A Phase I Study With a Personalized NeoAntigen Cancer Vaccine in Melanoma
Phase: PHASE1
Conditions: Melanoma
Sponsor: Patrick Ott, MD, PhD

Title: A Phase I Study With a Personalized NeoAntigen Cancer Vaccine in Melanoma 
NCT Number: NCT01970358 
IRB Approval Date: 10/22/2013 

A Personalized NeoAntigen Cancer Vaccine in Melanoma 
 
 
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Protocol Version Date: October 25, 2016  
 
Local Protocol #: 13-240  
 
 
Title: A Phase I Study with a Personalized NeoAntigen Cancer Vaccine in Melanoma  
 
Principal Investigator: Dr. Patrick A. Ott, Dana Farber Cancer Institute 
 
Site Principal Investigator:  Dr. Donald P. Lawrence, MD, Massachusetts General Hospital 
 
Coordinating Center: Dana Farber Cancer Institute 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Agent(s): 1) Personalized neoantigen peptides   IND15703 Dana Farber Cancer Institute 
                 2) Poly-ICLC (Hiltonol ®) DMF BB-MF 14,266 and BB-MF 14,322 Oncovir Inc. 

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Schema A:  
 
 
Schema B 
 
 
Start at dose 
level 1
Treat cohort
(N=5)
Number of 
DLTs
Treat 10 more 
patients
Treat cohort -1*
(N=5) 
Number 
of DLTs
Stop
0 or 1
≥2 of 5
Subject Identification, 
Consent,  Pre-Screen,  QACT 
Initial Registration
Melanoma resection, tumor 
procurement, pathology 
review
Whole exome sequencing, RNA sequencing, 
HLA typing, bioinformatics, vaccine 
preparation, Secondary Registration
* Cohort -1: Poly-
ICLC 50% dose 
reduction
Treat 10 more 
patients
0 or 1
≥2 of 5
Prime
Boost
Boost
Week
15
20
25
10
5
0
Primary safety
endpoint
Primary 
immunological
endpoint
Vaccinations

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TABLE OF CONTENTS 
A Personalized NeoAntigen Cancer Vaccine in Melanoma 
1. 
OBJECTIVES .................................................................................................................................................... 5 
1.1 
Study Design .................................................................................................................................................... 5 
1.2 
Primary Objectives .......................................................................................................................................... 6 
1.3 
Secondary Objectives ...................................................................................................................................... 6 
2 
BACKGROUND ................................................................................................................................................ 6 
2.1 
Study Agents .................................................................................................................................................... 6 
2.2 
Study Disease ................................................................................................................................................ 13 
2.3 
Rationale ....................................................................................................................................................... 14 
2.4 
Correlative Studies Background.................................................................................................................... 16 
3 
PATIENT SELECTION .................................................................................................................................. 16 
3.1 
Eligibility Criteria ......................................................................................................................................... 16 
3.2 
Exclusion Criteria ...................................................................................................................................... 19 
3.3 
Inclusion of Women, Minorities and Other Underrepresented Populations ................................................. 20 
4 
REGISTRATION PROCEDURES ................................................................................................................ 20 
4.1 
General Guidelines for DF/HCC and DF/PCC Institutions ......................................................................... 21 
4.2 
Registration Process for DF/HCC and DF/PCC Institutions ..................................................................... 21 
5 
TREATMENT PLAN ...................................................................................................................................... 22 
5.1 
Tissue procurement and sequencing ........................................................................................................... 23 
5.2 Preparation of NeoVax .................................................................................................................................. 23 
5.3 
Administration ............................................................................................................................................... 27 
5.4 
Dose schema and staggering of patients ....................................................................................................... 28 
5.5 
Definition of Dose-Limiting Toxicity .......................................................................................................... 29 
5.6 
General Concomitant Medication and Supportive Care Guidelines ............................................................. 30 
5.7 
Duration of Therapy ....................................................................................................................................... 30 
5.8 
Duration of Follow Up .................................................................................................................................. 30 
5.9 
Criteria for Removal from Study ................................................................................................................... 31 
6 
EXPECTED TOXICITIES AND DOSING DELAYS/DOSE MODIFICATIONS .................................... 31 
6.1 
Anticipated Toxicities .................................................................................................................................... 31 
6.2 
Toxicity Management .................................................................................................................................... 33 
6.3 
Dose Modifications/Delays ........................................................................................................................... 33 
7 
DRUG FORMULATION AND ADMINISTRATION ................................................................................. 33 
7.1 
Poly-ICLC (Hiltonol ®) ................................................................................................................................ 33 

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7.2 
Personalized neoantigen peptides ................................................................................................................. 37 
8 
CORRELATIVE/SPECIAL STUDIES .......................................................................................................... 38 
8.1 
Pharmacodynamic Studies ............................................................................................................................ 38 
9 
STUDY CALENDAR ...................................................................................................................................... 41 
10 
MEASUREMENT OF EFFECT..................................................................................................................... 43 
10.1 Antitumor Effect– Solid Tumors .................................................................................................................... 43 
11 
ADVERSE EVENT REPORTING REQUIREMENTS ............................................................................... 43 
11.1 Definitions ..................................................................................................................................................... 43 
11.2 Procedures for AE and SAE Recording and Reporting ................................................................................. 45 
11.3 Reporting Requirements ................................................................................................................................ 46 
11.4 Reporting to the Study Sponsor ..................................................................................................................... 46 
11.5 Reporting to the Institutional Review Board (IRB) ....................................................................................... 47 
11.6 Reporting to the Food and Drug Administration (FDA) ............................................................................... 47 
11.7 Reporting to Hospital Risk Management....................................................................................................... 47 
11.8 Monitoring of Adverse Events and Period of Observation ............................................................................ 47 
12 
DATA AND SAFETY MONITORING .......................................................................................................... 48 
12.1 Data Reporting .............................................................................................................................................. 48 
12.2 Safety Meetings ............................................................................................................................................. 49 
12.3 External Safety Monitoring Board ................................................................................................................ 49 
12.4 Monitoring ..................................................................................................................................................... 49 
12.5 Data sharing .................................................................................................................................................. 50 
13 
REGULATORY CONSIDERATIONS .......................................................................................................... 50 
13.1 Protocol Review and Amendments ................................................................................................................ 50 
13.2 Informed Consent .......................................................................................................................................... 51 
13.3 Ethics and Good Clinical Practice (GCP) .................................................................................................... 51 
13.4 Study Documentation .................................................................................................................................... 52 
13.5 Records Retention ......................................................................................................................................... 52 
14 
STATISTICAL CONSIDERATIONS ............................................................................................................ 52 
14.1 Study Design/Endpoints ................................................................................................................................ 52 
15 
PUBLICATION PLAN .................................................................................................................................... 57 
16 
REFERENCES ................................................................................................................................................. 58 
17 
APPENDICES .................................................................................................................................................. 63 
17.1 Appendix A: Performance Status Criteria ..................................................................................................... 63 

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1. OBJECTIVES 
 
1.1 
Study Design 
The study is an open label, phase I trial in which patients with high-risk melanoma will be 
immunized with up to 20 peptides that are both specific to the patient’s tumor cells (i.e. –  not 
found in their normal cells) and unique to the patient (i.e. – “personal”).  These peptides are 
encoded by missense mutations, in-frame gene fusions and novel open reading frame 
mutations (collectively known as “neoantigens”) that have occurred within that patient’s 
tumor cells and are identified through DNA and RNA sequencing.  Up to 20 peptides  about 
20-30 amino acids in length will be prepared for each patient and will be administered 
together with the immune adjuvant poly-ICLC.  Thus, the personalized NeoAntigen Cancer 
Vaccine consists of peptides + poly-ICLC and will be termed “NeoVax”. 
Eligible patients will be initially entered into the trial to undergo surgery with the intent to 
resect all melanoma detectable clinically and on radiographic scans. Patients will be 
reassessed for eligibility prior to the first vaccination and will continue onto the treatment 
phase of the trial if secondary eligibility criteria are met.  
Five patients will be entered for the initial safety evaluation (Cohort 1). If none or only 1 
patient experiences a DLT on Cohort 1, an additional 10 patients will be treated at this dose 
to increase the likelihood of detecting serious toxicities, to complete biologic correlative 
endpoints and to gain preliminary experience with clinical tumor activity.  If two or more 
patients in Cohort 1 experience dose limiting toxicity (DLT) during the first 7 weeks of 
treatment, then the dose of poly-ICLC will be reduced by 50% and 5 patients will be enrolled 
on Cohort -1.  If none or only 1 patient experiences a DLT on Cohort -1, then an additional 
10 patients will be enrolled as described above. If two or more patients in Cohort -1 
experience a DLT, then the study will be stopped.   
 
In parallel and under the same IND (#15703), a second phase I trial is being conducted with 
NeoVax (DF/HCC # 14-362 - A Phase I Study of a Personalized NeoAntigen Cancer Vaccine 
with Radiotherapy Among MGMT Unmethylated, Newly Diagnosed Glioblastoma Patients). 
Both trials utilize early stage patients who are otherwise healthy with competent immune 
systems. For both trials, the NeoVax personalized peptides are being produced by the same 
process and released to the same specifications, identical amounts are being used and the 
identical amount of Hiltonol® is added. In both trials the vaccination schedule is identical and 
the definition of Dose Limiting Toxicities is identical. In both trials, aninitial cohort of five 
patients is being evaluated for safety. 
 
For the purposes of evaluation of safety in the five patient cohort for either trial, the initial 
patients from both trials will be combined to achieve the required 5 patient cohort. Once 5 
patients from the combined trials have successfully reached the end of the DLT evaluation 
window (4 weeks after the last priming dose) and if fewer than two DLTs have been observed, 
treatment can initiate to complete the initial 5 patient cohort and for the additional 10 patients 
at the selected dose. 

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The primary objectives of the study are to test the safety and feasibility of administering 
NeoVax; the secondary objectives include evaluating the immune response to the 
personalized neoantigens and monitoring disease recurrence. 
 
 
1.2 
Primary Objectives  
1.2.1   To evaluate safety and tolerability of administering NeoVax in patients with high-
risk melanoma  
1.2.2 To determine the feasibility of generating and administering NeoVax in patients 
with high-risk melanoma 
 
Two feasibility endpoints will be evaluated: 
(1) 
The proportion of patients for whom sequencing and analysis leads to 
identification of at least 10 actionable peptides to initiate vaccine production  
(2) 
The proportion of patients for whom the time between surgery and vaccine 
availability is 12 weeks or less (excluding those patients who did not generate at 
least 10 actionable peptides) 
 
 
1.3 
Secondary Objectives 
1.3.1 
To assess the induction of specific cellular immune responses following administration 
of NeoVax 
1.3.2 
To determine the proportion of patients alive without progression at two years after 
surgery following administration of NeoVax 
 
2 BACKGROUND 
 
2.1 
Study Agents 
 
2.1.1 Personalized NeoAntigen Peptides   
Sequencing technology has revealed that each tumor contains multiple, patient-specific 
mutations that alter the protein coding content of a gene1.  Such mutations create altered 
proteins, ranging from single amino acid changes (caused by missense mutations) to 
addition of long regions of novel amino acid sequence due to frame shifts, read-through 
of termination codons or translation of intron regions (novel open reading frame 
mutations; neoORFs).  These mutated proteins are valuable targets for the host’s 

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immune response to the tumor as, unlike native proteins, they are not subject to the 
immune-dampening effects of self-tolerance. Therefore, mutated proteins are more 
likely to be immunogenic and are also more specific for the tumor cells compared to 
normal cells of the patient2.  
There have been several reports indicating that tumors expressing missense mutations 
or neoORFs can be prevented, and in some cases eradicated, by immunization with 
peptides corresponding to the mutated protein3-7. For example, CD8+ cytotoxic T 
lymphocytes (CTLs) directed at missense mutations in the RNA helicase protein6 or the 
ß2-spectrin protein8 found in particular murine tumors have been identified.  
Expression of these mutated proteins by the tumor was found to correlate with tumor 
control or progression.   
Correspondingly, several human studies of spontaneous regression and long-term 
survival have shown that powerful CD8+ T cell responses against mutated epitopes 
correlate with good clinical responses9-12. These observations were made in both 
melanoma and lung cancer.  Most of these CD8+ T cell responses show specificity 
toward the mutated missense epitope compared to the native epitope, represent a high 
proportion of circulating T cells, and result in cells that are more abundant and active 
than CD8+ T cell responses in the same patients directed toward over-expressed native 
antigens.   
Two studies in humans have directly assessed the immunotherapeutic potential of 
mutated antigens.  Follicular lymphoma is characterized by uncontrolled growth of a B 
cell expressing rearranged immunoglobulin.  Purification of that rearranged 
immunoglobulin and use as a vaccine may improve disease-free survival13.  The 
induced CD8+ T cells showed reactivity to the rearranged, mutated portion of the 
immunoglobulin molecule (the idiotype) and not the germline framework14.  Moreover, 
a mix of peptides corresponding to the oncogenic proteins of HPV (a neoORF for 
humans) has been shown to result in significant remission of premalignant lesions 
induced by HPV15-17. 
Thus, in animals and in humans, immune responses to both discrete mutated antigens 
(such as missense mutations) and expansive novel antigen (neoORF) are 
observationally correlated with regression and long-term remission and, in two cases, 
were shown to control disease following immunization.  Importantly, escape from host 
immunosurveillance (“immunoediting”) can be tracked to alterations in expression of 
dominant mutated antigens in mice8,18 and man19. 
Most cancer vaccines employing peptides as immunogens have utilized “short” 
peptides. These peptides are typically 9 – 10 amino acids in length and capable of direct 
binding to the HLA molecule on the surface of HLA-expressing cells.  “Long” 
peptides, ~20-30 amino acids in length, have recently been shown to produce a more 
robust and more durable immune response15,16.  Long peptides require internalization, 
processing and cross-presentation in order to bind to HLA molecules; these functions 

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only occur in professional antigen-presenting cells, such as dendritic cells, which can 
induce strong T cell responses. 
 
Many studies in humans have demonstrated the safety of peptide vaccines.  These 
include studies with multiple short peptides33 as well as multiple long peptides, 
including neoORFs.  In particular, two studies have been conducted with a mixture of 
10 overlapping long peptides derived from p5328,29  and three separate studies with a 
mixture of 13 long peptides derived from the oncogenic proteins of HPV17-19.  In these 
studies, no toxicity higher than grade 2 was observed and most adverse events were of 
limited duration and severity.  Additionally, many heterologous antigen preparations 
have been tested in humans.  Such preparations include irradiated cell vaccines20,21 and 
tumor cell lysates22.  These heterogeneous vaccines contain mutated antigens, in the 
form of intact proteins, partially degraded intracellular protein, and peptides found on 
the surface bound to MHC I.  Moreover, they contain over-expressed and selectively-
expressed molecules as well as many additional native proteins.  In addition, purified 
heat shock protein (HSP) 96 peptide complexes have been used as antigen23; such 
complexes also contain many mutated peptides.  None of these studies have reported 
significant safety issues directly attributable to the immunogens of the vaccines.   
 
GMP peptides will be prepared by synthetic chemistry, purified by Reverse Phase 
High-Performance Liquid Chromatography (RP-HPLC), mixed in small groups (see 
Sect 5.2.1.2 and Appendix B; Section 7.3.4) and combined with the immune adjuvant 
poly-ICLC, a stabilized double-stranded RNA (see Section 2.1.2 below). The mixtures 
of peptides and poly-ICLC will be used for vaccination with the intention to induce 
cellular immune responses directed at these patient/tumor specific mutations.  Each 
patient will receive the full complement of peptides at each immunization.   
 
 
 
 
 
 
2.1.2 
Poly-ICLC 
2.1.2.1 TLR agonists as adjuvants for cancer vaccines 
Toll like receptors (TLRs) are important members of the family of pattern recognition 
receptors (PRRs) which recognize conserved motifs shared by many micro-organisms, 
termed “pathogen-associated molecular patterns” (PAMPS).  Recognition of these 
“danger signals” activates multiple elements of the innate and adaptive immune system.  
TLRs are expressed by cells of the innate and adaptive immune systems such as 
dendritic cells (DCs), macrophages, T and B cells, mast cells, and granulocytes and are 
localized in different cellular compartments, such as the plasma membrane, lysosomes, 
endosomes, and endolysosomes24. Different TLRs recognize distinct PAMPS. For 
example, TLR4 is activated by LPS contained in bacterial cell walls, TLR9 is activated 

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by unmethylated bacterial or viral CpG DNA, and TLR3 is activated by double 
stranded RNA25.  TLR ligand binding leads to the activation of one or more 
intracellular signaling pathways, ultimately resulting in the production of many key 
molecules associated with inflammation and immunity (particularly the transcription 
factor NF-κB and the Type-I interferons). TLR mediated DC activation leads to 
enhanced DC activation, phagocytosis, upregulation of activation and co-stimulation 
markers such as CD80, CD83, and CD86, expression of CCR7 allowing migration of 
DC to draining lymph nodes and facilitating antigen presentation to T cells, as well as 
increased secretion of cytokines such as type I interferons, IL-12, and IL-6. All of these 
downstream events are critical for the induction of an adaptive immune response. 
Among the most promising cancer vaccine adjuvants currently in clinical development 
are the TLR9 agonist CpG and the synthetic double-stranded RNA (dsRNA) TLR3 
ligand poly-ICLC.  In preclinical studies poly-ICLC appears to be the most potent TLR 
adjuvant when compared to LPS and CpG due to its induction of pro-inflammatory 
cytokines and lack of stimulation of IL-10, as well as maintenance of high levels of co-
stimulatory molecules in DCs26. Furthermore, poly-ICLC was recently directly 
compared to CpG in non-human primates (rhesus macaques) as adjuvant for a protein 
vaccine consisting of human papillomavirus (HPV)16 capsomers.  Poly-ICLC was 
found to be much more effective in inducing HPV specific Th1 immune responses27. 
2.1.2.2 Poly-ICLC – a synthetic TLR3 agonist with strong vaccine adjuvant 
properties 
 
Poly-ICLC is a synthetically prepared double-stranded RNA consisting of polyI and 
polyC strands of average length of about 5000 nucleotides, which has been stabilized to 
thermal denaturation and hydrolysis by serum nucleases by the addition of polylysine 
and carboxymethylcellulose.  The compound activates TLR3 and the RNA helicase-
domains of MDA5 and RIG3, both members of the PAMP family, leading to DC and 
natural killer (NK) cell activation and production of a “natural mix” of type I 
interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts a more direct, 
broad host-targeted anti-infectious and possibly anti-tumor effect mediated by the two 
IFN-inducible nuclear enzyme systems, the 2’5’-OAS and the P1/eIF2a kinase, also 
known as the PKR (4-6), as well as RIG-I helicase and MDA5. 
 
In rodents and non-human primates, poly-ICLC was shown to enhance T cell responses 
to viral antigens28-31, cross-priming, and the induction of tumor-, virus-, and 
autoantigen-specific CD8+ T-cells32-34.  In a recent study in non-human primates, poly-
ICLC was found to be essential for the generation of antibody responses and T-cell 
immunity to DC targeted or non-targeted HIV Gag p24 protein, emphasizing its 
effectiveness as a vaccine adjuvant.  
 
In human subjects, transcriptional analysis of serial whole blood samples revealed 
similar gene expression profiles among 8 healthy human volunteers receiving one 
single s.c. administration of poly-ICLC and differential expression of up to 212 genes 
between these 8 subjects versus 4 subjects receiving placebo35. Remarkably, 

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comparison of the poly-ICLC gene expression data to previous data from volunteers 
immunized with the highly effective yellow fever vaccine YF17D36 showed that a large 
number of transcriptional and signal transduction canonical pathways, including those 
of the innate immune system, were similarly upregulated at peak time points.  
 
More recently, an immunologic analysis was reported on patients with ovarian, 
fallopian tube, and primary peritoneal cancer in second or third complete clinical 
remission who were treated on a phase 1 study of subcutaneous vaccination with 
synthetic overlapping long peptides (OLP) from the cancer testis antigen NY-ESO-1 
alone or with Montanide-ISA-51, or with 1.4 mg poly-ICLC and Montanide.   The 
generation of NY-ESO-1-specific CD4+ and CD8+ T-cell and antibody responses were 
markedly enhanced with the addition of poly-ICLC and Montanide compared to OLP 
alone or OLP and Montanide37. 
 
2.1.2.3 Pre-clinical toxicology of poly-ICLC 
Complete information on the pre-clinical toxicology studies can be found in the poly-
ICLC (Hiltonol®) Investigator Brochure (IB).  The results of these toxicology studies 
are summarized in brief below: 
  
Single dose toxicity: 
All administrations of poly-ICLC were intravenous (IV).   
Rodents. The median lethal dose (LD50) values for single dose poly-ICLC were 
approximately 15-18.3 mg/kg in rats and 25-30 mg/kg in mice. No necropsies were 
conducted. 
Dogs. In beagle dogs, the lethal dose of single dose poly-ICLC administration was 4.0 
mg/kg. Necropsy indicated toxic effects in the gastrointestinal, hepatic, cardiovascular, 
renal, endocrine, and lymphatic systems. Sublethal doses caused reversible 
nephrotoxicity and hepatotoxicity. The non-toxic dose for dogs was 0.25 mg/kg.  
Primates. In rhesus monkeys, single-dose administration of poly-ICLC was non-toxic at 
a dose of .5 mg/kg, but lethal at 20 mg/kg. Toxic effects were seen in the 
gastrointestinal, hepatic, cardiovascular, renal, endocrine, and lymphatic systems.  
 
Repeated dose toxicity: 
Rodents. Mice treated with four daily IV doses of up to 3.66 mg/kg poly-ICLC 
exhibited no toxicity.   
Cats. One of two cats treated with the same regimen at 3.33 mg/kg poly-ICLC 
demonstrated vomiting, an episode of diarrhea, and moderate weight loss. 
Non-human Primates. Three rhesus monkeys and 6 macaque monkeys were treated 
with 5 mg/kg IV of poly-ICLC over 2 days and 3 mg/kg 3 times weekly for 16 weeks, 
respectively; no significant toxicity was observed other than a single instance of emesis 
and shivering. Necropsy of the macaques showed no drug-related changes by gross or 
microscopic pathology of all organs.  In two additional studies, macaque monkeys 

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were treated daily with up to 3 mg/kg IV for 12 days and chimpanzees were treated 
daily with 3 mg/kg IV for two 6-day periods, then every other day for a total of 7 
weeks. At the 3 mg/kg doses, a decrease in hematocrit was seen in both studies.   No 
change in hematocrit was seen at 0.3 mg/kg. In the chimpanzee study, in addition to a 
decrease in hematocrit, leukocytosis and elevations in alanine aminotrasferase (ALT) 
and aspartate aminotrasferase (AST) were observed and normalized after termination of 
dosing.  No other significant toxicity was observed in these studies. 
 
 
2.1.2.4 Clinical toxicology of poly-ICLC 
 
Poly-ICLC is the TLR3 agonist formulation most extensively tested in patients with 
infectious diseases and in subjects with a variety of different tumor types. 
 
Prior to the availability of recombinant interferon, poly-ICLC was used clinically at 
high doses ≥ 6mg/m2 (about 170 μg/kg) in patients with a variety of solid tumors and 
leukemia38.  Fever, often above 40oC, was a common adverse event and the primary 
dose-limiting factor. Other common adverse events were flu-like symptoms (nausea, 
vomiting, arthralgia. myalgia and fatigue) and hypotension, thrombocytopenia and 
leukopenia. Once recombinant interferon became clinically available, the need to 
pursue high dose poly-ICLC was eliminated, and it became recognized that lower doses 
(10 – 50 μg/kg) were highly effective at stimulating host defense and as an immune 
adjuvant.  
 
By now, more than 400 patients with malignant gliomas have been entered on 7 clinical 
trials using low dose (1-2 mg total dose) poly-ICLC either as monotherapy or in 
conjunction with chemotherapy, radiation, or vaccine (Table 1). Furthermore, patients 
with various other solid tumors (prostate, colorectal, pancreatic, hepatocellular, breast, 
and ovarian cancers), in addition to patients with HIV/AIDS and multiple sclerosis have 
been treated on more than 10 additional clinical phase I and phase II studies.  Overall, 
the drug has been well-tolerated across all studies and spectrum of diseases. The most 
common adverse events attributed as at least possibly related to poly-ICLC have 
included: 
 
- bone marrow toxicity (leukopenia, neutropenia, thrombocytopenia, and anemia)  
- reversible liver toxicity (AST/ALT elevations, LDH and alkaline phosphatase 
elevation) 
- transient discomfort at the injection site 
- transient fatigue/malaise 
- transient flu-like symptoms 
In a recently published phase I clinical trial, 11 patients with ovarian, fallopian tube, or 
primary peritoneal cancer were treated with poly-ICLC at a total dose of 1.4 mg in 
combination with Montanide-ISA-51 and synthetic  over-lapping long peptides from 
NY-ESO-1. The vaccine was generally well-tolerated with grade 1-2 injection site 

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adjuvant treatment strategies are critical in order to improve recurrence rates. Importantly, 
preventing metastatic disease by effectively reducing recurrence rates in the adjuvant setting 
(patients with no evidence of disease) may be seen as surrogate for curing patients with 
disease.  Immunotherapy has been recognized decades ago as a potentially effective 
treatment in melanoma. In the adjuvant setting, the purpose of vaccination is to initiate or re-
stimulate endogenous, melanoma-specific T and B cells to react against and eradicate 
minimal residual disease. 
 
2.3 
Rationale 
This phase 1 study seeks to demonstrate that the combination of personalized neoantigen 
peptides and poly-ICLC is safe, feasible, and induces strong tumor-specific T cell immunity.  
DNA sequencing, particularly next-generation sequencing technology, has revealed a genetic 
landscape of cancer that contains many protein coding mutations found uniquely in the tumor of 
an individual patient.   These mutations include both single-amino acid missense mutations 
(the predominant type) and novel open reading frames created by frame-shift or read-through 
mutations (neoORFs) varying in length from 1 to up to 100’s of amino acids.  There is 
evidence in both animals and humans demonstrating that such mutated epitopes are effective at 
inducing an immune response47.  Importantly, strong CD8+ T cell responses against mutated 
epitopes have been found in patients with melanoma and non small cell lung cancer who either 
had a spontaneous regression or a dramatic response after adoptive T cell transfer, suggesting that 
there may be an association with CD8+ T cell activity and good clinical responses9-12,48,49.  Most 
of these CD8+ T cell responses showed very good specificity toward the mutated epitope 
compared to the native epitope, represented a high proportion of circulating T cells, and induced 
cells that were more abundant and active than CD8+ T cells in the same patients directed toward 
over-expressed native antigens.  
Two studies in humans have directly assessed the immunotherapeutic potential of mutated 
antigens. Purified rearranged immunoglobulin expressed by malignant B cells in follicular 
lymphoma has been used as vaccine and has led to good clinical outcomes in some phase 2 and 
phase 3 studies (possibly dependent on amounts of residual disease)50.  More recently, a mix of 
peptides corresponding to the oncogenic proteins of HPV (directly analogous to the neoORF type 
of mutation) has been shown to result in significant remission of premalignant lesions induced by 
HPV15-17. Thus, in animals and in humans, immune responses to both discrete mutated antigens 
(such as missense mutations) and expansive novel antigen (neoORF) are observationally 
correlated with regression and long-term remission and, in two clinical studies, have been shown 
to control disease following therapeutic vaccination. The direct and comprehensive identification 
of the many mutated epitopes found in cancer genomes creates the opportunity to use this class of 
immunogen to improve the immune response and efficacy of cancer vaccines.   
 
Translating sequencing information into a therapeutic vaccine further requires:  
 
(1) Prediction of mutated peptides that can bind to personal HLA molecules. Efficiently choosing 
which particular mutations to utilize as immunogen requires identification of the patient HLA 

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type and the ability to predict which mutated peptides would efficiently bind to the patient’s HLA 
alleles. Recently, neural network based learning approaches with validated binding and non-
binding peptides have advanced the accuracy of prediction algorithms for the major HLA-A and 
-B alleles51.   In a pilot study, we prepared seventy-four 9-mer and sixty-three 10-mer peptides 
predicted by the algorithm NetMHCpan to have affinities to particular HLA alleles below 1000 
nM, with most being below 500 nM.  Binding of these peptides to their cognate HLA alleles was 
experimentally determined using a well-established competitive binding assay52.  Seventy-five 
percent of 9-mer peptides predicted to have an affinity below 150 nM were experimentally 
verified to be below 150 nM and 90% of these peptides were shown to have an affinity below 
500 nM, a generally accepted threshold .  Fifty percent of 9-mer peptides predicted to have an 
affinity between 150 and 500 nM were shown to have an affinity below 500 nM.  Ten-mer 
peptide predictions were slightly less precise.  For predicted high affinity 10-mer peptides 
(below 150 nM), 70% were shown to have an affinity below 500 nM.  However, only 35% of 
10-mer peptides predicted to have an affinity between 150 and 500 nM were shown to have 
an affinity below 500 nM. Thus, 10-mer peptides with predicted affinities above 150 nM will 
not be utilized. These predictions provide a reliable tool, when correctly applied, to assist in 
the selection of potentially immunogenic peptides. 
 
(2) Formulating the drug as a multi-epitope vaccine of long peptides. For each patient, multiple 
neoantigens will be targeted.  Targeting as many mutated epitopes as practically possible takes 
advantage of the enormous capacity of the immune system, prevents the opportunity for 
immunological escape53  by down-modulation of a particular immune targeted gene product, and 
compensates for the known inaccuracy of epitope prediction approaches.  Synthetic peptides 
provide a particularly useful means to prepare multiple immunogens efficiently and to rapidly 
translate identification of mutant epitopes to an effective vaccine.  Peptides can be readily 
synthesized chemically and easily purified utilizing reagents free of contaminating bacteria or 
animal substances.  The small size allows a clear focus on the mutated region of the protein and 
also reduces irrelevant antigenic competition from other components (unmutated protein or viral 
vector antigens). “Long” peptides, ~20-30 amino acids in length (as opposed to “short” 
peptides, ca 8-10 amino acids) have recently been shown to produce a more robust and more 
durable immune responses15,16.   
 
(3) Combination with a strong vaccine adjuvant.  Effective vaccines require a strong adjuvant to 
initiate an immune response. As described in Section 2.1.2.2, poly-ICLC, an agonist of TLR3 
and the RNA helicase domains of MDA5 and RIG3, has shown several desirable properties for 
a vaccine adjuvant.  These properties include the induction of local and systemic activation 
of immune cells in vivo, production of stimulatory chemokines and cytokines, and 
stimulation of antigen-presentation by DCs. Furthermore, poly-ICLC can induce durable 
CD4+ and CD8+ responses in humans. Importantly, striking similarities in the upregulation of 
transcriptional and signal transduction pathways were seen in subjects vaccinated with poly-
ICLC and in volunteers who had received the highly effective, replication-competent yellow 
fever vaccine35. Furthermore, >90% of ovarian carcinoma patients immunized with poly-
ICLC in combination with a NY-ESO-1 peptide vaccine (in addition to Montanide) showed 
induction of  CD4+ and CD8+ T cell,  as well as antibody responses to the peptide in a recent 

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phase 1 study37.  Moreover, poly-ICLC has been extensively tested in more than 25 clinical trials 
to date and exhibited a relatively benign toxicity profile.  
 
2.4 
Correlative Studies Background 
 
The primary immunological endpoint of this study will be the assessment of T cell response 
measured by ex vivo IFN-γ ELISPOT. IFN-γ secretion occurs as a result of the recognition 
of cognate peptides or mitogenic stimuli by CD4+ and/or CD8+ T –cells. A multitude of 
different CD4+ and CD8+ determinants will likely be presented to T cells in vivo since the 
20-30-mer peptides used for vaccination should undergo processing into smaller peptides by 
antigen presenting cells. We hypothesize that the combination of personalized neoantigen 
peptides, which are novel to the immune system and thus not subject to the immune-
dampening effects of self-tolerance, and the powerful immune adjuvant  poly-ICLC will 
induce strong CD4+ and/or CD8+ responses. The expectation is therefore that T cell 
responses are detectable ex vivo, i.e. without the need for in vitro expansion of epitope 
specific T cells through short-term culture.   Patients will initially be evaluated using the total 
pool of peptide immunogens as stimulant in the ELISPOT assay.  For patients demonstrating 
a robust positive response, the precise immunogenic peptide(s) will be determined in follow-
up analyses. 
 
The IFN-γ ELISPOT is generally accepted as a robust and reproducible assay to detect ex 
vivo T cell activity and determine specificity. In addition to the analysis of the magnitude 
and determinant mapping of the T cell response in peripheral blood monocytes, other 
aspects of the immune response induced by the vaccine are critical and will be assessed. 
These evaluations will be performed in patients who exhibit an ex vivo IFN-γ ELISPOT 
response in the screening assay. They include the evaluation of T cell subsets (Th1 versus 
Th2, T effector versus memory cells), analysis of the presence and abundance of regulatory 
cells such as T regulatory cells or myeloid derived suppressor cells, and cytotoxicity assays if 
patient-specific melanoma cells lines are successfully established.   
 
3 PATIENT SELECTION 
 
3.1 Eligibility Criteria 
 
Eligibility to participate will be assessed at two timepoints: prior to surgery (Initial Registration) 
and prior to the first vaccination (Secondary Registration).   
3.1.1  Eligibility Criteria for Initial Registration 
Patients must meet the following criteria prior to surgery to be eligible for initial registration 
onto the study: 
3.1.1.1 Patient is willing and able to give written informed consent. 

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3.1.1.2 Patient is agreeable to allow tumor and normal tissue samples to be submitted for 
complete exome and transcriptome sequencing. 
3.1.1.3 Pathologically confirmed, clinically evident (by physical examination or radiographic 
imaging) stage IIIB, IIIC, and IVM1a and b cutaneous melanoma (anatomic stages T1-
4b N1a and T1-4b N2a not included).  The current diagnosis may be the patient’s first 
diagnosis of melanoma or recurrent melanoma after previous diagnosis of an earlier 
stage melanoma. 
3.1.1.4 Complete surgical resection of metastatic disease (lymph node, in transit, satellite 
lesion(s), distant metastases) with negative margins on resected specimens as confirmed 
by pathologic review has not been performed, but is deemed feasible by the treating 
surgical oncologist. Surgical resection of the primary melanoma may or may not have 
been performed.  
3.1.1.5 The patient must be free of unresectable metastatic disease within 4 weeks prior to the 
surgery being performed with the intention to remove all melanoma. This pre-surgery 
baseline assessment must be documented by complete physical examination and 
imaging studies.  Imaging studies must include a total body PET-CT in conjunction 
with a brain MRI (or head CT if brain MRI is contraindicated). If a PET/CT scan cannot 
be done, a CT of the neck, chest, abdomen, and pelvis should be performed.  
3.1.1.6 Patients may have received prior interferon alpha (IFN-α), but must not have received 
IFN-α in the 4-week period prior to initiation of first vaccination with NeoVax. Patients 
who have not received prior adjuvant therapy should be informed of the potential 
therapeutic benefit of IFN-α. Previous radiation therapy, including after the surgical 
resection, is allowed as long as 14 days have elapsed between the radiation and 
initiation of first vaccination with NeoVax. 
3.1.1.7 Age ≥ 18 years.   
3.1.1.8 ECOG performance status  <1.  
3.1.1.9 Normal organ and bone marrow function as defined below: 
• Leukocytes 
 
 
 
 
 
 
≥  3,500/mcL 
• Absolute lymphocyte count  
 > 800/mcL   
• Absolute neutrophil count   
> 1,500/mcL    
• Platelets   
 
 
 
 
 
 
> 100,000/mcL   
• Hemoglobin  
 
 
 
 
 
> 9.0 g/dL   
• Total serum bilirubin   
 
 
< 1.0 x institutional upper limit of normal 
• AST (SGOT)/ALT (SGPT)   
< 2.0 x institutional upper limit of normal 
• Serum creatinine   
 
 
 
< 1.5 x institutional upper limit of normal 

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3.1.1.10 Women of childbearing potential (WOCBP) must have a negative pregnancy test 
(minimum sensitivity 25 IU/L or equivalent of HCG) before entry onto the trial, 
because the effects NeoVax on the developing human fetus are unknown.   
3.1.1.11 Female patients enrolled in the study, who are not free from menses for >2 years, post 
hysterectomy / oophorectomy, or surgically sterilized, must be willing to use either 2 
adequate barrier methods or a barrier method plus a hormonal method of contraception 
to prevent pregnancy or to abstain from sexual activity throughout the study, starting 
with visit 1 through 4 weeks after the last dose of study therapy. Approved 
contraceptive methods include for example; intra uterine device, diaphragm with 
spermicide, cervical cap with spermicide, male condoms, or female condom with 
spermicide.   Spermicides alone are not an acceptable method of contraception.    
3.1.1.12 Male patients must agree to use an adequate method of contraception starting with the 
first dose of NeoVax through 4 weeks  after the last dose of study therapy.  
 
3.1.2 Eligibility Criteria for Secondary Registration 
3.1.2.1 ECOG performance status  <1.  
3.1.2.2 Normal organ and bone marrow function as defined below: 
• Leukocytes 
 
 
 
 
 
 
≥  3,500/mcL 
• Absolute lymphocyte count  
 > 800/mcL   
• Absolute neutrophil count   
> 1,500/mcL    
• Platelets   
 
 
 
 
 
 
> 100,000/mcL   
• Hemoglobin  
 
 
 
 
 
> 9.0 g/dL   
• Total serum bilirubin   
 
 
< 1.0 x institutional upper limit of normal 
• AST (SGOT)/ALT (SGPT)   
< 2.0 x institutional upper limit of normal 
• Serum creatinine   
 
 
 
< 1.5 x institutional upper limit of normal 
 
 
3.1.2.3 Women of childbearing potential (WOCBP) must have a negative pregnancy test 
(minimum sensitivity 25 IU/L or equivalent of HCG) within 7 days prior to start of 
study medication, because the effects NeoVax on the developing human fetus are 
unknown.  It is the investigators’ responsibility to repeat the pregnancy test should start 
of treatment be delayed. 
3.1.2.4 Female patients enrolled in the study, who are not free from menses for >2 years, post 
hysterectomy / oophorectomy, or surgically sterilized, must be willing to use either 2 
adequate barrier methods or a barrier method plus a hormonal method of contraception 

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to prevent pregnancy or to abstain from sexual activity throughout the study, starting 
with visit 1 through 4 weeks after the last dose of study therapy. Approved 
contraceptive methods include for example; intra uterine device, diaphragm with 
spermicide, cervical cap with spermicide, male condoms, or female condom with 
spermicide.   Spermicides alone are not an acceptable method of contraception.    
3.1.2.5 Male patients must agree to use an adequate method of contraception starting with the 
first dose of NeoVax through 4 weeks  after the last dose of study therapy.  
3.2 Exclusion Criteria 
 
Patients who exhibit any of the following conditions at any point prior to first vaccination  
will not be eligible for admission to or continuation on the study: 
 
3.2.1 
Prior treatment with immune-modulatory agents including, but not limited to: IL-
2, CTLA-4 blockade, PD-1/PD-L1 blockade, CD40 stimulation, CD137 
stimulation with the exception of INF-α given as adjuvant treatment for high-risk, 
surgically resected melanoma 
3.2.2 
Prior investigational melanoma-directed cancer vaccine therapy 
3.2.3 
Prior chemotherapy, including targeted therapy such as BRAF or MEK inhibition 
3.2.4 
Treatment with other investigational products within the last 2 months prior to entry 
into this study 
3.2.5 
Previous bone marrow or stem cell transplant 
3.2.6 
Concomitant therapy with any anti-cancer agents, other investigational anti-cancer 
therapies, or immunosuppressive agents; chronic use of systemic corticosteroids  
3.2.7 
Use of a non-oncology vaccine therapy for prevention of infectious diseases during the 
4 week period prior to first dose of NeoVax administration.  Patients may not receive 
any non-oncology vaccine therapy during the period of NeoVax administration and 
until at least 8 weeks after the last dose of study therapy. 
3.2.8 
 History of severe allergic reactions attributed to any vaccine therapy for the 
prevention of infectious diseases  
3.2.9 
Mucosal melanoma and uveal melanoma 
3.2.10 Active, known, or suspected autoimmune disease or immunosuppressive conditions 
with the exception of vitiligo, type 1 diabetes, residual autoimmune-related 
hypothyroidism requiring hormone replacement, or psoriasis not requiring systemic 
treatment 
3.2.11 Concomitant treatment with corticosteroids greater than physiologic doses (used in 
the management of cancer or non-cancer-related illnesses). Topical (if not including 
the proposed vaccination sites) or inhalational steroids are allowed. 
3.2.12 Known chronic infections with HIV, hepatitis B or C 

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3.2.13 Uncontrolled intercurrent illness including, but not limited to ongoing or active 
infection, symptomatic congestive heart failure, unstable angina pectoris, 
cardiac arrhythmia 
3.2.14 Any underlying medical condition, psychiatric condition or social situation that in the 
opinion of the investigator would compromise study administration as per 
protocol or compromise the assessment of AEs 
3.2.15 Pregnant women are excluded from this study because personalized neoantigen 
peptides and poly-ICLC are agents with unknown risks to the developing fetus.  
Because there is an unknown but potential risk of adverse events in nursing infants 
secondary to treatment of the mother with personalized neoantigen peptides and poly-
ICLC, nursing women are excluded from this study.  
3.2.16 Individuals with history of an invasive malignancy except for the following 
circumstances: a) individuals with a history of invasive malignancy are eligible if they 
have been disease –free for at least 3 years and are deemed by the investigator to be at 
low risk for recurrence of that malignancy; b) individuals with any of the following 
cancers are eligible if diagnosed and treated: carcinoma in situ of the breast, oral 
cavity or cervix and basal cell or squamous cell carcinoma of the skin. 
 
3.3 Inclusion of Women, Minorities and Other Underrepresented Populations 
This protocol does not exclude women, minorities, or other underrepresented 
populations.  The inclusion and exclusion criteria are not expected to have a negative 
effect on the recruitment or retention of underrepresented populations. 
 
 
4 
REGISTRATION PROCEDURES 
Eligibility will be confirmed and participants registered on 2 separate, serial occasions on 
this trial: 
1. Prior to surgical resection: 
Prior to initial surgical resection, the patient is screened for eligibility using the initial 
eligibility checklist.  Once overall trial initial eligibility is confirmed, patient is 
registered, undergoes surgical resection and preparation  of the participant’s vaccine is 
initiated.   
 
2. Prior to first vaccination:  
As participant’s 1st study vaccine dose may not be administered until 10-17 weeks from 
initial study registration, there will be a secondary “screen” conducted prior to first 
vaccination to ensure s/he meets criteria to receive study treatment. 
If confirmed eligible for this next phase of the study (eligible to treat as per sections 3.1.2 
and 3.2), participant will proceed to receive vaccines on study.   
 
 

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4.1 General Guidelines for DF/HCC and DF/PCC Institutions 
 
The study team will register eligible patients with the DF/HCC Quality Assurance Office 
for Clinical Trials (QACT) central registration system. Initial Registration must occur 
prior to surgical resection. Any patient not registered to the protocol before surgical 
resection begins will be considered ineligible and registration will be denied. Secondary 
Registration must occur prior to initiation of study vaccine administrations. Any patient 
not registered to the protocol before receiving protocol vaccine treatment will be 
considered ineligible and registration will be denied.  
 
For both registration timepoints, an investigator will confirm eligibility criteria and a 
member of the study team will complete the appropriate protocol-specific eligibility 
checklist: 
• Initial Eligibility Checklist for Overall Study Participation (for Initial Registration) 
• Secondary Eligibility Checklist to Initiate Treatment (for Secondary Registration) 
 
Following each registration stage, participants may begin the relevant protocol therapy. 
Issues that would cause treatment delays beyond this timeframe should be discussed with 
the Principal Investigator (PI). If a participant does not receive study therapy as outlined in 
protocol following either registration, the participant’s registration must be canceled. Notify 
the QACT Registrar of registration cancellations as soon as possible. 
 
4.2 
Registration Process for DF/HCC and DF/PCC Institutions 
 
The Initial Registration procedures are as follows: 
 
1. Obtain written informed consent from the participant prior to the performance of any 
protocol-specific screening procedures or assessments. 
2. Complete the QACT protocol-specific eligibility checklist:  
Initial Eligibility Checklist for Overall Study Participation (for Initial 
Registration) 
using the initial eligibility assessment documented in the patient’s medical/research 
record.  
To be eligible for initial registration to the study, the participant must meet each 
inclusion and exclusion criteria listed on the initial eligibility checklist.  
Reminder: Confirm eligibility for ancillary studies at the same time as eligibility for the 
treatment study. Registration to both treatment and ancillary studies will not be 
completed if eligibility requirements are not met for all studies. 
3. Fax the eligibility checklist(s) and all pages of the consent form(s) to the QACT at 
. 
For Phase I protocols, attach participant dose level assignment confirmation from the 
sponsor. 
4. The QACT Registrar will (a) review the initial eligibility checklist, (b) initially 
register the participant on the study, and (c) randomize the participant when 
applicable. 

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5. An email confirmation of the registration and/or randomization will be sent to the PI, 
study coordinator(s) from QACT immediately following the registration and/or 
randomization. 
  
The Secondary Registration procedures are as follows: 
 
1. Complete the QACT protocol-specific eligibility checklist:  
Secondary 
Eligibility 
Checklist 
to 
Initiate 
Treatment 
(for 
Secondary 
Registration) 
using the secondary eligibility assessment documented in the patient’s medical/research 
record.  
To be eligible for secondary registration to the study, the participant must meet 
each inclusion and exclusion criteria listed on the secondary eligibility checklist.  
Reminder: Confirm eligibility for ancillary studies at the same time as eligibility for the 
treatment study. Registration to both treatment and ancillary studies will not be 
completed if eligibility requirements are not met for all studies. 
2. Fax the eligibility checklist(s) and all pages of the consent form(s) to the QACT at 
. 
For Phase I protocols, attach participant dose level assignment confirmation from the 
sponsor. 
3. The QACT Registrar will (a) review the secondary eligibility checklist, (b) register 
the participant on the study, and (c) randomize the participant when applicable. 
4. An email confirmation of the registration and/or randomization will be sent to the PI, 
study coordinator(s) from QACT immediately following the registration and/or 
randomization. 
 
5 TREATMENT PLAN  
 
Treatment will be administered on an outpatient basis. Expected toxicities and potential risks 
as well as dose modifications for NeoVax are described in Section 6 (Expected Toxicities and 
Dosing Delays/Dose Modification). No investigational or commercial agents or therapies 
other than those described below may be administered with the intent to treat the patient’s 
malignancy. 
 
Patients with clinically evident locoregional metastatic disease or fully resectable distant 
metastatic disease as defined in 3.1.3 will be identified and enrolled on the study. After 
signing consent, the patients will undergo surgery with the intention to remove all melanoma 
(curative intent). Tissue procurement will be performed as described in 5.1. After 
confirmation of complete tumor resection by pathologic review and determination of 
adequacy of DNA and RNA yield from tumor tissue for sequencing, the generation of the 
vaccine as described in 5.2.1 will be initiated. Patients with tumors that are not completely 
resected and/or that provide inadequate DNA and/or RNA for sequencing will be removed 
from the trial and replaced.   
 

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5.1 Tissue procurement and sequencing 
5.1.1 Tumor and normal tissue harvest 
Patients will undergo complete resection of their primary melanoma (if not already 
removed) and all regional metastatic disease with the intent of rendering them free of 
melanoma. Dr. Patrick Ott, the principal investigator 
 
 should be contacted at the time of the initial consent to ensure that ancillary 
studies are coordinated. The study investigators and members of the study team will 
coordinate tissue acquisition with the surgical staff of the participating institution and the 
staff of the DFCI/Cell Manipulation Core Facility.    
After adequate tumor for pathological assessment has been harvested as deemed by the 
surgeon, remaining tumor tissue will be placed in sterile media in a sterile container and 
transferred to the DFCI/Cell Manipulation Core Facility for disaggregation.  Portions of 
the tumor tissue will be used for whole-exome and transcriptome sequencing and cell 
line and tumor infiltrating lymphocyte generation and any remaining tumor will be 
cryopreserved or given to the sponsor for protocol-related studies.  In the event that 
sequence analysis yields no results or sub-optimal results, tumor cell line cells or 
paraffin embedded samples retained by the participating institutions pathology 
department may be used to prepare additional nucleic acid for sequencing.  No saved 
samples will be used to prepare cellular products for future clinical use. Peripheral 
blood mononuclear cells from a blood draw will be utilized for a normal tissue sample.  
 
5.1.2  Nucleic acid will be extracted from the tissue samples and sequencing will be 
conducted at the CLIA-certified laboratory at Broad Institute.  For tumor and normal 
DNA samples, whole exome capture will be conducted prior to sequencing on Illumina 
HiSeq.  For tumor RNA, a cDNA library will be prepared on poly-A selected RNA prior 
to sequencing on Illumina HiSeq.  If the quantity or quality of DNA or RNA isolated 
from the tissue sample is inadequate for exome or cDNA library preparation and 
sequencing, then DNA or RNA may be extracted from the patient-specific tumor cell line 
(if generated) or paraffin embedded samples.   
 
5.2 Preparation of NeoVax  
 
5.2.1   Preparation of personalized neoantigen peptides 
5.2.1.1 Identification of target epitopes for peptide design 
 
 
 
 
 
 
 

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5.2.1.2 Peptide synthesis 
 
 
 
 
 
 
 
 
 

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5.2.2 Preparation of NeoVax peptide pools 
 
 
 
 
  
 
 
 
 
  
 
 
 
 
 
 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
    
 
  
5.2.3   Pretreatment criteria 
Preparation of the final NeoVax product will begin upon confirmation that the patient is 
medically cleared to undergo vaccine administration (confirmed arrival in the clinic, 

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stable vital signs, no new acute medical issues or laboratory abnormalities potentially 
interfering with vaccine administration).  
5.2.3.1 Day 1 
Laboratory parameters must be reviewed and must be within the ranges as listed in 
3.1.1.9 prior to administration of the study agents. No specific prophylactic or 
supportive care is necessary prior to administration of the study agents.  
5.2.3.2 Subsequent treatment days  
Laboratory parameters must be reviewed and must be within the ranges as listed in 
3.1.1.9 prior to administration of the study agents. No specific prophylactic or 
supportive care is necessary prior to administration of the study agents, however, 
patients who have developed flu-like symptoms that were controlled with 
acetaminophen or non-steroidal anti-inflammatory drugs during previous treatments 
may be pre-treated with acetaminophen (650 mg PO) or ibuprofen (400-600mg PO) as 
per the investigator’s discretion. 
5.2.4    Preparation of the dosage form 
 
 
 
 
 
  
 
    
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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5.3 
Administration 
 
Treatment Description 
Agent 
Treatment 
phase 
Schedule 
Premedications
Precautions 
Dose 
Route 
NeoVax 
(peptides + 
poly-ICLC) 
 Prime 
Days 1, 4, 
8, 15, 22* 
None 
Poly-ICLC:  
4 x 0.5 mg (total 
dose 2 mg) 
Peptides: 
x g per 
peptide 
Vol = 4 x 1 ml 
injections into 
4 different 
anatomic sites 
(0.5 ml i.d. & 
0.5 ml s.c.) 
NeoVax 
(peptides + 
poly -ICLC) 
Boost 
Days 78 
and 134** 
None 
Poly-ICLC:  
4 x 0.5 mg (total 
dose 2 mg) 
Peptides: 
4 x 300g  per 
peptide 
Vol = 4 x 1 ml 
injections 
into 4 
different 
anatomic 
sites (0.5 ml 
i.d. & 0.5 ml 
s.c.) 
*± 3 day treatment window is allowed except for days 4 and 8 (± 1 day)  
** ± 7 day treatment window is allowed 
5.3.1 
Injections 
Each of the up to 4 NeoVax syringes will be assigned to one of four extremities. At 
each immunization, each NeoVax syringe will be administered to the assigned 
extremity as follows: using one syringe/needle per site, 0.5 mL will be administered 
intradermally (i.d.) and 0.5 mL will be administered subcutaneously (s.c.) using the 
same syringe/needle at the same injection site (i.e. NeoVax A will be injected i.d. and 
s.c. into left arm on day 1, 4, 8 etc., NeoVax B will be injected i.d. and s.c. into right 
arm on days 1, 4, 8 etc.). Alternative anatomical locations for patients who are status 
post complete axillary or inguinal lymph node dissection or other contraindications 
that prevent injections to a particular extremity are the left and right midriff, 
respectively. 
5.3.2 
Treatment window 
NeoVax may be administered within 1 day of the scheduled administration date for days 
4 and 8, within 3 days of the scheduled administration date for days 15 and 22 and 
within 7 days for days 78 and 134. 
5.3.3 
Observation post vaccination 
Patients should be observed in the clinic for at least 60 minutes after the last of the 4 
injections and vital signs should be checked once between 30 and 90 minutes after the 

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last injection for each clinic visit. Monitoring for immediate adverse events should 
include attention to possible injections site reaction or a systemic reaction. In the 
absence of the occurrence of an adverse event, patients will be discharged from the 
outpatient clinic. 
5.4 
Dose schema and staggering of patients 
5.4.1  DLT and expansion cohorts 
Five patients will be entered for the initial safety evaluation (Cohort1). If two or more 
patients in Cohort 1 experience dose limiting toxicity (DLT) during the first 7 weeks of 
treatment, then that cohort will be determined to have unacceptable toxicity; otherwise, 
an additional 10 patients will be treated at the initial dose to increase the likelihood of 
detecting serious toxicities, to complete biologic correlative endpoints, and to gain 
preliminary experience with clinical tumor activity. If Cohort 1 is found to have 
unacceptable toxicity, then 5 patients may be enrolled on cohort -1 shown below.   For 
Cohort -1, the dose of poly-ICLC is dropped by 50% relative to Cohort 1 but the dose of 
peptides remains the same.  If two or more patients in Cohort -1 experience a DLT, then 
the study will be stopped.  If none or only one patient experiences a DLT on Cohort -1, 
then 10 additional patients will be enrolled for additional toxicity evaluation, to complete 
biologic correlative endpoint, and to gain preliminary experience with clinical tumor 
activity. If four or more patients in the expansion cohort experience dose limiting toxicity 
(DLT) during the first 7 weeks of treatment, then that cohort will be determined to have 
unacceptable toxicity and further dosing of all patients will be stopped. 
 
Dose De-escalation Schedule 
1 cycle = 22days 
 
Dose Level 
Dose of poly-ICLC 
 
- 1 
0.25 mg per 
injection site (4 
injections, total dose 
1 mg 
Vol = 4 x 0.875 ml 
Starting Dose → 
  1 
0.5 mg per injection 
site (4 injections, 
total dose 2 mg 
5.4.2 Staggering of patients in DLT cohorts 
5.4.3 The first three patients of Cohort 1 and Cohort -1 will be enrolled in a staggered 
fashion: Patients 2, 3 and 4 will not start treatment until the week after the previous 

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patient has completed the priming phase (scheduled for day 22). Prime/boost 
schedule 
Vaccine will be administered following a prime/boost schedule.  Priming doses of 
vaccine will be administered on days 1, 4, 8, 15, and 22 as shown above (Section 5.3, 
Administration and Schema B).  In the boost phase, vaccine will be administered on days 
78 (week 12) and 134 (week 20). 
5.4.4 Definition of evaluable patients 
All patients receiving at least one dose of vaccine will be evaluable for toxicity. Patients 
will be evaluable for immunologic activity if they have received all vaccinations during 
the priming phase and the first boost vaccination. 
A minimum of 3 vaccinations and absence of DLTs are required for a patient to complete 
the 7-week DLT observation period successfully. 
 
5.5 Definition of Dose-Limiting Toxicity 
 
Dose-limiting toxicity (DLT) is based on the CTEP Active Version (version 4.0) of the 
NCI Common Terminology Criteria for Adverse Events (CTCAE). DLT refers to 
toxicities experienced within 49 days (7 weeks) of treatment initiation (“DLT 
window”). If treatment has to be delayed because of a toxicity that does not fulfill the 
criteria of a DLT, the DLT window should be extended by that time period of delay. A 
DLT will be defined as follows:  
1. Grade 3 or 4 toxicity that is definitely, probably, or possibly related to the 
administration of vaccine, excluding: 
  - Transient (≤ 72 hours) flu-like syndrome;  
  - Grade 3 nausea, vomiting, constipation, or diarrhea that returns to grade 2 (or 
lower) level within 48 hours; 
  - Grade 3 rash that resolves to grade 2 or grade 1 within < 14 days.  
2. Grade 3 or 4 abnormal laboratory value that is definitely, probably, or 
possibly related to the administration of vaccine if it persists for more than 7 
days, requires hospitalization or medical intervention (except for Grade 3 
electrolyte abnormality that lasts ≤ 72 hours, is not clinically complicated, and 
resolves spontaneously or responds to conventional medical intervention). 
3. Any grade 3 or grade 4 toxicity that is considered, in the opinion of the 
investigator, to be dose-limiting. 
4. Any death related to study treatment. 
 

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Management and dose modifications associated with the above adverse events are 
outlined in Section 6 (Expected Toxicities and Dosing Delays/Dose Modifications). 
 
5.6 
General Concomitant Medication and Supportive Care Guidelines 
 
Acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDS) may be used 
for prevention or treatment of flu-like illness symptoms. If clinically necessary, 
corticosteroids, antihistamines and non-steroidal anti-inflammatory drugs, including 
COX-2 inhibitors may be used at the discretion of the treating physician. Investigators 
may prescribe all other concomitant medications or treatments deemed necessary to 
provide adequate patient care. 
 
Management of more serious toxicities will be directed by a study investigator and will 
be in accordance with standard of care clinical practice. 
 
 
5.7 Duration of Therapy 
 
Duration of therapy will depend on tolerability of the immunizations and evidence of 
disease recurrence as judged by the treating investigator. In the absence of treatment 
delays due to adverse events, treatment will be given until the day 134 vaccination 
(the 2nd booster vaccination) or until one of the following criteria applies: 
 
• Disease recurrence, if it is deemed by the treating investigator to be in the 
best interest of the patient to discontinue study treatment;  
• Intercurrent illness that prevents further administration of treatment 
• Unacceptable adverse event(s); 
• Patient demonstrates an inability or unwillingness to comply with protocol 
requirements; 
• Patient decides to withdraw from the study; or 
• General or specific changes in the patient's condition which render the patient 
unacceptable for further treatment in the opinion of the treating investigator. 
 
5.8 Duration of Follow Up  
 
Patients removed from study for unacceptable adverse events will be followed until 
resolution or stabilization of the adverse event. 
 
Every 3 months (“close observation”) follow-up visits should continue until 
initiation of a new therapy for recurrent disease or withdrawal of consent, whichever 
occurs first, for a maximum of 2 years after the surgery.  Patients will undergo 
disease restaging scans every 6 months for 2 years after the surgery.   
 
  
 

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5.9 Criteria for Removal from Study  
 
Patients will be removed from study when any of the criteria listed in Section 5.7 
applies. The reason for study removal and the date the patient was removed must 
be documented in the study-specific case report form (CRF).  
 
In the event of unusual or life-threatening complications, participating investigators 
must immediately notify the Principal Investigator, Patrick Ott, MD, PhD at DFCI 
. 
 
 
6 
EXPECTED TOXICITIES AND DOSING DELAYS/DOSE MODIFICATIONS  
 
 
Dose delays and modifications will be made using the following recommendations. Toxicity 
assessments will be done using the CTEP Active Version (version 4.0) of the NCI Common 
Terminology Criteria for Adverse Events (CTCAE) which is identified and located on the 
CTEP website at: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.  
 
If possible, symptoms should be managed symptomatically. In the case of toxicity, 
appropriate medical treatment should be used. 
 
All adverse events experienced by patients will be collected from the time of the first dose 
of study treatment, through the study and until the final study visit. Patients continuing to 
experience toxicity at the off study visit may be contacted for additional assessments until 
the toxicity has resolved or is deemed irreversible. 
  
 
6.1 Anticipated Toxicities 
A list of the adverse events and potential risks associated with the agents administered 
in this study appears below and will determine whether dose delays and modifications 
will be made or whether the event requires expedited reporting in addition to routine 
reporting. 
 
6.1.1 Adverse Events for poly-ICLC 
Poly-ICLC is an investigational agent; however there has been significant experience in 
previous clinical trials as discussed in 2.1.2.4. The safety profile has been acceptable. 
 
These reactions have been generally mild to moderate and transient for most patients in 
previous studies. 

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Localized reactions:  
• Transient (<24 h) injection fluid accumulation   
• Erythema 
• Induration 
• Pruritus 
• Edema 
• Localized rash 
Systemic reactions: 
Flu-like symptoms including malaise, fatigue, myalgia/arthralgia, headache, fever and 
chills. 
 
Hepatobiliary Disorders: 
• Alanine aminotransferase (ALT) increased 
• Aspartate aminotransferase (AST) increased 
• Lactate dehydrogenase (LDH) elevated   
• Alkaline  Phosphatase increased 
 
Bone Marrow: 
• Transient Leukocyte count decreased, Neutrophil count decreased, Platelet count 
decreased, and Anemia 
6.1.2    Adverse Events for Personalized NeoAntigen Peptides 
 
Localized reactions or systemic reactions are not expected, but may occur: 
Localized reactions:  
 
• Erythema 
• Induration 
• Pruritus 
• Edema 
• Localized rash 
Systemic reactions: 
Flu-like symptoms including malaise, fatigue, myalgia/arthralgia, headache, fever and 
chills. 
 
Autoimmune diseases: 

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This is a theoretical possibility for which patients will be monitored. In principle, the 
mechanisms that allow the immune system to recognize tumor antigens could also lead 
to breakdown of tolerance to native or self-antigens, generating an autoimmune 
reaction. 
6.2 Toxicity Management 
 
6.2.1 Treatment with corticosteroids and antihistamines are not recommended for initial 
treatment, but may be instituted at the discretion of the treating physician if clinically 
necessary.  
6.2.2 Local applications of cold compresses and moisturizing creams are preferable over 
systemic agents for the management of local reactions. 
6.2.3 Patients who develop flu-like symptoms that are controlled with acetaminophen or non-
steroidal anti-inflammatory drugs given at the discretion of the investigator may be pre-
treated with acetaminophen (650 mg PO) or ibuprofen (400-600 mg PO). 
 
6.3 Dose Modifications/Delays 
 
 
6.3.1 If a patient develops grade 3 toxicity attributable to the vaccine, additional treatment 
will be withheld until the toxicity has resolved or improved to grade 1. If the toxicity 
constitutes a DLT as defined in 5.5, the patient will be removed from the study. If the 
toxicity does not fulfill the criteria of a DLT, the next vaccine administration may be 
delayed for a maximum duration of 14 days. If vaccine dosing has to be delayed more 
than 2 times because of a toxicity that is not considered a DLT, the patient will be 
removed from the study. 
6.3.2 If a patient develops grade 4 toxicity attributable to the vaccine (with the exception of  
certain grade 4 laboratory abnormalities as defined in section 5.5), he or she will be 
removed from treatment.  
6.3.3 If two or more grade 4 or greater toxicities attributable to the vaccine are observed, the 
trial will be suspended to investigate the causes of these (unexpected) toxicities. 
 
  
7 
DRUG FORMULATION AND ADMINISTRATION  
 
 
7.1 Poly-ICLC (Hiltonol ®)  
 
7.1.1   Description 

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Poly-ICLC is a synthetic, nuclease resistant, hydrophilic complex of polyinosinic and 
polycytidylic acid, stabilized with poly-L-lysine and carboxymethylcellulose.   
 
 
 
 
 
 
 
Figure 1: Structure of poly-ICLC 
 
 
 
 
 
-Lysine  
 
 

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        Carboxymethylcellulose 
7.1.2   Form 
Poly-ICLC is supplied by Oncovir in single-dose vials containing 1 mL of 2 mg/mL 
opalescent white suspension. Each mL of poly-ICLC for injection contains 2 mg of 
poly-IC, 1.5 mg poly-L-lysine, and 5 mg sodium carboxymethylcellulose in 0.9% 
sodium chloride solution, adjusted to pH 7.6-7.8 with sodium hydroxide. 
 
7.1.3   Storage and Stability 
Poly-ICLC is stable at room temperature for brief periods (days). For long term 
storage, it should be refrigerated at ca. 40ºF (2-8ºC). Poly-ICLC should NOT be 
frozen. 
 
7.1.4 
  Compatibility 
Poly-ICLC will be mixed with personalized neoantigen peptides just prior to use to 
prepare the final product (NeoVax). 
 
7.1.5 
  Handling 
There are no specific handling instructions for poly-ICLC. 
 
7.1.6 
  Availability 
Poly-ICLC is an investigational agent and has been purchased from Oncovir, Inc.  
Vials of poly-ICLC sufficient for this clinical trial have been shipped from the 
Hiltonol® storage facility (Bellwyck Packaging, Mississauga, Ontario, Canada) to the 
DFCI Pharmacy and are currently stored under conditions appropriate for this 
investigational agent. 
 
7.1.7 
  Preparation 

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Poly-ICLC is supplied as an opalescent white suspension. It will be directly 
withdrawn from the vial under sterile conditions and mixed with the personalized 
neoantigen peptides (see section 5.2.1.3).  
 
7.1.8 
  Administration 
Following mixing with personalized neoantigen peptides, the vaccine (peptide + poly-
ICLC) will be administered as follows: using one syringe/needle per site 0.5 mL will 
be administered intradermally and 0.5 mL will be administered subcutaneously.  
 
 
 
7.1.9   Ordering 
Poly-ICLC is an investigational agent that has been obtained from Oncovir, Inc 
under a Clinical Trials Agreement between DFCI and Oncovir, Inc, dated May 2012. 
Under that agreement Oncovir, Inc will grant access to the Drug Master File for 
poly-ICLC for regulatory purposes and provides DFCI information necessary to 
pursue the clinical use of poly-ICLC. Poly-ICLC can be ordered directly from 
Oncovir, Inc, by submitting a purchase order to: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
7.1.10 Accountability 
The investigator, or a responsible party designated by the investigator, must maintain 
a careful record of the inventory and disposition of the agent (investigational or free of 
charge) using the NCI Drug Accountability Record or another comparable drug 
accountability form. (See the CTEP website at 
http://ctep.cancer.gov/protocolDevelopment for the “Policy and Guidelines for  
Accountability and Storage of Investigational Agents” or to obtain a copy of the drug 
accountability form.) 
 
7.1.11 Destruction and Return 
 
At the end of the study, unused supplies of poly-ICLC will be held by the IND holder 
for future use until they are out of date at which point they will be destroyed according 
to institutional policies. Destruction will be documented in the Drug Accountability 
Record Form. 
 

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7.2 Personalized neoantigen peptides  
 
7.2.1 Description 
Personalized neoantigen peptides are comprised of up to 20 distinct peptides unique to 
each patient.  Each peptide is a linear polymer of ~20 - ~30 L-amino acids joined by 
standard peptide bonds.  The amino terminus is a primary amine (NH2-) and the 
carboxy terminus is a carbonyl group (-COOH).  Only the standard 20 amino acids 
commonly found in mammalian cells are utilized (alanine, arginine, asparagine, 
aspartic acid, cysteine, glutamine, glutamic acid , glycine, histidine, isoleucine, 
leucine lysine, methionine, phenylalanine,  proline, serine, threonine, tryptophan, 
tyrosine, valine).  The molecular weight of each peptide varies based on its length and 
sequence and is calculated for each peptide.  
 
7.2.2 
Form 
 
 
 
 
 
 
 
 
  
.     
 
7.2.3 
Storage and Stability 
Personalized neoantigen peptides are stored frozen at -70 to -80oC. The thawed vials 
and the final mixture of personalized neoantigen peptides and poly-ICLC can be kept 
at room temperature but should be used within 6 hours after preparing the peptide + 
poly-ICLC mixture.                                                                                                                    
  
7.2.4 Compatibility 
Personalized neoantigen peptides will be mixed with poly-ICLC just prior to use to 
prepare the final product (NeoVax).   
7.2.5 Handling 
 
There are no specific instructions for handling personalized neoantigen peptides. 
 
7.2.6 Availability 
 
The personalized neoantigen peptides are prepared by CSBio, Inc under contract with 
DFCI and will be supplied directly to the DFCI.  It will be supplied from DFCI to 
other participating institutions. 

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7.2.7 Preparation 
The final step in the preparation of NeoVax (mixing with poly-ICLC ) is conducted on 
the day of the scheduled vaccine administration as described in 5.2.1.1.  Briefly, one 
vial each of the frozen personalized neoantigen peptides pools will be thawed the 
personalized neoantigen peptides will be combined and mixed with poly-ICLC within 
a properly certified biosafety cabinet. 
 
7.2.8 Administration 
Following mixing with personalized neoantigen peptides, the vaccine (peptide + poly-
ICLC) will be administered as follows: using one syringe/needle per site 0.5 mL will 
be administered intradermally and 0.5 mL will be administered subcutaneously.  
 
  
7.2.9 Ordering 
The order for personalized neoantigen peptides to CSBio, Inc will be placed by the 
DFCI / Cell Manipulation Core Facility separately for each individual patient.  Delivery 
time is anticipated to be 4 – 8 weeks. 
 
7.2.10 Accountability 
The investigator, or a responsible party designated by the investigator, must 
maintain a careful record of the inventory and disposition of the agent (investigational 
or free of charge) using the NCI Drug Accountability Record or another comparable 
drug accountability form. (See the CTEP website at 
http://ctep.cancer.gov/protocolDevelopment  for the “Policy and Guidelines for  
Accountability and Storage of Investigational Agents” or to obtain a copy of the drug 
accountability form.) 
7.2.11 Destruction and Return 
 
Unused supplies of personalized neoantigen peptides will be released to the sponsor 
for further research or destroyed according to institutional policies. If unused 
personalized neoantigen peptides are destroyed this will be documented in the 
Drug Accountability Record Form.  
  
8 CORRELATIVE/SPECIAL STUDIES 
8.1 
Pharmacodynamic Studies 
The immunization strategy is a “prime-boost” approach, involving an initial series of 
closely spaced immunizations to induce an immune response followed by a period of 

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rest to allow memory T-cells to be established. This will be followed by a booster 
immunization, and the T-cell response 4 weeks after this boost (16 weeks after the first 
vaccination) is expected to generate the strongest response and will be the primary 
immunological endpoint. Immune monitoring will be performed in a step-wise fashion 
as outlined below to characterize the intensity and quality of the elicited immune 
responses. Peripheral blood will be collected and PBMC will be frozen at two separate 
time points prior to the first vaccination (baseline) and at different time points 
thereafter as illustrated in Schema B and specified in the study calendar. Immune 
monitoring in a given patient will be performed after the entire set of samples from the 
induction phase and the maintenance phase, respectively, have been collected. 
 If sufficient tumor tissue is available, a portion of the tumor will be used to develop 
autologous melanoma cell lines for use in cytotoxic T-cell assays.  
 
8.1.1 Screening ex vivo IFN-γ ELISPOT  
For each patient, a set of screening peptides will be synthesized. The screening peptides 
will be 15-17 amino acids in length, overlapping by 11 amino acids and covering the 
entire length of each peptide or the entire length of the neoORF for neoORF-derived 
peptides.  The entire set of patient-specific screening peptides will be pooled together at 
approximately equal concentration and a portion of each peptide will also be stored 
individually.  Purity of the peptide pool will be ascertained by testing PBMC from 5 
healthy donors with established low background in ex vivo IFN-γ ELISPOTs.   Initially, 
PBMC obtained at baseline and at week 16 (the primary immunological endpoint) will 
be stimulated for 18 hours with the complete pool of overlapping 15-mer peptides (11 
amino acids overlap) to examine the global response to the peptide vaccine.  Subsequent 
assays may utilize PBMC collected at other time points as indicated.    
If no response is identified at the primary immunological endpoint using the ex vivo 
IFN-γ ELISPOT assay, PBMC will be stimulated in vitro with the peptide pool for a 
longer time period (up to 10 days) and re-analyzed. 
 
We are broadly interested in the immunogenic potential of the predicted neoantigens that 
have been used to formulate the study subject's personal vaccines. Given that patients 
will have completed vaccine dosing, subsequent profiling of the subject's immune 
responses necessarily will include analysis of antigen-specific immunity to the precise 
vaccine antigens that had been administered. The unused peptides are thus valuable 
reagents to enable these informative immune studies. For this reason, and under the 
request of the Overall PI, personalized neoantigen peptides may be transferred from the 
pharmacy department to the research lab. 
8.1.2 Deconvolution of epitopes in follow-up ex vivo IFN-γ ELISPOT assays 
Once an ex vivo IFN-γ ELISPOT response elicited by a overlapping peptide pool is 
observed (defined as at least 55 spot forming units / 106 PBMC or increased at least 3 

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times over baseline), the particular immunogenic peptide eliciting this response will be 
identified by de-convoluting the peptide pool based into sub-pools based on the 
immunizing peptides and repeating the ex vivo IFN-γ ELISPOT assays.   
For some responses, an attempt will be made to precisely characterize the stimulating 
epitope by utilizing overlapping 8-10 mer peptides derived from confirmed, 
stimulating peptides in IFN-γ ELISPOT assays 
8.1.3 Additional assays conducted on a case-by case basis for appropriate samples 
 
• The entire 15mer pool or sub-pools will be used as stimulating peptides for 
intracellular cytokine staining assays to identify and quantify antigen-specific 
CD4+, CD8+, central memory and effector memory populations 
• Similarly, these pools will be used to evaluate the pattern of cytokines secreted by 
these cells to determine the TH1 vs TH2 phenotype 
• Extracellular cytokine staining and flow cytometry of unstimulated cells will be 
used to quantify Treg and myeloid-derived suppressor cells (MDSC). 
• If a melanoma cell line is successfully established from a responding patient and 
the activating epitope can be identified, T-cell cytotoxicity assays will be 
conducted using the mutant and corresponding wild type peptide 
• PBMC from the primary immunological endpoint will be evaluated for “epitope 
spreading” by using known melanoma tumor associated antigens as stimulants 
and by using several additional identified mutated epitopes that were not selected 
to be among the immunogens 
• Peptide ELISAs will be used to monitor humoral responses to the immunizing 
peptides 
8.1.4 Immunohistochemistry 
Immuno-histochemistry of tumor samples will be conducted to quantify CD4+, CD8+, 
MDSC, and Treg infiltrating populations.   
 
 

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9 
 STUDY CALENDAR 
 
1All study procedures should be performed within a +/- 3 day window unless specified 
otherwise. 
2History and Physical examination include ECOG Performance Status and Vital Signs 
3Initial screening scans (PET/CT or CT neck, chest, abdomen, and pelvis, and brain MRI for 
baseline assessment must be performed <4 weeks prior to surgery. Day 50 re-staging scans 
can be done between day 43 and 50; day 190 scans can be done between day 183 and day 
190. 
4ANA, anti-thyroglobulin antibodies, rheumatoid factor.  
5Women of child-bearing potential must have a negative serum -HCG pregnancy test during 
screening and within 7 days of starting treatment. 
6Glucose, urea nitrogen, creatinine, sodium, potassium, calcium, total and direct bilirubin, AST, 
ALT, alkaline phosphatase 
7 HBSAg, HBcAb, HCV, HTLV I/II, RPR, HIV 1 and 2 Ab test 
8Molecular HLA typing to be performed by Tissue Typing laboratory 

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910 ml (one purple capped tube) of blood will be drawn and transmitted to the Broad Institute for 
whole exome sequencing.  
10Resection of locoregional metastatic disease (regional lymph nodes, in transit metastases, 
satellite lesions, limited distant metastases) and primary tumor (if not yet removed) should 
occur within 3 weeks of study enrollment. 
11Vaccine preparation should begin immediately after the surgical specimen is deemed adequate 
for vaccine preparation and it has been determined that the primary melanoma and metastatic 
disease have been completely removed. Vaccine preparation should be completed within 8-10 
weeks after this assessment. 
12Patients will undergo leukapheresis procedure to collect sufficient amounts of PBMCs to allow 
comprehensive immune monitoring. The pre-treatment leukapheresis should be performed no 
sooner than 2 weeks after the surgery, prior to the first vaccine administration.  A +/- 7 day 
window is allowed for the day 106 leukapheresis. If a patient is discontinued from active 
treatment (during Priming or Boosting Phase) and has not yet undergone the day 106 
leukapheresis, the leukapheresis or a 50 ml blood draw should be performed no later than 14 
days after the day of discontinuation. 
13135 ml of blood should be drawn if pre-treatment leukapheresis cannot be performed. If pre 
treatment Leukapheresis was performed 20 ml should be drawn. 
14If pre-treatment leukapheresis cannot be performed, there is no blood draw.  If leukapheresis 
was performed, 60 ml of blood will be collected. 
15200 ml 
16200 ml of blood should be drawn if week-16 leukapheresis cannot be performed.  If week -16 
leukapheresis can be performed, there is no blood draw.  
17 Follow-up: Every 3 months re-staging follow-up visits (+/- 1 week) should continue until 
initiation of a new therapy for recurrent disease, withdrawal of consent, or death whichever 
occurs first for a total of 2 years after treatment initiation.   
18 Every 6 months 
19 Assessments such as: Medical History / Physical Examination, lab tests and Con Meds can be 
done on Day 1 visit (previous starting treatment) if needed 
 
 

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10 MEASUREMENT OF EFFECT  
 
10.1 Antitumor Effect– Solid Tumors 
For the purposes of this study, patients should be re-evaluated for disease recurrence 
every 12 weeks. 
10.1.1   Definitions 
Evaluable for toxicity. All patients who receive at least one dose of study treatment 
will be evaluable for toxicity from the time of their first treatment. 
Evaluable for disease recurrence. All patients who receive at least one dose of 
study treatment will be evaluable for disease recurrence from the time of their first 
treatment.  
Disease recurrence: The finding of a new lesion on clinical examination or on imaging 
should be unequivocal (i.e. not due to difference in scanning technique, imaging 
modality, or findings thought to represent something other than tumor (ex: new bone 
lesions may be healing or flare of pre-existing lesions). However, a lesion identified on 
a follow-up scan in an anatomical location that was not scanned at baseline is 
considered new and will indicate disease recurrence. If a new lesion is equivocal 
(because of small size, etc.), follow-up evaluation will clarify if it truly represents new 
disease and if disease recurrence is confirmed, recurrence should be declared using the 
date of the initial scan on which the lesion was discovered.  
 
11 ADVERSE EVENT REPORTING REQUIREMENTS 
11.1 Definitions 
11.1.1 Adverse Event (AE) 
 
An adverse event (AE) is any undesirable sign, symptom or medical condition or 
experience that develops or worsens in severity after starting the first dose of study 
treatment or any procedure specified in the protocol, even if the event is not considered 
to be related to the study.  
 
Abnormal laboratory values or diagnostic test results constitute adverse events only if 
they induce clinical signs or symptoms or require treatment or further diagnostic tests.  
 
 
 

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11.1.2 Serious adverse event (SAE) 
 
A serious adverse event (SAE) is any adverse event, occurring at any dose and 
regardless of causality that:  
• Results in death 
• Is life-threatening. Life-threatening means that the person was at 
immediate risk of death from the reaction as it occurred, i.e., it does not 
include a reaction which hypothetically might have caused death had it 
occurred in a more severe form. 
• Requires or prolongs inpatient hospitalization (i.e., the event required at 
least a 24-hour hospitalization or prolonged a hospitalization beyond the 
expected length of stay). Hospitalization admissions and/or surgical 
operations scheduled to occur during the study period, but planned prior to 
study entry are not considered SAEs if the illness or disease existed before 
the person was enrolled in the trial, provided that it did not deteriorate in 
an unexpected manner during the trial (e.g., surgery performed earlier than 
planned). 
• Results in persistent or significant disability/incapacity. Disability is 
defined as a substantial disruption of a person’s ability to conduct normal 
life functions. 
• Is a congenital anomaly or birth defect; or 
• Is an important medical event when, based upon appropriate medical 
judgment, it may jeopardize the patient and require medical or surgical 
intervention to prevent one of the outcomes listed above. Examples of 
such medical events include allergic bronchospasm requiring intensive 
treatment in an emergency room or at home; blood dyscrasias or 
convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse. 
 
Events not considered to be serious adverse events are hospitalizations for: 
• routine treatment or monitoring of the studied indication, not associated 
with any deterioration in condition, or for elective procedures 
• elective or pre-planned treatment for a pre-existing condition that did not 
worsen 
• emergency outpatient treatment for an event not fulfilling the serious 
criteria outlined above and not resulting in inpatient admission 
• respite care 
 
11.1.3 Expectedness 
 
Adverse events can be 'Expected' or 'Unexpected.'  
 
 

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11.1.3.1   Expected adverse event 
 
For the purposes of this study, an adverse event is considered expected when it appears 
in the current adverse event list, the Investigator’s Brochure, or is included in the 
informed consent document as a potential risk.   
 
Refer to Section 6.1 for a listing of expected adverse events associated with the study 
agent(s). 
11.1.3.2   Unexpected adverse event 
 
 
For the purposes of this study, an adverse event is considered unexpected when it varies 
in nature, intensity or frequency from information provided in the current adverse event 
list, the Investigator’s Brochure, the package insert or when it is not included in the 
informed consent document as a potential risk.   
11.1.4 Attribution 
 
Attribution is the relationship between an adverse event or serious adverse event and 
the study treatment. Attribution will be assigned as follows: 
 
• Definite – The AE is clearly related to the study treatment. 
• Probable – The AE is likely related to the study treatment. 
• Possible – The AE may be related to the study treatment.  
• Unlikely - The AE is doubtfully related to the study treatment. 
• Unrelated - The AE is clearly NOT related to the study treatment. 
11.2 Procedures for AE and SAE Recording and Reporting 
 
Participating investigators will assess the occurrence of AEs and SAEs at all patient 
evaluation time points during the study.  
 
All AEs and SAEs whether reported by the patient, discovered during questioning, 
directly observed, or detected by physical examination, laboratory test or other means, 
will be recorded in the patient’s medical record and on the appropriate study-specific 
case report forms.  
 
The descriptions and grading scales found in the revised NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All 
appropriate treatment areas should have access to a copy of the CTCAE version 4.0. 
A copy of the CTCAE version 4.0 can be downloaded from the CTEP website at: 
 
 http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.  
 
 

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11.3 Reporting Requirements 
 
The study must be conducted in compliance with FDA regulations, local safety 
reporting requirements, and reporting requirements of the principal investigator.  
 
It is the responsibility of each participating investigator to report serious adverse events 
to the study sponsor and/or others as described below.  
11.4 Reporting to the Study Sponsor 
 
11.4.1 Serious Adverse Event Reporting 
All serious adverse events that occur after the initial dose of study treatment, during 
treatment, or within 30 days of the last dose of treatment must be reported to the 
DF/HCC Overall Principal Investigator on the local institutional SAE form. This 
includes events meeting the criteria outlined in Section 11.1.2, as well as the following: 
 
• Grade 2 (moderate) and Grade 3 (severe) Events – Only events that are 
unexpected and possibly, probably or definitely related/associated with the 
intervention. 
 
• All Grade 4 (life-threatening or disabling) Events – Unless expected AND 
specifically listed in the protocol as not requiring reporting. 
 
• All Grade 5 (fatal) Events – When the patient is enrolled and actively 
participating in the trial OR when the event occurs within 30 days of the 
last study intervention.  
 
Note: If the patient is in long term follow up, report the death at the time of 
continuing review.  
 
Participating investigators must report each serious adverse event to the DF/HCC 
Overall Principal Investigator within 24 hours of learning of the occurrence. In the 
event that the participating investigator does not become aware of the serious adverse 
event immediately (e.g., patient sought treatment elsewhere), the participating 
investigator is to report the event within 24 hours after learning of it and document the 
time of his or her first awareness of the adverse event. Report serious adverse events by 
telephone, email or facsimile to: 
 
Patrick Ott, MD  
 
 
 
 
 
  
 
 
 
 
 
Within the following 24-48 hours, the participating investigator must provide follow-up 
information on the serious adverse event. Follow-up information should describe 

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whether the event has resolved or continues, if and how the event was treated, and 
whether the patient will continue or discontinue study participation. 
 
Oncovir, Inc. will be provided with a simultaneous copy of all adverse events filed 
with the FDA. 
 
11.4.2 Non-Serious Adverse Event Reporting  
Non-serious adverse events will be reported to the DF/HCC Overall Principal 
Investigator on the toxicity Case Report Forms. 
   
11.5 Reporting to the Institutional Review Board (IRB) 
Investigative sites within DF/HCC will report all serious adverse events directly to the 
DFCI Office for Human Research Studies (OHRS). 
 
11.6 Reporting to the Food and Drug Administration (FDA) 
Patrick Ott, MD, PhD as IND sponsor, will be responsible for all communication with 
the FDA. She will report to the FDA any adverse event that is serious, unexpected and 
reasonably related (i.e., possible, probable, definite) to the study treatment. 
 
Unexpected fatal or life-threatening experiences associated with the use of the 
investigational agent will be reported to FDA as soon as possible but in no event later 
than 7 calendar days after initial receipt of the information. 
 
All other serious unexpected experiences associated with the use of the study treatment 
will be reported to FDA as soon as possible but in no event later than 15 calendar days 
after initial receipt of the information. 
 
  
Events will be reported to the FDA by telephone (1-800-FDA-1088) or by paper mail  
using Form FDA 3500A (Mandatory Reporting Form for investigational agents). If a 
faxed copy of Form FDA 3500A is requested by the agency, a paper copy will also be 
submitted as a follow-up.  Forms are available at 
http://www.fda.gov/medwatch/getforms.htm.  
 
11.7 Reporting to Hospital Risk Management 
Participating investigators will report to their local Risk Management office any subject 
safety reports or sentinel events that require reporting according to institutional policy. 
 
11.8 Monitoring of Adverse Events and Period of Observation 
All adverse events, both serious and non-serious, and deaths that are encountered from 
initiation of study intervention, throughout the study, and within 30 days of the last 

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study intervention should be followed to their resolution, or until the participating 
investigator assesses them as stable, or the participating investigator determines the 
event to be irreversible, or the patient is lost to follow-up. The presence and resolution 
of AEs and SAEs (with dates) should be documented on the appropriate case report 
form and recorded in the patient’s medical record to facilitate source data verification.  
 
For some SAEs, the study sponsor or designee may follow-up by telephone, fax, and/or 
monitoring visit to obtain additional case details deemed necessary to appropriately 
evaluate the SAE report (e.g., hospital discharge summary, consultant report, or 
autopsy report).  
 
Patients should be instructed to report any serious post-study event(s) that might 
reasonably be related to participation in this study. Participating investigators should 
notify the DF/HCC Overall Principal Investigator and their respective IRB of any 
unanticipated death or adverse event occurring after a patient has discontinued or 
terminated study participation that may reasonably be related to the study.   
 
12 DATA AND SAFETY MONITORING 
12.1 Data Reporting 
 
12.1.1 Method  
The QACT will collect, manage, and monitor data for this study.  
12.1.2 Data Submission  
The schedule for completion and submission of case report forms (paper or electronic) 
to the QACT is as follows: 
 
Form 
Submission Timeline 
Eligibility Checklist 
Complete prior to registration with QACT 
On Study Form 
Within 14 days of registration 
Baseline Assessment Form 
Within 14 days of registration 
Treatment Form 
Within 10 days of the last day of the cycle 
Adverse Event Report Form 
Within 10 days of the last day of the cycle 
Response Assessment Form 
Within 10 days of the completion of the cycle 
required for response evaluation 

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Off Treatment/Off Study Form Within 14 days of completing treatment or 
being taken off study for any reason 
Follow up/Survival Form 
Within 14 days of the protocol defined follow 
up visit date or call 
 
12.2 Safety Meetings 
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor 
toxicity and accrual data from this trial. The committee is composed of clinical 
specialists with experience in oncology and who have no direct relationship with the 
study. Information that raises any questions about patient safety will be addressed with 
the Principal Investigator and study team. 
 
The DSMC will meet quarterly and/or more often if required to review toxicity and 
accrual data.  Information to be provided to the committee may include:  up-to-date 
patient accrual; current dose level information; DLT information; all grade 2 or higher 
unexpected adverse events that have been reported; summary of all deaths occurring 
within 30 days for Phase I or II protocols; for gene transfer protocols, summary of all 
deaths while being treated and during active follow-up; any response information; audit 
results, and a summary provided by the study team. Other information (e.g. scans, 
laboratory values) will be provided upon request.  
12.3 External Safety Monitoring Board 
 
DFCI will be subject to independent monitoring conducted by ESMB. 
 
DFCI shall make reasonable efforts to ensure that the integrity of the research is not 
directly influenced by its interest in the technology being investigated by ensuring that 
the trial will be subject to independent data monitoring and auditing by an independent 
board, the cost of which shall be borne by DFCI. This board shall consist of three 
members.  None of the independent board members have any relationship to either 
DFCI or Neon. The ESMB shall be review subject enrollment practices to ensure 
compliance with any ICOI management plan; review and evaluate the accumulated 
DFCI study data for accuracy, participant safety, study conduct and compliance with 
any ICOI management plan; monitor individual and institutional compliance with ICOI 
management plan. The ESMB shall report to the ICOIC bi-annually for each trial. 
 
The ESMB may interact with DSMC and/or CTO if deemed necessary to accomplish 
their functions. 
 
12.4 Monitoring  
Involvement in this study as a participating investigator implies acceptance of 
potential audits or inspections, including source data verification, by representatives 
designated by the DF/HCC Overall Principal Investigator (or Protocol Chair) or 

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DF/HCC. The purpose of these audits or inspections is to examine study-related 
activities and documents to determine whether these activities were conducted and 
data were recorded, analyzed, and accurately reported in accordance with the protocol, 
institutional policy, Good Clinical Practice (GCP), and any applicable regulatory 
requirements.  
 
Monitoring plan for 13-240 protocol has been developed in conjunction with the DFCI 
CTO. The monitoring for 13-240 will be completed by a CTO monitor. Monitoring 
plan for this study will follow a risk-based monitoring approach with a focus on 
consent/eligibility documentation, safety, endpoints and full source data verification on 
approximately 50% of participants. 
 
All data will be monitored for timeliness of submission, completeness, and adherence 
to protocol requirements. Monitoring will begin at the time of patient registration and 
will continue during protocol performance and completion.  
 
12.5 Data sharing 
 
DFCI may share patient specific data with Neon Therapeutics, Inc. This data may 
contain PHI, limited identifiable patient’s information as well as de-identified 
information. DFCI will release only the minimum necessary identifiable health care 
information to Neon Therapeutics, Inc. Informed consent will be requested and 
obtained from former participants, active participants and future participants about the 
sharing of their information with Neon Therapeutics, Inc.   
 
Data submission will be performed via secure cloud-based service for document 
transfer and storage. Implemented security of the cloud-based system includes 
encrypted data transfer, two factor authentication and integration with Active 
Directory security groups. Neon ensures limited system access to authorized 
individuals through the use of security groups to agreed-upon members. Data will be 
backed up as part of the cloud based document storage service and the same security 
setting will apply to the cloud backup. 
 
13 REGULATORY CONSIDERATIONS 
 
13.1 Protocol Review and Amendments 
This protocol, the proposed informed consent and all forms of patient information 
related to the study (e.g., advertisements used to recruit patients) and any other 
necessary documents must be submitted, reviewed and approved by a properly 
constituted IRB governing each study location.  
Any changes made to the protocol must be submitted as amendments and must be 
approved by the IRB prior to implementation. Any changes in study conduct must be 

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reported to the IRB. The DF/HCC Overall Principal Investigator (or Protocol Chair) 
will disseminate protocol amendment information to all participating investigators.  
All decisions of the IRB concerning the conduct of the study must be made in 
writing. 
13.2 Informed Consent 
All patients must be provided a consent form describing this study and providing 
sufficient information for patients to make an informed decision about their 
participation in this study. The formal consent of a patient, using the IRB 
approved consent form, must be obtained before the patient is involved in any 
study-related procedure. The consent form must be signed and dated by the 
patient or the patient’s legally authorized representative, and by the person 
obtaining the consent. The patient must be given a copy of the signed and dated 
consent document. The original signed copy of the consent document must be 
retained in the medical record or research file.  
13.3 Ethics and Good Clinical Practice (GCP) 
This study is to be conducted according to the following considerations, which 
represent good and sound research practice: 
• 
US Code of Federal Regulations (CFR) governing clinical study conduct and 
ethical principles that have their origin in the Declaration of Helsinki 
o Title 21 Part 50 – Protection of Human Subjects 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr50_02.html  
o Title 21 Part 54 – Financial Disclosure by Clinical Investigators 
www.access.gpo.gov/nara/cfr/waisidx 02/21cfr54 02.html  
o Title 21 Part 56 – Institutional Review Boards 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr56_02.html  
o Title 21 Part 312 – Investigational New Drug Application 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr312_02.html  
• 
State laws 
• 
DF/HCC research policies and procedures        
http://www.dfhcc.harvard.edu/clinical-research-support/clinical-research-unit-
cru/policies-and-procedures/  
It is understood that deviations from the protocol should be avoided, except when 
necessary to eliminate an immediate hazard to a research patient. In such case, the 
deviation must be reported to the IRB according to the local reporting policy.  
 

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13.4 Study Documentation 
The investigator must prepare and maintain adequate and accurate case histories 
designed to record all observations and other data pertinent to the study for each 
research patient. This information enables the study to be fully documented and the 
study data to be subsequently verified. 
 
Original source documents supporting entries in the case report forms include but are 
not limited to hospital records, clinical charts, laboratory and pharmacy records, 
recorded data from automated instruments, microfiches, photographic negatives, 
microfilm or magnetic media, and/or x-rays.  
13.5 Records Retention 
All study-related documents must be retained for the maximum period required by 
applicable federal regulations and guidelines or institutional policies.  
 
14 STATISTICAL CONSIDERATIONS 
14.1 Study Design/Endpoints 
 
The primary goals of this Phase I trial are: a) to assess safety of the vaccine and determine 
the recommended dose of poly-ICLC and b) to assess the feasibility of making vaccine 
containing an adequate number of actionable peptides and in a timely manner. 
 
A. To evaluate safety and tolerability of administering NeoVax in patients with high-risk 
melanoma. 
 
 
Dose-limiting toxicity (DLT) will be defined as any of the following toxicities observed: 
1. Grade-3 or -4 toxicity that is definitely, probably, or possibly related to the 
administration of vaccine, excluding: 
  - Transient (≤ 72 hours) flu-like syndrome  
  - Grade-3 injection site reaction (localized rash, erythema, edema, induration) 
that resolves to grade 2 (or lower) within 72 hours  
  - Grade-3 nausea, vomiting, constipation, or diarrhea that returns to grade 2 (or 
lower) within 48 hours 
  - Grade-3 rash that resolves to grade 2 or grade 1 within 14 days  
2. Grade-3 or -4 abnormal laboratory value that is definitely, probably, or 
possibly related to the administration of vaccine if it persists for more than 7 
days, requires hospitalization or medical intervention (except for Grade-3 
electrolyte abnormality that lasts ≤ 72 hours, is not clinically complicated, and 
resolves spontaneously or responds to conventional medical intervention) 

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3. Any grade-3 or grade- 4 toxicity that is considered, in the opinion of the 
investigator, to be dose-limiting 
4. Any death related to study treatment 
 
Five patients will be entered for the initial safety evaluation (Cohort 1).  We have chosen 
dose escalation cohorts of 5 patients, rather than a more traditional 3+3 design, because the 
determination of whether to treat at a different dose in this trial is made over a longer period 
of time due to the staggered enrollment of patients, the time required for vaccine preparation, 
and the extended period for safety evaluation.  These larger cohort sizes help us avoid having 
to pause and then restart enrollment to a dosing cohort several months later.  If two or more 
patients in Cohort 1 experience dose limiting toxicity (DLT) during the first 7 weeks of 
treatment, then that cohort will be determined to have unacceptable toxicity; otherwise, an 
additional 10 patients will be treated at this dose level to increase the likelihood of detecting 
serious toxicities, to complete biologic correlative endpoints, and to gain preliminary 
experience with clinical tumor activity. If Cohort 1 is found to have unacceptable toxicity, 
then 5 patients will be enrolled on Cohort -1. If two or more patients in Cohort -1 experience 
a DLT, then the study will be stopped. If zero or one patient experiences a DLT in Cohort -1, 
then 10 additional patients will be enrolled in an expansion cohort.  
 
 
The table below shows the probability of moving to the dose expansion phase (0 or 1 patients 
with DLT out of 5 patients) for various possible values of the true, but unknown, toxicity 
rate. 
 
True rate of DLT (%) 
Probability of Dose Expansion (%) 
10 
92 
20 
74 
30 
53 
40 
34 
50 
19 
60 
9 
 
 
If the true probability of DLT is 10% or less, then the probability of proceeding to dose 
expansion is at least 92%.  The probability of dose expansion is less than 50% if the true rate 
of dose-limiting toxicities is 32% or greater.   
 
Minimum vaccination requirement to pass DLT observation period: A minimum of 3 
vaccinations and absence of DLTs are required for a patient to complete the 7-week DLT 
observation period successfully. 
 
Expansion Cohort:  When the recommended dose has been determined, an expansion 
cohort of 10 patients will be enrolled to assess the performance of the vaccine and to gain 
additional information about toxicities. 

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Stopping rule for Expansion Cohort: If four or more patients in the expansion cohort 
experience dose limiting toxicity (DLT) during the first 7 weeks of treatment, then the dose 
expansion cohort will be determined to have unacceptable toxicity and the study will be 
stopped prior to completion of enrollment of the expansion cohort. The table below 
summarizes the probability of observing 4 or more DLTs within the 10 patients of the 
expansion cohort for varying underlying rates of toxicity. 
 
 
 
True, but unknown, toxicity rate 
5% 
10% 
20% 
30% 
36% 
40% 
Probability 
of observing 
4 or more 
patients with 
DLT 
0.001 
0.013 
0.121 
0.350 
0.513 
0.618 
 
If the true probability of DLT in the expansion cohort is higher than 35%, then the 
probability is at least 50% of observing 4 or more patients with DLT within the 10 patients of 
the expansion cohort. 
 
 
Total Sample Size:  The final sample size for this trial will depend upon the number of dose-
escalation cohorts; however, the number of patients enrolled will be between 4 and 20.  The 
minimum number of patients to be enrolled is based on observing DLTs after the first dose of 
NeoVax in the first two patients of cohort 1 and similarly in cohort-1.  The maximum would 
occur if Cohort 1 and Cohort -1 were enrolled, with an additional 10 patients at the 
recommended dose. 
 
 
B:  To determine the feasibility of generating and administering NeoVax in patients with high-
risk melanoma 
 
Two feasibility endpoints will be evaluated: 
 
1) 
The proportion of patients for whom sequencing and analysis leads to identification of 
at least 10 actionable peptides to initiate vaccine production;  
2) 
The proportion of patients for whom the time between surgery and vaccine availability 
is 12 weeks or less (excluding those patients who did not generate at least 10 actionable 
peptides).   
 
We will base the primary assessment of feasibility on all patients enrolled with a 
confirmation of completely resected tumor and with adequate DNA and RNA for 
sequencing.  The statistical considerations for feasibility that follow are based on 15 patients.  

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However, the decision rule used to assess feasibility will be adjusted to maintain the 
operating characteristics if the number of patients is greater than 16.  
 
We would consider NeoVax to be feasible if no more than 50% of patients have fewer than 
10 actionable peptides or have NeoVax available more than 12 weeks after confirmation of 
complete resection.  If 4 or fewer of the first 15 patients have insufficient vaccine or first 
dose delays of more than 12 weeks, then the upper bound of the one-sided 90% exact 
confidence interval will be less than 50% and we will consider the process feasible.  If 5 or 
more of the first 15 patients have insufficient or delayed vaccine, we will consider the 
vaccine not feasible.   
 
The planned vaccination regimen will be administered even if the feasibility endpoints are 
not met. However, the treatment will not be started in a given patient if three or fewer 
actionable peptides are identified.  
 
14.2 Analysis of Secondary Endpoints 
 
Secondary endpoints in this clinical trial will: (a) assess the induction of IFN-γ T-cell 
response to the total peptide pool, and (b) estimate the proportion of patients alive without 
disease progression at two years after resection.  Analyses of these secondary endpoints will 
be based on the 15 patients treated at the recommended vaccine dose. 
 
The induction of IFN-γ T-cell response will be based on ELISPOT assessments taken prior to 
vaccine administration and at week 16.  The proportion of patients who achieve more than 55 
SFU/106 PBMC or 3 times their baseline level will be presented with a 90% exact binomial 
confidence interval. Based on a cohort of size 15, the confidence interval will be no wider 
than 0.46.   
 
The proportion of patients alive, without disease progression at two years after resection will 
be estimated using the method of Kaplan-Meier.  The endpoint will be calculated as the time 
between pathological confirmation of complete resection and the first of disease recurrence 
or death.  Patients who are disease-free at the time of study reporting will have their follow-
up censored at the time of the last study visit.  Patients who were disease-free at the time of 
their last assessment and who have either been lost to follow-up or withdrawn consent will 
have their follow-up censored at the time of the last study assessment.  The proportion of 
patients alive and disease-free at two years will be presented with 90% confidence intervals 
estimated using log(-log(DFS)) methodology.   
 
14.3 Analysis of Correlative Endpoints   
 
Longitudinal analyses of PBMC immune response kinetics to the peptide pool will be 
presented graphically and descriptively at each time point.  Changes in the magnitude of the 
response relative to pre-treatment at several different time points after vaccination will be 
summarized descriptively.  Changes in response between pre-treatment and 16 weeks post-
vaccination, which is the time of the primary immunologic endpoint,  will be assessed using 
the Wilcoxon signed-rank test.  

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Immune studies of T cell subpopulations (CD4+, CD8+, central/effector memory cells, 
Th1/Th2 cells, Tregs, etc.), myeloid-derived suppressor cells (MDSC), CTL response, and T 
cell activation status will be summarized using descriptive statistics. 
  
14.4 Analysis of Safety and Toxicity Data 
 
Analysis set and grouping for the analyses:  All patients who receive one or more doses of 
NeoVax will be included in the safety analysis. All listings and tables will be presented by 
dose level. 
 
Adverse events (AEs):  All adverse events recorded during the study will be summarized. 
The incidence of treatment emergent adverse events will be summarized by dose level 
according to primary system organ class, severity based on the National Cancer Institute’s 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, type of adverse 
event, and relationship to the vaccine. Deaths reportable as SAEs and non-fatal serious 
adverse events will be listed by patient and tabulated by primary system organ class, type of 
adverse event, and dose level. 
 
DLTs will be listed and their incidence summarized by primary system organ class, worst 
grade based on the CTCAE version 4.0, type of adverse event, and by dose level.  Any other 
information collected (e.g. start/end dates and duration of adverse event, severity or 
relatedness to study medication) will be listed, as appropriate. 
 
Laboratory abnormalities:  All laboratory values will be converted into SI units, as 
appropriate, and the severity grade calculated using CTCAE, version 4.0.  Parameters for 
which a grading does not exist will be classified into low/normal/high group by means of 
laboratory normal ranges. For each laboratory test (e.g., hematology, biochemistry) a listing 
of laboratory values will be provided by laboratory parameter, patient and treatment group. 
The frequency of notable lab abnormalities (i.e., newly occurring CTCAE grade-3 or -4 
laboratory toxicities), will be displayed by parameter, cycle, and dose level. Similarly, the 
frequency of all laboratory abnormalities will be displayed by parameter, worst CTCAE 
version 4.0 grade experienced and dose level. A separate listing will display notable 
laboratory abnormalities (i.e., newly occurring CTCAE grade-3 or -4 laboratory toxicities). 
Laboratory data will be summarized by presenting grade shift tables for those parameters for 
which CTCAE version 4.0 allows classification. All remaining data will be summarized by 
presenting shift tables based on normal ranges. 
 
Laboratory data will be also be displayed by presenting summary statistics of raw data and 
change from baseline values (means, medians, standard deviations, ranges). 
 
 
 

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15 PUBLICATION PLAN 
 
This study is intended for publication, even if terminated prematurely. Publication may 
include any or all of the following: posting of a synopsis online, abstract and/or 
presentation at a scientific conference, or publication of a full manuscript. The Sponsor will 
work with the Principal Investigator and authors to submit a manuscript describing study 
results within 24 months after the last data become available; which for vaccine trials, may 
take up to several months after the last patient visit.  
 
 

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

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D:6/13/2013 MML/F: 8/30/2013 MML/CK; R: 8/14/2014( amd.#3)/MML; R: 5/19/2016 (amd. #8) MML 
 
DANA-FARBER CANCER INSTITUTE 
Nursing Protocol Education Sheet 
 
Protocol Number: 
13-240 
Protocol Name: 
Ph I Study with Personalized NeoAntigen Cancer Vaccine in Melanoma 
DFCI Site PI: 
Patrick Ott, MD 
DFCI Research Nurse: 
; Kristina Kelley, RN; Christine Wong, RN; Jennifer Rowan, RN 
       Page the DFCI research nurse or DFCI site PI if there are any questions/concerns about the protocol.   
Please also refer to ONC 15: Oncology Nursing Protocol Education Policy 
This is a PI Initiated Study & Therefore There is No Alert Page 
                      SPECIAL NURSING CONSIDERATIONS UNIQUE TO THIS PROTOCOL 
Study 
Design 
This personalized NeoAntigen Cancer Vaccine approach has not been done in humans before. The vaccine is a tumor 
antigen and will hopefully induce immune responses against tumor cells that are much stronger than what has 
previously been achieved with traditional tumor vaccine approaches. The anticipated strong Immune responses 
should allow for clinical benefit. Please refer to Section 1.1                                  One Cycle = 22 Days (Section 5.4.1) 
Dose 
Calc 
 
***Please refer to Section 5.2.4 
Study Drug Administration 
***Agent Administration Guidelines are found in Sections 5.3, 5.2.3.1, 5.2.3.2, 5.3.1, 5.2.4, & 7.1.8 
Priming Doses : NeoVax (peptides + poly-ICLC) 
• Laboratory parameters must be reviewed and must re-meet Eligibility Criteria. (Section 5.2.3.1) 
• Subsequent treatment days, Laboratory parameters must continue to re-meet Eligibility Criteria.(Section 5.2.3.2)    
• 4 syringes containing 1 ml each of mixture for Neovax A, B, C, &D will be prepared for sub cu injections.  
• Administered Days 1, 4, 8, 15, & 22 via sub cu injections into 4 different anatomic sites.  
• Each of the 4 NeoVax syringes will be assigned to one of four extremities. (Example; NeoVax A will be injected into 
left arm on day 1, 4, 8 etc., NeoVax B will be injected into right arm on days 1, 4, 8, etc.) 
• NeoVax may be administered within 1 day of the scheduled administration date for days 4 & 8, within 3 days of the 
scheduled administration date for days 15 & 22 and 7 days for days 78 & 134  (Section 5.3.2) 
• For EACH clinic visit Participants are to be observed in the clinic for at least 60 minutes after the last of the 4 
injections. (Section 5.3.3) 
• At EACH clinic visit Vital Signs are to be checked once between 30 and 90 minutes after the last injection for each 
clinic visit. (Section 5.3.3) 
Boost Phase: 
• NeoVax will be administered on Days 78 (week 12) and-134 (week 20) (Section 5.4.3) 
Dose Mod  
& Tox 
Dose Modifications/Dosing Delay for Toxicity are outlined in Section 6.0, 6.3 
• DLT observation period is 7 weeks. (Section 5.4.4) 
• Toxicities will be graded using the NCI CTCAE version 4.0 (Section 5.5) 
• Maximum delay of NeoVax vaccine is 14 days. (Section 6.3.1) 
• Maximum number of times NeoVax may be delayed is 2. (Section 6.3.1) 
Concom 
Meds 
***Concomitant Therapy Guidelines are in Section 5.6 
• Participants who have developed flu-like symptoms that were controlled with acetaminophen or non-steroidal anti-
inflammatory drugs during previous treatments, Per Investigator’s discretion, may be pre-treated with acetaminophen
or NSAID. (Sections 5.2.3.2 & 6.2.3) 
Req 
 Data  
Study Calendar and Assessment Required data are outlined in Section 9.0 
• All Study procedures should be performed within a +/- 3 day window unless specified otherwise. (Section 9) 
Charting 
Tips 
Please be sure to DOCUMENT study medication actual UP/DOWN times in medical record 
• 
If there is a discrepancy in the infusion time, delay in administration, or the infusion takes longer than is 
permitted by the guidelines of the protocol, please document the reason for the discrepancy in the medical 
record. 
Please be sure to also DOCUMENT any required observation periods, any additional vital signs, routes of 
administration, or injection sites 
 
 

 
1 
ONCOVIR  
 
3203 Cleveland Ave, NW       Phone: (202) 342-1726 
Washington, DC, 20008 
Fax:  (202) 248-2324 
USA 
 
Email: asalazar@oncovir.com 
 
 
 
Note To Dispensors: Poly-ICLC 
Suspension Inspection prior to use 
To: 
Pharmacy 
From: 
Andres M. Salazar, MD,   Scientific Director 
 
cc: 
Pharmacy Sites 
Date: 
28 February 2017 
Re: 
Visual inspection of Poly-ICLC vials lot PJ 215-1-10-01 prior to use 
 
Poly-ICLC presents as an opalescent, or white, viscous uniform coarse particulate suspension in a 2 
mL glass vial.  Recently it has been observed that some product vials exhibit some settling of the 
suspension around the lower rim of the product vial as well as some larger clumps as seen in the 
photo below . 
 
Upon allowing the vial to reach room temperature (approximately 1-2 hours), the vial should be 
gently inverted about 10 times to insure suspension of the formulation.  Gently tap the vial to gather 
the liquid suspension to the bottom of the vial to aid in extracting the required volume for dose 
preparation. If visible particulates or aggregates are present when measured against a black 
background and are not resuspendable or the settled material fails to re-suspend, the vial should be 
placed in quarantine and not used for clinical administration.  Obtain a new vial of Poly-ICLC for 
dosage preparation. 
 
Please report to the sponsor and/or Oncovir, Inc any instances of Poly-ICLC failures to re-suspend 
and/or observations of insoluble particulates or agglomerates. 
 

 
2 
 
 
Signature:  
 
 
Andres M. Salazar, M.D. 
CEO, Scientific Director, 
Oncovir, Inc.