Open Label, Randomized, Phase I/II Study of a Long Peptide Vaccine Plus TLR Agonists for Resected Stage IIb-IV Melanoma. (MEL60)
Phase: PHASE1
Conditions: Melanoma, Metastatic Melanoma, Mucosal Melanoma
Sponsor: Craig L Slingluff, Jr

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1 
 
 
 
 
 
 
 
 
 
 
 
 UNIVERSITY OF VIRGINIA 
HUMAN IMMUNE THERAPY CENTER 
 
 
 
PHASE I/II TRIAL OF A LONG PEPTIDE VACCINE (LPV7) PLUS TLR AGONISTS  
FOR RESECTED STAGE IIB – IV MELANOMA 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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’s Representative 
 
 
 
______________________   _______________________ 
  _______________ 
Name              Signature            Date 
 
 
 
 
 
 
Investigator 
 
 
                        
_______________________ 
 _________________________  ________________ 
Name             Signature            Date 
 
 
 

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3 
UNIVERSITY OF VIRGINIA 
HUMAN IMMUNE THERAPY CENTER 
 
 
PHASE I/II TRIAL OF A LONG PEPTIDE VACCINE (LPV7) PLUS TLR AGONISTS  
FOR RESECTED STAGE IIB – IV MELANOMA 
 
Principal Investigator (UVA): 
Craig L. Slingluff, Jr., M.D. 
University of Virginia 
434-924-1730 
cls8h@virginia.edu 
 
Principal Investigator (MDA) 
Sapna Patel, M.D. 
The University of Texas MD Anderson 
Cancer Center 
713-792-2921 
sppatel@mdanderson.org 
 
Co-Investigators: 
Patrick Hwu, M.D., Ph.D. 
The University of Texas MD Anderson 
Cancer Center 
713-563-1728 
phwu@mdanderson.org 
 
Merrick Ross, M.D. 
The University of Texas MD Anderson 
Cancer Center 
713-792-0025 
mross@mdanderson.org 
 
Elizabeth Gaughan, M.D. 
Unviersity of Virginia 
434-924-1904 
emg5x@virginia.edu 
 
William W. Grosh, M.D. 
University of Virginia 
434-924-1904 
wwg9u@virginia.edu 
 
Carmel Nail, F.N.P. 
University of Virginia 
434-982-4042 
cjn2r@virginia.edu 
 
Kathleen Haden, RN MSN ANP-C 
University of Virginia 
434-924-1730 
km3s@virginia.edu 
 
 

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Walter C. Olson, Ph.D. 
University of Virginia 
434-243-9688 
wco3j@virginia.edu 
 
 
Kimberly A. Bullock, Ph.D. 
University of Virginia 
434-924-0180 
kb9d@virginia.edu 
 
Willem Overwijk, Ph.D. 
The University of Texas MD Anderson 
Cancer Center 
713-563-5294 
WOverwijk@mdanderson.org 
 
 
Biostatisticians: 
Gina R. Petroni, Ph.D.  
University of Virginia 
434-924-8363 
grp4c@virginia.edu 
 
Mark Smolkin, M.S. 
University of Virginia 
434-982-1032 
mes6r@virginia.edu 
 
 
 
Research Nurses: 
 
Carmel J. Nail, F.N.P. 
Kathleen Haden, RN MSN ANP-C 
 
Clinical Research Coordinators 
and Data Management- 
University of Virginia 
 
 
Emily Allred, PhD 
434-982-1902 
eh4m@virginia.edu 
 
Jessica Zareno, MS 
434-982-1901 
jhz4f@virginia.edu  
 
Alexandra Carney, MPH 
434-982-6714 
alc6a@virginia.edu  
 
Kendall Pettaway, BS 
434-982-6584 
kp8ac@virginia.edu  
 
Annika Shuali, MS 
434-243-7242 

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ah2ze@virginia.edu 
 
Sarah Lewis, BA 
434-924-8709 
set5v@virginia.edu 
 
 
Authors: 
 
Craig L. Slingluff, Jr., M.D. 
Jade Homsi, M.D. (Part 1) 
Willem Overwijk, Ph.D. (Part 1) 
Gina R. Petroni, Ph.D. 
Kimberly A. Bullock, Ph.D. 
Walter Olson, Ph.D. 
Sapna Patel, M.D. (Part 1) 
 
 
 
 

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1.0 INTRODUCTION .............................................................................................................. 14 
Abstract ..............................................................................................................................................................................14 
1.1 Study Rationale...........................................................................................................................................................14 
1.2 Vaccine Design and Surgical Excision .....................................................................................................................16 
2.0 STUDY OBJECTIVES ...................................................................................................... 23 
2.1 Part 1 (completed) .....................................................................................................................................................23 
2.2 Part 2 ............................................................................................................................................................................24 
3.0 STUDY OUTLINE ............................................................................................................. 24 
3.1 Type of Study...............................................................................................................................................................24 
4.0 PARTICIPANT SELECTION ............................................................................................ 24 
4.1 Inclusion Criteria .........................................................................................................................................................24 
4.2 Exclusion Criteria ........................................................................................................................................................25 
4.3 Demographics of Participant Population  ...............................................................................................................27 
4.4 Registration, Randomization, and Management of Participants ......................................................................27 
5.0 STUDY MEDICATION FORMULATION, DOSAGE, ACCOUNTABILITY AND 
ADMINISTRATION ................................................................................................................. 28 
5.1 Study Medication Formulation ................................................................................................................................28 
5.2 Preparation, Dosage, and Storage of Study Drug.................................................................................................29 
5.3 Drug Accountability and Distribution .....................................................................................................................31 
5.4 Administration of Vaccines .......................................................................................................................................31 
5.5 Modification/Discontinuation of Treatment .........................................................................................................33 
5.6 Concomitant Medications.........................................................................................................................................35 
6.0 CLINICAL AND LABORATORY EVALUATION............................................................. 36 
6.1 Screening......................................................................................................................................................................36 
6.2 Treatment ....................................................................................................................................................................37 
6.3 Tumor tissue collection..............................................................................................................................................41 
7.0 REGULATORY AND REPORTING REQUIREMENTS .................................................. 41 
7.1 Definitions....................................................................................................................................................................42 
7.2 Attribution Assessment .............................................................................................................................................43 
7.3 Data collection ............................................................................................................................................................44 
7.4 Risks and Safety ..........................................................................................................................................................44 
7.5 Adverse Event Classifications ...................................................................................................................................47 
7.6 Reporting Adverse Events .........................................................................................................................................49 
7.7 Adverse Event Review and Monitoring...................................................................................................................52 

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7.8 Adverse Event Stopping Guidelines .........................................................................................................................53 
7.9 Responsibility ..............................................................................................................................................................54 
7.10 Endpoint Data ...........................................................................................................................................................54 
8.0 EVALUATION OF IMMUNOLOGIC RESULTS............................................................... 54 
8.1 Immunohistochemistry and Immunofluorescence of skin biopsy specimens  ..................................................55 
8.2 ELIspot assay ...............................................................................................................................................................55 
8.3 Tetramer assay and multiparameter flow cytometry..........................................................................................56 
8.4 Proliferation assay / Cytokine Analysis...................................................................................................................56 
8.5 Antibody responses ....................................................................................................................................................56 
8.6 Evaluation of tumor ...................................................................................................................................................57 
9.0 STATISTICAL CONSIDERATIONS ................................................................................ 57 
9.1 Study design Part 1 (completed)..............................................................................................................................57 
9.2 Combination allocation Part 1 (completed)...........................................................................................................58 
9.3 Statistical properties Part 1 (completed) ...............................................................................................................60 
9.4 Study design Part 2 ....................................................................................................................................................61 
9.5 Sample size/accrual ...................................................................................................................................................61 
9.6 Stopping rules: ............................................................................................................................................................62 
9.7 Analyses .......................................................................................................................................................................63 
10.0 Appendices.................................................................................................................... 64 
10.1 Appendix 1: X-page ..................................................................................................................................................65 
10.2 Appendix 2: AJCC Staging System, 7th Edition .....................................................................................................66 
10.3 Appendix 3: ECOG Performance Status ................................................................................................................68 
10.4 Appendix 4: New York Heart Association Disease Classification .....................................................................69 
10.5 Appendix 5: Lot Testing...........................................................................................................................................70 
10.6 Appendix 6: NCI Common Terminology Criteria for Adverse Events v4.03  ....................................................72 
10.7 Appendix 7:  Summary of Changes .......................................................................................................................73 
11.0 Reference List ............................................................................................................... 78 
 
 
 
 
 
 
 
 
 
 

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Protocol Precis  
 
Title: Phase I/II trial of a long peptide vaccine (LPV7) + TLR agonists for resected stage IIB-
IV melanoma 
Objectives: 
Part 1 (completed) 
(1) To test safety of vaccination with a mixture of long peptides for patients with high-risk 
melanoma, in each of 7 adjuvant preparations, including TLR agonists and/or 
incomplete Freund’s adjuvant (IFA) 
(2) To estimate the immunogenicity of 7 long peptides in each of 7 adjuvant preparations 
 
Exploratory objective: 
(3) To evaluate the molecular and cellular changes at the vaccine site and systemically  
 
Part 2 
(4) To assess safety of vaccination of the optimal combination identified in Part 1 when 
administered in the same skin site for all 6 vaccines (same site vaccination). 
(5) To estimate the immunogenicity of the optimal combination identified in Part 1 when 
administered in the same skin site for all 6 vaccines (same site vaccination). 
 
Design:  This is an open-label, randomized, phase I/II study, using an adaptive design, of 
the safety and immunogenicity of a novel melanoma vaccination approach using long 
peptides plus TLR agonists, along with characterization of cellular and molecular events at 
the cutaneous sites of immunization and systemically.   
This trial involves two parts, which differ in the location of vaccine injections.  For part 1, 
each vaccine was administered in a skin site that differs on each vaccine date (rotating 
vaccine sites).  For part 2, each vaccine will be administered in the same skin site for all 6 
vaccines (same site vaccination). 
 
Table 1A. Clinical Trial Design for Part 1 
 
For part 2, the vaccine regimen is selected based on available data and the study decision 
rules from Part 1 which was determined to be Arm E. For Part 2, accrual will be to Study 
Arm E2.  However, a modification is made to exclude the tetanus helper peptide, due to (a) 
lack of availability of that peptide component, (b) the finding that, in part 1, LPV7 peptides do 
Zone 
Study 
Arm 
Peptide 
vaccine 
Adjuvant  
Dose 
of 
TLR 
agonist 
Route 
vax 
injection 
Admin of  
TLR agonist 
1 
A 
LPV7 + tet 
IFA 
 --- 
Id/sq 
-- 
1 
B  
LPV7 + tet 
PolyICLC 
1 mg 
Id/sq 
Id/sq 
1 
C 
LPV7 + tet 
Resiquimod 
1000 mcg 
Id/sq 
Topical 
2 
D 
LPV7 + tet 
PolyICLC + resiquimod 
1 mg/ 1000 mcg 
Id/sq 
Id/sq (PolyICLC) & 
topical (resiquimod) 
2 
E 
LPV7 + tet 
IFA + PolyICLC 
1 mg 
Id/sq 
Id/sq 
2 
F 
LPV7 + tet 
IFA + resiquimod 
1000 mcg 
Id/sq 
Topical 
3 
G 
LPV7 + tet 
IFA + PolyICLC + resiquimod 
1 mg/ 1000 mcg 
Id/sq 
Id/sq (PolyICLC) & 
topical (resiquimod) 

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induce helper T cell responses themselves (unpublished data), and prior data that adding 
the tetanus helper peptide did not add significantly to immunologic or clinical outcome of a 
peptide vaccine designed to induce CD8 T cells1. 
Table 1B. Clinical Trial Design for Part 2 
 
Regimen:  Patients will be registered on this phase I/II trial and will be vaccinated with 
LPV7 at 300 mcg peptide/dose every week x 3 then every 3 weeks x 3.   
For Part 1, the adjuvants are described, and will be administered to patients in 7 study 
groups as shown in Table 1A.  This incorporates an adaptive design with sequential 
initiation of accrual to study Zones with increasing numbers of adjuvants (Arms A-C, then D-
F, then G). Details are in Section 9.0 and Appendix 1 (X-page). 
For Part 2, the adjuvants will be administered to patients in 1 study group as shown in 
Table 1B. 
Vaccine site biopsies:  In each patient, three 4-mm punch biopsies will be performed of 
the skin at vaccine sites at two time points: 1 week after vaccine #1 (day 8) and one week 
after vaccine #3 (day 22). As negative controls, 3 punch biopsies will be obtained of 
clinically normal skin (not at the vaccine site) in the first 6 patients in Part 1 at the same 
time points (days 8 and 22). Details are provided in section 5.4.6.2. 
Leadership: This trial will be performed at two institutions in patients with high-risk 
resected stage IIB-IV melanoma. The lead institution for this trial will be the University of 
Virginia, and the PI will be Dr. Craig Slingluff. The institutional PI at M.D. Anderson 
Cancer Center will be Dr. Patrick Hwu. Part 2 will be conducted at the University of 
Virginia only.  
Population:  Criteria for inclusion include: 
 
age 18 years or older 
 
expression of HLA-A1, -A2, -A3, -B35, or -B51 
 
histologically or cytologically proven Stage IIB-IV melanoma rendered clinically free 
of disease by surgery, other therapy, or spontaneous remission.  Small (<1cm) 
findings that are equivocal on radiologic or clinical imaging will not be a basis for 
exclusion. 
 
ECOG performance status 0-1 (Appendix 3) 
 
Adequate hepatic and renal function 
 
Weight at least 50 kg (110 lbs) 
NOTE: Patients will be excluded if they are pregnant or are immunosuppressed, or 
have previously received vaccines containing an incomplete Freund’s adjuvant. 
Accrual: For Part 1, the maximum accrual to the study is estimated from the simulations to 
be approximately 52 patients. Adjusting for 10% ineligibility/dropout/withdrawal rate, 
maximum total target accrual is estimated at 58 patients. For Part 2, target accrual is 
estimated to be 16 eligible patients. Adjusting for 10% ineligibility/dropout/withdrawal rate, 
maximum total accrual for part 2 is estimated at 18 patients.  
Zone 
Study 
Arm 
Peptide 
vaccine 
Adjuvant  
Dose 
of 
TLR 
agonist 
Route 
vax 
injection 
Admin of  
TLR agonist 
2 
E2 
LPV7 
IFA + PolyICLC 
1 mg 
Id/sq 
Id/sq 

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Clinical outcome:  The primary goals are to assess safety and immunogenicity.  The study is 
not powered for differences in clinical outcome, but we will track disease-free survival, and 
overall survival. Significant differences in clinical outcome are not anticipated among the 
study arms, but trends in clinical outcome will be assessed in the context of the immunologic 
data to support decisions about the preferred vaccination strategy to test further in 
combination with other immune therapies. 
Vaccine composition:  The peptides described in Table 2 will be used in the vaccines. 
Table 2: 7 long Melanoma Peptides Restricted by MHC Class I and the corresponding 
defined minimal epitope 
 
 
Endpoints: 
Primary: 
 
 
Safety and toxicity following vaccination with 7 long peptides in melanoma patients 
with and without TLR agonists 
 
Levels of peptide-reactive CD8+ T cells in the peripheral blood (peak and durable at 
26 weeks) and vaccine site 
Secondary: 
 
CD4+ T cell responses to peptides in the vaccine, and their function. 
Exploratory: 
 
To analyze cellular infiltrates in the VSME 
 
To identify cytokine profiles of the VSME as a function of TLR signaling 
 
To identify toll-like receptor signaling in the VSME 
 
To understand regulatory processes in the replicate immunization sites 
o Regulatory T cells (CD4+CD25hiFoxP3+) 
o Myeloid-derived suppressor cells 
 
Overall Survival and disease-free survival 
 
 
 
 
 
 
Short Peptides 
Long Peptide (30-mers) in LPV7 for the present proposal 
Allele 
Minimal Epitope  
Sequence (minimal epitope underlined) 
Source  (# residues) 
HLA-A1 
Tyrosinase 240-251S* 
FTIPYWDWRDAEKSDICTDEYMGGQHPTN 
Tyrosinase 231-259 S* (29) 
HLA-A2 
 
 
Tyrosinase 369-377  
SMHNALHIYMDGTMSQVQGSANDPIFLLHH 
Tyrosinase 361-390  (30) 
gp100 209-217-2M  #  
VPLAHSSSAFTIMDQVPFSVSVSQLRALDG  
gp100 198-227 #  (30) 
MAGE-A10 254-262 
VIWEALNMMGLYDGMEHLIYGEPRKLLTQD 
MAGE-A10 245-274 (30)  
HLA-A3 
 
gp100 17-25 
LLHLAVIGALLAVGATKVPRNQDWLGVSRQL 
gp100 9-39  (31) 
MAGE-A1 96-104 
SREEEGPSTSCILESLFRAVITKKVADLVG 
MAGE-A1 82-111 (30) 
   
HLA-B35/B51 
NY-ESO-1 94-102 
GARGPESRLLEFYLAMPFATPMEAELARRS 
NY-ESO-1 79-108 (30) 
*(substitution of S for C at residue 244);   (post-translational change of N to D at residue 371);   #(209-2M, substitution of M for T 
at position 210)    

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List of Abbreviations  
Abbreviation 
Full text 
HCG 
Beta Human chorionic gonadotropin (pregnancy test) 
12-MP 
12 melanoma-derived class I MHC-restricted peptides 
AE 
adverse event 
AGC 
absolute granulocyte count 
AJCC 
American Joint Committee on Cancer 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
APC 
antigen presenting cell 
AST 
aspartate aminotransferase 
BTRF 
Biorepository and Tissue Research Facility 
CC 
Cancer Center 
cc 
cubic centimeter 
CFR 
Code of Federal Regulations 
cm 
centimeter 
CO2 
carbon dioxide 
CRF 
case report form 
C3TO 
Cancer Center Clinical Trials Database 
CT 
computed tomography 
CTA 
cancer-testis antigens 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTL 
cytotoxic T lymphocyte 
CTO 
Clinical Trials Office 
DC 
dendritic cells 
DFS 
Disease-free survival 
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 
FITC 
Fluorescein isothiocyanate 
g 
gram 
GLP 
good laboratory practice 
GM-CSF 
granulocyte-macrophage colony-stimulating factor 
GMP 
good manufacturing practice 
Hgb 
hemoglobin 
HGBA1C 
hemoglobin a1c 
HITC 
Human Immune Therapy Center 
HIV 
human immunodeficiency virus 
HLA 
human leukocyte antigen 
HPLC 
high performance liquid chromatography 
HR 
Hazard ratio 
IBW 
ideal body weight 
id 
intradermal 

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IFA 
incomplete Freund’s adjuvant 
IFN 
interferon 
IHC 
immunohistochemistry 
IL-2 
interleukin-2 
IL-7 
interleukin-7 
IL-15 
interleukin-15 
IML 
Immune Monitoring Laboratory 
in 
inch 
IND 
investigational new drug 
ip 
intraperitoneal 
IRB 
Institutional Review Board 
IRR 
immunologic response rate 
IU 
international unit 
IV 
intravenous 
kg 
kilogram 
KLH 
keyhole limpet hemocyanin 
LC 
Langerhans cells 
LDH 
lactate dehydrogenase 
LNLA 
Lymph-node-like aggregate 
LPV7 
Long peptide vaccine with 7 melanoma peptides 
LyNeo 
lymphoid neogenesis 
m 
meter 
mcCi 
microcurie 
mcg 
microgram 
mcl 
microliter 
MDACC 
M.D. Anderson Cancer Center 
MDP 
melanocyte differentiation proteins 
mg 
milligram 
MHC 
major histocompatibility complex 
mIU 
million international units 
ml 
milliliter 
mm 
millimeter 
MRI 
magnetic resonance imaging 
NBT/BCIP 
Nitro 
blue 
tetrazolium 
chloride/5-Bromo-4-Chloro-3-Indolyl 
Phosphate 
NED 
No evidence of disease, i.e. no clinical evidence of melanoma 
NCI 
National Cancer Institute 
NOS 
not otherwise specified 
NSAID 
non-steroidal anti-inflammatory drug 
OS 
overall survival 
PBL 
peripheral blood lymphocytes 
PBMC 
peripheral blood mononuclear cells 
PBS 
phosphate buffered saline 
PET 
positron emission tomography 
PHA 
phytohemagglutinin 
PI 
Principal Investigator 
PMA 
phorbol myristate acetate 
PolyICLC 
polyinosinic-polycytidylic 
acid 
stabilized 
with 
carboxymethylcellulose, and poly-L-lysine double-stranded RNA 

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ppm 
parts per million 
PRC 
protocol review committee 
RCC 
renal cell carcinoma 
RPMI 
Roswell Park Memorial Institute 
SD 
standard deviation 
SIN 
sentinel immunized node 
SQ 
subcutaneous 
TAA 
tumor associated antigens 
tet 
tetanus helper peptide 
TFA 
trifluoroacetic acid 
Th 
CD4+ helper T cells  
TIL 
tumor infiltrating lymphocytes 
TLR 
Toll-like receptor 
TNF 
tumor necrosis factor 
ULN 
upper limits of normal 
USP 
United States Pharmacopeia 
UVA 
University of Virginia 
VSME 
Vaccine site microenvironment 
WBC 
white blood cell 
w/v 
weight to volume 
v/v 
volume to volume 
 

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1.0   INTRODUCTION 
 
Abstract 
Identification of minimal peptide epitopes for melanoma reactive T cells in the early 1990s 
has provided useful tools for evaluating T cell responses to melanoma; however, the 
immune responses to vaccines using short peptides are often transient and of low  
magnitude, and they have had limited clinical activity.  Recent work suggests that 
vaccination with longer (30-mer) peptides that encompass these short peptides may be a 
more effective strategy and has been associated with clinical regressions of squamous 
vulvar neoplasia1. The most common adjuvant for peptide vaccines for melanoma has 
been a form of incomplete Freund’s adjuvant (IFA).  However, Toll-like receptor (TLR) 
agonists offer the potential to improve the magnitude and persistence of antitumor T cell 
responses, and findings from a few clinical trials support this potential2-4. However, there 
has been very limited characterization of the molecular and cellular effects of TLR 
agonists at the vaccine site microenvironment (VSME). A randomized trial of high-dose 
interleukin-2 (IL2) vs. IL2 + peptide vaccine showed improved response rate and 
progression free survival with addition of the peptide vaccine 5. Other preclinical studies 
show improved tumor control by adding vaccines to adoptive T cell therapy and to 
blockade of immunologic checkpoints. An optimized vaccine strategy, relevant across the 
large range of HLA alleles is needed. 
The proposal is for a clinical trial, performed in a collaborative multicenter setting 
involving 2 academic institutions. This trial will accrue approximately 52 eligible patients 
(Part 1) and  approximately eligible 18 patients (Part 2) in a phase I/II design with a long 
peptide vaccine (LPV7) in one of 7 adjuvant strategies of IFA alone, TLR agonist(s) 
alone, or a combination of IFA plus TLR agonist(s). Goals of this trial will include a safety 
assessment and measures of immunogenicity, and characterization of the cellular and 
molecular events induced by each TLR agonist in the VSME.  This will be a first-in-
humans evaluation of the safety and immunogenicity of 7 long 30-mer peptides (LPV7) 
for melanoma, which incorporate 7 well-characterized minimal epitopes restricted by 
HLA-A1, A2, and A3 (approximately 80% of melanoma patients) and a well-characterized 
epitope restricted by HLA-B35/B51.  We propose to test these peptides in IFA alone, an 
adjuvant combining IFA with each of the following polyICLC/Hiltonol (TLR3 agonist), 
resiquimod (TLR7/8 agonist), and both polyICLC and resiquimod, to be administered 
locally at the site of vaccination.  
 
1.1   Study Rationale 
Cancer immunotherapy for solid tumors is coming of age, with FDA-approved immuno-
therapeutics now available for the treatment of multiple tumor types.  In melanoma, 
checkpoint inhibitors specific for CTLA-4 and PD-1 have been shown to induce durable 
clinical regressions and improve overall survival. There is excitement about the growing 
armamentarium 
of 
systemic 
immunotherapeutics, 
whose 
effects 
are 
mediated 
predominantly by T lymphocytes.  However, despite the effectiveness of those therapies, 
checkpoint inhibitors fail in about 70-80% of patients, and these patients often succumb 
to their disease. There is a need for new combination approaches that build on the 
demonstrated clinical value of immune therapy. 
Cancer vaccines inducing antigen-specific T cell responses are emerging as a 
component of combination immunotherapy.  In the past few years, a cancer vaccine has 
been approved for prostate cancer, based on two randomized trials showing improved 

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survival and a randomized prospective trial showed that adding a peptide vaccine to high-
dose IL-2 significantly prolonged progression-free survival (PFS) when compared to IL-2 
alone.83;5  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 therapy.  
 
There is a need to improve defined antigens for melanoma vaccines.  Peptide vaccines for 
melanoma offer the promise of inducing T cells reactive to well-characterized tumor antigens 
and also enabling assessment of effectiveness of the vaccinations by monitoring antigen-
specific T cell responses.  However, clinical experience with peptide vaccines has been 
mixed.  On one hand, we and others have found that selected peptides can induce 
circulating T cell responses in a majority of patients,8;9 and that vaccination with a mixture of 
peptides can be immunogenic in up to 100% of patients.8  In some trials, circulating immune 
responses have correlated with clinical outcome8;10, but this has not been the rule.  Also, the 
magnitude of T cell responses sometimes is substantial, with 1-5% of circulating CD8 T cells 
reactive to a single antigen 4;11;12; however, responses in most patients represent T cell 
percentages that are 1-2 orders of magnitude lower, which may or may not be adequate for 
clinical benefit. The T cell responses to vaccines may be very durable, for months or years, 
but are at least as likely to be transient, sometimes declining even while still receiving 
vaccines.13 Clinically, there have been durable clinical responses in some patients receiving 
melanoma vaccines, suggesting that there is clinical activity.14  However, the overall clinical 
response rates are only about 3-5%.15  Thus, vaccines are not optimized, and both the 
antigens and the adjuvants may be improved. 
Long peptides.  Recent work with longer (30-mer) peptides that encompass short minimal 
epitope peptides suggests that these longer peptides may be more effective immunogens 
than the minimal peptides. The extra length contributes to a tertiary structure that may 
protect from peptidases, and they are too long to be presented directly on MHC; so 
intracellular processing is required.  Importantly, this further ensures that the peptides are 
presented just on professional APC. A vaccine using long peptides for squamous vulvar 
neoplasia has been associated with high rates of clinical regressions.1  This 
proposal 
evaluates long peptides as immunogens for melanoma vaccines. It is for a first-in-human 
evaluation of safety and immunogenicity of 7 long peptides for melanoma, which 
incorporate well-characterized minimal epitopes restricted by HLA-A1, A2, and A3 and 
HLA-B35/51.   
Adjuvants for cancer vaccines. Some data have challenged the effectiveness of the 
current formulation of incomplete Freund’s adjuvant (IFA) with a peptide vaccine, 
especially for induction of Th1/Tc1 T cell responses,16 while data from our own 
experience supports its adjuvanticity17. However, data from our own experience, and from 
our collaborators also show that vaccines using short peptides in IFA induce chronic 
inflammation at the site of vaccination that attracts and retains antigen-specific T cells at 
the vaccine site and may cause T cell dysfunction 18;19. Thus, there is a need to optimize 
adjuvants; there also is reason to expect that IFA will function differently with long than 
with short peptides, as the epitopes derived from processing the long peptides will be 
presented in the draining nodes, rather at the sites of vaccination. 
Toll-like receptor (TLR) agonists offer the potential to improve the magnitude and 
persistence of antitumor T cell responses 4; however, most trials of TLR agonists have 
been limited to use of one TLR agonist formulation, and have not defined the molecular 
and cellular effects at the vaccine site microenvironment (VSME).  In the present study, 
we propose to test each of 2 TLR agonists in parallel and in combination, with or without 
IFA, and to evaluate the added value over IFA alone. These include agonists for TLR3, 
TLR7, and TLR8, to be administered locally at the site of vaccination.  

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Combination immune therapy: This proposal will enable selection of an effective 
immunization strategy for combination with other effective systemic immune therapies in 
future studies.  Vaccination with a single HLA-A2 restricted peptide in IFA improved 
response rate and progression-free survival when combined with HD IL2 in a randomized 
prospective trial for Stage IV melanoma5. Progression-free survival was the primary 
endpoint for that study. Despite the fact that the study was not powered to detect 
differences in survival, the vaccine-treated patients had a strong trend to improvement in 
overall survival (p = 0.06)5. However, vaccination with HLA-A2 peptides plus IFA alone in 
another study had no impact on clinical response.20 The trial that produced the 
encouraging positive finding in combination with HD IL2 needs to repeated both for 
confirmation of benefit and to evaluate impact on survival in an adequately sized study.  
Recent concerns with the inadequacy of IFA alone as an adjuvant have prevented 
investigators from moving to a confirmatory trial until a more optimal adjuvant is available.   
Other preclinical studies show that adding a vaccine to adoptive T cell therapy increases 
its therapeutic efficacy.21-24 Also, vaccination can synergize with PD1/PDL1 blockade to 
increase immune responses and tumor control.25  Thus, there is evidence to support the 
value of combining vaccines plus any of several effective immune therapies.  However, 
vaccines are not yet optimized.  The present application will test a vaccine comprised of 
multiple long peptides and multiple adjuvants. The use of peptides restricted by HLA-A1, 
A2, A3, and B alleles has a significant advantage for speeding accrual for a future 
definitive trial by almost doubling potential eligibility over HLA-A2 restriction alone (more 
than 80% vs. 45% HLA appropriate). 
Vaccine site selection:  Preliminary data from analysis of several clinical trials of peptide 
vaccines at the University of Virginia suggests that patients who have been vaccinated at 
the same skin site each week (for at least 3 vaccines, same site vaccination) have had 
stronger T cell responses than those with vaccines administered to a different skin site 
with each vaccine (rotating vaccine sites).  However, those data were based on 
retrospective assessments among trials with varied vaccine adjuvants and over extending 
time periods.  Also, the differences were not definitive enough to be assured that site 
makes a difference in outcome, We hypothesize that same site vaccination with LPV7 will 
be safe and will result in stronger CD4+ and CD8+ T cell responses to those peptides.  
Preliminary data from Part 2 will assess whether there may be a basis for exploring this 
question in a definitive randomized trial, and will provide a basis for powering such a 
study. 
 
1.2   Vaccine Design and Surgical Excision  
 
1.2.1   Antigen-Specific Immune Responses Induced by Peptide Vaccines  
We have substantial experience in studies with multipeptide vaccines, and with high 
antigen-specific immune response rates in the peripheral blood and in vaccine-draining 
nodes.8;9;12  These provide a strong foundation for evaluation of the multipeptide vaccines 
proposed in this application, and for the immunologic analyses. 
1.2.2   Melanoma Peptides Restricted by MHC Class I Molecules and Incorporated in the 
prior vaccine trials, that are selected for use in the LPV7 mixture 
Antigens representing the majority of melanoma-associated antigens identified thus far can 
be divided into two groups.  The first group consists of melanocytic differentiation proteins 
(MDP); such antigens are only expressed on cells of the melanocytic lineage and include 
MART-1/MelanA, gp100/Pmel17, tyrosinase, TRP-1/gp75 and TRP-2 proteins.  The second 
group consists of cancer-testis antigens (CTA) such as MAGE proteins, LAGE-1, and NY-
ESO-1.  These proteins are encoded by genes expressed in several tumors of different 

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histologic types, but not in normal tissues, other than testis and placenta.  Peptides derived 
from these proteins are recognized by CTL in the context of various HLA alleles.  
 
Melanocytic differentiation proteins: Gp100 is an enzyme involved in melanin synthesis.  
Multiple epitopes restricted by Class I MHC molecules have been identified from this protein, 
including peptides restricted by HLA-A2, and HLA-A3 26-28, and we chose to incorporate in 
the present proposal long peptides that represent the following short epitopes: gp100209-217-
2M IMDQVPFSV (substitution of M for T at position 210; HLA-A2), and ALLAVGATK (HLA-
A317-25). The parent gp100209-217 epitope (ITDQVPFSV) has been modified to incorporate a 
methionine residue at position 210 of the peptide (IMDQVPFSV).  This modification 
increases the binding affinity of the IMDQVPFSV peptide for HLA-A2 and increases its 
immunogenicity in vivo when compared to the native peptide29;30.  Another gp100 derived 
epitope, ALLAVGATK, is also naturally processed and presented by melanoma cells, and 
holds promise for induction of HLA-A3 restricted responses in vivo 28.  CD8+ T cell 
responses to the ALLAVGATK peptide have been detected in multiple prior melanoma 
vaccine trials 12;31-34. T-cell lines cultured from lymphocytes infiltrating human melanomas, 
when administered adoptively to participants with metastatic melanoma, have induced 
partial or complete remissions in a large subset of participants. Clinical response to this 
therapy has been reported to be increased when the T-cells used for therapy recognize 
peptides derived from Pmel-17/gp100 35.  Thus, there is substantial evidence to support the 
use of the gp100-derived peptides in the melanoma vaccines. 
 
Tyrosinase is an enzyme involved with melanin synthesis.  Epitopes derived from tyrosinase 
have been identified for HLA-A1, HLA-A2, and other class I alleles 26.  In the proposed 
study, we are including long peptides incorporating one epitope (DAEKSDICTDEY) 
restricted by HLA-A1 and one epitope (YMDGTMSQV) restricted by HLA-A2.  The parent 
epitope of tyrosinase240-251, DAEKCDICTDEY, has been modified to incorporate a serine 
residue at position 244 of the peptide (DAEKSDICTDEY). This modification prevents 
disulfide bond formation within the peptide, but does not interfere with HLA-A1 binding or 
with T cell recognition 36. In phase II clinical trials conducted by the UVA HITC (UVA-Mel31, 
Mel36, Mel39, Mel43, and Mel44), immunological responses against the DAEKSDICTDEY 
peptide were detected as the dominant peptide target for participants who expressed HLA-
A1 and who were immunized with a synthetic peptide mixture containing the 
DAEKSDICTDEY peptide12;31-34. CTL cultured from a vaccine draining node (sentinel 
immunized node, SIN) after vaccination with DAEKSDICTDEY were capable of lysing HLA-
A1+ tumor cells naturally expressing tyrosinase37.   The naturally occurring HLA-A2 restricted 
epitope from tyrosinase, tyrosinase369-377 YMDGTMSQV, contains a post-translational 
modification of an asparagine to aspartic acid at residue 371 38.  In phase II clinical trials 
conducted by the UVA HITC, immunological responses to the YMDGTMSQV peptide have 
been detected frequently in stage III and IV melanoma participants who expressed HLA-A2, 
and were immunized with a synthetic peptide mixture containing this peptide 12;31-34.   
 
MAGE-A1 and MAGE-A10 are members of the MAGE gene family and are expressed in the 
testes, as well as in several different tumor types such as melanoma, breast, prostate, 
esophagus, colon, and lung 26. One epitope derived from MAGE-A1 (SLFRAVITK) is 
included in the proposed study.  The MAGE-A196-104 epitope (SLFRAVITK) was identified by 
Chaux et al. 39, and was first tested in humans in UVA-Mel39, in which immunogenicity and 
safety have been demonstrated 33.  It is a highly immunogenic peptide in the Mel39, Mel43, 
and Mel44 trials 8;12;40. 
 

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The MAGE-A10254-262 epitope (GLYDGMEHL) was identified by Huang et al., and was 
shown to be naturally processed and presented by HLA-A2+/MAGE-A10+ melanoma cell 
lines.41  In a report by Valmori et al., CTL responses to this epitope were readily detected in 
two-thirds of participants with melanoma whose tumors tested positive for the expression of 
MAGE-A10.42  However, the ability of MAGE-A10 synthetic peptide to stimulate immunologic 
responses in humans had not been tested until UVA-Mel39, in which immunogenicity and 
safety have been demonstrated and this was found to be very immunogenic33. Followup 
studies further support immunogenicity of this MAGE-A10 peptide12;34. 
 
The cancer testis antigen NY-ESO-1 has been extensively studied in human clinical trials, 
including peptide, protein, and DNA 2;43-46.  Recently, a set of 4 overlapping long (30-mer) 
peptides have been tested for safety and immunogenicity in patients with NY-ESO-1+ 
ovarian cancer, using either IFA alone, or IFA plus polyICLC (1.4 mg) 2.  That study 
incorporated the NY-ESO-1 peptide to be used in the current trial (NY-ESO-179-108; CD4+ 
and CD8+ T cell responses were induced to a mixture of peptides covering the NY-ESO-1 
sequence over residues 79-130, in a majority of evaluated patients. 
 
For the present study, the 7 peptides have been selected for use as long peptides. Their 
sequences are listed in Table 2 in the protocol précis.  The reason for selecting six of them 
is that their minimal epitopes are among the most immunogenic in the set of 12 peptides 
included in our prior trials 
12;33;34: DAEKSDICTDEY, YMDGTMSQV, IMDQVPFSV, 
GLYDGMEHL, ALLAVGATK, SLFRAVITK, and they have been administered safely 12;33;34. 
We had also selected the peptide EVDPIGHLY from those prior trials, but the long peptide 
encompassing EVDPGIHLY could not be synthesized with adequate purity; the remaining 6 
peptides were selected for use.  
 
The NY-ESO-179-108 peptide was included based on the encouraging immunogenicity and 
safety findings referred to above with four NY-ESO-1 peptides2.  Among the four peptides 
used in that trial, this one was available in adequate quantity as GMP-grade preparation.  
 
Selection and synthesis of long peptides encompassing the short peptide antigens:  The 
NY-ESO-179-108 peptide incorporates a defined nonamer epitope restricted by HLA-B35 and 
HLA-B51; the sequence was one of 4 previously used as the 30-mer peptide in a phase I 
trial.  For the present study, the 7 long peptides selected for use have been designed to 
incorporate the minimal epitope sequences above, plus extension at the N and C terminal 
ends, with attention to avoid N-terminal glutamic acid or glutamine residues, to minimize 
cysteine residues, and to maximize incorporation of polar residues to aid in aqueous 
solubility. Their sequences are listed in Table 2 in the protocol précis.   
 
1.2.3   Montanide ISA-51 as a Vaccine Adjuvant.    
Montanide adjuvant has been effective at inducing immune responses against murine viral 
antigens when administered with a synthetic peptide epitope 47;48.  Montanide ISA-51 has 
also been used with peptides plus GM-CSF-in-adjuvant vaccine useful for enhancing both 
cellular and humoral immunity.49  The product consists of a mineral oil base comparable to 
incomplete Freund's adjuvant.  However, the Arlacel A emulsifying agent of incomplete 
Freund's, which has caused reactions in the past, has been replaced with a purified 
mannoside 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 500 participants.  Immunological responses against the 

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immunizing peptides have been detected in most participants.  Our recent experience with 
the current formulation of this adjuvant is published.17;50    
 
1.2.4   PolyICLC (Hiltonol, Oncovir Inc.), TLR3 agonist.  
Polyinosinic-Polycytidylic acid (PolyIC) is a double-stranded RNA (dsRNA) that acts as a 
TLR3 agonist. However, its short half-life limits its usefulness. To increase half-life and its 
practical use in clinical settings, it has been stabilized with polylysine and 
carboxymethylcellulose as polyICLC. Like polyIC, polyICLC is a TLR3 agonist.  TLR3 is 
expressed in the early endosome of myeloid DC; thus polyICLC preferentially activates 
myeloid dendritic cells, favoring a Th1 cytotoxic T-cell response.51;52 PolyICLC also activates 
NK cells and induces cytolytic potential.52 It has been administered in the emulsion with 
Montanide ISA-51 plus long peptides, with increased immunogenicity over Montanide ISA-
51 plus peptides alone; the combination was safe, with some significant local reactions but 
no DLTs 2. 
 
Source:  Hiltonol is provided by the Ludwig Institute for Cancer Research and its Cancer 
Vaccine Consortium at no charge; as they have purchased a lot of it from Dr. Andres 
Salazar from Oncovir, Inc. (Washington, D.C.; letter provided). It is provided as a clinical 
grade reagent for experimental use in single-use vials containing 1 mL of a 2 mg/mL 
solution.  
 
Dosing and preparation: It has been used safely in cancer patients, with intravenous doses 
up to 300 mcg/kg.53 We will administer 1 mg (0.5 mL) per vaccine, as used in other trials 
(e.g.: NCT01008527).   
 
1.2.5   Resiquimod: TLR7 and 8 agonist.  
This agent is an agonist for both TLR7 and TLR8. TLR8 activation may reduce regulatory T 
cells.54 It has been safe when administered topically as a 0.05% gel once a week; more 
frequent administration led to dose-limiting local skin toxicity.55 Thus, w eekly dosing is 
selected for this trial. Other human experience with resiquimod is that oral dosing at 0.01 
mg/kg (700 mcg/70kg) caused increases in serum IFN-alpha with associated side effects, 
but it was well tolerated.56   It has been used (NCT00821652) safely at 500 mg 0.2% 
resiquimod gel per application up to 3x/week after each vaccine, with the dose spread over 
a 25 cm2 area each time. 
 
Source: Resiquimod is provided by 3M Pharmaceuticals, St. Paul, MN.  It is not yet FDA 
approved, but is available as a clinical grade reagent for topical use.  We have approval 
from Mark Tomai at 3M to provide it for clinical use as a vaccine adjuvant.  
Dosing and preparation: Multi-dose tubes contain approximately 3 grams of 0.2% 
resiquimod gel.  The dose of resiquimod in 500 mg of 0.2% resiquimod gel is 1000 mcg. 
Immediately after vaccine injection, measure out approximately 500 mg of 0.2% resiquimod 
on dosing paper and apply resiquimod gel with a gloved finger over a 25 cm2 area of the 
skin at the vaccine site.  Using this dosing method, the dose range of resiquimod will be 
approximately 400-600 mg.  Rub the gel into the skin for 1 minute until it vanishes.  
Following application, the site may be covered with an occlusive dressing, such as 
Tegaderm, for about 8 hours.  Participants are encouraged to wash the area at 8 hours 
after application.The tubes may be retained in the investigational pharmacy between 
uses. 
 

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1.2.6   Integration of Peptide-Based Vaccines in the Adjuvant Setting  
High-dose interferon (IFN-alpha therapy) is approved by the FDA for use in patients with 
resected stage IIB or III melanoma treated in the adjuvant setting.  Approval was based 
largely on survival improvement observed following a one-year regimen with IFN as 
described in ECOG 1684.57  However, survival improvement is modest and data from a 
subsequent trial, ECOG 1690, failed to reproduce differences in survival.58  Data from a third 
trial, ECOG 1694, reveal a statistically significant, but still modest, survival improvement with 
IFN therapy compared to a ganglioside vaccine.59  Thus, any patient with resected stage IIB 
or III melanoma should be offered IFN therapy, which is routine practice at UVA and 
MDACC. However, the toxicity of high-dose IFN therapy is substantial.  Approximately 25-
30% of patients withdraw from therapy because of toxicity, and approximately 60% of 
patients require dose reductions during treatment.57   
 
Ipilimamb 10mg/kg is approved by the FDA for use in the adjuvant setting for patients who 
have undergone complete resection of cutaneous melanoma with pathological involvement 
of regional lymph nodes greater than 1 mm. Approval was based largely on results from the 
the Phase 3 EORTC 18071/CA184029 trial.84 The ipilimumab 10mg/kg treatment yielded 
statistically significant prolonged distant metastasis-free survival, with five-year distant 
metastasis-free survival rate 9% higher than the placebo.  However, the toxicty of 
ipilimumab 10mg/kg is is substantial, with approximately 50% of patients who started 
ipilimumab withdraw ing as a result of ipilimumab related toxicities. 
 
The experience at UVA is that most patients who are candidates for therapy refuse IFN 
because of concerns with toxicity, despite being informed IFN therapy is one of the few FDA 
approved adjuvant therapies for melanoma and that therapy may provide a survival 
advantage.  In addition, many patients are not eligible for the current approved dose of 
ipilimumab (10 mg/kg) or decline treatment with ipilimumab because of the potential for 
serious immune-related toxicities.  Recent data from the E1609 study presented at ASCO  
showed that there was no difference in disease-free-survival between ipilimumab 3 mg/kg 
and ipilimumab 10 mg/kg (E1609); however, ipilimumab 10 mg/kg was associated with 
greater toxicity.85  Thus, future approved treatment options may include ipilimumab 
administered at the lower dose of 3 mg/kg, although even the lower dose carries risk of 
death.  The same may be true for other checkpoint inhibitors that are currently being 
explored in the adjuvant setting (e.g. PD-1 blockade). 
 
1.2.7   Toxicology 
There is no reason to expect direct toxicity of the melanoma peptides; they are not directly 
cytotoxic in vitro and are not expected to have functional implications.  On the other hand, 
because some of these peptides are identical or similar to a portion of a normal protein, risks 
of autoimmunity in humans are reasonable to evaluate.  Unfortunately, there is no murine 
system adequately modeling the human immune response to these peptides.  The most 
meaningful evaluation of this peptide vaccine mixture is in patients with melanoma.  This trial 
is for participants with resected stage IIB/C, III, or IV NED melanoma.  These individuals 
face a high risk (> 40%) of death from melanoma, 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 participants.  The 
potential implications of autoimmunity against cells of melanocytic lineage are illustrated by 
reported cases of vitiligo occurring coincident with regressions of melanoma.60  Most of 
these are limited, often occurring in skin surrounding the regressing melanoma, but 

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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 and hair pigment can be striking in cases of vitiligo, 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 reported either as a 
complication of successful immunotherapy with melanoma-reactive CTL or spontaneous 
vitiligo.  However, some ocular toxicity has been reported that resolved with steroid 
treatment.61   
 
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).   
 
Thus far, 10-15% of participants receiving peptide-based vaccines in the studies at UVA 
were diagnosed with definite vitiligo.  In all cases, the vitiligo was asymptomatic.  No major 
visual/ocular toxicity was reported for participants on these prior trials. 
 
Toxicities Previously Reported for Participants Receiving the 12-MP vaccine administered in 
Montanide ISA-51 adjuvant 
Participants have been vaccinated with a mixture of 12 short melanoma peptide mixture, 
plus tetanus peptide, in Montanide ISA-51 adjuvant (MELITAC 12.1) in 62 participants on 
the UVA-Mel43 trial, to about 80 participants in the UVA-Mel44 trial, and to about 40 
participants on the UVA-Mel48 trial.  Toxicity data for the first 102 participants on the Mel43 
and Mel44 trials were graded using the NCI Common Terminology Criteria for Adverse 
Events v3.0 and are listed regardless of attribution.  Maximum Grade 2-4 toxicities 
experienced by at least 10% of participants are listed in Table 3: 
 
Table 3.  Toxicities related to administration of MELITAC 12.1 melanoma 
vaccine administered with Montanide ISA-51 (IFA) in 102 patients on 
UVA-Mel43 and UVA-Mel44 clinical trials 
Toxicity 
Grade 2 
Grade 3 
Fatigue (lethargy, malaise, asthenia) 
17% 
3% 
Injection site reaction 
87% 
2% 
Ulceration 
7% 
3% 
 
The grading of injection site reactions was modified from CTC v2.0 to CTCAE v3.0, and now 
to v4.03.  CTC v2.0 did not have an ulceration Adverse Event Term, therefore participants 
with ulceration in conjunction with an injection site reaction were graded as experiencing a 
Grade 3 injection site reaction (ulceration or necrosis that is severe; operative intervention 
indicated).  In prior UVA HITC studies, an injection site reaction with ulceration ≤ 2 cm in 
maximal diameter was an expected adverse event in a subset of participants.  However, an 
injection site reaction that included ulceration >2 cm in diameter, or an injection site reaction 
requiring debridement, narcotic analgesics for pain, or surgery was not an expected toxicity 
and was considered a dose-limiting toxicity (DLT) and was graded as a Grade 3 injection 
site reaction. UVA-Mel43 used the CTC v2.0 grading system whereas UVA-Mel44 used the 
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therefore, ulceration and injection site reactions can be graded separately.  For this study, 
participants who experience induration at their injection site, both injection site reaction and 
induration will be graded and reported.  The data in Table 3 have been corrected to be 
consistent with CTCAE v3.0. The criteria for each of these toxicity categories (fatigue, 
injection site reaction, and ulceration) have changed from v3.0 to v4.03, but are very similar.  
Thus, they are probably comparable, but the exact details for assessing them in v4.03 
require information that was not collected at the time those studies were performed. In the 
proposed study, we will grade based on v4.03, and we do not expect Grade 3 injection site 
reactions; ulceration and injection site reaction toxicities will be expected at Grade 2.   
 
1.2.8   Patient acceptance of biopsies of skin at the Vaccine Site  
This study will include three punch biopsies 4mm in diameter of the vaccine site, on two 
dates.  We have previously performed much larger excisions of skin and subcutaneous 
tissue approximately 2 x 4-6 cm in size.50  There were questions, prior to that study, of 
whether that size excision would be too large to expect participants to tolerate or to accept it.  
As a surgeon, the Principal Investigator (PI) routinely performs excisions 2-4 cm wide (and 
about 6-12 cm long) in patients when they have wide excisions of melanoma, and these can 
reliably be closed primarily, without a skin graft, when taken from the upper thigh and upper 
arm.  Thus, excisions of a 2x4 cm skin section (regardless of depth) can be closed primarily 
with minimal morbidity on those locations where the vaccines would be given.   
 
Also, we surveyed a convenience sample of 52 of our patients during clinic visits for their 
willingness (on a scale of 1 to 9) to participate in a clinical trial of a melanoma vaccine with 
or without a skin biopsy.  Forty-three (43) of the 52 patients (83%), answered some of the 
questions, and 37 (71%) answered all questions.  Findings are summarized in Table 4 below 
and have been published62.  Willingness to enter such a trial was diminished by any biopsy 
requirement, but interest in accrual remained high even with a biopsy.  Interestingly the size 
of the biopsy had a much smaller impact than the fact that any biopsy was required.  In prior 
trials, we have included excision of a vaccine-draining lymph node (sentinel immunized 
node), which requires a 3 cm incision, and we have done over 150 of such biopsies on 
vaccine patients.31-33;37    Based on that questionnaire, patients were expected to be about 
70-80% as likely to enroll on a trial with a 6 cm excision vs. a 3 cm excision as without a 
biopsy.  The actual accrual matched closely the predicted accrual.62 
 
The present proposal is to remove much less skin that was done in that prior protocol 
(Mel48).   Thus, we anticipate rapid accrual and high patient acceptance. We typically have 
accrued 40-60 patients per year on trials with sentinel immunized node biopsies; so if we 
accrue at even half that rate (20-30 per year), we expect to meet accrual goals.   
 
Table 4.  Findings from vaccine biopsy questionnaire (n = 91) 
Willingness to enter 
vaccine trial with  or 
without skin excision 
N  
Likely (5) 
to highly likely  (9) 
Moderately likely (7) 
to highly likely (9) 
Highly likely 
(score 9) 
No skin excision 
43 
95 % 
69 % 
54 % 
3 cm scar 
37 
74 % 
47 % 
28 % 
 
1.2.9   Regulatory Issues 
The peptides for this trial have been synthesized under GMP conditions by a commercial 
provider.  Prior experience has been with peptides made by Polypeptide Group, San Diego, 
California.  They have been vialed under GLP conditions in our HITC lab clean room, which 

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has limited access, an entry room for gowning and gloving, and positive pressure ventilation 
and laminar flow hoods. Lot release testing of the final vialed peptides will be completed in 
accord with CFR guidelines.  Stability testing will be conducted regularly, in accord with the 
Chemistry and Manufacturing section of the IND application. 
 
Montanide ISA-51 is produced by Seppic, Inc. (Fairfield, NJ and Paris, France).  A master 
drug file for Montanide ISA-51 is filed with the FDA.  This agent has been used in hundreds 
of patients in our own experience and in thousands of patients worldwide.   
 
This study will be approved by the Institutional Review Board (IRB) of the University of 
Virginia and by the IRBs of M.D. Anderson Cancer Center.  This protocol will be submitted to 
the FDA as an IND application.  Records of all study review and approval documents will be 
kept on file by the Principal Investigator, and are subject to FDA inspection during or after 
completion of the study.  The Institutional Review Board will receive notification of study 
closure within three months of study termination or completion.   
           
This study will be conducted in compliance with Good Clinical Practices and regulations. 
 
1.2.10    Same site vaccination (Part 2) 
The clinical trials group at the University of Virginia has extensive experience in several 
hundred patients with administering peptide vaccines in IFA, with or without other immune 
modulators, at the same skin site for each vaccine. This has been well-tolerated 34;77-99.  
Later trials with rotating vaccine sites were performed for several reasons, but with the 
expectation that vaccine site reactions would be less prominent; however, overall vaccine 
site reactions have been similar with both approaches (Table 5): 
 
Table 5:  Vaccine Site Reactions 
 
Clinical trial 
N 
CTCAE 
Grade 1 
Grade 2 
Grade 3 
Injection site reaction 
Mel44 
170 
V3 
17% 
75% 
4% 
Mel58 
51 
V4 
35% 
61% 
0% 
Ulceration 
Mel44 
170 
V3 
1% 
19% 
6% 
Mel58 
51 
V4 
0% 
4% 
2% 
Note: data are from the published data from the Mel44 trial 2 and the 2017 IND report for the Mel58 
trial. 
 
The LPV7 vaccine has been well-tolerated in the patients on Part 1.  Same site vaccination 
will be assessed for safety/tolerability and immunogenicity in a pilot cohort in Part 2. 
 
 
2.0   STUDY OBJECTIVES 
 
2.1   Part 1 (completed) 
(1) To assess safety of vaccination with a mixture of long peptides for patients with high-
risk melanoma, in each of 7 adjuvant preparations, including TLR agonists and/or 
incomplete Freund’s adjuvant (IFA) 
(2) To estimate the immunogenicity of 7 long peptides in each of 7 adjuvant preparations 
Exploratory 
(3) To evaluate the molecular and cellular changes at the vaccine site and systemically  

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2.2   Part 2 
(4) To assess safety of vaccination of the optimal combination identified in Part 1 when 
administered in the same skin site for all 6 vaccines (same site vaccination). 
(5) To estimate the immunogenicity of the optimal combination identified in Part 1 when 
administered in the same skin site for all 6 vaccines (same site vaccination). 
 
3.0   STUDY OUTLINE 
3.1   Type of Study 
This is an open-label, randomized, phase I/II study, using an adaptive design, to test the 
safety and immunogenicity of a novel melanoma vaccination approach using long peptides 
plus TLR agonists, along with characterization of cellular and molecular events at the 
cutaneous site of immunization.   
 
Regimen:   
Part 1:  Patients will be registered on this phase I/II trial and will be vaccinated with LPV7 
at 300 mcg peptide/dose, and tetanus peptide at 200 mcg/dose, every week x 3 then 
every 3 weeks x 3.  The adjuvants are described, and will be administered to patients in 7 
study groups as shown in Tables 1 and 5.  This incorporates an adaptive design with 
sequential initiation of accrual to study Zones with increasing numbers of adjuvants (Arms A-
C, then D-F, then G) and a 3 week interval between subjects 1 and 2 of each arm of the 
study.  The design allows, during that 3 week interval, randomization to arms that remain 
open. 
Part 2:  Patients will be registered on this phase I/II trial and will be vaccinated with LPV7 
at 300 mcg peptide/dose every week x 3 then every 3 weeks x 3.  For part 2, the vaccine 
regimen is selected based on available data and the study decision rules from Part 1 which 
was determined to be Arm E. For Part 2 accrual will be to Study Arm E2. However, a 
modification is made to exclude the tetanus helper peptide, due to (a) lack of availability of 
that peptide component, (b) the finding that, in part 1, LPV7 peptides do induce helper T cell 
responses themselves (unpublished data), and prior data that adding the tetanus helper 
peptide did not add significantly to immunologic or clinical outcome of a peptide vaccine 
designed to induce CD8 T cells1. 
 
4.0   PARTICIPANT SELECTION 
 
All questions regarding eligibility should be directed to the Human Immune Therapy Center 
(HITC) at the University of Virginia. 
 
4.1   Inclusion Criteria 
4.1.1   Histologically or cytologically proven Stage IIB-IV melanoma (at diagnosis or at the 
time of recurrence) rendered clinically free of disease by surgery, other therapy, or 
spontaneous remission within 6 months prior to registration; patients with treated brain 
metastases may be eligible if they meet the criteria in 4.1.4.  Small radiologic or clinical 
findings of an indeterminate nature will not be a basis for exclusion, and brain metastases 
treated in accord with section 4.1.4 will not be a basis for exclusion. 
 
Staging of cutaneous melanoma will be based on the 7th edition AJCC staging system 
(Appendix 2)63. 
 

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Staging of mucosal melanomas will be based on the following system modified from the 
cutaneous melanoma staging system:  2.01- 4mm primary with ulceration or > 4mm 
primary= stage IIB, lymph node metastases = stage III, distant metastases = stage IV.  
 
4.1.2    Patients may have had multiple primary melanomas. 
4.1.3   Patients may have had or may have a metastasis from a cutaneous primary site, 
mucosal primary site, or unknown  primary site. 
4.1.4   Patients with brain metastases may be eligible if all of the following are true: 
 
The total number of brain metastases ever is less than or equal to 3. 
 
The brain metastases have been completely removed by surgery or 
have 
been 
treated 
completely 
by 
stereotactic 
radiotherapy.  
Stereotactic radiotherapy, such as gamma knife, can be used up to 1 
week prior to study entry.  In the absence of concerning clinical 
findings, repeat scans are not required after stereotactic radiotherapy if 
the patient enrolls within 8 weeks of completing the stereotactic 
therapy. 
 
There has been no evident growth of any brain metastasis since 
treatment. 
 
No treated brain metastasis is greater than 2 cm in diameter at the time 
of protocol entry. 
4.1.5   Patients must have at least one axillary and/or inguinal lymph node basin that is 
intact (no prior excisional biopsy of a node or complete lymph node dissection). 
4.1.6   All patients must have: 
 ECOG performance status of 0 or 1. 
 Ability and willingness to give informed consent. 
4.1.7   Laboratory parameters as follows: 
1) HLA-A1, A2, A3, B35, or B51.    
2) ANC > 1000/mm3, and Platelets > 100,000/mm3 and Hgb > 9 g/dL 
3) AST and ALT up to 2.5 x upper limits of normal (ULN). Patients 
known to have Gilbert’s disease may be eligible with AST and ALT 
up to 5 x ULN. 
4) Bilirubin up to 2.5 x ULN 
5) Alkaline phosphatase up to 2.5 x ULN.  
6) Creatinine up to 1.5 x ULN 
7) Hgb-A1C level of ≤ 7.5% 
4.1.8   Patients must be 18 years or older at study entry. 
 
4.2    Exclusion Criteria 
 
4.2.1   Patients who have had brain metastases unless they meet the criteria outlined in 
section 4.1.4. 
4.2.2   Patients with melanoma arising from uveal/ocular primary sites 
4.2.3   Patients who are currently receiving systemic cytotoxic chemotherapy, radiation, 
targeted molecular therapy (e.g. vemurafenib, other inhibitor of mutant BRAF, MEK, or cKit), 
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weeks (except as specified in section 4.2.4).  Gamma knife or stereotactic radiosurgery may 
be administered within the prior 4 weeks, but must not be administered less than one week 
prior to study registration.  Patients who are currently receiving nitrosoureas or who have 
received this therapy within the preceding 6 weeks are excluded.   
4.2.4   Patients who are receiving or have been treated with antibody to CTLA4 (e.g. 
ipilimumab), PD-1, PD-L1, CD137, or CD27 within the prior 12 months.  Any patient who has 
had one or more of these therapies greater than 12 months prior and is clinically free of 
disease and totally recovered from toxicities related to those therapies can be eligible.  
4.2.5   Patients with known or suspected allergies to any component of the vaccine. 
4.2.6   HIV positivity or evidence of active Hepatitis C virus. 
4.2.7   Patients receiving the following medications at study entry or within the preceding 4 
weeks are excluded: 
1) Agents with putative immunomodulating activity (with the 
exception of non-steroidal anti-inflammatory agents and topical 
steroids (see section 4.2.7 (3)). 
2) Allergy desensitization injections. 
3) Systemic corticosteroids, administered parenterally or orally.  
Inhaled steroids (e.g. Advair®, Flovent®, Azmacort®) are not 
permitted.  Topical corticosteroids are acceptable, including 
steroids with very low solubility administered nasally for local 
effects only (e.g. Nasonex®). 
4) Any growth factors (e.g. GM-CSF, G-CSF, erythropoietin). 
5) Interferon therapy. 
6) Interleukin-2 or other interleukins. 
7) For immune modulating agents referred to in 4.2.3 and 4.2.4, it 
may be necessary for more than 4 weeks to have elapsed 
since completion of that therapy (as specified in those 
sections). 
 
4.2.8   Prior melanoma vaccinations may be an exclusion criterion in the following 
circumstances: 
1) Patients who have received, within the prior 5 years, 
melanoma 
vaccines 
or other vaccines containing an 
incomplete Freund’s adjuvant (IFA; such as Montanide ISA-
51).   
2) Patients who have received any melanoma vaccine within the 
past 12 months. 
3) Patients who have been vaccinated in the past with any of the 
melanoma peptides in the LPV7 vaccine included in this study. 
 
4.2.9   Other investigational drugs or investigational therapy if the patient is currently taking 
those drugs/therapy, or if they have received the drugs/therapy within 1 month. 
4.2.10   Pregnancy or the possibility of becoming pregnant during vaccine administration.   
Female patients of child-bearing potential must have a negative pregnancy test (urinary or 
serum beta-HCG) prior to administration of the first vaccine dose.  Males and females must 
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vaccination.  Women must also not be breast feeding.  This is consistent with existing 
standards of practice for vaccine and chemotherapy protocols. 
4.2.11   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 
 
Hypothyroidism of any etiology on stable thyroid hormone 
replacement therapy. 
 
Other forms of depigmenting illness 
 
Mild arthritis requiring NSAID medications 
 
4.2.12   Patients in whom there is a medical contraindication or potential problem in 
complying with the requirements of the protocol, in the opinion of the investigator. 
 
4.2.13   Patients classified according to the New York Heart Association classification as 
having Class III or IV heart disease (Appendix 4). 
 
4.2.14   Patients with a body weight < 110 lbs (50 kg) because of the amount and frequency 
with which blood will be drawn, and because of the skin biopsies required. 
 
4.3   Demographics of Participant Population 
4.3.1   Age:  18 years or older.   
4.3.2   Sex:  Both male and female participants are eligible.  
4.3.3   Race:  All races and ethnic backgrounds are eligible. 
 
4.4   Registration, Randomization, and Management of Participants 
 
4.4.1   Participant Registration 
All participants must sign the consent form prior to determination of eligibility for this study.  
Participants eligible for interferon therapy will complete the interferon education packet and 
review their answers with the study team and convince the study team that they answered 
the questions accurately or understand the correct answers.  Registration information, the 
eligibility checklist with supporting documentation, and the on-study case report form for 
potential participants at institutions other than UVA must be received by the UVA HITC prior 
to registration and randomization.  All participants who meet the inclusion/exclusion criteria 
may be registered to the study.  Registration will occur following verification of eligibility by 
the treating physician or institutional PI.   
4.4.2   Randomization  
Randomization will be discussed with participants during the process of informed consent, 
and informed consent must be documented prior to randomization.  Randomization will 
occur after registration and no sooner than 5 days prior to the start of treatment.  In part 1, 
randomization will be based on equal allocation among allowable arms unless a weighted 
allocation scheme is triggered (section 9.2.1). Randomization will not be stratified by 

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institution. The randomization codes are generated by the study statisticians and stored in 
the Cancer Center Clinical Trials Database.  The UVA HITC will communicate with the 
outside site to let them know which study arm a participant has been randomized to.  
Participants should receive the first scheduled vaccination or biopsy (if applicable) within 2 
weeks of registration. Part 2 will not include randomization. 
4.4.3   Management of Participants 
This study will be conducted on an outpatient basis, with participants scheduled to be 
evaluated on days 1, 8, 15, 22, 36, 43, 57, 78, and 85, and week 26 (or more often if needed 
for testing or medical reasons).  As needed, there may also be follow -up within 1-3 weeks 
after biopsies performed on days 8 or 22 for wound evaluation.  Participants will also be 
follow ed-up at 52 weeks (1 year) and at 2 years with blood draws for studies of immune 
response durability.  During off treatment follow -up, participants will be followed yearly for 
survival and disease status. 
 
 
5.0   STUDY MEDICATION FORMULATION, DOSAGE, ACCOUNTABILITY AND 
ADMINISTRATION 
 
5.1   Study Medication Formulation 
5.1.1   Peptide Synthesis and Storage 
All peptides are synthesized directly from amino acids, and then are purified by high 
performance liquid chromatography (HPLC).  The identity of the synthetic peptides will be 
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 
Chemistry and Manufacturing section of the IND application. Each bulk peptide is supplied 
as lyophilized powder without excipients and stored at a temperature ≤ -70°C and protected 
from light. 
 
5.1.2   Preparation of peptide vaccine vials for patient use. 
For the 6 peptides prepared at UVA, the bulk peptides are solubilized in aqueous solution, 
sterile-filtered, and mixed to provide a final concentration of 600 mcg/ml for each of the 6 
peptides.  The peptide mixture is then aliquoted at 1 ml per vial into sterile borosilicate vials 
in the HITC clean room.  The mixture is slightly cloudy, as the pH of the mixture supports 
solubility of most of the peptides, but some peptides are partially in suspension at that pH.  
The 7th peptide, from NY-ESO-1 is provided as single use vials of lyophilized peptide, at 600 
mcg/vial, from the Ludwig Institute for Cancer Research, prepared under GMP conditions. 
 
5.1.3   Storage of Vialed Peptides 
The vials of peptide are stored by the HITC at a temperature ≤ -70°C and protected from 
light.  On the day of use, they are thawed at room  temperature or in a water bath up to 
37°C.  They should be swirled gently as they are thawed and moved to a refrigerator as 
soon as they are thawed if not being injected immediately. Once thawed, the vial(s) must be 
used for preparation of the vaccine within 24 hours.  
 
5.1.4   Lot Testing 
Each lot of peptide vaccine is evaluated as required by the FDA to confirm identity, sterility, 
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studies of stability will be performed over time.  The details of these tests are outlined in 
Appendix 5. 
 
5.1.5   Labeling 
The labeling of vials will occur as specified below.  
 
Each vial of lyophilized peptide is labeled with the following information: 
Short name of the product:  “LPV6” 
Product number   
Proper name of the product:  “6 long melanoma peptides” 
Name and address of the vialing facility:  “UVA-HITC” 
Lot number 
Date of manufacture (the date of vialing the reconstituted peptides) 
Serial number 
Quantity of each peptide per vial:  600 mcg/ml 
Vial contains no preservative, store at ≤ -70°C 
“Caution:  New Drug – Limited by US Federal law to investigational use” 
 
5.2   Preparation, Dosage, and Storage of Study Drug 
5.2.1   Dosage and Preparation of Peptide Vaccine  
Part 1: 
The peptides will be prepared by combining the LPV6 mixture of 6 peptides with one NY-
ESO-1 long peptide.  The long melanoma peptides (LPV6) will be available as single-use 
vials of 1 ml volume containing 600 mcg/ml of each peptide. The NY-ESO-1 peptide is 
provided as lyophilized preparation with 600 mcg of peptide in single use vials.  The tetanus 
peptide is vialed as 300 mcg lyophilized peptide per vial.  For each vaccine, the full 1 ml of 
the peptide solution of LPV6 (600 mcg) will be withdrawn and injected into the vial of 
lyophilized NY-ESO-1 peptide, creating a solution/suspension of all 7 long peptides, each at 
600 mcg/ml.  Then 0.75 ml of this suspension/solution will be withdrawn and injected into 
one vial of lyophilized tetanus peptide, to create a suspension/solution of 450 mcg of each of 
the 7 long peptides, and 300 mcg of tetanus peptide in 0.75 ml. Then, the full 0.75 ml of this 
solution/suspension (450 mcg of all 7 long peptides, plus 300 mcg tetanus peptide) will be 
withdrawn into a syringe for injection as specified below for each study arm.  Details of 
making emulsions with IFA (Montanide ISA-51) are provided in the Investigator’s Brochure 
and Study Manual.   
 
Part 2: 
The peptides will be prepared by combining the LPV6 mixture of 6 peptides with one NY-
ESO-1 long peptide.  The long melanoma peptides (LPV6) will be available as single-use 
vials of 1 ml volume containing 600 mcg/ml of each peptide. The NY-ESO-1 peptide is 
provided as lyophilized preparation with 600 mcg of peptide in single use vials.  For each 
vaccine, the full 1 ml of the peptide solution of LPV6 (600 mcg) will be withdrawn and 
injected into the vial of lyophilized NY-ESO-1 peptide, creating a solution/suspension of all 7 
long peptides, each at 600 mcg/ml.  Then 0.75 ml of this solution/suspension (450 mcg of all 
7 long peptides) will be withdrawn into a syringe for injection as specified below for each 
study arm.  Details of making emulsions with IFA (Montanide ISA-51) are provided in the 
Investigator’s Brochure and Study Manual.   
 
 

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Study Arm A (IFA):  Sterile saline solution 0.75ml will be added to 0.75 ml of LPV7 + 
tetanus peptide (total 1.5 ml). This will be combined with 1.5 ml of Montanide ISA-51VG to 
create an emulsion. Two (2) ml will be injected half-intradermally and half-subcutaneously in 
one skin location. 
Study Arm B (polyICLC):  PolyICLC (Hiltonol) is provided from Oncovir, Inc. (Washington, 
D.C.) as a clinical grade reagent for experimental use in single-use vials containing 1 mL of 
a 2 mg/mL solution. We will administer 1 mg (0.5 ml) per vaccine. PolyICLC (Hiltonol) 0.75 
ml will be added to 0.75 ml of LPV7 + tetanus peptide. One (1) ml of this will be injected half-
intradermally and half-subcutaneously in one skin location. 
Study Arm C (resiquimod):  Resiquimod is provided by 3M (Minneapolis, MN) and is 
packaged in multi-dose tubes (typically 3 g of 0.2% gel). Sterile saline solution 0.75ml will be 
added to 0.75 ml of LPV7 + tetanus peptide. One (1) ml of this will be injected half-
intradermally and half-subcutaneously in one skin location. Immediately after vaccine 
injection, apply approximately 500 mg of 0.2% resiquimod gel with a gloved finger over a 
25 cm2 area of the skin at the vaccine site. Rub the gel into the skin for 1 minute until it 
vanishes. Following application, the site may be covered with an occlusive dressing, such 
as Tegaderm, for about 8 hours.  Participants are encouraged to wash the area at 8 
hours after application. 
Study Arm D (polyICLC + resiquimod):  PolyICLC (Hiltonol) 0.75 ml will be added to 0.75 
ml of LPV7 + tetanus peptide. One (1) ml of this will be injected half-intradermally and half-
subcutaneously in one skin location Immediately after vaccine injection, apply 
approximately 500 mg of 0.2% resiquimod gel with a gloved finger over a 25 cm2 area of 
the skin at the vaccine site. Rub the gel into the skin for 1 minute until it vanishes. 
Following application, the site may be covered with an occlusive dressing, such as 
Tegaderm, for about 8 hours.  Participants are encouraged to wash the area at 8 hours 
after application. 
Study Arm E (IFA + polyICLC):  Part 1:  PolyICLC (0.75 ml) will be added to 0.75 ml of 
LPV7 + tetanus peptide (total 1.5 ml). This will be combined with 1.5 ml of Montanide ISA-
51VG to create an emulsion. Two (2) ml will be injected half-intradermally and half-
subcutaneously in one skin location.   
 
Study Arm E2 (IFA + polyICLC):  Part 2:    PolyICLC (0.75 ml) will be added to 0.75 ml of 
LPV7 (total 1.5 ml). This will be combined with 1.5 ml of Montanide ISA-51VG to create an 
emulsion. Two (2) ml will be injected half-intradermally and half-subcutaneously in one skin 
location (as in study arm A).   
 
Study Arm F (IFA + resiquimod):  Sterile saline solution 0.75ml will be added to 0.75 ml 
of LPV7 + tetanus peptide (total 1.5 ml). This will be combined with 1.5 ml of Montanide ISA-
51VG to create an emulsion. Two (2) ml will be injected half-intradermally and half-
subcutaneously in one skin location (as in study arm A).  Immediately after vaccine 
injection, apply approximately 500 mg of 0.2% resiquimod gel with a gloved finger over a 
25 cm2 area of the skin at the vaccine site. Rub the gel into the skin for 1 minute until it 
vanishes.  Following application, the site may be covered with an occlusive dressing, 
such as Tegaderm, for about 8 hours.  Participants are encouraged to wash the area at 8 
hours after application. 
Study Arm G (IFA + polyICLC + resiquimod): PolyICLC (0.75 ml) will be added to 0.75 
ml of LPV7 + tetanus peptide (total 1.5 ml). This will be combined with 1.5 ml of Montanide 
ISA-51VG to create an emulsion. Two (2) ml will be injected half-intradermally and half-
subcutaneously in one skin location (as in study arm A).  Immediately after vaccine and 
polyICLC injection, apply approximately 500 mg of 0.2% resiquimod gel with a gloved 
finger over a 25 cm2 area of the skin at the vaccine site. Rub the gel into the skin for 1 

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minute until it vanishes.  Following application, the site may be covered with an occlusive 
dressing, such as Tegaderm, for about 8 hours.  Participants are encouraged to wash the 
area at 8 hours after application. 
 
Storage of Prepared Peptide Vaccines 
The prepared peptide vaccines will be stored in a plastic syringe and     delivered to the 
clinicians in a plastic bag.  This bag with the syringe will be stored at room temperature until 
the vaccine is administered.  Ideally, the vaccine should be administered within 1-2 hours 
after mixing.  If the vaccine is not administered within 4 hours after mixing, it should be 
discarded. 
 
5.3   Drug Accountability and Distribution 
Study drug will be accounted for and distributed to UVA patients and the outside sites using 
the HITC InvestMed Database.  The InvestMed Database is an Access database that 
interfaces with the Cancer Center Clinical Trials clinical database so that drug may be 
allocated and dispensed on a per patient basis to UVA patients.  For the outside sites, drug 
is dispensed to the corresponding institution and tracked on a per institution basis.  Study 
drug will be sent to the outside sites by HITC personnel. 
 
5.4   Administration of Vaccines 
5.4.1   Designation of Vaccine Sites 
Evidence suggests nodes proximal to a tumor site may be relatively immunosuppressed 64; 
therefore, the vaccination sites will be distant from sites of known tumor whenever possible.  
In general, participants will be vaccinated in upper arm or thigh locations.  If a participant 
has received a prior experimental vaccine, vaccines should not be administered at the same 
site as the prior experimental vaccine, but they may be administered on the same extremity 
as prior vaccines.   
5.4.2   Regimen  
Part 1:  Each vaccine will be administered at a different site, rotating among available 
extremities when possible.   
 
Part 2:  The same skin location will be used for all vaccines.  If the vaccine site has severe 
inflammation or ulceration after multiple vaccines, the next vaccine may be placed adjacent 
to the original site. 
 
Chronic inflammatory reactions at the vaccine sites are expected to occur in all participants 
who receive Montanide ISA-51.  Induration may persist for days to months, but is not 
expected to require additional therapy.  Sterile abscesses may occur in some participants.  
These are not a basis for discontinuation of the vaccines.  However, if the inflammatory 
reactions develop significant ulceration of the skin (> 2 cm), then this would be considered a 
dose-limiting toxicity and the patient would be taken off the study. 
5.4.3   Vaccination doses:   
Part 1: 
For each vaccine, 300 mcg of each of the long peptides in LPV7 and 200 mcg of the tetanus 
helper peptide will be administered, plus 1 ml IFA or the dose of TLR agonists as specified 
in Table_1A and section 5.2.1. 
 
Part 2: 

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For each vaccine, 300 mcg of each of the long peptides in LPV7 will be administered, plus 1 
ml IFA and the dose of TLR agonists as specified in Table 1B and Section 5.2.1. 
   
5.4.4   Route of administration:   
At each injection site for emulsions with IFA, a single needle puncture is to be performed, 
with delivery of 1 cc volume into the subcutaneous tissue and 1 cc volume into the dermis.  
This usually will require advancing the needle into the subcutaneous tissue to deliver the 
first 1 cc over an area within reach of that needle, then pulling the needle back to near the 
skin puncture site, and advancing it into the intradermal tissue for delivery of the remaining 1 
cc.  For injections with peptides in polyICLC without IFA, the total volume will be 1 ml, which 
will be divided between subcutaneous tissue and dermis as above. 
 
5.4.5   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. 
 
5.4.6   Biopsies of Vaccine site and normal skin:   
Each participant in Part 1 and Part 2 will undergo biopsy of the vaccine site at two time 
points (days 8 and 22; one week after vaccines 1 and 3).  Control biopsies of clinically 
normal skin (three 4-mm punch biopsies of the dermis and subcutaneous tissue) will be 
taken at the same time points (days 8 and 22), for the first 6 participants enrolled in Part 1.   
 
The biopsies will consist of three 4-mm punch biopsies of skin. Note that vaccine sites will 
be rotated to different skin sites for each vaccination; so there will not be repeat injection of 
vaccines into the prior skin biopsy sites. 
 

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5.4.6.1   The punch biopsies of normal skin will be taken from an upper thigh or upper 
arm, and will be preserved/processed as follows: 
5.4.6.1.1Normal 1:  quick-frozen in liquid nitrogen. 
5.4.6.1.2Normal 2:  preserved in formalin, then paraffin-embedded 
5.4.6.1.3Normal 3:  preserved in RNA later. 
5.4.6.2   The punch biopsy specimens of the VSME will be preserved/processed as 
follows: 
5.4.6.2.1VSME sample 1: quick-frozen in liquid nitrogen. 
5.4.6.2.2VSME sample 2: preserved in formalin, then paraffin-embedded 
5.4.6.2.3VSME sample 3: preserved in RNA later. 
5.4.6.3   Their evaluation is summarized in the Study Manual.   
5.4.6.4   The specimens should be put into liquid nitrogen, formalin, or RNA later within 
15 minutes if at all possible, and ideally within 5 min of excision.  Delays beyond 15 
minutes should be documented.  This information should be documented on the Skin 
Biopsy Submission Form. 
 
5.5   Modification/Discontinuation of Treatment 
 
5.5.1   Dose Modifications 
There will be no dose modifications of the vaccine components. 
 
5.5.2   Discontinuation 
If participants are discontinued prior to one or more scheduled skin biopsies, they will not 
receive those biopsies.  Protocol treatment will be discontinued for any of the following 
reasons: 
5.5.2.1   Any dose-limiting toxicity as defined in Section 7.4. 
5.5.2.2   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/biopsy schedule may be interrupted for up to 7 days.  Delay of 
one vaccine administration, biopsy, or evaluation visit by up to 7 days will not be 
considered a protocol violation, regardless of attribution.  If more than one vaccine is 
delayed by 8 days or more, treatment must be discontinued. 
 
5.5.2.3   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. 
 

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5.5.2.4   Initiation of cytotoxic chemotherapy, radiation therapy, steroid therapy as 
detailed under Section 4.2.5, or other immunosuppressive therapy. 
 
5.5.2.5   Any other potential adverse reaction deemed sufficiently serious to warrant 
discontinuation of therapy by the Principal Investigator or one of the sub-investigators. 
 
5.5.2.6   Noncompliance with the requirements of the study. 
 
5.5.2.7   Therapy may be discontinued at the participant's request. 
 
5.5.2.8   Therapy may be discontinued at the discretion of an investigator.  
 
5.5.2.9   Pregnancy.  Pregnant participants will continue to be followed for the duration 
of the pregnancy. 
 
5.5.3   Elective Withdrawal 
A participant who is enrolled but neither receives any study drug nor has a skin biopsy on 
protocol may be replaced.  Every attempt will be made to evaluate any data from these 
participants for endpoint assessment.   
 
5.5.4   Delayed Visit 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 the 
subsequent vaccinations are given in the appropriate intervals.  Treatment may be 
continued for an additional time period, if needed.  Participants who are vaccinated outside 
of the established schedule should return to the original schedule as soon as possible. 
 
The table below defines what constitutes a delayed visit, whether the participant should 
continue to be treated, and whether a protocol violation/deviation should be reported and 
recorded.  The range of days is counted from the original scheduled date.   
 
 
Table 6.  Delayed Visit for Reasons other than Toxicity 
Treatment Period 
Range of Days 
Participant Treatment 
Protocol 
Deviation 
Vaccine 1 
 
 
 
Day 1 
 2 days 
Vaccine/Labs 
No 
        
 3 to 7 days 
Vaccine/Labs 
Yes 
 
 8 or more days 
Labs 
Yes 
Vaccine 2/Biopsy* 
 
 
 
Day 8 
 2 days 
Vaccine/Labs/Biopsy 
No 
        
 3 to 7 days 
Vaccine/Labs/Biopsy 
Yes 
 
 8 or more days 
Labs 
Yes 
Vaccine 3* 
 
 
 
Day 15 
 2 days 
Vaccine/Labs 
No 
        
 3 to 7 days 
Vaccine/Labs 
Yes 

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 8 or more days 
Labs 
Yes 
Assessment/Biopsy 
 
 
 
Day 22 
 2 days 
Biopsy/Labs 
No 
 
 3 to 7 days 
Biopsy/Labs 
Yes 
 
 8 or more days 
Labs 
Yes 
 
 
 
 
Assessment 
 
 
 
Day 43 
 
 
 
 
 2 days 
Labs 
No 
 
 3 to 7 days 
Labs 
Yes 
 
 8 or more days 
Labs 
Yes 
 
 
 
 
Vaccines 4-6* 
 
 
 
Days 36, 57, 78 
 7 days 
Vaccine/Labs 
No 
 
 8 to 14 days 
Vaccine/Labs 
Yes 
 
 15 or more days 
Labs 
Yes 
Assessment 
 
 
 
Day 85 (Week 12) 
 7 days 
Labs/Scans 
No 
 
 8 to 14 days 
Labs/Scans 
Yes 
 
 15 or more days 
Labs 
Yes 
Follow-up 
 
 
 
Week 26, Year 1 and 2 
 7 days 
Scans/Labs 
No 
 
 8 to 30 days 
Scans/Labs 
Yes 
* 
A participant will be taken off protocol treatment if more than one vaccination is 
delayed [ 3 to 7 days] during the treatment period. 
 
5.6   Concomitant Medications 
Medications taken in the month prior to randomization (Part 1) or arm assignment (Part 2) 
should 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. 
5.6.1   Non-permitted medications or treatments (within 30 d of study treatment) 
5.6.1.1   Systemic cytotoxic chemotherapy 
5.6.1.2   Interferon therapy (e.g. Intron-A®) 
5.6.1.3   Radiation therapy 
5.6.1.4   Nitrosureas 
5.6.1.5   Allergy desensitization injections 
5.6.1.6   Systemic corticosteroids, administered parenterally or orally.  Inhaled steroids 
(e.g. Advair®, Flovent®, Azmacort®) are not permitted.   
5.6.1.7   Growth factors (e.g. Procrit®, Aranesp®, Neulasta®) 
5.6.1.8    Interleukins (e.g. Proleukin®) 
5.6.1.9   Other investigational medications 

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5.6.1.10   Street drugs 
5.6.1.11   Antibodies to CTLA-4, PD-1, PD-L1, or CD137 
5.6.1.12   Targeted molecular therapy (e.g. vemurafenib, other inhibitor of mutant 
BRAF, MEK, or cKit). 
 
5.6.2   Permitted medications or treatments 
 
5.6.2.1   Nonsteroidal anti-inflammatory agents 
5.6.2.2   Anti-histamines (e.g. Claritin®, Allegra®) 
5.6.2.3   Topical corticosteroids are acceptable, including steroids with very low 
solubility administered nasally for local effects only (e.g. Nasonex®). 
5.6.2.4   Short-term therapy for acute conditions not specifically related to melanoma 
(except those listed under Section 5.6.1) 
5.6.2.5   Chronic medications except those listed in Section 4.2.7 or 5.6.1. 
5.6.2.6   Vaccines, including live vaccines (e.g. FluMist) are permitted, but should be 
administered at least 2 weeks prior to or at least 2 weeks after a study vaccine. 
5.6.2.7   Topical forms of chemotherapeutic reagents (e.g. Efudex®) are permitted, but 
should be administered at least 2 weeks after a study vaccine. 
 
 
6.0   CLINICAL AND LABORATORY EVALUATION 
 
6.1   Screening 
The following studies should be completed within 6 weeks prior to randomization unless 
otherwise noted: 
 
6.1.1   Class I HLA-typing (any time prior to randomization; typing for HLA-A and HLA-B is 
sufficient). If the patient is known to express HLA-A1, A2, or A3, HLA-B testing is not 
required).   
6.1.2   Review of pathology at the treating and participating institution (any time prior to 
randomization). Central pathology review at UVA is not required for pathology reviewed at 
other participating institutions. 
6.1.3   CBC with differential, including automated lymphocyte count if available (0.3 ml) 
6.1.4   Comprehensive chemistry panel to include sodium, potassium, creatinine, fasting 
glucose, calcium, total bilirubin, AST, ALT, and alkaline phosphatase.  NOTE:  fasting blood 
sugars may be evaluated 4h or more after last eating (0.9 ml) 
6.1.5   Lactate Dehydrogenase (LDH) (0.3 ml) 
6.1.6   Urinalysis 
6.1.7   β-HCG for women of childbearing potential (within two weeks prior to randomization) 

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6.1.8   HgB-A1C (only required at baseline) 
6.1.9   HIV and HCV screening (within 6 months prior to randomization) 
6.1.10   Chest x-ray or chest CT, abdominal and pelvic CT, and head CT or MRI.  PET/CT 
fusion scan may replace scans of the chest, abdomen, and pelvis. 
6.1.11   Complete history and physical examination, including: 
 
Vital signs 
 
Weight 
 
Performance Status 
 Assessment of skin and nodal basins for evidence of disease 
recurrence or metastasis 
 
Designation of vaccination sites 
 
Medication review  
 
6.2   Treatment  
The following evaluations will be performed on an outpatient basis on each of the listed 
days.  Blood for these evaluations should be obtained prior to the vaccine injection if a 
vaccine is scheduled to be administered.  Results of those tests are not required prior to 
administering the vaccine on that date. 
 
6.2.1   Day 1 
 
Interval history and physical examination directed at new signs and symptoms. 
 
Vital signs 
 
Weight 
 
Performance status 
 
Visual acuity exam using a Snellen chart, and color vision exam using an 
Ishihara eye chart  
 
Neurologic function, general. 
 
Note hair and eye color  
 
Assessment of skin for vitiligo 
 
Anti-nuclear antibody and rheumatoid factor (4 ml total)  
 
Baseline symptoms, codable in the NIH CTCAE v.4.03, to be recorded as pre-
existing conditions unrelated to the research protocol interventions 
 
Distribute participant toxicity diary 
 
Medication review  
 
Blood collection for immunologic testing: The following blood samples for 
research will be provided to UVA and processed by the UVA BTRF and stored 
by the UVA-HITC IML: 
o 120 cc blood collected in heparinized green top tubes for 
lymphocytes. This may be decreased to 100 cc if more blood must be 
drawn for safety or screening labs, in order to comply with institutional 
limits on total blood drawn in a day. 
o 
20 cc blood collected in red top tubes for serum 
 
Vaccine administration 
 
6.2.2   Day 8 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included.. 

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 
Vital signs 
 
Weight 
 
Performance status 
 
Review of adverse events 
 
Review of participant toxicity diary and distribute new toxicity diary 
 
Medication review  
 Blood collection for immunologic testing: The following blood samples for 
research will be provided to UVA and processed by the UVA BTRF and 
stored by the UVA-HITC IML: 
o 80 cc blood collected in heparinized green top tubes for 
lymphocytes.  
o 20 cc blood collected in red top tubes for serum 
 
Biopsies of 1st vaccine site (all patients), and biopsies of normal skin (patients 
1-6 only in Part 1).  Details in section 5.4.6 and in Study Manual.     
 Vaccine administration 
 
6.2.3   Day 15  
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included. 
 
Vital signs 
 
Weight 
 
Performance status 
 
Review of adverse events 
 
Review of participant diary and distribute new toxicity diary 
 
Medication review  
 Vaccine administration 
 
6.2.4   Day 22 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included.  
 
Vital signs 
 
Weight 
 
Performance status 
 
Review of adverse events 
 CBC with differential, including automated lymphocyte count if available (0.3 
ml) 
 Comprehensive chemistry panel to include sodium, potassium, creatinine, 
glucose, calcium, total bilirubin, AST, ALT, and alkaline phosphatase.  (0.9 
ml) 
 
Lactate Dehydrogenase (LDH) (0.3 ml) 
 
Anti-nuclear antibody and rheumatoid factor (4 ml total)  
 
Assessment of skin for vitiligo 
 Neurologic function 
 
Vaccine Site Biopsy (3) 
 
Review of participant diary and distribute new toxicity diary 
 
Medication review  

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 
Blood collection for immunologic testing: The following blood samples for 
research will be provided to UVA and processed by the UVA BTRF 
and stored by the UVA-HITC IML: 
 
80 cc blood collected in heparinized green top tubes for lymphocytes.  
 
20 cc blood collected in red top tubes for serum 
 
Biopsies of 3rd vaccine site (all patients), and biopsies of normal skin (patients 
1-6 only, in Part 1).  Details in section 5.4.6 and in Study Manual.     
 
6.2.5   Day 36 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included. 
 
Vital signs 
 
Weight 
 
Performance status 
 
Review of adverse events 
 
Review of participant diary and distribute new toxicity diary 
 
Medication review  
 Vaccine administration 
 
6.2.6   Day 43 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included. 
 
Vital signs 
 
Weight 
 
Performance status 
 
Review of adverse events 
 
Review of participant diary and distribute new toxicity diary 
 
Medication review  
 
Blood collection for immunologic testing: The following blood samples for 
research will be provided to UVA and processed by the UVA BTRF and 
stored by the UVA-HITC IML: 
o 80 cc blood collected in heparinized green top tubes for 
lymphocytes.  
o 20 cc blood collected in red top tubes for serum 
 
6.2.7   Day 57 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included. 
 
Vital signs 
 
Weight 
 
Performance status 
 
Review of adverse events 
 
Review of participant diary and distribute new toxicity diary 
 
Medication review  
 Vaccine administration 
 

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6.2.8   Day 78 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included. 
 
Vital signs 
 
Weight 
 
Performance status 
 
Review of adverse events 
 CBC with differential, including automated lymphocyte count if available (0.3 
ml) 
 Comprehensive chemistry panel to include sodium, potassium, creatinine, 
glucose, calcium, total bilirubin, AST, ALT, and alkaline phosphatase.  (0.9 
ml) 
 
Lactate Dehydrogenase (LDH) (0.3 ml) 
 
Anti-nuclear antibody and rheumatoid factor (4 ml total)  
 
Review of participant diary and distribute new toxicity diary 
 
Medication review  
 
Vaccine administration 
 
6.2.9   Day 85 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included.  
 
Vital signs 
 
Weight 
 
Performance status 
 
Review of adverse events 
 CBC with differential, including automated lymphocyte count if available (0.3 
ml) 
 Comprehensive chemistry panel to include sodium, potassium, creatinine, 
glucose, calcium, total bilirubin, AST, ALT, and alkaline phosphatase.  (0.9 
ml) 
 
Lactate Dehydrogenase (LDH) (0.3 ml) 
 
Chest X-ray or chest CT 
 
Visual acuity exam using a Snellen chart, and color vision exam   
      using an Ishihara eye chart  
 
 Neurologic function 
 
 Note hair and eye color  
 
 Assessment of skin for vitiligo 
 
 Review of participant diary 
 
 Medication review  
 
Blood collection for immunologic testing: The following blood samples for 
research will be provided to UVA and processed by the UVA BTRF and 
stored by the UVA-HITC IML: 
o 80 cc blood collected in heparinized green top tubes for lymphocytes.  
o 20 cc blood collected in red top tubes for serum 
 
6.2.10   Week 26 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included.  

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 
Vital signs 
 
Weight 
 
Performance status 
 
Anti-nuclear antibody and rheumatoid factor (4 ml total)  
 
Chest x-ray or chest CT 
 
Neurologic function 
 
Assessment of skin for vitiligo  
 
Medication review  
 
Blood collection for immunologic testing: The following blood samples for 
research will be provided to UVA and processed by the UVA BTRF and 
stored by the UVA-HITC IML: 
o 80 cc blood collected in heparinized green top tubes for lymphocytes.  
o 20 cc blood collected in red top tubes for serum 
 
6.2.11   Weeks 52 and 104 (Year 1 and 2) 
 Interval history and physical examination directed at new signs and 
symptoms.  Examination of the vaccine site(s) should be included.  
 
Vital signs 
 
Weight 
 
Performance status 
 
Medication review  
 
Blood collection for immunologic testing: The following blood samples for 
research will be provided to UVA and processed by the UVA BTRF and 
stored by the UVA-HITC IML: 
o 80 cc blood collected in heparinized green top tubes for lymphocytes.  
o 20 cc blood collected in red top tubes for serum 
 
6.3   Tumor tissue collection  
If during the study, participants develop metastases or recurrences, these may be removed, 
and following receipt by pathology, may be evaluated by the study research team.  Tissue 
samples may be screened for antigen expression or protein profiles using tests such as 
Western blots, immunohistochemistry, PCR, flow cytometry or gene chip analysis.  Tumor 
escape mechanisms may also be evaluated.  Specimens will be used in immunological 
assays to assess T cell 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 and intracellular cytokine staining.  Specimens may be used to study the 
immunologic aspects of the tumor microenvironment or as targets or controls in laboratory 
assays.  Specimens may be used to establish cell lines for long-term studies. 
 
This tissue may also be compared to lesions resected prior to enrollment, which will be 
requested from the pathology department of each institution as paraffin-embedded tissue 
samples, and these tissues may be banked for use in future studies.  If participants are 
removed from the study or progress during or after follow -up, tissue may be collected for use 
as part of this study, as described above, or banked for use in future studies 
 
 
7.0   REGULATORY AND REPORTING REQUIREMENTS 
Adverse event reporting requirements for this trial, as specified in this section, apply to 
patients accrued at all participating sites.  There will be accrual at UVA and at MD Anderson 

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Cancer Center; UVA will be the coordinating site, and responsibility for data coordination 
and reporting for the study as a whole falls to the Human Immune Therapy Center (HITC).  
Throughout this section, when referring to 'Cancer Center Database', 'Cancer Center 
DSMC', 'CRC', 'PI' or, 'nurse practitioner', they are preceded by ‘site’ when it refers to 
reporting within either of the participating institutions, or are preceded by ‘UVA’ when it 
refers to central reporting to the coordinating UVA center.  
  
In addition to the reporting requirements for the study, specified in this section of the 
protocol, investigators should also follow their institutional policy for documentation in their 
database and reporting to the IRB locally at their own site. 
 
7.1   Definitions 
7.1.1   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. 
 
7.1.2   Unexpected AE –  
Any adverse event not listed in Section 7.4.3. 
 
7.1.3   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 
subject 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, a hospital visit 
where a patient is admitted for observation or minor treatment (e.g. hydration) and 
released in less than 24 hours would not be considered serious.  Furthermore, 
hospitalization for pharmacokinetic sampling is not an AE, and therefore is not to be 
reported either as a routine AE or in an expedited report. 
 

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7.1.4   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 subject 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. (see section 7.2) 
 
The incident suggests that the research placed the subject or others at greater 
risk of harm than was previously known or recognized OR results in actual harm 
to the subject or others. 
 
7.1.5   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. 
 
7.1.6   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. 
 
7.2   Attribution Assessment 
7.2.1   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 the vaccine.  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 subject’s clinical state, environmental or 
toxic factors, or other modes of therapy administered to the 
subject. 
 
It disappears or decreases on cessation or reduction in dose 
with re-exposure to drug.  (Note: This is not to be constructed 
as requiring re-exposure of the subject; 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 

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of the test drug. 
 It could not be reasonably explained by the known 
characteristics of the subject’s clinical state, environmental or 
toxic factors, or other modes of therapy administered to the 
subject. 
 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 subject’s clinical 
state, environmental or toxic factors, or other modes of therapy 
administered to the subject. 
 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 subject’s clinical 
state, environmental or toxic factors, or other modes of therapy 
administered to the subject. 
 It does not follow a known pattern of response to the test drug. 
 It does not reappear or worsen when the drug is re-
administered. 
Unrelated – Applies to those adverse events, which after careful consideration, 
are clearly and incontrovertibly due to extraneous causes (disease, environment, 
etc.). 
 
7.3   Data collection  
Data will be collected using a centralized electronic case report form called ON-line Clinical 
Oncology Research Environment = Oncore. 
 
7.4   Risks and Safety 
7.4.1   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 (see Appendix 6). 
 

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7.4.2   Time Span for Reporting Adverse Events 
Reporting of AEs will begin when the subject is administered the study drug or has a study 
related biopsy. Events occurring through 30 days after administration of the last vaccine, 
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 the vaccine will be recorded until the 
subject completes treatment follow -up.  If, during treatment follow -up, the subject receives 
additional treatment, subjects will be off treatment follow -up and will be followed yearly for 
disease progression and survival. 
 
7.4.3   Agent-Specific Expected Adverse Events List: 
Any AE not on this list will be considered an unexpected AE. 
 
Expected toxicities related to peptide vaccines.  This list of expected AEs for the LPV7 
vaccine is based on prior data from the UVA-Mel 44 clinical trial in which 41 participants in 
Arm A received a mixture of 12 Class I peptides plus a tetanus helper peptide in an 
emulsion with Montanide ISA-51 adjuvant.  AEs are listed only if observed in ≥ 5% of treated 
participants. 
 
Table 7:  Summary Toxicity Data from UVA-Me44 
Grade 2:     
CTCAE Category 
Description 
Investigations 
Lymphocyte count decreased 
General 
disorders and administration 
site conditions 
Fatigue 
General 
disorders and administration 
site conditions  
Fever  
Skin and subcutaneous tissue disorders 
Hyperhidrosis 
Skin and subcutaneous tissue disorders 
Skin induration 
Skin and subcutaneous tissue disorders 
Skin ulceration 
General disorders and administration 
site conditions 
Injection site reaction 
Gastrointestinal Disorders 
Nausea 
Metabolism and nutrition disorders 
Hyperglycemia 
 General 
disorders and administration 
site conditions 
Flu-like symptoms 
Skin and subcutaneous disorders 
Other:  any rash 
*NOTE:  Hyperglycemia ≥ Grade 3 is not an expected event.  If a non-fasting 
hyperglycemia adverse event occurs at a level ≥ Grade 3, a fasting serum 
glucose test will be ordered and the event will be re-graded appropriately.  The 
fasting serum glucose measurement will be used for the purpose of 
determining reporting requirements. 
 
Injection site reaction/induration was experienced at maximum grade 3 in 5% of patients in 
Arm A of Mel44.  Ulceration was experienced as maximum grade 3 in 7% of patients in Arm 
A of Mel 44.  By the rule above (observed in ≥5% of treated patients), injection site reaction, 
induration, and ulceration would be expected at grade 3.  Despite this and in the interest of 
the patients, we would consider grade 3 injection site reaction with significant ulceration of 
the skin (> 2 cm) as a DLT (see section 7.8).  

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Adverse Events Expected with Resiquimod application at the vaccine site. 
The investigator brochure specifies the following adverse events in more than 1800 patients 
treated with topical resiquimod in 25 clinical studies.  Reported adverse events were 
primarily local adverse events including protocol-defined local skin reactions (including, 
erythema, edema, ulceration, vesicles, weeping, scabbing, and flaking); and protocol-
defined local symptoms at the site of administration (including pain, burning, pruritus, and 
numbness/tingling). Other adverse events at the application site include drainage, infection, 
and pigmentation changes. Systemic adverse events that may be related to resiquimod due 
to a systemic induction of cytokines and/or release of locally induced cytokines into the 
circulation include influenza-like symptoms (e.g., fever, headache, arthralgia, myalgia, chills, 
asthenia, fatigue), and transient decreases in neutrophils and total leukocytes that may be 
related to cellular trafficking to the application site. Other infrequent adverse events that 
were considered possibly or probably related to study medication included liver function test 
abnormalities, abdominal cramping, nausea, vomiting, diarrhea, backache, neck pain, 
rhinitis, urticaria, and dizziness.    
 
The formal list, below, of expected AEs for resiquimod, for this study, is based on published 
data related to its clinical use 65;66  The table below is based on a study applying resiquimod 
3 times per week for 4 weeks, for treatment of actinic keratosis. This study had 4 treatment 
arms, using 0.01, 0.03, 0.06, and 0.1% resiquimod gel.  We will be using 0.2% resiquimod 
gel, only once a week; so toxicities from 3x/weekly administration with 0.06% resiquimod 
use may exceed what we observe65.  Overall in that study, no deaths or serious adverse 
events were reported, but some adverse events are described as severe.  We expect grade 
2 toxicities in these categories. Listed in the table below are those categories where adverse 
events were recorded in 10% of patients or more, in any study group, but the percentages 
listed are those for patients receiving resiquimod 0.06% three times per week. 
 
Table 8:  Toxicity Data for Resiquimod 
Grade 2: 
CTCAE Category 
Description 
Percent with  
possibly or probably 
related AEs at 0.6% 
resiquimod three times 
per week 
General 
disorders 
and 
administration 
site 
conditions 
 
Application site burning 
25% 
Application 
site 
erythema 
6% 
Application site pain 
16% 
Application site pruritus 
25% 
Application site reaction 
None 
Fatigue 
9% 
Rigors/chills 
16% 
Musculoskeletal and connective tissue disorders 
Arthralgia 
16% 
Myalgia 
6% 
Nervous system disorders 
 
Headache 
19% 
Lethargy/fatigue 
13% 
Psychiatric disorders 
Not specified 
16% 
Skin and subcutaneous tissue disorders 
Not specified 
13% 
 
  Mild and transient leukopenia and thrombocytopenia have also been reported with use of  
 
  resiquimod, but these are expected only to be grade I toxicities. 

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Toxicities at sites of resiquimod application should be limited to grade 1, 2, or 3 (with 
ulceration ≤ 2 cm) dermatologic toxicities, and should be manageable either by continuation 
of  resiquimod if the patient is tolerating the toxicities and if the toxicities are relatively stable, 
or they may be managed by holding vaccination for one to several days (up to 7 days 
without being a DLT).   
 
Adverse events expected with polyICLC (Hiltonol) 
The following expected toxicities for polyICLC are based on the data from 45 patients 
treated with 20 mcg/kg 3x/week.  This would be about 1.4 mg per dose and 4.2 mg per week 
(much higher than we will use). 
 
Table 9:  Toxicity Data for PolyICLC 
Category 
Toxicity 
Grade 
Comment 
Nervous system disorders 
 
Headache 
Grade 2 
1 of 22 (<5%) 
Tremors 
Grade 3 
2 of 45 (4%) poss related 
Musculoskeletal 
and 
connective tissue disorders 
Muscle weakness 
Grade 2 
1 of 45 (<2%) experienced at 
Grade 3 
Respiratory, 
thoracic 
and 
mediastinal disorders 
 
Dyspnea 
Grade 2 
1 of 45 (<2%) experienced at 
Grade 3 
Hypoxia 
Grade 2 
1 of 45 (<2%) experienced at 
Grade 3 
Metabolism 
and 
nutrition 
disorders 
Hypernatremia  
Grade 2 
1 of 45 (<2%) experienced at 
Grade 3 
Investigations 
 
Elevated 
transaminases (GPT) 
Grade 3 
4 of 45 (9%) 3 cases possibly 
related; 
one 
case probably 
related.  Typically transient. 
Elevated 
Alkaline 
phosphatase 
Grade 3 
7% of patients, in IB 
Leukocytopenia 
Grade 3 
2 of 45 (<4%) in published 
work; 20% in IB 
Thrombocytopenia 
Grade 3 
14% of patients in IB 
Neutropenia 
Grade 3 
10% of patients in IB 
Blood/lymph disorders 
Anemia 
Grade 3 
13-31% of patients in IB 
General 
disorders 
and 
administration site conditions 
 
Vaccine site reaction 
Grade 2 
1 of 22 (<5%) 
Fever 
Grade 3 
14% of patients in RCC trial 
Chills 
Grade 3 
10% of patients in RCC trial 
Fatigue 
Grade 3 
10% of patients in RCC trial 
 
Adverse events expected from vaccine and normal skin biopsies.   
Below is a list of expected AEs related to skin biopsies: 
 
        Table 10:  Toxicity Data for Skin Biopsies 
 
 
 
 
Grade 2: 
CTCAE Category 
Description 
Pain 
Pain - Skin 
 
7.5   Adverse Event Classifications 
The determination under which adverse event classification an AE is reported.  The adverse 
event classifications are specified in the CTCAE v4.03.  For specific classifications 
pertaining to the protocol, we specify the following: 

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Hematologic/Metabolic- Any AE coded under one of the following CTCAE v4.03 categories 
should be reported under the Hematologic/Metabolic adverse event classification: 
 
 
 
Table 11:  Hematologic/Metabolic Classifications 
Section 
AE 
Blood and lymphatic 
Anemia 
Leukocytosis 
Investigations 
ALL EXCEPT:  
Carbon monoxide diffusing capacity decreased 
Ejection fraction decreased 
Forced expiratory volume decreased 
Vital capacity abnormal 
Weight gain 
Weight loss 
Metabolism and nutrition 
disorders 
ALL EXCEPT: 
Alcohol intolerance 
Anorexia 
Dehydration 
Glucose intolerance 
Iron overload 
Obesity 
Tumor lysis syndrome 
 
Non-hematologic/Non-Metabolic- Any AE not reported under hematologic/metabolic, ocular, 
or allergic/autoimmune, should be reported under the non-hematologic/non-metabolic 
adverse event classification. 
 
Ocular – Any AE coded under one of the following CTCAE v4.03 Adverse Event Terms 
should be reported under the Ocular adverse event classification: 
1) 
A single treatment-related experience of the following adverse events 
will be classified as a DLT: 
 
Eye Disorders: Night blindness (nyctalopia) 
 
Eye Disorders: Papilledema 
 
Eye Disordersl: Retinopathy 
 
Participants will be referred for an ophthalmologic exam if any of these 
ocular adverse events occur. 
 
2) 
A prolonged treatment-related experience (e.g., lasting > 5 days) of 
the following non-severe adverse events will be classified as a DLT: 
 
Eye Disorders: Blurred vision 
 
Eye Disorders: Flashing lights 
 
Eye Disorders: Floaters 
 
Participants will be referred for an ophthalmologic exam if any of these 
ocular adverse events occur. 
 
Allergic/Autoimmune – Only AEs coded as Immune System Disorder: Allergic reaction, 
autoimmune disorder, or anaphylaxis should be reported under the Allergic/Autoimmune 

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adverse event classification.  Other AEs coded under Immune System Disorder should be 
reported under  Non-hematologic/Non-metabolic adverse event classification. 
 
7.6   Reporting Adverse Events 
7.6.1   Process for Reporting AEs: 
 
The process of reporting AEs will occur as follows: 
 
A. Identify the type of event using the NCI CTCAE v4.03 (Appendix 6).  The CTCAE 
provides descriptive terminology and a grading scale for each adverse event listed. 
B. Grade the event using the NCI CTCAE v4.03. 
C. Determine whether the AE is related to the vaccine.  Attribution groups are defined 
in Section 7.2.1. 
D. Determine the prior experience of the AE.  Expected events are those that have 
been previously identified as resulting from administration of the vaccine.  An AE is 
expected when it is listed in Section 7.4.3. 
E. Determine the adverse event classification of the AE, as specified and defined in 
Section 7.5. 
F. Determine whether the AE is a dose-limiting toxicity and report as specified below . 
G. Determine whether the AE is an SAE and report as specified below . 
H. AEs determined to require expedited reporting to the Sponsor, UVA HITC, must 
also be reported to your own institution according to the local policy and 
procedures.  
 
Reporting Dose Limiting Toxicities 
All DLT’s (defined in Section 7.8) should be reported by phone or fax to the HITC 
within 24 hours of when the site is notified of the event and entered into Oncore 
within 5 calendar days of when the site is notified of the event. 
 
DLT’s that are deemed serious and unexpected should be submitted to to a site’s local IRB 
per their institutional guidelines. 
 
SAE reporting  
 
Report SAEs by phone or fax to the HITC within 24 hours of of when the 
site is notified of the event.  
 
Send completed FDA MedWatch 3500a form and relevant supporting 
documentation (e.g. clinic notes, hospital case records) to the Sponsor 
within 3 calendar days for deaths or life-threatening events and 5 calendar 
events for other SAEs. 
 
Enter SAE into Oncore per guidelines specified in the table below. 
 
DLT and SAE Reporting Information: 
UVA HITC 
Phone: (434) 982-6714 
Fax: (434) 982-6608 
Box 801457, 1352 Jordan Hall 
Charlottesville, VA 22908 
 
In addition to DLT’s and SAE’s, all other AEs must be recorded into the University of Virginia 
Cancer Center OnCore database per the following guidelines: 

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Table 12:  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 w ithin 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 w ill be recoreded in the Cancer Center Database; how ever, regardless 
of attribution, these events do not have to be reported. 
 
 
 
 
Pregnancy 
 
Report pregnancy to the Sponsor within 5 calendar days of when the site is 
notified of the event. 
 
Report the pregnancy using the Safety Reporting Cover Sheet. 
 
 
 
Pregnancy Reporting Information: 
 
UVA HITC 
 
Phone: (434) 982-6714 
 
Fax: (434) 982-6608 
 
Box 801457, 1352 Jordan Hall 
 
 
Charlottesville, VA 22908 
 
7.6.2   IRB Reporting Requirements 
Participating sites should follow their institutional policies for reporting adverse events and 
serious adverse events to their local Institutional Review Board. 
 
The University of Virginia is responsible for reporting to the UVA IRB-HSR per the following 
guidelines: 
 
Table 13:  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 
IRB-HSR 
Within 24 hours 
IRB Online and phone call 
 
www.irb.virginia.edu/ 
 

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Type of Event 
To whom will 
it be reported: 
Time Frame for 
Reporting 
How reported? 
(Note: 
 An internal 
event 
is 
one 
that 
occurs in a subject 
enrolled 
in 
a 
UVa 
protocol.) 
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/vprg
s/irb/HSR_docs/Forms/Rep
orting_Requirements-
Unanticipated_Problems.do
c ) 
 
  
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/vpr/i
rb/hsr/   
 
 
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/s
ecurity/reporting.html 
 
 
Phone- (434) 924-7166 
 
 

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7.6.3   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 to the Sites 
The sponsor will report serious and unexpected suspected adverse reactions to the outside 
sites no later than 15 calendar days after the sponsor determines that the requirements for 
an IND safety report have been met. 
 
7.6.4   Reporting of Subject Withdrawals/Dropouts Prior to Study Completion 
Subjects 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.  These data should be 
reported to the sponsor within 30 days of subject withdrawal or dropout. 
 
7.7   Adverse Event Review and Monitoring 
7.7.1   In addition to clinic notes, adverse events will be initially captured using study-specific 
tools and participant toxicity diaries.  
 
As specified in the study flow chart (Appendix 1), 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, blood collection, re-
assessment for vitiligo, and examination of the skin and nodal basins for evidence of 
metastasis.   
 
Participants will keep a daily diary of toxicities for days 1 through 85.  The diaries will be 
reviewed by a research clinician prior to the next scheduled vaccine.  During clinic visits, 
participants will also be asked about subjective symptoms including headache, malaise, 
fatigue, dyspnea, nausea, rash, diarrhea, abdominal discomfort, peripheral nerve pain, 
visual changes, appetite, tremors, night sweats, and ability to concentrate.  Additional 
toxicities will be captured from laboratory tests.  For each AE (with the exception of Grade 1 
hematologic/metabolic events), date of onset, duration, grade, and attribution will be noted 
in the participant’s study chart, on study documents, or in the clinic note, and will be entered 
into the UVA Cancer Center database.   
 
After each vaccination, 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.  Subjects will be instructed on how to reach their provider should 
they have any questions and/or problems during the study. 
 
In the event of an AE, appropriate action will be taken to ensure adequate care for the 
participant.  If the participant is still on protocol, treatment delay or withdrawal from the 
protocol will be considered according to the protocol guidelines. 
 

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7.7.2   Individual AEs will be reviewed at each site by the Principal Investigator, nurse 
practitioner(s), and clinical research coordinator(s) (CRC),.   
 
7.7.3   SAEs experienced by participants at all sites will be reviewed twice a month by the 
sponsor during the UVA Melanoma Team meeting.  This meeting will occur at least 20 times 
in a calendar year.  Those present at the meeting include the sponsor/overall study PI, sub-
investigators, protocol development staff, biostatisticians, research nurses, research 
coordinators, laboratory specialists, and laboratory research managers.  These meetings 
may 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. 
 
7.7.4   Monthly conference calls will be held with the Sponsor and the site designee to 
review protocol status, accruals, and adverse events. 
 
7.7.5   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 7.6: 
1. Alk Phosphatase 
2. ALT (GPT) 
3. ANA 
4. AST (GOT) 
5. Bilirubin, total  
6. Creatinine 
7. Eosinophil #  
8. Hepatitis C serology 
9. beta-HCG 
10. HgB 
11. HIV 
12. HLA type 
13. LDH 
14. Potassium 
15. RF 
16. Urinalysis 
17. WBC 
 
Any abnormal laboratory values captured which are not included in the above list, but are 
considered to be pertinent positive clinical signs/symptoms, and laboratory results obtained 
as part of routine care of patients will be recorded in the UVA Cancer Center database and 
reported as described in Section 7.6.  If there is any doubt on the part of study personnel 
concerning what constitutes a pertinent positive finding, the sponsor is to be consulted. 
 
7.8   Adverse Event Stopping Guidelines  
Based on results from previous vaccinations with 12-MP and tetanus peptide preparations, 
and with findings from use of resiquimod and polyICLC, significant toxicity is not anticipated.  
The study will be monitored continuously for treatment-related adverse events within each 
vaccine preparation by the sponsor.  Data from the UVA- Mel44 vaccine trial (Arm A) (using 
12-MP and tetanus peptide) indicate an 8% rate of treatment-related unexpected Grade 3 
adverse events.   

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Adverse events will be described and coded based upon the NCI CTCAE v4.03.   
 
A DLT is defined as any unexpected adverse event that is possibly, probably 
or definitely related and: 
 
≥ Grade 3, with the exception of grade 3 injection site reaction with 
ulceration ≤ 2 cm 
 
≥ Grade 1 ocular adverse events as defined under Section 7.5 
 
≥ Grade 2 allergic/autoimmune reactions 
 
7.9   Responsibility 
7.9.1   The University of Virginia Cancer Center Data and Safety Monitoring Committee 
(DSMC) will provide oversight of the conduct of this study.  The CC DSMC will report to the 
UVA Protocol Review Committee (PRC). 
7.9.2   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 
7.9.3   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 sponsor is requested.   Per the UVA Cancer Center 
NIH approved institutional plan, this study will be audited approximately every 6 months. 
 
7.10   Endpoint Data 
7.10.1   Endpoint data will be collected using HITC IML data forms, participant-specific 
binders, and the HITC laboratory database. 
 
7.10.2   The HITC laboratory database, which has password-restricted access, is stored on 
the UVA Health System Computing Services secure server. 
 
 
8.0   EVALUATION OF IMMUNOLOGIC RESULTS 
 
A primary goal is to evaluate immunogenicity of LPV7 in each of 7 adjuvant preparations, 
including IFA, with or without TLR agonists.  The primary immunological response of interest 
is the CD8+ T cell response to the defined minimal peptides epitopes contained within each 
of the 7 long peptides (Table 2).  This will be assessed primarily by ELIspot assay as 
outlined below. Associated analyses will include the durable immune response (week 26), 
and the peak magnitude of the CD8+ T cell responses. Details related to study design are in 
Section 9. 
 
Studies in the peripheral blood will also assess CD4 T cell responses, T cell persistence, 
antibody response, Th1/Tc1 deviation and cytokine functionality. The antitumor immune 
response to vaccination with LPV7 with TLR agonists will be compared to vaccination with 

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IFA alone, and the highest immune response rate will be selected. Blood will be collected as 
described in Section 6.   
 
Skin biopsy specimens from the vaccine sites after 1 (day 8) and 3 vaccines (day 22) (and 
from normal skin controls) will be evaluated for changes induced by TLR agonists, and these 
will be evaluated as we have for a prior study 50: immunohistochemistry of the vaccine-site 
immune cells to define the cell types and function; multiparameter flow cytometry to identify 
activation status of antigen specific cells, regulatory T cells, and MDSC; ELIspot assays for 
the ability to produce IFN; and Luminex studies to define the cytokine and chemokine 
profiles of the VSME.   
  
The follow ing laboratory criteria will be used to evaluate effects of the vaccines and the TLR 
agonists in circulating peripheral blood mononuclear cells (PBMC) and in the vaccine sites.  
In general, immunological evaluations will be performed in batch.  
 
8.1   Immunohistochemistry and Immunofluorescence of skin biopsy specimens 
For the immunohistochemical studies of the VSME, we will employ stains with single 
antibodies (eg CD8), with multiple antibodies (eg CD8 and FoxP3), and a technique 
developed by our colleague Jim Mandell, which we have working our lab now. This 
technique, called SIMPLE, enables repeated IHC stains on the same section, with whole-
slide imaging using an Aperio or Leica system, and multiparameter evaluation of these cells 
in situ by combining the multiple images with each antibody.67 Immunofluorescence studies 
will also be available in the event they are helpful.  
 
We will obtain CD8/FoxP3 ratios to evaluate whether higher (more favorable) ratios are 
induced by adjuvants that are associated with improved circulating immune responses. 
 
For optimization of antibodies in IHC, we have used positive control tissues, which will 
continue to be useful controls in the proposed studies.  These include,  but are not limited to, 
normal human peripheral blood lymphocytes (PBL), PBL activated with CD3/CD28 beads 
(activated PBL), human lymph node tissue from non-melanoma patients (LN), and inflamed 
human tissue from a surgical specimen of Crohn’s disease (Crohn’s). 
 
8.2   ELIspot assay 
We will evaluate CD4+ and CD8+ T cell responses in the peripheral blood using a direct (ex 
vivo) ELIspot assay for IFN as reported.12  This will be performed in a core laboratory 
dedicated to immune monitoring, and with SOPs, controls, and quality assurance measures, 
which we have reported.12. Vaccination site infiltrating lymphocytes will be evaluated by 
ELIspot as well.  Vaccine site infiltrating lymphocytes will be compared to those in peripheral 
blood.   ELIspot assays after one in vitro stimulation (stimulated ELIspot) may also be 
employed if appropriate, as reported 33. 
 
CD4+ T cell responses will be assessed by IFN ELIspot assay using the mixture of 7 long 
peptides and also pools of 15-mer peptides (overlapping by 5 residues) from each peptide 
(4 peptides for each long peptide) as antigen sources.  
 
Durability of CD8 and helper T cell responses will be assessed by measuring T cell activity 
at 26 weeks, and for longer intervals when available, with the hypothesis that there will be 
increased durability of T cell responses when compared to prior experience with short 
peptides13 and will be improved with TLR agonists when compared to IFA alone in this 
study.  

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We have extensive stores of cryopreserved T cells reactive to minimal epitope peptides 
included in the 12 short peptides (12MP) mixture, and detectable by IFN-gamma ELIspot 
assay 8;12 and by cytotoxicity assays.68;69.  These cells will serve as positive controls for 
reactivity to the minimal epitopes contained within the long peptides used in this trial.     
 
8.3   Tetramer assay and multiparameter flow cytometry 
Peripheral blood lymphocytes and VSME lymphocytes will be evaluated by flow cytometry 
after incubation with MHC-peptide tetramers or pentamers for the number of peptide-
reactive T-cells. We have validated tetramers for all 5 of the minimal CD8 epitopes restricted 
by HLA-A2 or A3 among the LPV7 peptides.  We will use these MHC multimers with which 
we also have substantial experience.12 Tetramers will likely detect more antigen-specific T 
cells than are detected in the functional ELIspot assay, but the primary measure of CD8 T 
cell response will be the ELIspot assay, on which the statistical power was based.  
Tetramers will also be used in multiparameter flow cytometry to determine the 
effector/memory status of the antigen-specific T cells 70 (CD45RA, CD27, CD28, CCR7, 
CD62L), and evaluation for multiple functions of the CD8 T cells (degranulation (CD107a), 
IFN, MIP-1β, TNF, and IL-2) 71  
 
We will also use multiparameter flow cytometry to measure myeloid suppressor cells 
(CD14+ HLA-DR lo/neg), and several subtypes of MDSC in PBMC to determine if they are 
increased or decreased by any of these vaccine approaches, compared to our current 
experience with MDSC numbers using our short 12MP vaccine (data not shown). 
 
8.4   Proliferation assay / Cytokine Analysis 
To characterize responding T cells more completely, we will evaluate with multiparameter 
flow cytometry after short-term culture with antigen. CD4+ T cells responding to antigen by 
CFSE dilution will be assayed for intracellular cytokine production to evaluate Th1/Th2/Th17 
bias (Th1: IL-2, IFN, TNF; Th2: IL4, IL-5, IL-10; Th17: IL-17a).  Antigen specific T-regs will 
also be assayed as described 72.  
 
We will also assay supernatants of PBMC, and of immune cells infiltrating the VSME, 
cultured 2 and 5 days with the 7 long peptides to define secreted cytokines and chemokines 
representative of the whole cell population.  We anticipate that the long peptides will induce 
Tc1 and Th1 dominant responses, with a high proportion of memory phenotype after several 
vaccines.  
 
Peripheral blood mononuclear cells (PBMC) and vaccine-site inflammatory cells from 
participants in this clinical trial may also be evaluated for CD4 T cell reactivity, using a 3H-
thymidine uptake assay, CFSE dilution or other assays as summarized above. 
 
Responses to the tetanus helper peptide will be assayed as well as responses to the 30-mer 
melanoma peptides and 15-mer pools comprising those peptides.  Either or both of two 
peptides will be used as negative controls for CD4+ T cell responses: one being the PADRE 
peptide (aK(X)VAAWTLKAa), where X = L-cyclohexylalanine 73;74. 
 
8.5   Antibody responses 
Antibody responses to 30 mer peptides (and to pools of 15-mer peptides) will be measured 
by ELISA assay using methods described by Sabbatini et al 2. 
 

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8.6   Evaluation of tumor 
Tumor tissue collected prior to enrollment or at the time of progression will be evaluated by 
routine histology and immunohistochemistry.  In addition, in vitro evaluations of tumor tissue 
and tumor infiltrating lymphocytes may be completed. 
 
9.0   STATISTICAL CONSIDERATIONS   
This is an open-label, Phase I/II study evaluating the safety and immunogenicity of a long 
peptide vaccine (LPV7) + TLR for resected stage IIB – IV melanoma. There are two parts to 
the study.  
 
For Part 1, the trial was designed to find the range of optimal combination therapies, defined 
as combinations with high immunologic response and an acceptable level of toxicity. The 
primary outcomes included the frequency of treatment related dose limiting toxicities (DLTs) 
and frequency of immunologic response. For Part 2, the goal is to obtain preliminary safety 
and immunogenicity data on the optimal combination identified in Part 1 when administered 
in the same skin site for all 6 vaccines (same site vaccination). 
  
For part 2, the vaccine regimen is selected based on available data from Part 1, in which the 
strongest immune responses were observed in participants enrolled on Arms E and G 
(Table 1A).  Resiquimod is no longer available; so the regimen for Arm G is not currently 
feasible.  Also, the total enrollment on Arm E is larger (15 vs 6) so that outcomes from Part 2 
will be evaluable in a similar sample size as for Part 1, Arm E.  This group will be called 
Study Arm E2. 
 
9.1   Study design Part 1 (completed) 
In order to assess the first two objectives, the co-primary objective is to determine a range 
of optimal combination therapies, among the combinations provided in the following table 
where an optimal combination is a combination that is estimated to have an acceptable 
toxicity profile as measured by DLT’s and a high rate of immunologic response (IRR) as 
measured by peak immune response.  
 
 
Table 14:  Study Design 
Zone 
Arm/Combination 
Peptide vaccine 
Adjuvant preparation 
1 
A 
LPV7 + tet 
IFA  
1 
B 
LPV7 + tet 
PolyICLC  
1 
C 
LPV7 + tet 
Resiquimod  
2 
D 
LPV7 + tet 
PolyICLC + Resiquimod  
2 
E 
LPV7 + tet 
IFA + PolyICLC  
2 
F 
LPV7 + tet 
IFA + Resiquimod  
3 
G 
LPV7 + tet 
IFA + PolyICLC + Resiquimod 
 
DLTs are defined in Section 7.8 of the protocol. 
 
Within the range of optimal doses a co-primary objective is if more than one combination is 
contained within the range of optimal combinations, to estimate the difference in 
immunologic response rates between combinations, among those in the range of optimal 
combinations. 
 

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9.2   Combination allocation Part 1 (completed) 
The goal is to determine the range of optimal combinations, where optimal combination 
allocation will occur in two stages. The initial stage will accrue eligible participants in cohorts 
of one on each arm per the allocation process in 9.2.1 until a participant experiences a DLT. 
The second stage will allocate eligible participants based upon a continual reassessment 
method (CRM)81 for combinations of agents82.  Randomization will be based on equal 
allocation among allowable arms unless a weighted allocation scheme is triggered (Section 
9.2.1). Randomization will not be stratified by institution. Participants must be observed for a 
minimum of 3 weeks after the initial vaccine for initial escalation between zones, and 
between the 1st and 2nd participants within an arm. 
9.2.1   Combination allocation in Stage 1: 
The initial stage will accrue eligible participants on combinations in cohorts of one until a 
participant experiences a dose-limiting toxicity (DLT). The escalation plan for the first stage 
is based on grouping treatment combinations into “zones.” With this dose escalation design 
participants can be accrued and assigned to other open combinations within a zone but 
escalation will not occur outside the zone until the minimum follow -up period is observed for 
the first participant accrued to a combination.  Allocation will begin in Zone 1.  
 
 
Allocation in Zone 1: 
a) The first eligible participant in Zone 1 will be entered onto combination A, B or C, 
chosen at random.  
b) The second eligible participant in Zone 1 will be randomized to one of the two 
remaining combinations in the zone that have not yet been tried.  
c) The third eligible participant will be entered onto the combination in Zone 1 that 
has not yet been tried.  
d) Escalation to a Zone 2 occurs only when all combinations in Zone 1 have 
accrued at least 1 participant and no participants have experienced a DLT. 
 
 
Allocation in Zone 2: 
a) After completion of Zone 1, the first eligible participant in Zone 2 will be entered 
onto combination D, E or F, chosen at random.  
b) The second eligible participant in Zone 2 will be randomized to one of the two 
remaining combinations in the zone that have not yet been tried. 
c) The third eligible participant will be entered onto the combination in Zone 2 that 
has not yet been tried.  
d) Escalation to a Zone 3 occurs only when all combinations in Zone 2 have 
accrued at least 1 participant and no participants have experienced a DLT. 
 
 
Allocation in Zone 3: 
a) Note Zone 3 contains a single combination, so the first eligible participant on 
Zone 3 will be entered onto combination G.  
b) If 0/1 participants experience a DLT, the next eligible participant will be entered 
onto combination G. If 0/2 participants experience DLT, the next eligible 
participant will be randomized to any combination.  
c) In the absence of DLT’s, participants will continue to be randomized to 
combination A through G until a DLT occurs (upon which the modeling stage 
begins), or until at least 3 participants have been treated at every combination, at 
which point allocation will be based on which combination has the highest IRR. In 
the absence of DLT’s, participants will continue to be entered on the combination 
with the highest IRR until sufficient information has been obtained regarding the 

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optimal combination, according to the stopping rules described below. Once a 
DLT has been observed, Stage 2 using CRM modeling begins.  
9.2.2   Combination allocation in Stage 2: 
The second stage will allocate eligible participants based upon a continual reassessment 
method for combinations of agents (R package, pocrm). The modeling stage uses (a) a 
selected set of possible orderings for the DLT probabilities and (b) a working model for the 
DLT probabilities under each ordering.  
 
Table 15:  Possible Orderings of DLT Probabilities 
Possible orderings of DLT probabilities 
1) A-B-C-D-E-F-G 
2) A-C-B-D-F-E-G 
3) B-A-C-E-D-F-G 
4) B-C-A-E-F-D-G 
5) C-A-B-F-D-E-G 
6) C-B-A-F-E-D-G 
 
 
 
Table 16:  Working Model of DLT Probabilities Under Each Ordering 
Working model of DLT probabilities under each ordering** 
Order 
A 
B 
C 
D 
E 
F 
G 
1 
0.01 
0.05 
0.12 
0.20 
0.28 
0.36 
0.45 
2 
0.01 
0.12 
0.05 
0.20 
0.36 
0.28 
0.45 
3 
0.05 
0.01 
0.12 
0.28 
0.20 
0.36 
0.45 
4 
0.12 
0.01 
0.05 
0.36 
0.20 
0.28 
0.45 
5 
0.05 
0.12 
0.01 
0.28 
0.36 
0.20 
0.45 
6 
0.12 
0.05 
0.01 
0.36 
0.28 
0.20 
0.45 
**Working models were chosen according to the algorithm of Lee and Cheung (2009) 
 
Within each ordering, the continual reassessment method (CRM) is fit, using the working 
model and the accumulated data. The second stage will accrue eligible participants in 
cohorts of one and use a CRM model fit to estimate DLT probabilities at each arm 
combination. For each order, 𝑚= 1, ⋯,6, in Table 16, the DLT probabilities for each arm 
combination 
𝑖= 1, ⋯,7, 
are 
modeled 
via 
a 
one-parameter 
power 
model, 
𝑃𝑟⁡(DLT at combination⁡𝑖) ≈𝑝𝑚𝑖
𝜃𝑚, where the 𝑝𝑚𝑖 are the working model values for order 𝑚 
given in Table 16. After accrual of each participant into the trial, the parameter 𝜃𝑚 is 
estimated for each ordering by maximum likelihood estimation where the likelihood is given 
by ∏
(𝑝𝑚𝑖
𝜃𝑚)
𝑦𝑖
(1 −𝑝𝑚𝑖
𝜃𝑚)
𝑛𝑖−𝑦𝑖
7
𝑖=1
where 𝑦𝑖= the number of DLTs and 𝑛𝑖=the number of treated 
participants in arm combination 𝑖. The order with the largest likelihood is chosen and, within 
this ordering, DLT probability estimates are updated for each combination. If there is a tie 
between the likelihood values of tw o or more orderings, then the selected order is randomly 
chosen from among the tied orderings. These DLT probabilities will be used to define a set 
of “acceptable” combinations, defined as any dose with estimated DLT probability less than 
or equal to 33%. This toxicity tolerance of 33% was chosen based on the expectedness of 
adverse events.  

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9.2.3   Combination recommendation:  
Once the set of “acceptable” combinations is determined, the recommended combination 
will be based upon how many participants have been entered into the study to that point. If 
less than 3 participants have been treated on any acceptable dose, then the recommended 
combination for the next entered participant will be chosen at random from the acceptable 
combinations. If at least 3 participants have been treated at every acceptable combination, 
the recommended combination for the next participant entered is defined as the 
“acceptable” combination with the highest observed IRR. After each participant, a new 
recommended combination is obtained, and the next entered participant is allocated to the 
recommended combination. Part 1 of the trial will stop once sufficient information about the 
optimal dose range has been obtained, according to the stopping rules outlined in Section 
9.6.1.  
 
9.3   Statistical properties Part 1 (completed) 
Simulation results were run (R package, pocrm) to display the performance of the design 
characteristics. For each scenario, 1000 simulated trials were run. Each table reports the 
true DLT probability at each combination, the true IRR at each combination, the percentage 
of trials in which each combination was recommended as the optimal combination, and the 
average number of participants treated in a simulated study. 
9.3.1   Scenario 1: 
All true DLT probabilities are safe (i.e. less toxic than 33%) and one combination has the 
highest IRR. The true optimal combination is indicated in italicized bold type.  
 
Table 17:  Study Scenario 1 
Zones 
1 
2 
3 
Combination 
A 
B 
C 
D 
E 
F 
G 
True DLT probabilities 
1% 
2% 
5% 
7% 
11% 
14% 
20% 
True IRR 
35% 
35% 
40% 
55% 
60% 
65% 
80% 
% optimal combination recommendation 
0% 
1% 
1% 
7% 
10% 
18% 
62% 
% participant allocation 
8% 
8% 
9% 
12% 
14% 
17% 
33% 
Average trial size: 45 participants (25th, 50th, 75th percentile = 40, 43, 48) 
0.4% of simulated trials were stopped for safety 
The overall toxicity rate was 11.8%. The overall IRR was 60.9%. 
 
9.3.2   Scenario 2: 
One combination (G) has true DLT probability more toxic than 33% and the three 
combinations in Zone 2 have the highest IRR among safe combinations. The true range of 
optimal combinations is indicated in italicized bold type.  
 
Table 18:  Study Scenario 2 
Zones 
1 
2 
3 
Combination 
A 
B 
C 
D 
E 
F 
G 
True DLT probabilities 
5% 
6% 
8% 
22% 
23% 
25% 
45% 
True IRR 
45% 
50% 
50% 
65% 
65% 
65% 
80% 
% optimal combination recommendation 
6% 
10% 
10% 
25% 
22% 
20% 
5% 
% participant allocation 
12% 
14% 
13% 
18% 
18% 
17% 
8% 
Average trial size: 45 participants (25th, 50th, 75th percentile = 39, 44, 52) 

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2.2% of simulated trials were stopped for safety 
The overall toxicity rate was 18.3%. The overall IRR was 60.0%. 
 
9.3.3   Scenario 3: 
Combinations in Zones 2 and 3 have true DLT probabilities more toxic than 33% and the two 
combinations in Zone 1 have the highest IRR among safe combinations. The true range of 
optimal combinations is indicated in italicized bold type. 
 
Table 19:  Study Scenario 3 
Zones 
1 
2 
3 
Combination 
A 
B 
C 
D 
E 
F 
G 
True DLT probabilities 
18% 
20% 
25% 
45% 
48% 
54% 
64% 
True IRR 
65% 
70% 
70% 
80% 
80% 
80% 
90% 
% optimal combination recommendation 
19% 
28% 
25% 
1% 
1% 
0% 
0% 
% participant allocation 
25% 
29% 
29% 
6% 
6% 
5% 
1% 
Average trial size: 29 participants (25th, 50th, 75th percentile = 21, 33, 39) 
25.2% of simulated trials were stopped for safety 
The overall toxicity rate was 25.4%. The overall IRR was 70.6%. 
 
9.3.4   Scenario 4: 
All true DLT probabilities are unsafe (i.e. more toxic than 33%). 
 
Table 20:  Study Scenario 4 
Zones 
1 
2 
3 
Combination 
A 
B 
C 
D 
E 
F 
G 
True DLT probabilities 
50% 
55% 
56% 
65% 
66% 
74% 
80% 
True IRR 
70% 
70% 
70% 
80% 
80% 
80% 
90% 
% optimal combination recommendation 
1% 
1% 
3% 
0% 
0% 
0% 
0% 
% participant allocation 
32% 
28% 
35% 
2% 
2% 
1% 
0% 
Average trial size: 7 participants (25th, 50th, 75th percentile = 2, 3, 6) 
96% of simulated trials were stopped for safety 
The overall toxicity rate was 45.6%. The overall IRR was 70.3%. 
 
9.4   Study design Part 2 
A fixed sample size design will be used in part 2. The vaccine regimen is selected based on 
available data from Part 1, in which the recommended combination was Arm E. For Part 2, 
all accruals will be to Study Arm E2. 
 
9.5   Sample size/accrual 
9.5.1   Part 1 (completed) 
Maximum sample size is based upon acquiring sufficient information to assess the objective 
of selecting the arm with the highest IRR, assuming at least one optimal combination has 
been found.  
 
Data from our prior Mel43 trial in the arms not getting GM-CSF 12 indicated a high T cell 
response (5-fold increase in reactivity, at least 1200 cells per 100,000 CD8, and no overlap 

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62 
of 2 SD) to the 12 peptide vaccine in PBMC in 30 of 60 participants (50%, 95%CI (37%, 
63%)). Using the range as an approximate guideline, maximum sample size is established 
to ensure that if IRR among all seven arms differ by 30%, with the smallest expected rate of 
50%, then the arm with the higher IRR would be selected with high probability, P=0.90. The 
initial maximum target accrual of 20 eligible participants per arm is based upon the ranking 
and selection procedure, BSH 75, which allows selection of the best treatment worthy of 
further investigation in single-factor Bernoulli response experiments. However, it is not 
anticipated that all arms will fall in the range of optimal combinations, thus, sample size is 
estimated from the simulations and will be determined by the stopping rules below. Although 
maximum accrual is set at 140, in the simulation results below, on average a total of 
between 7 and 46 participants were required to complete the study. 
  
Accrual is estimated at 2 participants per month at each site (4 per month overall). The 
maximum accrual to the study is estimated from the simulations to be approximately 52 
participants. Adjusting for 10% ineligibility/dropout/withdrawal rate, maximum total target 
accrual for Part 1 is estimated at 58 participants. 
9.5.2   Part 2 
Immune response was observed in 6/16 (37.5%, 90%CI [17.8, 60.9%]) from Part 1. For Part 
2 target accrual is set at 16 eligible participants. At study conclusion, if we observe that at 
least 12/16 (a doubling of the response rate) eligible participants satisfy the definition of 
immune response then the lower limit of a one-sided 80%CI around the observed immune 
response rate for Arm E2 would be 61.5% which we would conclude supports that the 
immune response rate is likely to be higher for single site injections.  If fewer responses are 
observed then additional studies will be needed to better understand the impact of site 
rotation with  the use of the tetanus helper peptide. 
 
Accrual is estimated at 2 participants per month. Adjusting for 10% dropout rate, maximum 
total target accrual for Part 2 is estimated at 18 participants. 
 
9.6   Stopping rules: 
9.6.1   Part 1 (completed) 
The study will be stopped according to the following rules: 
1. The study will stop early for safety if the first three entered participants in Zone 1 
experience DLT on combinations in Zone 1 in Stage 1.  
2. If at any point in Stage 2, the set of acceptable combinations is empty, the trial will stop 
for safety.  
3. The study will stop if the recommendation is to assign the next participant to a 
combination that already has 20 participants treated at that combination.  
9.6.2   Part 2 
Safety will be assessed by monitoring the number of participants who experience a DLT. In 
Part 1, 1/16 (6.3%, 95%CI [0.2, 30.2%]) participants treated on Arm E experienced a DLT. 
For Part 2 the upper boundary of a sequential probability ratio test (SPRT) based upon a 
binomial test of proportions for DLT will be used for monitoring to protect against excessive 
DLT rates.  The stopping boundary is for a SPRT contrasting a 5% versus 30% DLT rate, 
with nominal type I and II errors of 5% and 20%, respectively. Safety stopping guidelines for 
the occurrence of DLTs are detailed in the following table. 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. 

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           Table 21: Incidence of Treatment-Related DLTs  
Incidence of treatment related DLTs 
Number of participants Stopping boundary 
2-4 
≥ 2 
5-11 
≥ 3 
12-18 
≥ 4 
 
9.7   Analyses 
9.7.1   Safety: 
All participants who receive any study intervention will be monitored for adverse events. 
Adverse events will be described and coded based upon the NCI CTCAE v4.03. A DLT is 
defined as any unexpected adverse event that is possibly, probably or definitely related to 
treatment and satisfies the criteria in section 7.8.  Occurrence of DLTs will guide escalation 
and stopping decisions.  At study conclusion frequency, proportion and severity of adverse 
events, and DLTs by arm will be tabulated.  
 
9.7.2   Immunologic response: 
Subjects are evaluable for immune response if at least one post treatment sample is 
measurable for response. Peak immunogenicity will be evaluated by direct ELIspot assay 
against the defined nonamer peptides in the peripheral blood. The presence or absence of 
an immune response will be defined by induction of at least a 2-fold increase in IFN-gamma-
secreting cells over background, and over pre-existing responses, and at least 30 IFN-
gamma secreting spots per 100,000 CD8 T cells, as described 34.  This assessment will be 
performed on blood through week 12, although a minimum of 4 weeks of data will be used 
to guide decisions about the range of optimal dose combinations.  IRR is defined as the 
proportion of participants where presence (as defined above) of an immune response has 
been observed. 
 
Durable immunogenicity will be evaluated week 26 by ELIspot assay against the defined 
nonamer peptides in the peripheral blood. The presence or absence of an immune response 
at that time will be defined by induction of at least a 2-fold increase in IFN-gamma-secreting 
cells over background, and over pre-existing responses, and at least 30 IFN-gamma 
secreting spots per 100,000 CD8 T cells, as described 34.  This assessment will be 
performed on blood through week 26.  
 
9.7.3   At study conclusion: 
Frequency and magnitude of adverse events will be summarized by arm. Point estimates 
and confidence intervals will be calculated for all dichotomous endpoints. If more than one 
combination is contained within the range of optimal combinations, estimates and 
confidence intervals will be used to estimate the difference in IRR between pairs of 
combinations among those in the range of optimal combinations.  
 
Immunogenicity will be evaluated by direct ELIspot assay against the defined nonamer 
peptides in vaccine site biopsies using the above criteria. Disease-free survival is defined as 
the duration of time from start of treatment to time of recurrence/progression or death from 
any cause, whichever occurs first. Participants who do not experience an event 
(recurrence/progression or death) will be censored at date of last contact. Overall survival is 
defined as the duration of time from start of treatment to time of death from any cause. 
Participants who do not experience an event (death) will be censored at date of last contact.  

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Disease-free survival and overall survival distributions will be estimated by the product limit 
method of Kaplan and Meier. 
 
10.0   Appendices 
 

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10.1   Appendix 1: X-page 
 
Studies & Tests 
 
 
Active Treatment 
Follow-up 
Day 
Pre  
1 
8 
15 
22 
36 
43 
57 
78 
85 
183 
365 
730 
Week 
 
0 
1 
2 
3 
5 
6 
8 
11 
12  
26 
52 
104 
Vax # 
 
1 
2 
3 
 
4 
 
5 
6 
 
 
 
 
Informed consent a 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
Class I HLA-typing a 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
Pathology review a 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
-HCG c  
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
HIV / Hepatitis C d 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
HGBA1C b 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
Urinalysis b 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
CBC with differential b 
 
X 
 
  
  
 X 
  
  
  
X 
X 
  
 
 
Comprehensive chemistry b, f 
 
Xe 
 
  
  
 X 
  
  
  
X 
X 
  
 
 
LDH b 
 
X 
 
  
  
 X 
  
  
  
X 
X 
  
 
 
Anti-nuclear antibody / Rf 
factor 
 
  
X 
  
  
 X 
  
  
  
X 
 
X  
 
 
Chest x-ray / CT b, g 
 
X 
  
  
  
  
  
  
  
  
X 
X 
 
 
Abdominal CT b, g 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
Pelvic CT b, g 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
Head MRI / CT b, g 
 
X 
  
  
  
  
  
  
  
  
  
  
 
 
History & physical b 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
120cc green top tubes  
 
  
X 
  
  
  
  
  
  
  
  
  
 
 
80cc green top tubes 
 
  
 
X 
 
X 
  
X 
  
 
X 
X 
X 
X 
20cc red top tubes  
 
  
X 
X 
 
X 
  
X 
  
 
X 
X 
X 
X 
Vaccination  
 
  
X 
X 
X 
 
X 
 
X 
X  
  
  
 
 
Biopsy normal skin,  pts 1-6, 
in Part 1 
 
 
 
X i 
 
X i 
 
 
 
 
 
 
 
 
Vaccine Site Biopsies  
 
  
 
X 
  
X 
  
  
  
 
 
  
 
 
Visual 
acuity, 
and color 
acuity examinations 
 
  
X 
  
  
 
  
  
  
 
X 
  
 
 
Note hair and eye color 
 
 
X 
 
 
 
 
 
 
 
X 
 
 
 
Note vitiligo 
 
 
X 
 
 
X 
 
 
 
 
X 
X  
 
 
Note neurologic function 
 
 
X 
 
 
X 
 
 
 
 
X 
X 
 
 
Distribute/review participant 
toxicity diary 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Xh 
 
 
 
 
a Any  point prior to randomization 
 
b Pre-study  within 6 weeks of  randomization 
c Within 2 weeks of  randomization (f or childbearing women) 
d Within 6 months of  randomization 
e   To include f asting glucose.  Fasting blood sugars may  be ev aluated 4h or more af ter last eating.   
f   Comprehensiv e chemistry  panel to include sodium, potassium, creatinine, glucose, calcium, total bilirubin, AST, ALT, and alkaline phosphatase  
g   PET-CT may  replace CT of  the chest, abdomen, and/or pelv is. New abnormalities of  AST, ALT, or LDH lev els, or new nodules on CXR will 
warrant more complete staging with CT scans of  the chest, abdomen and pelv is or PET-CT and with CT or MRI of  the brain as appropriate. 
h     Rev iew only   
i   Biopsies of  normal skin to be done just f or the f irst 6 patients enrolled in Part 1. 

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10.2   Appendix 2: AJCC Staging System, 7th Edition 
 
Melanoma TNM Classification 
T Classification 
Thickness 
Ulceration Status 
T1 
≤ 1.0 mm 
a:  without ulceration and level II/III 
b:  with ulceration or mitoses ≥1/mm2 
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 metastasis 
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 
N2 
N3 
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. 
 
 
Addendum:  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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10.3   Appendix 3: ECOG Performance Status 
 
Grade 
Performance 
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 self-care, but unable to carry out any work activities. Up and 
about more than 50% of waking hours 
3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any self-care.  Totally confined to bed or chair 
5 
Dead 
* As published in Am. J. Clin. Oncol.:  Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., 
McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. 
Am J Clin Oncol 5:649-655, 1982. 

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10.4   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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10.5   Appendix 5: Lot Testing   
A. Preparation of the synthetic melanoma and tetanus peptides 
All peptides have been synthesized under GMP conditions by Polypeptide Group (San 
Diego, CA) or comparable provider.  Certificates of analysis and technical summaries for 
each of the peptides will be included in the chemistry and manufacturing portion of the 
IND application. 
 
Peptide preparation and vialing has been performed under GMP conditions by 
Clinalfa/Merck for the tetanus peptide, and Bachem for the NY-ESO-1 long peptide, and 
will be performed under GLP conditions by the UVA Human Immune Therapy Center 
laboratory for the 6 peptides in the LPV6 long peptide mixture.  Documentation relating 
to the procedures used to prepare and vial the peptides will comply with the CFR and 
will be included in the Chemistry and Manufacturing Section of the IND application.   
 
B. Lot Release Quality Assurance Testing 
Prepared peptides will be subjected to the following tests: 
 
1. Identity:  Identity will be confirmed by structural studies.  The individual peptides will 
be tested for identity by mass spectrometry (to define molecular mass and amino 
acid sequence) and high performance liquid chromatography (HPLC, to confirm 
purity) in a GMP laboratory (Polypeptide Group). Amino acid analysis will also be 
performed to confirm identity further. 
 
Before mixing, the amino acid sequence of each individual peptide preparation will 
be reconfirmed by mass spectrometry or co-elution.  
 
After combining the peptides, the mixture will be subjected to HPLC analysis. 
 
2. Purity:  Purity will be assessed before and after vialing the peptide mixtures.  Before 
vialing the peptide mixtures, each synthetic peptide will be evaluated for the 
presence of a single dominant species by high performance liquid chromatography 
(HPLC).  Purity of each peptide component will exceed 90% (expected 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.  After vialing the peptide mixture, purity will be 
reconfirmed by HPLC. 
 
3. Aggregation:  Aggregation of peptides will be assessed by HPLC.  Aggregation is not 
expected. 
 
4. Trifluoroacetic acid (TFA):  The amount of total fluorine in each peptide preparation 
will be less than 0.5% or 5000 ppm. 
 
5. Potency:  The net peptide content will be calculated for each synthesis.  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. 
The amount of peptide in each sterile filtered peptide solution will be measured by 
amino acid analysis. The final vials will also be tested for quantity of total peptide by 
amino acid analysis.  Lot release criteria are defined in the Chemistry and 
Manufacturing section of the IND application. 
 

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6. Endotoxin:  Endotoxin testing will be conducted in accordance with CFR guidelines. 
 
7. General Safety:  General safety testing will be conducted in accordance with US 
CFR guidelines. 
 
8. Sterility:  Sterility testing w ill be conducted in accordance with CFR guidelines. 
 
C. Ongoing Quality Assurance Testing 
Stability:  We will follow guidelines for quality assurance studies to assure stability of the 
peptide preparation over time during the trial76.   The peptide preparations will be 
assayed for stability every six months for 2 years and every year thereafter.  The 
following analyses will be performed to confirm stability: 
1. HPLC:  HPLC will be performed to confirm purity.  A 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 intended peptide represents at least 75% of the total peptide 
species.  With liquid formulations of this peptide preparation, we have observed 
dimerization 
of 
cysteine-containing 
peptides, 
yet 
this 
peptide 
retained 
immunogenicity even when as little as 10% remains as monomer.  For the present 
study, dimerization of cysteine-containing peptides will be acceptable as long as at 
least 25% remains as monomer by HPLC.  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.  If a second measure is required, 
the lot will be held until the second reading is obtained and shown to be acceptable. 
 
2. Aggregation.  Aggregation will also be assessed by HPLC. 
 
3. Mass spectrometry.  This will be performed on each peptide peak to define the 
molecular sequence and mass if there is any ambiguity about the identity of the 
peptides based on HPLC.   New peaks on HPLC, or significant change in the elution 
profiles will warrant determination of the peptide identities by mass spectrometry on 
each peak. 
 
4. 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).  
Sterility testing will be conducted in accord with FDA guidelines in 21 CFR part 
610.12.  
 
 

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10.6   Appendix 6: NCI Common Terminology Criteria for Adverse Events v4.03 
 
http://evs.nci.nih.gov/ftp1/CTCAE 
 
 
 
 

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10.7   Appendix 7:  Summary of Changes 
 
07-31-17 
1) Updated Table of Contents. 
2) Section 1.1: updated to address current treatments for resected 
melanoma in the adjuvant setting. 
3) Section 1.2.6: updated to address ipilimumab approval for treatment of 
resected melanoma in the adjuvant setting. 
4) Section 4.1: removed reference to interferon packet. 
5) Section 5.6.1: added targeted molecular therapy (e.g. vemurafenib, other 
inhibitor of mutant BRAF, MEK, or cKit) as nonmerpitted treatments. 
6) Section 5.6.2.6: edited to indicate that any live vaccines (e.g. FluMist) are 
permitted, but should be administered at least 2 weeks prior to or at least 
2 weeks after a study vaccine. 
7) Updated reference list. 
06-26-17 
1) Updated cover sheet 
2) Updated study personnel 
3) Updated formatting and numbering throughout study document 
4) Additional administrative changes and typographical errors corrected 
throughout the document to improve readability. 
5) Table of Contents:  Updated 
6) Protocol precis 
 
Design: The study has been divided into Part 1 and Part 2.  Part 1 
required rotating vaccine sites and accrual to this part was 
completed.  Part 2 of the study has been added and the regimen 
requires that each vaccine is administered in the same skin site for 
all 6 vaccines. 
 
The objectives have been revised to add objectives specific for part 
2. 
 
Table 1 has been re-labeled as Table 1A.  The title of the table has 
been revised to specify that Table 1A is relevant for Part 1 of the 
study. 
 
Addition of Table 1B:  Study Arm E2 has been specified for Part 2 of 
the study. 
 
The rationale for choosing the vaccine regimen from study arm E 
has been added. 
 
Regimen:  Revised to describe regimen for Parts 1 and 2 of the 
study. 
 
Vaccine site biopsies:  revised to specify that 3 punch biopsies of 
normal skin will be completed in the first 6 patients enrolled in Part 1 
of the study. 
 
Leadership:  Revised to specify that Part 2 of the study will be 
conducted just at the University of Virgina. 
 
Accrual:  Adjusted to allow for an additional patients to be enrolled 
to part 2 of the study. 
 
7) Section 1.0:  Updated accrual information. 
8) Section 1.1:  Updated information describing incidence of melanoma and 
deaths from melanoma in 2017 and added a section on vaccine site 
selection to provide a rationale for evaluating same site vaccinations. 
9) Section 1.2.6:  Updated information on approved adjuvant therapies for 
melanoma. 
10) Section 1.2.10:  Added prior experience with same site vaccinations. 
11) Added Table 5 to describe vaccine site reactions for prior Mel44 and 
Mel58 studies. 
12) Section 2.0:  Revised objectives to add objectives for Part 2. 
 

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13) Section 3.1:  Regimen-added the regimen for Part 2, which includes 
same site vaccinations. 
14) Section 4.2.7(1):  Corrected section reference. 
15) Section 4.4.2:  Revised randomization section and specified that Part 2 of 
the study will not include randomization. 
16) Section 5.2.1: Added a description for preparing drug for Study Arm E2. 
17) Section 5.4.2:  Deleted prior text describing the vaccine regimen and 
replaced with a description for the administration of vaccines for Part 1 
and Part 2 and a description of expected vaccine site toxicities. 
18) Section 5.4.3:  A section describing doses for part 2 of the study has 
been added. 
19) Section 5.4.6 , 6.2.2, 6.2.4, Appendix 1:  Clarified that biopsies of normal 
skin will be completed in the first 6 participants who are enrolled in Part 1. 
20) Section 5.6:  Clarified that patients will be randomized in Part 1 and 
assigned to an arm in Part 2 of the study. 
21) Section 7.4.3:  Clarified that grade 3 injection site reactions with 
significant ulceration of the skin (> 2 cm) will be considered DLTs.   
Clarified dermatologic toxicities for resiquimod. 
22) Section 7.8:  Added grade 3 injection site reaction with ulceration ≤ 2 cm 
as an exception to the DLT rule 
23) Section 9.0:  Revised introduction to statistical considerations to add 
information about Part 2 of the study. 
24) Section 9.1-removed the DLT rule from this section and revised to refer to 
section 7.8 of the protocol. 
25) Sections 9.2, 9.3, 9.4, 9.5, 9.6:  clarified which sections pertain to Part 1 
and added statistical language for Part 2. 
26) Reformatted headings for the appendices 
27) Appendix 7:  added a summary of changes list for prior versions of the 
protocol 
06-17-14 
1) Sapna Patel’s role was changed from co-investigator to PI at MD 
Anderson Cancer Center. 
2) Table of Contents page numbers were updated. 
 
06-06-14 
1) Section 1.2.3:  Editorial change for clarification 
2) Section 1.2.4:  Editorial change for clarification 
3) Section 1.2.5:  The dosing for resiquimod was revised to include an 
acceptable dose range of 400-600 mg. Instructions about coverage and 
washing the affected area have been incorporated into the study protocol. 
4) Section 1.2.8:  Reference added 
5) Table 3:  Reformatted 
6) Table 4:  Updated with new data 
7) Section 4.1.4:  Clarification of inclusion criterion and whether repeat scans 
are needed after stereotactic radiotherapy. 
8) Section 4.1.5:  Clarification of inclusion criterion and the description of 
required lymph node basins. 
9) Section 4.1.8:  Clarification of inclusion criterion a) specifications for patients 
with Gilbert’s disease and b) re-defined acceptable limit for Hgb-A1C level. 
10) Section 4.2.11:  Addition of the following to allowable autoimmune disorders: 
       Hypothyroidism of any etiology on stable thyroid hormone replacement 
therapy. 
11) Section 5.2.1:  Arms E and G:  Change in mixing instructions for PolyICLC.  
PolyICLC will be incorporated into the vaccine mixture rather than 
administered as a separate injection.   
12) Section 5.2.1:  Clarification of vaccine components for Study Arms C, D, F 
and G.  The dose of resiquimod was adjusted from 500 mg to approximately 
500 mg.  Instructions aoubt coverage and washing the affected area have 

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been incorporated into the study protocol. 
13) Reference List:  Updated. 
 
03-25-14 
1) Dr. Geoffrey Weiss and Dr. Elizabeth Gaughan have been added to the list of 
co-investigators. 
2)  The Table of Contents was updated.   
3) The dose of resiquimod has been corrected throughout the study document.  
The correct dose is 1000 mcg and the drug will be supplied in multi-dose tubes.  
Section 1.2.6 also includes information on prior usage with this dose. 
4) Section 4.1.4:  An editorial change was made and also a clarification that the 
brain metastases in this section refer to treated brain metastases. 
5) Section 4.1.8:  The lab parameters inclusion criteria were reformatted.  HIV 
and HCV requirements were moved to the exclusion criteria (Section 4.2.6). 
6) Section 4.2.3:  The reference to Section 4.2.3 within this section was corrected 
to Section 4.2.4. 
7) Section 4.4.1:  Information about the interferon education packets was 
changed to include a statement that participants mus t convice the study team 
that the answered the questions accurately or understand the correct answers. 
8) Section 5.4.1:  A clarification was made to this section to specify that chronic 
inflammation is expected in subjects who receive Montanide ISA-51.  Also, 
editorial changes were made to this section. 
9) Section 6.2.2:  “Vaccine site biopsy (3)” was removed from the Day 8 schedule 
of events as the biopsy procedures were listed twice for this day. 
10) Sections 6.2.1, 6.2.2, 6.2.3, 6.2.4, 6.2.5, 6.2.6, 6.2.7, 6.2.8:  Clarfication was 
made to the language pertaining to the interval history and physical exam.  
Examination of vaccine sites was added to each of the study visit days, when 
applicable.  
11) Section 7:  Editorial correction was made to change “or” to “for”. 
12) Section 7.4.3:  A redundant section related to AEs that do not require 
expedited reporting.  Reporting guidelines are provided in Table 12. 
13) Section 7.4.3:  A clarification was made to  the dosing regimen that was used 
for resiquimod  in prior studies. 
14) Section 7.6.1:  A clarification was made to the process of reporting AEs.  AEs 
determined to require expedited reporting to the sponsor, must also be reported 
to a site’s institution according to local policy and procedures. 
15) Section 7.6.3:  A clarification was made regarding reporting AEs to the sites.  
Serious and unexpected suspected adverse reactions will be reported to the sites 
no later than 15 calendar days after the Sponsor determines that the the 
requirements for an IND safety report have been met. 
16) Section 7.7.2:  The phrase “and at UVA by the UVA melanoma team” was 
removed from this section as it did not apply to the outside sites.  Individual AEs 
will be reviewed at each site. 
17) Section 7.7.3:   The term “weekly” was removed when describing the 
melanoma team meetings.  The melanoma team meets 20 times a year, as 
specified in section 7.7.4 of the DSMP.  The term “may” was included in the 
description of the events that are reviewed at each meeting as the agenda varies 
depending upon study accruals. 
18) Section 7.7.4 :  The DSMP was modified to specify that month conference 
calls will be held between the Sponsor and the sites. 
19) Section 9.2.2:  “R-package, pocrm” was added for clarification.  Additional 
statistical methods were added to clarify the model of DLT probabilities. 

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20) 9.6.3: The endpoints of disease-free survival and overall survival have been 
defined. 
21) Appendix 1:  editorial change made to subscript “i”. 
22) Appendix 5:  Clarifications were made to the descriptions of the peptide 
preparation and vialing. 
10-10-13 
 
1) Cover sheet has been updated  
2. New study staff members have been added and the formatting for contact 
information has been corrected.  
3. Zone information has been added to the précis (Table 1) and Table 5 and the 
description of the regimen has been modified to reflect changes in the statistical 
section.  
4. Table of Contents has been updated.  
5. List of abbreviations has been updated.  
6. The timing of the negative control biopsies has been clarified throughout the 
protocol. The negative control biopsies will be completed on days 8 and 22 in the 
first 6 patients.  
7. Table 3: An explanation of the grading criteria are provided.  
8. Section 1.2.6 and 5.2.1: additional directions for applying the resiquimod have 
been added.  
9. Section 4.4.1: Replaced the section that participants will have to complete the 
interferon packet questions satisfactorily with a statement that answers to 
questions in the packet will be reviewed with the participant.  
10. Section 4.4.2: A description of when randomization is to take place in relation 
to registration and the start of treatment has been added.  
11. Section 4.4.3: “suture removal” was removed from the evalution following the 
biopsies as punch biopsies will not require sutures.  
12. Section 5.2 and 5.4.3: The dosage of and preparation of the vaccines were 
clarified.  
13. Section 5.4.6.4: Information on specimen collection delays and the associated 
documentation are provided.  
14. Section 5.6.2.6: clarification made to permitted influenza vaccines  
15. Section 6.2 Treatment: The schedules for the immunologic bloods, vaccines, 
and biopsies have been moved to this section.  
16. Section 7.0 Regulatory and Reporting Requirements: This entire section has 
been revised and reformatted to clarify the reporting requirments for the study. 
The original reporting tables have been replaced with a template table from the 
UVA Cancer Center DSMP and an IRB reporting table has been added. In 
addition, reporting criteria for reporting to the FDA have been replaced.  
17. Section 7.1.3: A clarification for the definition of hospitalization for expedited 
AE reporting has been added.  
18. Section 7.5: Adverse event classifications have been updated and the 
hospitalization category has been moved to the description for an SAE.  
19. Section 7.5: AE classifications have been updated to include 
Hematologic/Metabolic and Non-Hematologic/Non-metabolic categories. 
Autoimmune has been added to the allergic category.  
20. Section 7.7.1: Replaced section stating that someone from the research team 
would be available 24 hours a day 7 days a week for questions with the statement 
that subjects would be given information on how to contact their providers if they 
have questions or problems.  
21. Section 9.1: The table has been modified to correct the description of the 
adjuvant preparation for each arm/combination.  
22. Section 9.2: A description of the randomization has been added.  

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23. Appendix 1: Administrative changes have been made to the X-page to 
improve readability.  
24. Additional administrative changes and typographical errors corrected 
throughout the document to improve readability.  
 
08-06-13 
1) An additional study group has been added in response to the 
recommendations of the FDA and of our collaborators at MD Anderson Cancer 
Cente 
2) The study design has been changed to incorporate an adaptive design in order 
to minimize data with the least number of patients. This reduces the target 
sample size by half. 
3) Section 4.4 of the study protocol has been modified to include additional details 
regarding the registration and randomization of study patients. 
4) A section for study drug accountability and distribution has been added 
(section 5.3) 
5) Table of Contents has been updated. 
6) The source of Hiltonol (polyICLC) has been updated, in section 1.2.5. 
7) Updated section 1.2.10 to specify that the peptides for the vaccines have been 
synthesized under GMP conditions, rather than GLP. 
8) Appendices about vaccine preparation, immunologic assays, and details of 
tissue handling with the biopsies has been removed from the protocol and will be 
included ina Study Manual and Investigator Brochure. 
9) Numerous small administrative changes have been made to improve clarity, 
consistency and readability and to provide appropriate citations. 
10) Details about the quality assurance studies to be done on the peptides have 
been added to respond to the FDA after our preIND meeting. 
11) Skin biopsies have been amended: Patients 1-6 will have 3 skin punch 
biopsies rather than 2, on day 1.  The 3rd sample from skin on day 1 in those 
patients and from all patients on days 8 and 22 will be stored in RNA later rather 
than be collected for viable cell studies. 
12) Signature page and study staff contact information has been added. 
13) X-page has been corrected with regards to the timing of the normal skin 
biopsies. 
 
12/05/11 
1) Section 4.4 of the study protocol has been modified to include additional 
details regarding the registration and randomization of study patients. 
2) A section for study drug accountability and distribution has been added 
(section 5.3) 
3) Table of Contents has been updated. 
 
 
 

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11.0   Reference List 
 
 
 
(1)  Kenter GG, Welters MJ, Valentijn AR et al. Vaccination against HPV-16 oncoproteins 
for vulvar intraepithelial neoplasia. N Engl J Med 2009;361:1838-1847. 
 
(2)  Sabbatini P, Tsuji T, Ferran L et al. Phase I Trial of Overlapping Long Peptides from 
a Tumor Self-Antigen and Poly-ICLC Shows Rapid Induction of Integrated Immune 
Response in Ovarian Cancer Patients. Clin Cancer Res 2012;18:6497-6508. 
 
(3)  Gnjatic S, Saw hney NB, Bhardwaj N. Toll-like receptor agonists: are they good 
adjuvants? Cancer J 2010;16:382-391. 
 
(4)  Speiser DE, Lienard D, Rufer N et al. Rapid and strong human CD8+ T cell 
responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J 
Clin Invest 2005;115:739-746. 
 
(5)  Schwartzentruber DJ, Lawson DH, Richards JM et al. gp100 peptide vaccine and 
interleukin-2 in patients with advanced melanoma. N Engl J Med 2011;364:2119-
2127. 
 
(6)  Ascierto PA, Gogas HJ, Grob JJ et al. Adjuvant interferon alfa in malignant 
melanoma: an interdisciplinary and multinational expert review. Crit Rev Oncol 
Hematol 2013;85:149-161. 
 
(7)  Agarwala SS, Lee SJ, Flaherty LE et al. Randomized phase III trial of high-dose 
interferon alfa-2b (HDI) for 4 weeks induction only in patients with intermediate- and 
high-risk melanoma (Intergroup trial E 1697) . [abstract]Agarwala SS, Lee SJ, 
Flaherty LE et al. J Clin Oncol 2011;29 suppl:Abstract 8505 
 
(8)  Slingluff CL, Jr., Petroni GR, Chianese-Bullock KA et al. Immunologic and clinical 
outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma 
in the adjuvant setting. Clin Cancer Res 2007;13:6386-6395. 
 
(9)  Slingluff CL, Jr., Chianese-Bullock KA, Bullock TN et al. Immunity to melanoma 
antigens: from self-tolerance to immunotherapy. Adv Immunol 2006;90:243-95.:243-
295. 
 (10)  Kirkwood JM, Lee S, Land S et al. E1696: Final analysis of the clinical and 
immunological results of a multicenter ECOG phase II trial of multi-epitope peptide 
vaccination for stage IV melanoma with MART-1 (27-35), gp100 (209-217, 210M), 
and tyrosinase (368-376, 370D) (MGT) +/- IFNa2b and GM-CSF. [abstract]Kirkwood 
JM, Lee S, Land S et al. J Clin Oncol (Meeting Abstracts) 2004;22:7502 
 (11)  Rosenberg SA, Sherry RM, Morton KE et al. Tumor progression can occur despite 
the induction of very high levels of self/tumor antigen-specific CD8+ T cells in 
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