overall survival (OS), defined as the time from the date of start of 
treatment to the date of death from any cause. Participants who do 
not experience an event (death) will be censored on the date of last 
follow-up/contact.  
(Cohort 1) 
Disease control as assessed by the RECIST 1.1 (CR, PR, SD at 12 
weeks) criteria.  
Clinical response by irRC criteria. 
Progression-free survival (PFS), defined as the time from the date of 
start of treatment to the date of progression or death from any cause, 
whichever occurs first. A participant who dies without a reported 
progression will be considered an event on the date of death. 
Participants  who have neither progressed nor died will be censored 
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(Cohort 2 and 3) 
Disease-free survival (DFS), defined as the time from the date of start of 
treatment to the date of progression or death from any cause, 
whichever occurs first. A participant who dies without a reported 
progression will be considered an event on the date of death. 
Participants  who have neither progressed nor died will be censored 
on the date of last evaluable tumor assessment. 
 
 Sample Size and Accrual 
Target sample size is based upon having sufficient information to assess 
immunogenicity overall cohort conditional on safety bounds not being crossed. 
Observed immune response from arm D from our most current 6MHP protocol 
Mel63, provides the basis for sample size determination. Define immune response 
as 'at least two consecutive 10 fold' responses for 6MHP pool assessed in the 
blood. Results for Mel63 arm D indicate a immune response rate of 55% (12/22) 
with 95%CI(32, 76%) satisfy this definition. Accrual of a total of 24 eligible 
participants provides 80% power to test for a null immune response rate of 55% vs 
an alternative immune response rate of 80% with a one-side 5% level binomial test. 
The alternative immune response rate of 80% was chosen to be above the upper 
limit of the CI to determine that the addition of ipilimumab improved immune 
response. At study conclusion the null hypothesis will be rejected and the treatment 
considered worthy of further study if at least 18/24 (75%) eligible participants 
experience an immune response. 
 
Maximum target sample size is estimated to be 27 participants, which assumes a 
10% dropout/ineligibility/lost to follow-up adjustment. Accrual to cohort 3 is 
expected to accumulate the most participants, at approximately 10 per year, 
followed by cohort 2 at 3-5 per year, and cohort 1 at 1-3 per year.  Therefore, total 
accrual is estimated at approximately 16 participants per year, therefore, accrual to 
the study should be completed in less that two years. 
 
 Cohort and post entry classification 
At study entery participants will be stratified by cohort. In addition. Participants will 
be monitored for post entry classification by whether or not biopsies are obtained at 
baseline and one week after the third vaccine; (yes/no).  Target sample size for 
participants for whom biopsies are obtained is set at a minimum of 14 eligible 
participants.  If increased infiltration of CD8+ T cell response from day 1 compared 
to day 22 tumor is observed in 10/14 participants then the lower limit of a one-sided 
90% confidence interval exceeds 50% and would be considered to support further 
study of the treatment regimen. Similar criteria apply for CD4+FoxP3neg T cells 
response.  
 
 Safety Monitoring 
Toxicities will be monitored using CTCAE 4.03 criteria, with a focus on recording 
immune-related adverse events (irAE) (9). Stopping rules will be based on an 
expected irAE rate of 15-20%, where 10-15% had grade 3-4 irAE in one study (38) 
and in another 19% had grade 3, and 3.2% grade 4 irAEs (59).  Significant 
increases over these rates are not expected but would be a reason for early 
discontinuation. Thus, formal safety bounds based upon the observed number of 
participants who experience a DLT as defined in Section 9.9 will guide decisions 
about early stopping due to potential safety concerns (Table 8). The definition of 


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DLT considers both irAEs and previously reported DLTs related to vaccine. If a 
stopping bound is crossed then accrual to the study will be suspended until the 
study PI, co-investigators and the DSMC can review the data, and determine if the 
study should  continue, be amended or be closed to further accrual. The rate of 
DLTs will be monitored up to 30 days after the last vaccination. A sequential 
probability ratio test (SPRT) based upon a binomial test of proportions for DLTs will 
be used.  Only the upper boundary will be used for monitoring to protect against 
excessive failure rates.  The stopping boundary are for a SPRT contrasting a 15% 
versus 30% DLT rate, with nominal type I and II errors of 10% and 10%, 
respectively.  The slope of the parallel lines for monitoring is 0.219 and the 
intercept for the upper bound is 2.476. 
 
Table 8:  Stopping Guidelines for DLTs 
Number of participants 
Boundary 
4-6 
≥ 4 
7-11 
≥ 5 
12-15 
≥ 6 
16-20 
≥ 7 
21-25 
≥ 8 
26-27 
≥ 9 
 
 Analyses 
Study populations and evaluation criteria are noted in Section 8.2.  Adverse events 
will be summarized by frequency and magnitude of event. Incidence of DLTs will be 
monitored against the safety bounds above. The bounds are non-binding, and 
provide a guideline that may result in study modification or closure. Participants in 
cohorts 1 and 2 will be assessed for clinical response and will be classified by 
disease control by week 12 and at final assessement. DCR will be estimated along 
with a 95% confidence interval around the point estimate. 
 
Immune response will be measured based upon the methods in Section 7.9.  The 
criteria for defining immune responses have been reported for ELIspot assays (34).   
The circulating CD4+ T cell response rate to 6MHP peptides in participants treated 
with 6MHP vaccine plus ipilimumab will be assessed at each time noted in the 
study calendar.  Maximum response over all time periods will be used to estimate 
the overall CD4+ T cell response. Other measures assessed over time in the blood 
include epitope-spreading for circulating CD8+ T cells reactive to a panel of defined 
melanoma antigens, and production of serum IgG reactive to 6MHP.  Assessments 
in the tumor microenvironment include assessment for of CD8+ and/or 
CD4+FoxP3neg T cells into melanoma. Each participant will be assessed for the 
endpoints of interest and the proportion of participants experiencing an event 
(immune response, IgG antibody response to 6MHP, presence of CD8+ T cell 
infiltrates into the tumor (a subset), etc) will be estimated along with 95% 
confidence intervals. Repeated measure models will be explored to estimate 
changes over time in interval measurements in the blood and tissue.  
 
Additionally, for cohort 3 only, estimates of immune response within the cohort will 
be estimated. We expect accrual of at least 15 eligible participants to cohort 3.  At 
study conclusion, if  12/15 (80%) eligible pariticipants satisfy the definition of 
immune response in cohort 3 then a 90%CI for immune response rate of 80% is 
(56%, 94%) which would support that immune response rate has improved in that 
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PFS, DFS (within appropriate cohorts) and OS distributions will be estimated by the 
product limit method of Kaplan and Meier. Other summary measures for PFS, DFS 
and OS will include estimates and 95% CIs for median and 1-year estimates of 
PFS, DFS and OS to assess relative to published data. Clinical response by irRC 
criteria will be tabulated and cross classified by DCR. We acknowledge that 
participants who progress by RECIST criteria and then go onto other therapies may 
result in an underestimate of response by irRC criteria.  However, if deemed 
appropriate some of these participants may be followed for a longer period before 
start of other treatment given that they received immune therapy and their response 
by irRC may be captured. Exploratory proportional hazard models will be used to 
obtain preliminary estimates of potential associations with T cell and antibody 
responses to melanoma antigens and survival. 
 
9.0 ADVERSE EVENT DATA COLLECTION AND MONITORING 
 
 Definitions 
 Adverse event (AE) – Any unfavorable and unintended sign (including an 
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. 
 Unexpected AE – Any adverse event not listed in Tables 3-6. 
 Serious AE – Any adverse drug experience occurring at any dose that 
results in 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 
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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 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 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 event for which there is a reasonable possibility that the drug caused 
the adverse event. 
 
 Attribution Assessment 
 Attribution – The determination of whether an adverse event is related to a 
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. 
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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 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. 


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Unrelated – Applies to those adverse events, which after careful 
consideration, are clearly and incontrovertibly due to extraneous 
causes (disease, environment, etc.). 
 
 Data collection  
Data will be collected using a centralized electronic case report form called ON-line 
Clinical Oncology Research Environment = Oncore. 
 
 Risks and Safety 
 Adverse Event Descriptions and Grading Scales 
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 
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 vaccine or ipilimumab, 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: 
 
Any AE not in this list will be considered an unexpected AE. 
 
 
Expected toxicities related to 6MHP 
The list of expected AEs for the 6MHP vaccine is based on prior data from 
the UVA-Mel41 and Mel44 clinical trials, plus the ECOG trial E1602; from 
these trials, aggregated data are available from over 200 patients vaccinated 
with the 6MHP vaccine in an emulsion with Montanide ISA-51 adjuvant, with 
or without GM-CSF.  These data are summarized in Table 3 and Table 4 in 
section 1. 
 
 
Expected toxicities related to ipilimumab 
Adverse events reported for treatment of patients with the CTLA-4 antibody 
ipilimumab have been reported and are summarized in section 1 and Table 5 
and Table 6.  Interactions with the toxicities of the 6MHP vaccine are not 
expected, but toxicities will be tracked. 
 
 
Adverse events expected from tumor and node biopsies.   
 
 Below is a list of expected AEs related to tumor and SIN biopsies: 
 
bleeding 
 
bruising 
 
pain 


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 
infection 
 
lymphedema 
 
delayed wound healing 
 
scarring 
 
numbness 
 
 
 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: 
 
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 9:   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: 
 
 
Eye Disorders: Night blindness (nyctalopia) 
 
Eye Disorders: Papilledema 
 
Eye Disordersl: Retinopathy 
 
Eye Disorders: Blurred vision 
 
Eye Disorders: Flashing lights 
 
Eye Disorders: Floaters 
 


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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. 
 
 Reporting Adverse Events 
 Process for Reporting AEs: 
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 10) 
 
 
 
 
 
 
 
 
Table 10:  AE reporting 
 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. 
 
 
       Pregnant-Partner Outcomes 
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 


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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. 
 
 IRB Reporting Requirements 
The University of Virginia is responsible for reporting to the UVA IRB-HSR 
per the following guidelines (Table 11): 
 
Table 11:  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 
 
 


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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 
 
 
 Additional Reporting Requirements for the Sponsor (UVA) 
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 of Participant Withdrawals/Dropouts Prior to Study Completion 
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.   
 
 Adverse Event Review and Monitoring 
 Capturing Adverse Events 
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 FUv1.  The diaries will 
be reviewed by a research clinician prior to the next scheduled visit.  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.   


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After administration of each 6MHP vaccine, participants will be observed for 
AEs for at least 20 minutes. 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. 
 
 Review of Adverse Events by the Study Team 
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. 
 
 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 9.6: 
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. LDH 
14. Potassium 
15. RF 
16. Urinalysis 
17. WBC 
18. Cortisol 
19. TSH 
20. Free T4 
 
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 


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54 
Cancer Center database and reported as described in Section 9.6.  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. 
 
 Dose-limiting Toxicities (DLT) 
6MHP 
A DLT of the 6MHP vaccine is defined as any unexpected Grade 3 or 
greater hematologic or non-hematologic toxicity that is definitely, probably, or 
possibly related to the administration of the vaccine.  Small ulcerations of the 
skin at vaccine sites are expected.  Thus, ulcerations will be considered 
DLTs only if the ulcers are ≥2 cm in diameter, require antibiotics or surgical 
debridement. 
 
Ipilimumab 
A DLT of ipilimumab is defined as any toxicity that is definitely, probably, or 
possibly related to the administration of ipilimumab and requires permanent 
discontinuation of ipilimumab. 
 
If a DLT occurs and cannot be attributed to either the 6MHP or ipilimumab, 
then a DLT will be attributed to both. 
 
 Management of Toxicity 
The study will be monitored continuously for treatment-related adverse events.  
Expected treatment-related toxicities of ipilimumab will be managed per standard 
clinical practice and in accord with the package insert for YERVOY® (ipilimumab). 
 
 
 Data Collection 
 Endpoint Data 
 
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. 
 
 Monitoring Plan 
 The University of Virginia Cancer Center Data and Safety Monitoring 
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: 
 
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.  
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 


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sponsor is requested.  Per the UVA Cancer Center NIH approved institutional 
plan, this study will be audited approximately every 6 months. 
 
10.0   STUDY CONDUCT AND ETHICAL CONSIDERATIONS 
This study will be conducted in compliance 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. 
 
 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. 
 
 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. 
 
 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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11.0 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:  NCI Common Terminology Criteria for Adverse Events v4 
Appendix 7:  Vaccine Lot Release and Stability Testing 
Appendix 8:  Summary of Changes 
 
 


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Appendix 1:  Study Calendar 
  
Studies & Tests 
 
 
 
Active Treatment  
Follow-upm 
At 
Screening 
Day 
1 
8 
15 
22 
43 
57 
64 
85 
92 
FU
v1 
FU
v2 
FU
v3 
FU
v4 
FU
v5 
Week 
0 
1 
2 
3 
6 
8 
9 
12 
13 
18 
24 
27 
52 
104 
Informed consent 
Xa 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pathology review 
Xa 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential  
Xb 
 
Xf 
 
 
X 
X 
 
X 
X 
 
 
 
 
X 
X 
Comprehensive chemistry  
Xb,e 
 
Xf 
 
 
X 
X 
 
X 
X 
 
 
 
 
X 
X 
HGB-A1C 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LDH 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cortisol 
 
 
X 
 
 
X 
X 
 
X 
 
 
 
 
 
 
 
Free T4 
 
 
X 
 
 
X 
X 
 
X 
 
 
 
 
 
 
 
TSH 
 
 
X 
 
 
X 
X 
 
X 
 
 
 
 
 
 
 
Urinalysis  
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
-HCG 
Xc 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV / Hepatitis C  
Xd 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CT chest/abdomen/pelvis or  
PET-CT  
Xb 
 
 
 
 
 
 
X 
 
Xh  
 
Xi 
 
X 
Xj 
Xj 
Head MRI / CT  
Xb 
 
 
 
 
 
 
Xl 
 
Xh 
 
Xi 
 
X 
Xj 
Xj 
History & physical  
Xb 
 
Xf 
 X 
X 
X 
X 
 
X 
X  
X  
X 
X  
X 
X 
X 
Medication Review 
Xb 
 
X 
X 
X 
X 
X 
 
X 
X 
X 
Xn 
Xn 
Xn 
Xn 
Xn 
Record baseline symptoms 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
AE assessment 
 
 
 
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 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
X 
X 
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   
 
 
Xk 
 
 
 
 
 
 
 
 
 
 
 
 
 
80cc green top tubes 
 
 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 


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Studies & Tests 
 
 
 
Active Treatment  
Follow-upm 
At 
Screening 
Day 
1 
8 
15 
22 
43 
57 
64 
85 
92 
FU
v1 
FU
v2 
FU
v3 
FU
v4 
FU
v5 
Week 
0 
1 
2 
3 
6 
8 
9 
12 
13 
18 
24 
27 
52 
104 
20cc red top tubes  
 
 
X 
X 
X 
X 
X 
 
X 
X  
X  
X 
X  
X 
X 
X 
Anti-nuclear antibody / Rf factor 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
X 
Vaccination with 6MHP 
 
 
X 
X 
X 
 
X 
 
X 
X  
 
 
 
 
 
 
Tumor biopsyg 
 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Sentinel immunized node biopsy 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Participant diary reviewed and/or 
distributed  
 
 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
 
 
 
ipilimumab: 3 mg/kg IV 
 
 
X 
 
 
X 
X 
 
X 
 
 
 
 
 
 
 
 
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 
 
 
e To include fasting glucose 
 
f History & physical, comprehensive chemistry, and CBC with differential scheduled for Day 1 are not required if prestudy assessments were within 10 calendar days of day 1.  
However, hair and eye color assessment and assessment of skin for vitiligo should be performed if theses assessments were not completed as part of the pre-study visit. 
gFor patients who have tumor accessible to biopsy, biopsies will be completed 1) during screening or, once enrolled, prior to treatment on Day 1, and 2) at day 22, for subjects who 
have melanoma available for biopsy.  Additional optional biopsies may be performed for research purposes during the study or during follow-up if participants are removed from the 
study or experience progression. If surgical resection or biopsies are completed for clinical care, the tumor tissue may be collected for study related analyses. 
 
h Tumor imaging at day 85 should be performed only when clinically needed to verify objective clinical responses at day 57. 
 
iIf scans were completed at day 85, the scans do not have to be repeated at this visit. 
 
j Scans do not need to be repeated for the protocol if they have been performed recently (typically within 3 months). 
 
kBlood for HLA typing is included in the research bloods. 
l Required only for subjects with brain metastases.   
mFollow-up visits in the initial follow-up phase will occur as follows:  FUv1:  42 days from the last dose of 6MHP; FUv2 = 24 weeks from the first dose of 6MHP; FUv3 = 27 weeks 
from the first dose of 6MHP; FUv4 = 1 year from the first dose of 6MHP; FUv5 = 2 years from the first dose of 6MHP. 
nOnly information about anticancer therapy will be collected 
 
 


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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 (55) 
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 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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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 
(58). 
 


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 Appendix 6:  NCI Common Terminology Criteria for Adverse Events v4 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html 
 
 
 


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Appendix 7:  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 8:  Summary of Changes 
 
10-10-17 
1) Updated personnel list. 
2) Protocol Synopsis: clarified additional biopsies are optional, removed 
reference to baseline tumor collection in Figure 3 and biopsy details 
addressed in section 6.10. 
3) Section 6.10: clarified biopsy timepoints. 
4) Section 7.7.3: added clarifier “when possible” and shifted RNA later to 
the least priority. 
5) Section 7.7.5: added section describing optional biopsies. 
6) Section 7.7.6: added section to address tissue removed as part of 
clinical care. 
7) Section 9.7.1: removed reference to vital signs being collected after 20 
minute observation period. 
8) Section 9.10: removed non-functional link and reference to the Immune-
mediated Adverse Reaction Management Guide. 
9) Appendix 1: clarified that additional biopsies are optional and may be 
collected for research purposes or as part of clinical care in footnote “g”. 
10) Corrected version date in summary of changes for v05-18-17. 
05-18-17 
1) Revised study title from “A Phase I/II Trial to Evaluate the Safety, 
Immunogenicity and Clinical Activity of a Helper Peptide Vaccine plus 
CTLA-4 Blockade in Advanced Melanoma” to “A Phase I/II Trial to 
Evaluate the Safety and Immunogenicity of a Helper Peptide Vaccine 
plus CTLA-4 Blockade in Melanoma Patients” 
2) Updated personnel list 
3) Updated Table of Contents 
4) Updated List of Abbreviations 
5) Editorial and formatting changes made throughout document 
6) Protocol Synopsis – added information about FDA approved dosing for 
Ipilimumab 
7) Protocol Synopsis, Section 1.1, Section 2.0, and Section 8.2—revised 
study objectives and endpoints 
8) Protocol Synopsis, Section 1.8, Section 3.1.1, and Section 8.1 --
changed the study design to include 3 cohorts: Unresectable stage III/IV 
advanced melanoma (Cohort 1); Neoadjuvant therapy (Cohort 2); and 
Adjuvant therapy (Cohort 3) 
9) Protocol Synopsis and Section 8.3 changed maximum target sample 
size from 51 to 27 and the target accrual from 46 to 24 eligible 
participants 
10) Section 1.1 Added additional background information on combination of 
ipilimumab and nivolumab 
11) Section 1.5 Added footnote a to Table 4 to indicate that injection site 
reaction with ulceration <2cm is expected 
12) Section 1.6 revised clinical data for checkpoint blockade with CTLA-4 
antibody 
13) Section 3.1.3 and Section 6.10 Clarified that biopsies will be done on 
Day 22 for Cohorts 1 and 2 only 
14) Section 3.1.4 Revised inclusion criteria for subjects who have had brain 
metastases  
15) Section 3.2.4.2 Revised inclusion criteria to specify that subjects may 
have previously received CTLA-4 blocking antibody either as 


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monotherapy or as part of combination CTLA-4/PD-1 blockade 
16) Section 3.2.13 Revised the exclusion criteria to include an exception for 
participants who have another cancer diagnosis for any cancer without 
distant metastasis that has been treated successfully, without evidence 
of recurrence or metastasis for over 2 years instead of over 3 years. 
17) Section 8.4 changed section name from “Stratification” to “Cohort and 
Post Entry Classification” 
18) Appendix 1 Study Calendar by deleting Comprehensive Chemistry and 
CBC with differential from Day 8 and Day 15 
19) Updated Reference list 
05-06-16 
1) Updated signature page 
2) Updated personnel list. 
3) Updated table of contents 
4) Editorial corrections made throughout document. 
5) Section 3.2.13:  revised to change 5 years to 3 years for exclusion 
criterion related to other cancer diagnoses. 
6) Schema, Table 7, Section 9.7.1 Appendix 1; replaced Days 127, 269, 
190, 365, 730 with FUv1, FUv2, FUv3, FUv4, and FUv5, respectively. 
7) Section 6.2.1:  Revised wording to specify that vaccines 4-6 will be  
administered at a separate site than vaccines 1-3 and that vaccines 4-6 will 
be administered at the same site and not rotated.  
8) Section 6.2.4:  revised to specify that the vaccine should be 
administered within 1-2 hours after mixing.   
9) Section 6.6.1: this section has been added to clarify the procedures for 
safety follow-up visits. 
10) Section 9.7.1:  revised section and moved corrected text to section  
6.6.1.  
11) Appendix 1: Added CBC and comp chemistry labs to Days 8 and 15. 
12) Revised the date ordering of summary of changes so that the most  
recent changes are at the top of the list. 
09-10-15 
1) Updated Investigator’s Agreement to match updated protocol template. 
2) Updated Study Personnel 
3) Updated Header and Table of Contents 
4) Section 3.2.4.1:  Updated exclusion criteria to allow subjects who have 
received a prior PD-1 blocking antibody to be enrolled 3 weeks after 
receiving the last dose of that antibody.  This decrease in the waiting 
period from 6 weeks to 3 weeks following the last dose of PD-1 blocking 
antibody is considered a sufficient wash-out period by the clinicians. 
5) Section 3.2.4.2:  Updated exclusion criteria to specify in which instances 
prior treatment with a CTLA-4 blocking antibody is considered to be 
exclusionary.  The rationale for the change is to broaden the entry 
criteria and is based on the potential synergistic effects of peptide plus 
CTLA-4 therapy. 
6) Section 9.4.3:  Updated expected toxicity list for SIN and tumor biopsies 
to be consistent with risks described in the consent form. 
7) Section 9.5:  Removed Grade 1 ocular toxicities as DLTs as Gr1 
immune-mediated ocular disease is described in the package insert for 
ipilimumab. 
8) Editorial changes throughout the document are noted. 
04-04-15 
1) Investigator’s statement has been updated. 
2) Updated Investigator list 


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3) Updated Table of Contents 
4) Section 6.4: Clarified that dose modifications of immune-related adverse 
events for ipilimumab will be managed in accord with standard practice, 
the package insert for ipilimumab and the Immune-mediated Adverse 
Reaction Management Guide, which is available from BMS.  
5) Preicis, Section 6.10:  clarified timing for the baseline biopsy 
6) Tables 5 and 6: added CTCAE adverse event terms for clarification 
7) Section 8.5:  reference has been corrected; title of Table 8 has been 
corrected. 
8) Section 8.6:  Clarification of measurements of response by irRC and 
how progression evaluated by RECIST will impact evaluation of 
response by irRC. 
9) Section 9.9:  deleted reference to package insert.   
10) Section 9.10:  added the package insert for ipilimumab and the link to 
the Immune-mediated Adverse Reaction Management Guide 
11) Editorial changes made throughout document. 
12) Appendix 1:  
a) Clarified completion of hair and eye color assessments at baseline 
b) Clarified timing for the biopsies. 
c) Clarified that the Head MRI/CT at day 57 is required only for  
subjects with brain metastases. 
01-22-15 
1) Table of Contents and header:  Updated 
2) Investigator’s Statement has been added 
3) Section 4.6:  Reference to manual was removed.  Information is 
included in Appendix 7. 
4) Section 4.8:  Reference to manual was removed.  Information on 
preparation will be supplied on vaccine mixing forms. 
5) Section 6.2.4:  Reference to manual was removed. 
6) Section 11:  List of appendices was updated. 
7) Appendix 1:  editorial change. 
8) Appendix 6:  Appendix and link to NCI CTCAE v4 were added. 
9) Appendix 7:  Appendix for vaccine lot release and stability testing was 
added. 
10) Formatting changes throughout document. 
01-06-15 
1) List of personnel:  Elizabeth Gaughan’s role  was changed to PI.  Craig 
Slingluff’s role was changed to co-investigator. 
2) Section 6.2.1:  volume of vaccine was changed to 2 ml 
3) Section 6.2.2:  volume of vaccine was changed to 2 ml 
4) Section 6.2.4:  volume of vaccine was changed to 2 ml 
5) Section 7.10:  editorial change, corrected hyperlink 
 
 
 


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