Clinical trial NCT03495921
Conditions: Ewing Sarcoma Ewing Family of Tumors Ewing's Tumor Metastatic Ewing's Sarcoma Metastatic Ewing's Tumor Recurrent Rare Diseases Sarcoma Neoplasms, Connective and Soft Tissue Neoplasms by Histologic Type Neoplasms, Bone Tissue Neoplasms, Connective Tissue Sarcoma, Ewing Neoplasms
Keywords: ESFT Immunotherapy Phase 3 irinotecan temozolomide First Relapse Second Line Vigil vaccine orphan drug soft bone pediatric FLI EWS Molecular Mechanisms of Pharmacological Action Antineoplastic Agents
Sponsor: Gradalis, Inc.
Start date: 2018-08-21; Completion date: 2022-01-20
Enrollment: 32 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The goal of this clinical trial was to compare participants with first relapse or refractory Ewing's sarcoma when treated with investigational product (Vigil) in addition to the standard treatment of irinotecan and temozolomide compared to the standard treatment of irinotecan and temozolomide alone. The main question it aimed to answer is "Will participants who receive Vigil in addition to irinotecan and temozolomide have a prolonged time to progression and improved quality of life compared to the participants who receive irinotecan and temozolomide alone?".

Detailed description:
This was a multicenter, Phase III study in participants with metastatic Ewing's sarcoma Family of Tumors (ESFT) refractory/intolerant or recurrent to 1 prior line of chemotherapy. Participants who agreed to participation had tumor tissue harvested from a scheduled standard surgical procedure (e.g., tumor biopsy or palliative resection). The tumor tissue removed was shipped to Gradalis, Inc. to attempt to manufacture the investigational product, Vigil.

Subjects who met eligibility criteria including manufacture of a minimum of 4 doses of Vigil were randomized 1:1 to either Group A (Vigil + Irinotecan + Temozolomide (Tem/Iri)) or Group B (Irinotecan + Temozolomide). Screening for the main portion of the study occurred as early as one week but no later than 8 weeks following tumor procurement.

Subjects received repeat cycles of treatment until disease progression, unacceptable toxicity, withdrawal of consent or other criterion was met for discontinuation from study. Subjects randomized to Group A (Vigil + Tem/Iri) received up to 12 doses depending upon the quantity of Vigil manufactured from the surgical specimen. 1 cycle = 21 days. If irinotecan + temozolomide was administered beyond 12 cycles, it was administered off study. Subjects randomized to Group B (Tem/Iri) may have crossed over to receive single agent Vigil every 21 days following End of Treatment assessment and documented disease progression confirmed by central radiology vendor, for up to 12 doses of Vigil depending upon the quantity of Vigil manufactured.

Participants were managed in an outpatient setting. Hematologic function, liver enzymes, renal function and electrolytes will be monitored. Blood for immune function analyses including IFNγ-ELISPOT analysis of cytotoxic T cell activation in response to autologous tumor antigens will be collected at tissue procurement, post-procurement screening and Day 1 (prior to chemotherapy administration) at Cycles 2, 4, and 6, end of treatment (EOT), 3 months after EOT, and every 6 months thereafter. Blood for ctDNA analysis was collected at tissue procurement, prior to chemotherapy administration at baseline and on Day 1 prior to chemotherapy administration at Cycles 2, 3, 4, and 6, and EOT. After progression, participants were contacted quarterly for documentation of post study therapies and survival status information.

Arms: arm 1: Participants randomized to Group A received oral temozolomide 100 mg/m2 daily (Days 1-5), total dose 500 mg/m2/cycle) and oral irinotecan 50 mg/m2 daily (Days 1-5, total dose 250 mg/m2/cycle). Vigil immunotherapy was administered at a concentration of 1 X 10e6 cells/dose given via intradermal injection on Day 15 of each cycle. 1 cycle = 21 days

Participants continued treatment for a maximum of 12 cycles, or until disease progression, unacceptable toxicity, withdrawal of consent or other criterion is met for discontinuation from study. arm 2: Participants randomized to Group B received oral temozolomide 100 mg/m2 daily (Days 1-5), total dose 500 mg/m2/cycle) and oral irinotecan 50 mg/m2 daily (Days 1-5, total dose 250 mg/m2/cycle).

1 cycle = 21 days

Participants continued treatment for a maximum of 12 cycles, or until disease progression, unacceptable toxicity, withdrawal of consent or other criterion is met for discontinuation from study. arm 3: Participants randomized to Group B were able to receive Vigil immunotherapy at a concentration of 1 X 10e6 cells/dose given via intradermal injection on Day 15 of each cycle. Confirmation of progression by central radiologist and pre-approval from sponsor was required. 1 cycle = 21 days
Interventions: intervention 1: Vigil intervention 2: Irinotecan intervention 3: Temozolomide
Intervention descriptions: intervention 1: Vigil is composed of autologous tumor cells harvested from the patient at the time of initial de-bulking surgery which are then transfected extracorporeally, with a plasmid encoding for the gene for GM-CSF, an immune-stimulatory cytokine, and a bifunctional, short hairpin RNA which specifically knocks down the expression of furin, the critical convertase responsible for production of the two TGβ isoforms. intervention 2: Injectable formulation of irinotecan was distributed from central supplier. 1 Cycle (5 doses of 50 mg/m2 per syringe) was drawn into provided oral syringes and dispenses to the subject with instructions to refrigerate until administration. intervention 3: Dose: 100 mg/m2 daily, oral Schedule: Days 1-5, every 21 days Administered at least 1 hour before Irinotecan.

Protocol documents:
Prot_000.pdf: 
CL-PTL-130 
 
 
IND 014205 
 
 
 
 
Confidential and proprietary information of Gradalis, Inc. 
 
It is the user’s responsibility to verify adequate training before executing a document.  Do not make unauthorized copies. 
COVER PAGE 
OFFICIAL PROTOCOL TITLE: 
A Multi-Center Phase III, Randomized, 
Open-Label Trial of Vigil (bi-shRNAfurin 
and GMCSF Augmented Autologous 
Tumor Cell Immunotherapy) in 
combination with Irinotecan and 
Temozolomide as a Second-Line Regimen 
for Ewing’s Sarcoma  
 
 
PROTOCOL NUMBER:  
CL-PTL-130 
 
 
IND NUMBER: 
014205 
 
 
NCT NUMBER: 
NCT03495921 
 
 
PROTOCOL DATE:  
Amendment 2 dated December 5, 2018 
 
 
IRB APPROVAL DATE:  
December 14, 2018 
 
 
 
 
 


CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of 
Gradalis, Inc.  No person is authorized to make it public without written permission of Gradalis, Inc.  These restrictions on disclosure 
will apply equally to all future information supplied to you, which is indicated as privileged or confidential.  This material may be 
disclosed to and used by your staff and associates as may be necessary to conduct this clinical study. 
 
 
PROTOCOL TITLE: 
A Multi-Center Phase III, Randomized, Open-Label Trial of 
Vigil (bi-shRNAfurin and GMCSF Augmented Autologous 
Tumor Cell Immunotherapy) in combination with Irinotecan 
and Temozolomide as a Second-Line Regimen for Ewing’s 
Sarcoma 
IND NUMBER: 
014205 
PROTOCOL NUMBER: 
CL-PTL-130 
STUDY AGENT(S): 
Vigil 
bi-shRNAfurin and GMCSF Augmented Autologous Tumor 
Cell Immunotherapy 
SPONSOR: 
Gradalis, Inc. 
2545 Golden Bear Drive, Suite 110 
Carrollton, TX 75006 
Phone: (214) 442-8124 
Fax: (214) 442-8101 
PROTOCOL DATE: 
Amendment 2 dated December 5, 2018 
Amendment 1 dated June 20, 2018 
Initial Protocol dated September 20, 2017 
 
 
 


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INVESTIGATOR PROTOCOL SIGNATURE PAGE 
PROTOCOL TITLE: 
A Multi-Center Phase III, Randomized, Open-Label Trial of 
Vigil (bi-shRNAfurin and GMCSF Augmented Autologous 
Tumor Cell Immunotherapy) in combination with Irinotecan 
and Temozolomide as a Second-Line Regimen for Ewing’s 
Sarcoma 
 
PROTOCOL NUMBER: 
CL-PTL-130 
 
STUDY AGENT(S): 
Vigil 
bi-shRNAfurin and GMCSF Augmented Autologous Tumor 
Cell Immunotherapy 
 
SPONSOR: 
Gradalis, Inc. 
2545 Golden Bear Drive, Suite 110 
Carrollton, TX 75006 
Phone: (214) 442-8124 
Fax: (214) 442-8101 
 
PROTOCOL DATE: 
Amendment 2 dated December 5, 2018 
Amendment 1 dated June 20, 2018 
Initial Protocol dated September 20, 2017 
 
I have read and understand the contents of the indicated clinical protocol and will adhere to the 
trial requirements as presented, including all statements regarding confidentiality.  In addition, 
should I choose to participate as an investigator, I and my sub-investigator(s) agree to conduct 
the study as outlined herein, in accordance with Good Clinical Practices (GCPs), the 
Declaration of Helsinki, in compliance with the obligations and requirements of clinical 
investigators and all other requirements listed in Title 21 Code of Federal Regulations (CFR) 
Part 312. 
 
 
 
 
Name of Investigator (please print) 
 
Signature of Investigator 
 
 
 
Date 
 
 
 
 
 


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TABLE OF CONTENTS 
INVESTIGATOR PROTOCOL SIGNATURE PAGE .................................................................... 2 
TABLE OF CONTENTS ............................................................................................................... 3 
ABBREVIATIONS ........................................................................................................................ 6 
SYNOPSIS .................................................................................................................................... 8 
STUDY SCHEMA ....................................................................................................................... 15 
STUDY PROCESS FLOW .......................................................................................................... 16 
1.0 
INTRODUCTION .......................................................................................................... 17 
1.1 
Summary ................................................................................................................... 17 
1.2 
Relapsed and Refractory Ewing’s sarcoma .............................................................. 17 
1.3 
Salvage chemotherapy in Ewing’s sarcoma ............................................................. 18 
1.4 
Immunotherapeutic considerations in Ewing’s sarcoma ........................................... 20 
2.0 
VIGIL ............................................................................................................................ 22 
2.1 
Vigil Phase II Trial: Ovarian Cancer .......................................................................... 26 
2.2 
Vigil Phase I Trial: Ewing’s sarcoma Subset ............................................................. 29 
2.3 
Efficacy Evidence of Vigil in Ewing’s sarcoma .......................................................... 30 
2.4 
Vigil Phase II Trial: Ewing’s sarcoma (CL-PTL-121, Part 1) ..................................... 32 
2.5 
Vigil Phase II Trial: Ewing’s sarcoma (CL-PTL-121, Part 2) ..................................... 34 
3.0 
STUDY RATIONALE ................................................................................................... 38 
4.0 
OBJECTIVES ............................................................................................................... 39 
4.1 
Primary objective ...................................................................................................... 39 
4.2 
Secondary objective(s) ............................................................................................. 39 
5.0 
STUDY DESIGN ........................................................................................................... 40 
6.0 
STUDY POPULATION ................................................................................................. 41 
6.1 
Sample Size .............................................................................................................. 41 
6.2 
Tissue Procurement Inclusion Criteria ...................................................................... 41 
6.3 
Tissue Procurement Exclusion Criteria ..................................................................... 41 
6.4 
Study Enrollment Inclusion Criteria ........................................................................... 42 
6.5 
Study Enrollment Exclusion Criteria .......................................................................... 43 
7.0 
WITHDRAWAL ............................................................................................................ 43 
7.1 
Discontinuation from Study Treatment ...................................................................... 43 
7.2 
Treatment Delay ....................................................................................................... 44 
8.0 
INVESTIGATIONAL PLAN .......................................................................................... 44 
8.1 
Subject Screening and Registration .......................................................................... 44 
8.2 
Tumor Procurement .................................................................................................. 45 
8.3 
Vigil Immunotherapy Manufacturing ......................................................................... 45 


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8.4 
Study Treatment Administration ................................................................................ 45 
8.4.1 
Schedule, Dose and Administration ................................................................... 46 
8.5 
Concomitant Medications and Supportive Care ........................................................ 48 
8.6 
Contraception ............................................................................................................ 49 
8.7 
Toxicity ...................................................................................................................... 49 
9.0 
SCHEDULE OF ASSESSMENTS ................................................................................ 49 
10.0 
CROSS-OVER ............................................................................................................. 53 
10.1 
Inclusion Criteria for Cross-Over ............................................................................... 54 
10.2 
Exclusion Criteria for Cross-Over ............................................................................. 54 
11.0 
CONDUCT OF THE STUDY ........................................................................................ 55 
11.1 
Ethics and Regulatory Considerations ...................................................................... 55 
11.2 
IRB ............................................................................................................................ 55 
11.3 
Written Informed Consent ......................................................................................... 55 
11.4 
Confidentiality of Records ......................................................................................... 56 
11.5 
Modification of Protocol ............................................................................................. 56 
11.6 
Protocol Questions and Deviations ........................................................................... 56 
11.7 
Data Safety Monitoring Board ................................................................................... 57 
12.0 
EVALUATION OF TUMORS ........................................................................................ 57 
12.1 
Tumor Measurements and Response (RECIST 1.1) ................................................ 57 
13.0 
DRUG INFORMATION ................................................................................................. 57 
13.1 
Vigil Investigational Product ...................................................................................... 57 
14.0 
ADVERSE EVENTS ..................................................................................................... 59 
14.1 
Adverse Event and Serious Adverse Event Definitions ............................................ 59 
14.2 
Attribution of Causality .............................................................................................. 60 
14.3 
Vigil Expected Side Effects ....................................................................................... 60 
14.4 
Recording of an Adverse Event ................................................................................ 60 
14.5 
Serious Adverse Event Reporting ............................................................................. 61 
15.0 
PATIENT COMPLETION AND WITHDRAWAL .......................................................... 61 
15.1 
Indication for Taking Patients Off Study .................................................................... 61 
16.0 
STATISTICAL CONSIDERATIONS ............................................................................. 62 
16.1 
Sample Size Justification .......................................................................................... 62 
16.2 
Definition of Progression Free Survival (PFS) .......................................................... 62 
16.3 
Analysis Populations ................................................................................................. 63 
16.4 
Efficacy Analyses ...................................................................................................... 63 
16.4.1 
Primary Efficacy Analysis .................................................................................. 63 
16.4.2 
Secondary Efficacy Analyses ............................................................................ 63 


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16.4.3 
Sensitivity Analyses of the Primary Endpoint..................................................... 64 
16.4.4 
Sensitivity Analysis of Overall Survival .............................................................. 64 
16.4.5 
Subgroup Analyses of the Primary (PFS) and Key Secondary Endpoints (OS, 
ORR) 
64 
16.5 
Safety Analyses ........................................................................................................ 65 
16.6 
Interim Analysis ......................................................................................................... 65 
17.0 
DOCUMENTATION ...................................................................................................... 65 
17.1 
Case Report Form (CRF) Procedures ...................................................................... 66 
APPENDIX A KARNOFSKY AND LANSKY PERFORMANCE SCALES ................................. 67 
APPENDIX B SCHEDULE OF ASSESSMENTS ....................................................................... 68 
REFERENCES ........................................................................................................................... 69 
 
 
 


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ABBREVIATIONS 
Abbreviation 
Term 
AE 
Adverse event 
ALT 
Alanine transaminase (also referred to as SGPT) 
ANC 
Absolute neutrophil count 
AST 
Aspartate transaminase (also referred to as SGOT) 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CD 
Cluster of differentiation 
CO2 
Total carbon dioxide  
CR 
Complete response 
CRF 
Case report form 
CT 
Cancer testis 
CT 
Computerized Tomography 
CTCAE 
Common Toxicity Criteria for Adverse Events 
ctDNA 
Circulating tumor DNA 
DFS 
Disease-Free Survival 
DSMB 
Data Safety Monitoring Board 
DTIC 
dacarbazine 
EDC 
Electronic Data Capture 
EFS 
Event Free Survival 
ELISPOT 
Enzyme-Linked ImmunoSorbent Spot 
ERG 
ETS-Related Gene 
ESFT 
Ewing’s Sarcoma Family of Tumors 
ESPS 
Ewing’s Sarcoma Prognostic Scores 
ETS 
E26 transformation-specific or E-twenty-six 
FANG 
bi-shRNAfurin and GMCSF Augmented Autologous Tumor 
Cell Immunotherapy 
FISH 
Fluorescent in situ hybridization 
FLI-1 
Friend Leukemia Virus Integration 1 
GMCSF 
Granulocyte Macrophage-Colony Stimulating Factor 
GVAX 
GMCSF Secreting autologous or allogenic tumor vaccine 
HLA 
Human Leukocyte Antigen 
IDMC 
Independent Data Monitoring Committee 
IE 
Ifosfamide and etoposide 
IEC 
Independent Ethics Committee 
IFN 
Interferon 


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Abbreviation 
Term 
IRB  
Institutional Review Board 
ITT 
Intent to Treat 
KPS 
Karnofsky Performance Score 
LDH 
Lactate dehydrogenase 
LS 
Lansky Performance Score 
MRI 
Magnetic resonance imaging 
NCI 
National Cancer Institute 
NED 
No evidence of disease 
NGS 
Next Generation Sequencing 
NK 
Natural Killer 
NKT 
Natural Killer T cell(s) 
ORR 
Objective Response Rate 
OS 
Overall Survival 
PBMC 
Peripheral Blood Mononuclear Cells 
PCR 
Polymerase Chain Reaction 
PD-L1 
Programmed death-ligand 1 
PFS 
Progression Free Survival 
PI 
Principal Investigator 
PR 
Partial response 
PS 
Performance Status 
RECIST 
Response Evaluation Criteria in Solid Tumors 
RFS 
Recurrence Free Survival 
RT-PCR 
Real Time Polymerase chain reaction 
TGFβ 
Transforming growth factor-β 
TIL 
Tumor Infiltrating Lymphocytes 
TTP 
Time to Progression 
TTR 
Time to Recurrence 
VAC 
Vincristine, Actinomycin, and Cyclophosphamide 
 
 
 


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SYNOPSIS 
Summary: 
Ewing’s sarcoma is the second most frequently diagnosed primary malignant bone tumor in 
the US with an annual incidence, from birth to age 20, of 2.9 cases per million population.  
The survival rate for patients in first relapse is dismal (13% 5-year survival).  Temozolomide 
and irinotecan is the consensus standard of care treatment regimen after relapse.  Vigil 
immune response correlates with survival and time to relapse improvement in multiple solid 
tumors including Ewing’s sarcoma.  Limited data has demonstrated no adverse effect of the 
combination therapy with irinotecan, temozolomide and Vigil (1x106 - 1x107 cells/injection (x4 
or more)).  Therefore, the combination regimen of Vigil, irinotecan, and temozolomide is 
hypothesized to increase anti-tumor responses and prolong time to progression in patients 
with relapsed or refractory Ewing’s sarcoma (first relapse). 
 
In an effort to overcome limitations of first-generation immunostimulatory cancer vaccines, we 
designed a novel autologous whole cell vaccine, Vigil, incorporating the rhGMCSF transgene 
and the bifunctional shRNAfurin to: 
1) address the inability to fully identify relevant strong-affinity cancer specific (and 
associated) antigens, 
2) effect antigen recognition by the immune system (i.e. antigen→immunogen), 
3) enhance effector potency, and 
4) subvert endogenous cancer-induced immune resistance. 
 
A Phase I assessment of Vigil in advanced solid tumor patients including 16 Ewing’s sarcoma 
patients receiving ≥1 cycle (at a dose > 1.0 x 106 cells / injection / month for a minimum of 4 
and maximum of 12 vaccinations) demonstrated safety of the Vigil immunotherapy.  
Furthermore, mechanism of action (i.e., vector effectiveness) was established in the 
manufactured products with increased mean GMCSF expression and knockdown of TGFβ1 
and TGFβ2.  The Vigil induced immunomodulatory effects against Ewing’s sarcoma was 
reflected in positive IFNγ ELISPOT response to autologous tumor cells in 10/12 patients, one 
RECIST criteria partial response (PR), a second patient achieving complete response (CR) 
and a total 1-year overall survival (Kaplan Meier method) of 73% in comparison to expected 
1-year survival of <25%. 
Objective(s): 
Primary Objective: 
To compare the progression free survival (PFS) of subjects dosed with Vigil immunotherapy 
in combination with irinotecan and temozolomide vs. irinotecan and temozolomide. 
 
Secondary Objective(s): 
• 
To determine and compare the overall survival (OS) of subjects with relapsed or 
refractory Ewing’s sarcoma dosed with Vigil immunotherapy in combination with 
irinotecan and temozolomide with patients that are treated with irinotecan and 
temozolomide. 


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• 
To determine the objective response rate (RECIST 1.1) of patients with metastatic 
Ewing’s sarcoma refractory or intolerant to 1 prior line of systemic chemotherapy 
treated with Vigil immunotherapy dosed with Vigil immunotherapy in combination with 
irinotecan and temozolomide. 
• 
To determine the Vigil manufacturing success rate by gathering data from tumor tissue 
collection, Vigil construction, and test procedures. 
Methodology: 
 
This is a multicenter, 1:1 randomized Phase III study of intradermal autologous Vigil 
immunotherapy (1.0 x 106 cells/injection; minimum of 4 to a maximum of 12 administrations) 
in combination with irinotecan and temozolomide in subjects with metastatic Ewing’s sarcoma 
Family of Tumors (ESFT) refractory/intolerant or recurrent to 1 prior line of chemotherapy.  
Participants undergoing a standard surgical procedure (e.g., tumor biopsy or palliative 
resection) may have tumor tissue harvested for manufacture of the investigational product, 
Vigil. 
 
Subjects meeting eligibility criteria including manufacture of a minimum of 4 immunotherapy 
doses of Vigil will be randomized to receive either: 
 
Group A 
(i) 
oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), 
(ii) 
oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle), and 
(iii) 
Vigil 1.0 x 106 cells/injection cells/injection, intradermally on Day 15. 
 
or 
 
Group B 
(i) 
oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), and 
(ii) 
oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle). 
 
One cycle equals 21 days.  Screening for the main portion of the study may occur as early as 
one week but no later than 8 weeks following tumor procurement.  Vigil is typically released 
approximately 3 weeks after the completion of the two-day manufacturing process.  At the 
time of analysis the subjects will be stratified by initial response to frontline treatment 
(recurrent versus refractory (progressive)). 
 
Participants will be managed in an outpatient setting.  Hematologic function, liver enzymes, 
renal function and electrolytes will be monitored.  Blood for immune function analyses 
including IFNγ-ELISPOT analysis of cytotoxic T cell activation in response to autologous 
tumor antigens will be collected at tissue procurement, post-procurement screening and Day 
1 (prior to chemotherapy administration) at Cycles 2, 4, and 6, end of treatment (EOT), 3 
months after EOT, and every 6 months thereafter.  Blood for ctDNA analysis will be collected 


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at tissue procurement, prior to chemotherapy administration at baseline and on Day 1 prior to 
chemotherapy administration at Cycles 2, 3, 4, and 6, and EOT. 
Number of Patients: 
 
Approximately 140 subjects will have tissue procured so that 114 evaluable subjects will be 
registered for enrollment into the study. 
 
Tissue Procurement Inclusion Criteria: 
 
Subjects will be eligible for tissue procurement for the Vigil manufacturing process, if they 
meet all of the following criteria: 
 
1. Histologically confirmed Ewing’s sarcoma Family of Tumors (ESFT). 
2. Age ≥ 2 years. 
3. Estimated survival ≥ 6 months. 
4. Evidence of EWS translocation by FISH or RT-PCR or Next Generation Sequencing 
(NGS).  If available, NGS sequencing report should be submitted to Gradalis. 
5. Recurrence or refractory to 1 line of systemic chemotherapy including but not limited 
to doxorubicin, vincristine and ifosfamide. 
6. Planned standard of care surgical procedure (e.g., tumor biopsy or palliative resection 
or thoracentesis) and expected availability of a cumulative soft-tissue mass of ~10-30 
grams tissue (“grape” to “golf-ball” size / approximate 2 cm total diameter on imaging) 
and/or pleural fluid estimated volume ≥ 500mL (from a primary or secondary 
thoracentesis, yielding in a high volume of tumor cells) for immunotherapy 
manufacture. 
7. Tumor intended for immunotherapy manufacture is not embedded in bone and does 
not contain luminal tissue (e.g. bowel, ureter, bile duct). 
8. Ability to understand and the willingness to sign a written informed protocol specific 
consent for tissue harvest or a parental/guardian informed consent and pediatric 
assent when appropriate. 
 
Tissue Procurement Exclusion Criteria: 
 
Subjects meeting any of the following criteria are not eligible for tissue procurement for the 
Vigil manufacturing: 
 
1. Medical condition requiring any form of chronic systemic immunosuppressive therapy 
(steroid or other) except physiologic replacement doses of hydrocortisone or 
equivalent (no more than 30 mg hydrocortisone or 10 mg prednisone equivalent daily) 
for < 30 days duration. 
2. Known history of other malignancy unless having undergone curative intent therapy 
without evidence of that disease for ≥ 3 years except cutaneous squamous cell and 


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basal cell skin cancer, superficial bladder cancer, in situ cervical cancer or other in situ 
cancers are allowed if definitively resected. 
3. Brain metastases unless treated with curative intent (gamma knife or surgical 
resection) and without evidence of progression for ≥ 2 months. 
4. Any documented history of autoimmune disease with exception of Type 1 diabetes on 
stable insulin regimen, hypothyroidism on stable dose of replacement thyroid 
medication, vitiligo, or asthma not requiring systemic steroids. 
5. Known HIV or chronic Hepatitis B or C infection. 
6. Known hypersensitivity to any temozolomide component or to dacarbazine (DTIC). 
7. Known hypersensitivity to irinotecan or its excipients. 
8. Known history of allergies or sensitivities to gentamicin. 
9. History of or current evidence of any condition (including medical, psychiatric or 
substance abuse disorder), therapy, or laboratory abnormality that might confound the 
results of the study, interfere with the patient’s participation for the full duration of the 
study, or is not in the best interest of the patient to participate, in the opinion of the 
treating Investigator. 
 
Study Enrollment Inclusion Criteria: 
 
Subjects will be eligible for registration if they meet all of the following inclusion criteria: 
 
1. Completed manufacture of at least 4 vials of Vigil. 
2. Karnofsky performance status (KPS) / Lansky performance status (LS) ≥ 80%. 
3. Normal organ and marrow function as defined below: 
Absolute granulocyte count 
≥1,000/mm3 
Absolute lymphocyte count 
≥400/mm3 
Platelets 
≥75,000/mm3 
Hemoglobin 
≥8.0 g/dL 
Total bilirubin 
≤ institutional upper limit of normal* 
AST(SGOT)/ALT(SGPT) 
≤2x institutional upper limit of normal 
Creatinine 
<1.5 mg/dL 
*documented Gilbert’s syndrome may be considered after medical monitor review 
4. Subject has recovered to CTCAE Grade 1 (except for parameters noted in Item 3, 
above) or better from all adverse events associated with prior therapy or surgery.  Pre-
existing motor, sensory neurologic pathology or symptoms, or dermatologic toxicities 
must be recovered to CTCAE Grade 2 or better. 
5. If female of childbearing potential, has a negative urine or serum pregnancy test.  If 
the urine test is positive or cannot be confirmed as negative, a negative serum test will 
be required for study entry. 
6. Ability to understand and the willingness to sign a written informed protocol specific 
consent or a parental/guardian informed consent and pediatric assent when 
appropriate. 
 
Study Enrollment Exclusion Criteria: 


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In addition to the procurement exclusion criteria, subjects will NOT be eligible for study 
registration and randomization if meeting any of the following additional criteria: 
 
1. Any anti-neoplastic therapy between tissue procurement for Vigil manufacture and 
start of study therapy. 
2. Live vaccine used for the prevention of infectious disease administered < 30 days 
prior to the start of study therapy. 
3. Post-surgery complication that in the opinion of the treating investigator would 
interfere with the subject’s study participation or make it not in the best interest of the 
patient to participate. 
Medication and Doses: 
 
Autologous Vigil will be supplied by Gradalis, Inc.  Irinotecan will be supplied by Clinigen.  
Temozolomide will be ordered per standard institutional practice. 
 
Group A 
(i) 
oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), 
(ii) 
oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle), and 
(iii) 
Vigil 1.0 x 106 cells/injection or cells/injection, intradermally on Day 15. 
 
or 
 
Group B 
(i) 
oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), and 
(ii) 
oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle). 
 
One cycle equals 21 days. 
Duration: 
 
Subjects may receive repeat cycles of treatment until disease progression, unacceptable 
toxicity, withdrawal of consent or other criterion is met for discontinuation from study.  
Subjects randomized to Group A, Vigil will receive up to 12 doses depending upon the 
quantity of Vigil manufactured from the surgical specimen.  Within 6 weeks of second relapse 
or progression, subjects randomized to Group B, will be allowed to cross-over (see Section 
10.0) to receive single agent Vigil every 21 days.  Subjects who cross-over may receive up to 
12 doses of Vigil depending upon the quantity of Vigil manufactured.  Cross-over must occur 
within 2 years of End of Treatment assessments of Group B enrollment.  For subjects 
crossing over, no alternative therapies will be given between End of Treatment and cross-
over. 


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Efficacy Assessments: 
• 
Progression Free Survival (PFS) 
• 
Objective Response Rate (ORR) (RECIST 1.1) 
• 
Overall survival (OS) 
Safety Assessments: 
• 
Laboratory assessments 
• 
Physical examination, performance status, height, weight, temperature, blood 
pressure, and pulse 
• 
Toxicity:  CTCAE v 5.0 
Exploratory Assessments: 
 
Circulating EWS FLI1 DNA detection and intratumoral immune function analysis including, but 
not limited to IFNγ-ELISPOT analysis. 
 
Evaluation of baseline tumor microenvironment including, but not limited to 
immunohistochemical evaluation of tumor PD-L1 expression and tumor infiltrating lymphocyte 
(TIL) populations. 
Statistical Considerations: 
 
The planned sample size of 114 randomized subjects is based on the following assumptions 
for the PFS endpoint: 
• 
1:1 randomization; 
• 
Accrual period of 12 months; 
• 
Follow-up period of 6 months; 
• 
Assumed control group median PFS of 6 months from randomization; 
• 
Two-sided test at the alpha=0.0476 level of significance; 
• 
Power 90%; 
• 
Assumed hazard ratio (HR) of 0.45 
 
The planned number of events is 67. 
 
An interim analysis for efficacy, futility, and potential sample size re-estimation will be 
conducted by an independent data monitoring committee (IDMC) when 40 PFS events have 
been achieved (60% information fraction).  Based on the Lan-DeMets method for group 
sequential trials using O’Brien-Fleming boundaries (Reboussin, DeMets et al. 2000), the 
levels of significance for the interim and final analyses are alpha=0.00762 and alpha=0.0476, 
respectively. 
 
Based on the results of the interim analysis, the IDMC will make one of the following four 
recommendations to the sponsor: 
• 
Terminate the study for efficacy (p<0.00762 at the interim analysis); 


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• 
Continue the study as planned; 
• 
Increase the sample size to 120 events; 
• 
Terminate the study for futility. 
 
If the sample size is increased to 120 events, then the study will have approximately 90% 
power to detect a true HR of 0.55.  Note that if the sample size is increased to 120 events, 
then the accrual period will be extended to 24 months, with a 6-month follow-up period after 
randomization of the last patient.  Under these assumptions, it is estimated that approximately 
164 patients will need to be randomized in order to achieve 120 events. 
 
Provided that the IDMC does not recommend terminating the study early for efficacy, the 
conditional power of the planned final analysis at 67 events will be computed using the 
methodology of (Jennison and Turnbull 2000) under the assumption that the observed hazard 
ratio at the interim analysis represents the true effect.  In terms of the HR observed at the 
interim analysis, these rules can be translated (approximately) as follows: 
• 
If the HR is less than 0.43, terminate for efficacy 
• 
If the HR is in the range from 0.43 to 0.51, continue as planned. 
• 
If the HR is in the range from 0.51 to 0.61 (i.e., conditional power in the range from 
50% to 80%), increase the sample size to 120 events. 
• 
If the HR is greater than 0.61 (i.e., conditional power less than 50%), terminate for 
futility. 
 
 
 


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STUDY SCHEMA 
 
 
 


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STUDY PROCESS FLOW 
 
 
 


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1.0 
INTRODUCTION 
 
1.1 
Summary 
 
Ewing’s sarcoma is the second most frequently diagnosed primary malignant bone tumor in the 
US with an annual age-adapted (birth – 20 years) incidence of 2.9 cases per million population 
(Esiashvili, Goodman et al. 2008).  Approximately 20-30% of cases are diagnosed in the first 
decade and 10% after age 20.  The median age at diagnosis is 14 – 15 years (Cotterill, Ahrens 
et al. 2000, Stahl, Ranft et al. 2011).  In greater than 90% of patients there is an essentially 
specific translocation of the EWSR1 gene on chromosome 22q12 with FLI1 on chromosome 
11q24 or, less commonly, other genes (ERG) of the ETS transcription factor family (Marino-
Enriquez and Fletcher 2014).  Up to 85% of Ewing’s tumors are characterized by the EWS/FLI1 
Type 1 (EWS exon 7 to FLI-1 exon 6) or Type 2 (EWS exon 7 to FLI1 exon 5) translocation 
(Arvand and Denny 2001). 
 
Patients with initially diagnosed localized disease have a disease-free survival rate (DFS) of 60-
70% with standard 5-drug chemotherapy regimen (VAC +IE or VIDE), while patients with 
metastatic disease have a DFS of <20%.  The 5-year front line treatment survival rate for 
patients with localized disease approaches 70% with standard of care, but only ~30% for those 
with metastatic lesions isolated to the lung and <20% for those with bone or bone marrow 
involvement (Cotterill, Ahrens et al. 2000, Meyers, Krailo et al. 2001, Grier, Krailo et al. 2003).  
In a large analysis of 975 newly diagnosed Ewing’s sarcoma patients enrolled in trials from 1979 
to 1993, the 5-year recurrence free survival (RFS) for patients with primary metastatic disease 
(n=179) was 22%, which was significantly worse than localized disease (p<.0001) (Cotterill, 
Ahrens et al. 2000).  Although the SEER data survival for metastatic disease has improved (5-
year survival of 39%) with “current” standard of care it is achieved at the cost of an increased 
toxicity profile (Esiashvili, Goodman et al. 2008).  Second or greater line therapy regimens only 
show temporary benefit with dismal survival outcome. 
 
1.2 
Relapsed and Refractory Ewing’s sarcoma 
 
Thirty to forty percent (30-40%) of Ewing’s sarcoma patients who present with localized disease, 
and 60-80% presenting with primary metastatic disease, will experience relapse or progression 
at a median time to relapse of 1.3 years (Ozaki, Hillmann et al. 1996, Bacci, Picci et al. 1998, 
Klingebiel, Pertl et al. 1998, Leavey and Collier 2008, Stahl, Ranft et al. 2011).  Presentation 
with localized disease is associated with a 6-year event free survival (EFS) rate of 69% and 
overall survival (OS) rate of 72% (Leavey, Mascarenhas et al. 2008).  If patients present with 
metastatic disease at diagnosis, the 6-year event free survival in this analysis significantly 
deteriorated to 28% with OS 29% (Guerney, Swensen et al. 1999, Miser, Goldsby et al. 2007, 
Leavey, Mascarenhas et al. 2008).  Other reports have shown worse overall survival in patients 
who relapse with a marked reduction in overall survival of only 12% at 5 years, and 19% at 2 
years (Leavey, Mascarenhas et al. 2008).  Factors like site of recurrence (localized, metastatic, 
combined localized and metastatic) and time to relapse (<2 years or >2 years) play important 
prognostic factors for survival in recurrent Ewing’s sarcoma (Table 1).  For relapses that occur 


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within the first 2 years after initial diagnosis, which make up 72% of relapses (Stahl, Ranft et al. 
2011), the 2-year OS was 7% (Shankar, Ashley et al. 2003), 5-year EFS was 5% (Bacci, Ferrari 
et al. 2003), and 5-year OS was 7% (Stahl, Ranft et al. 2011).  In addition, response status to 
frontline therapy like recurrence (initial response followed by relapse) versus refractory (disease 
progression on first line regimen) appear to correlate with clinical outcome, while the survival 
rate is dismal for patients that do not respond to frontline therapy (Casey, Wexler et al. 2009). 
 
Table 1. Relapse from Time of Initial Treatment 
PI 
Number 
Patients 
Primary 
Metastatic 
Disease at 
Diagnosis 
5-year 
Overall 
Survival 
After First 
Relapse 
5-year OS: 
Relapse < 2 
years from 
Initial Diagnosis 
5-year OS: 
Relapse ≥ 2 
years from 
Initial Diagnosis 
Stahl 2011 
714 
35% 
13.0% 
7.0% 
29.0% 
Leavey 2008 
262 
46% 
12.0% 
7.0% 
30.0% 
Bacci 2003 
195 
0% 
13.8% 
2.5% 
14.3% 
Rodriguez-
Galindo 
2002 
71 
42% 
23.7% 
5.0% 
34.9% 
Shankar 
2003 
64 
0% 
10.0% 
7.0% 
45.0% 
Barker 2005 
55 
38% 
23.0% 
12.0% 
48.0% 
McTiernan 
2006 
114 
47% 
15.2% 
6.7%* 
31.6% 
*Relapse free interval defined as < or ≥ 18 months. 
 
The NCCN guidelines do not provide standard of care recommendations for second-line 
treatment and only 9-13% of patients achieve a second disease free remission (Bacci, Ferrari et 
al. 2003, Barker, Pendergrass et al. 2005, Rasper, Jabar et al. 2014).  In one large retrospective 
analysis of 714 patients in first relapse, the 1-year OS was 43%, 5-year OS was 13%, and 10-
year OS was 9% (Stahl, Ranft et al. 2011). 
 
1.3 
Salvage chemotherapy in Ewing’s sarcoma 
 
Regimens such as topotecan/cyclophosphamide, irinotecan/temozolomide, or 
docetaxel/gemcitabine have been utilized in second line or later treatment and may prolong life 
for those who respond (Merchant, Kushner et al. 1999, Saylors, Stine et al. 2001, Wagner, 
Crews et al. 2004, Wagner, McAllister et al. 2007, Navid, Billups et al. 2008, Navid, Willert et al. 
2008, Casey, Wexler et al. 2009, Mora, Cruz et al. 2009, Wagner 2010, Wagner, Perentesis et 
al. 2010, McGregor, Stewart et al. 2011, Rapkin, Qayed et al. 2012, Raciborska, Bilska et al. 
2013, Wagner, Turpin et al. 2013).  Although results of progression free survival (PFS) and OS 
do not significantly vary between dose regimens (Yildiz, Sen et al. 2014), PFS in patients 
treated with second-line irinotecan/temozolomide, the most commonly used regimen, varies 


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between 3 and 8 months with most commonly reported PFS of 6 months (Casey, Wexler et al. 
2009, Raciborska, Bilska et al. 2013, Palmerini, Jones et al. 2016). 
 
Clinical studies have demonstrated the combination of irinotecan and temozolomide to have 
synergy and significant antitumor activity. Casey et al. showed that the combination of 
temozolomide and irinotecan is well-tolerated and an active regimen for recurrent or refractory 
Ewing’s sarcoma patients (first relapse) with an overall response of 63% and median TTP of 8.3 
months (Casey, Wexler et al. 2009).  
 
A retrospective study of 22 patients with relapsed or refractory Ewing’s sarcoma treated with the 
combination of vincristine, irinotecan, and temozolomide reviewed a shorter treatment course of 
5 days for irinotecan instead of the protracted 10-day course. Results showed comparable 
response rates (68.1 %) with a median TTP of 3 months and suggests the use of the short 
schedule of administration (Raciborska, Bilska et al. 2013). In a similar study orally administered 
irinotecan, temozolomide and vincristine showed milder cumulative toxicities employing a 5-day 
treatment course rather than 10 days in sarcoma patients (Wagner, Perentesis et al. 2010).  A 
pediatric study has directly compared the efficacy of irinotecan with a daily for 5 days versus 
daily for 5 days for 2 weeks schedule (Mascarenhas, Lyden et al. 2010) in 89 evaluable patients 
with recurrent rhabdomyosarcoma were randomized to receive vincristine combined with 
irinotecan. The overall incidence of grade 3–4 adverse events was similar and no significant 
difference in efficacy was observed.  Casey and colleagues showed that irinotecan (iv dx5 x2) 
and temozolomide (orally dx5) was well tolerated and an active regimen in Ewing’s sarcoma in 
first recurrence (n=11), second recurrence (n=3) or refractory to primary chemotherapy (n=6) 
(Casey, Wexler et al. 2009). 
 
A more recent review of irinotecan of the past 15 years evaluated oral versus intravenous 
administration in pediatric sarcoma patients (Wagner, Perentesis et al. 2010, Wagner 2015).  
Even though the oral dosing requires a higher total dose of irinotecan (bioavailability is less) the 
metabolism of orally administered irinotecan appears to be more efficient, given that the 
intestinal tract contains high levels of carboxylesterases, which may pre-systemically metabolize 
irinotecan to the active moiety, SN-38 and increase the SN-38/irinotecan ratio by threefold or 
more (Drengler, Kuhn et al. 1999).  Although there have been no studies directly comparing the 
efficacy of the two routes of administration, at similar SN-38 lactone exposures (60 mg/m2/day 
oral vs. 20 mg/m2/day IV), response rates, and toxicity profiles appear to be equivalent. A phase 
1 dose escalation (Irinotecan 50mg/m2/dose escalated to a maximum of 90mg/m2/dose, and 
temozolomide 100mg/m2/dose escalated to a maximum of 150mg/m2/dose) study in pediatric 
patients with relapsed or refractory solid tumors of oral irinotecan, temozolomide and 
temsirolimus demonstrated that lower dose level of irinotecan (50mg/m2/dose) and 
temozolomide (100mg/m2/dose) was well tolerated with less toxicities (Bagatell, Norris et al. 
2014).  An additional study recommends 50mg/m2 per day for 5 consecutive days for phase II 
studies of oral irinotecan (Soepenberg, Dumez et al. 2005).  The benefit of oral administration 
includes not only greater patient convenience, shorter hospitalization and decrease in costs, but 
most importantly improved quality of life. Thus, summarizing studies of oral irinotecan and 
temozolomide and in agreement with participating investigators a lower dose regimen of 
50mg/m2/d irinotecan and 100mg/m2/d temozolomide administered orally appears to be an 
acceptable standard of care regimen for second-line Ewing’s sarcoma providing clinical activity 
with low toxicity profiles. In those occasional patients with difficulty taking oral medication (e.g. 
severe nausea, chronic gastrointestinal complaints, and age-related oral aversion) intravenous 


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administration may remain a reasonable and acceptable alternative standard of care option 
(Wagner 2015). 
 
1.4 
Immunotherapeutic considerations in Ewing’s sarcoma 
 
Ewing’s sarcoma demonstrates elements of the immune escape mechanisms determined to be 
integral to the natural history of cancer.  Patients with primary disseminated disease at 
presentation show an increased frequency of T-regulatory cells in the bone marrow compared to 
those with localized disease (Brinkrolf, Landmeier et al. 2009).  Factors associated with survival 
are increased number of tumor infiltrating CD8+ T-lymphocytes (p=0.05) which inversely 
correlates with tumor volume (<200 ml) at diagnosis (Berghuis, Santos et al. 2011). 
Furthermore, the unique, defining EWS/FLI-1 fusion protein may be an effective immunogenic 
driver neoantigen less likely to be adaptively downregulated due to its driver status (Liu, Huang 
et al. 2012, Peng, Huang et al. 2014).  Meyer-Wentrup et al. discovered the first immunogenic 
Ewing’s sarcoma specific peptide directly derived from the EWS/FLI1 fusion gene (Meyer-
Wentrup, Richter et al. 2005).  Ten (10) peptides derived from the 21-mer fusion region were 
used to prime T cell responses by pulsing single peptides onto mature dendritic cells.  Gamma 
interferon (IFNγ) production was measured in ELISPOT assays as a surrogate marker for T- 
and NK cell activation.  QQSSSYGQQN-pulsed dendritic cells induced significant IFNγ immune 
response while the other 9 peptide-induced IFNγ production remained unspecific. Interestingly, 
the response to QQSSSYGQQN was highly peptide specific and could be inhibited by HLA-DR-
blocking antibody, known to block HLA class II activation. Fusion-region specific neoantigens, 
such as described by Meyer-Wentrup and colleagues, could reveal new opportunities to induce 
and sustain cytotoxic immune responses. Re-activation of immunogenic antitumor response as 
a treatment strategy for EWS could be exploited either in a monotherapy or in combination with 
chemotherapy agents such as irinotecan and temozolomide, which produce lower rates of 
lymphopenia of shorter duration and allow for effective, concurrent immune responsiveness 
(Kushner, Kramer et al. 2006). 
 
Preliminary results in 83% of Ewing’s sarcoma patients treated with Vigil immunotherapy 
demonstrated CD8+ T-cell activation, as defined by IFNγ ELISPOT assay using unmanipulated 
autologous tumor cells (verified by Mt. Sinai and plates are assessed for spots by Zellnet, Fort 
Lee, NJ).  Notably, Vigil induced ELISPOT response has been shown to significantly correlate 
with survival and time to recurrence (TTR) in patients with a variety of solid tumors (Senzer, 
Barve et al. 2013, Oh, Barve et al. 2016) and specifically with tumor responses (1 CR/NED, 1 
PR) in two Ewing’s sarcoma patients one of whom is described in a prior publication (Ghisoli, 
Rutledge et al. 2017).  The relationship of the sequential IFNγ ELISPOT level to clinical course 
in this patient is shown in Figure 1.  Evidence of CD4+ immune activation has been identified by 
analysis of 24 different cancer testis (CT) antigens in Vigil treated Ewing’s sarcoma patients.  
Patient 062 (Figure 1) showed seroconversion of CT47 during Vigil treatment with persistence 
of antibodies after treatment completion. CT47 expression has been observed to be elevated in 
sarcoma patients suggesting antigen targeted therapies (Chen, Iseli et al. 2006).  Another 
Ewing’s sarcoma patient (PID 095) and long-term survivor (1033 days since procurement) 
showed similar CT47 CD4 response during Vigil treatment in addition to CD8 T cell activation 
(ELISPOT response). 
 


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Figure 1. Case Report 1 (F-062). Relapsed Ewing's sarcoma Response to Vigil. 


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2.0 
VIGIL 
 
Vigil immunotherapy comprises 1) autologous tumor cells as a source of the full matrix of a 
patient’s tumor-related antigens and 2) a DNA plasmid with two genetic modifications in order to 
optimize a “triad” functionality i) patient tumor-specific antigen presentation, ii) dendritic cell 
activation (GMCSF), and iii) tolerance escape (blocking TGFβ1, β2 activation) (Maples PB 
2009, Nemunaitis 2011).  To construct Vigil, autologous cancer cells are transfected with a 
multi-component expression vector encoding GMCSF and a downstream bi-functional small 
hairpin RNA for specific knockdown of furin, a proprotein convertase critical for maturational 
proteolytic processing of immune relevant TGFβ isoforms. 
 
Since the start of a Phase I trial of Vigil in advanced cancer patients on 06/08/2009, strong 
evidence for safety and benefit was initially seen in a sub-analysis of 35 adult and pediatric 
patients given 176 vaccinations (Senzer, Barve et al. 2013).  To determine optimal dosing 
regimen, analyses were carried out comparing survival, safety and ELISPOT response at 1 x 
107 cells/injection vs. 2.5 x 107 cells/injection and at a range of Vigil dosing schedules between 
4 and 12 monthly injections.  This analysis showed similar results between dose levels and 
number of doses; thus 1 x 107 cells/injection with a minimum of 4 and a maximum of 12 monthly 
injections was selected as the treatment regimen for subsequent studies.  Vector effectiveness 
was also confirmed via GMCSF transgene expression and knockdown of Furin, TGFβ1 and 
TGFβ2 expression (Senzer, Barve et al. 2012, Senzer, Barve et al. 2013). 
 
Interestingly, a small sub-cohort (n=15) with a dose level of 1 x 106 cells/injection was enrolled 
and treated on the Phase 1 study with Vigil and showed ELISPOT conversion (12/12 evaluable 
patients) from baseline to treatment start in a recent analysis (Figure 2 and Figure 3) (Manning 
2017). Six (6) of these patients enrolled with advanced, relapsed or refractory Ewing’s sarcoma 
and 5 of the 6 Ewing’s patients had prolonged survival of > 2 years (median OS 807.5 days, 
range 58-1090 days from time of procurement).  Four (4) of the low-dose Ewing’s patients were 
also analyzed for ELISPOT response and all 4 showed positive conversion post treatment with 
Vigil (Figure 4).  The median survival from time of procurement of these 4 patients was 1288 
days (range 840-1814 days).  This data supports a 1x106 dose administration of Vigil in Ewing’s 
sarcoma. 
 


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Figure 2. Low Dose Vigil Phase I Survival status.  Arrow indicates subjects still alive. 
 
 
Figure 3. Low Dose Vigil Phase I ELISPOT response status.  Gray lines indicates time 
points off treatment. 
 


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Figure 4. Low Dose Vigil Phase I Ewing’s Patients: ELISPOT response status.  Gray lines 
indicates time points off treatment. 
 
Eighty patients in total (54 females, aged 13-84 / 26 males, aged 12-76) entered into Phase I 
trial involving 19 solid tumor cancers.  Four hundred sixty-four (464) Vigil injections were 
administered and no ≥ Grade 3 toxic events attributed to Vigil have been observed.  Dose 
administered ranged from 1 x 106 cells/injection to 2.5 x 107 cells/injection via intradermal 
injection once a month for a minimum of 4 months.  The prolonged survival shown in Figure 5 
involving predominantly advanced stage, heavily pretreated patients is greater than historical 
expectation (Senzer, Barve et al. 2013). 
 


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Figure 5. Long term survival by cancer type of patients entered into Vigil Phase I trial. 
 
Moreover, correlation of survival benefit with recent follow up involving the first 30 Phase I 
patients to receive Vigil to γIFN ELISPOT response which measures Vigil turned on circulating 
immune cells (T effector cells) against autologous tumor suggests efficacy tied to predicted 
mechanism of Vigil (Figure 6). 
 
 


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Group 
N 
Median 
(mo.) 
p-value 
 
Group 
N 
Median 
(mo.) 
p-value 
ELISPOT (-) 
15 
17.2 
0.00015 
 ELISPOT (-) 
15 
6.4 
0.00010 
ELISPOT (+) 
52 
38.5 
 
 ELISPOT (+) 
52 
28.7 
 
Figure 6. Correlated survival and immune response and updated analysis of Vigil Phase 1 
patients.  78% ELISPOT conversion rate.  55% ELISPOT (+) patients are still alive vs. 13% 
ELISPOT (-) are deceased. (Analysis performed on 02/27/17) 
 
These results have recently been updated (Figure 6).  Sixty-seven (67) of these Phase I 
patients have now undergone IFNγ ELISPOT testing.  Fifty-two demonstrated change from 
negative at baseline (prior to Vigil treatment) to positive IFNγ ELIPSOT reactivity after Vigil 
(78% conversion rate).  However, fifteen failed to show upregulation of circulating active effector 
T cells against autologous tumor induction by Vigil.  Survival correlation continues to be 
demonstrated in this 4-year updated analysis. 
 
2.1 
Vigil Phase II Trial: Ovarian Cancer 
 
Based on positive Phase I results, a 2:1 (Vigil:Control) randomized Phase IIa open-label trial of 
Vigil (tumor harvested at the time of surgical debulking), in patients with Stage III/IV ovarian 
cancer, who achieved clinical complete response (cCR) following primary surgical debulking 
and standard chemotherapy (5-6 cycles, front-line maintenance setting), was initiated (CL-PTL-
105).  A 1.0 x 107 dose of transfected autologous tumor cells/intradermal injection was 
administered once a month for up to 12 doses to the treatment group.  Standard of care was 
provided for the control patients.  Cross-over was allowed at time of relapse for patients 
randomized to standard of care therapy.  Results have been published by Oh et al (Oh, Barve et 
al. 2016). 
 


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Twenty-one patients were randomized to Vigil, 11 to control standard of care.  Discussion of 
these results lead to a larger (currently ongoing) Phase II/III trial design.  Once the Phase II/III 
trial was initiated study CL-PTL-105 was closed to further accrual and patients not yet 
randomized in consolidation chemotherapy section entered the Vigil arm.  Thirty-one patients 
received Vigil as part of the Phase IIa trial (11 were randomized to control standard of care).  
Demographics revealed no significant differences between the 2 groups. 
 
No ≥ Grade 3 adverse events were observed related to Vigil.  Efficacy with respect to time to 
relapse induced immune response is shown in Figure 7. 
 
 
Group 
N 
Median (mo.) 
p-value 
Control 
11 
12.4 
0.025 
Vigil 
31 
19.8 
 
Figure 7. Correlated survival of Vigil compared to control in Ovarian Cancer (CL-PTL 105) 
with immune responses.  OV-1049 and OV-1074 were censored at time of cross-over.  
OV-1049 crossed-over and started Vigil 08/27/14 (5 cycles) and relapsed 4 months after 
treatment start (12/17/14). 
 


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ELISPOT Conversion 
 
At Baseline 
Post Vigil Treatment 
Vigil 
1/31 (0.3%) 
31/31 (100% Conversion) 
Median spots 134 (12-448) 
Control 
0/8 
7/8 (cross-over) 
Median spots 58 (43-105) 
Figure 8. ELISPOT response of Vigil in Ovarian Cancer (CL-PTL-105).  Thirty-one (31) 
patients are ELISPOT (+) post Vigil treatment. 
 
These positive results were associated with robust ELISPOT response (Figure 7).  Less than 
3% of ovarian patients had circulating T cells with capacity to release IFNγ and induce 
autologous tumor necrosis at baseline (prior to Vigil), however following Vigil treatment 31/31 
patients demonstrated brisk antitumor immune activity.  No control patients demonstrated 
circulating T cell response against autologous tumor during or after standard of care, or at time 
of relapse.  Ninety percent (90%, 7 of 8) demonstrated autologous tumoricidal activity of 
circulating T cells by IFNγ ELISPOT response, after cross-over.  Interestingly, ELISPOT 
reactivity response was only 43% of what was observed with Vigil prior to relapse (134 spots vs. 
58 spots) (Figure 8). 
 


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2.2 
Vigil Phase I Trial: Ewing’s sarcoma Subset 
 
Initial results of 12 Ewing’s sarcoma Vigil treated patients entered into CL-PTL-101 were 
published two years ago (Ghisoli, Barve et al. 2015).  A total of 30 Ewing’s sarcoma patients 
have now been enrolled into CL-PTL-101 with intent to treat and procurement for EWS Vigil 
manufacture.  All patients had late stage Ewing’s sarcoma (majority ≥ third line chemotherapy).  
Sixteen of these patients received Vigil.  Fourteen concurrent patients who did not receive Vigil 
after undergoing similar surgery and Vigil manufacture served as control.  Nine of the latter were 
unable to have vaccine released (6 contaminants, 3 insufficient viable tumor cells) and five 
chose other treatment management.  All products constructed fulfilled release criteria of 
GMCSF production and TGFβ1, β2 knockdown.  See demographics in Table 2. 
 
Table 2. Ewing’s sarcoma Phase I Demographics 
 
Vigil 
MC* 
Tumor Location Harvest (Lung/Soft Tissue/Other) 
13/0/3 
11/2/1 
Sex (M/F) 
12/4 
7/7 
Age median (range) 
19 (59-12) 
17 (30-12) 
Ethnicity (Caucasian/Other) 
13/3 
12/2 
Prior Systemic Treatment (Frontline/2nd/≥3rd) 
1/5/10 
3/4/7 
Surgical Candidate (Yes/No) 
16/0 
14/0 
Tissue Harvested (Yes/No) 
16/0 
14/0 
* Matched Control (MC); 3 insufficient viable tumor cells, 6 contaminants, 5 sought other 
management 
 
Consistent with Phase I data in adults and Phase II data in ovarian cancer, no significant toxicity 
was observed in Phase I Vigil treated Ewing’s sarcoma patients.  Specifically, no product related 
Grade 3, 4 toxic effects were demonstrated during treatment course and no long term “post 
treatment” toxicity has been observed.  Reported adverse events are shown in Table 3. 
 
Table 3. Phase I (CL-PTL-101):  Ewing's sarcoma patients who received Vigil, definitely or 
probably related adverse events 
Preferred Term 
CTC 
Grade 
Relationship to 
Study Drug 
Number of 
Subjects 
Number 
of Events 
Bruising 
1 
Definitely Related 
1 
1 
Erythema @ Injection Site 
1 
Definitely Related 
1 
1 
Fatigue 
1 
Probably Related 
1 
4 
Induration / Fibrosis 
Injection Site Reaction- 
Induration 
1 
Definitely Related 
2 
2 


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Preferred Term 
CTC 
Grade 
Relationship to 
Study Drug 
Number of 
Subjects 
Number 
of Events 
Injection Site Reaction – 
Induration 
1 
Definitely Related 
1 
2 
Injection Site Reaction- Erythema 
1 
Definitely Related 
11 
31 
Injection Site Reaction- 
Induration 
1 
Definitely Related 
12 
55 
Injection Site Reaction- Pain 
1 
Definitely Related 
3 
3 
Injection Site Reaction- Pruritus 
1 
Definitely Related 
1 
3 
Injection Site Reaction- Swelling 
1 
Definitely Related 
2 
3 
Injection Site Reaction- 
Tenderness 
1 
Definitely Related 
1 
1 
Joint-function 
1 
Probably Related 
1 
2 
Pain – Back 
1 
Probably Related 
1 
1 
 
No Grade 3 or 4 related adverse events were observed to Vigil.  There were 11 serious adverse 
events (SAEs) reported involving 7 participants.  None of the SAEs were related to Vigil. 
 
2.3 
Efficacy Evidence of Vigil in Ewing’s sarcoma 
 
A survival advantage was seen in the Vigil treated patients compared to control. 
 
Figure 9 shows the results of the Kaplan-Meier analysis of the survival data. Comparison of 
overall survival from time of procurement (for Vigil manufacture) between the two concurrent 
groups revealed a 17+ month improvement in survival in the Vigil treated patients.  From time of 
treatment the Vigil treated patients achieved a median survival of 689 days. 
 


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Group 
N 
Median (mo.) 
p-value 
Matched Control 
14 
6.8 
0.056 
Vigil 
16 
24.0 
 
Figure 9. Survival from surgical procurement of advanced Ewing’s patients in study CL-
PTL-101 successfully harvested for Vigil construction.  Comparing those who received 
Vigil (ORR 12.5% (1NED, 1PR)) vs. those who did not receive Vigil censored alive as of 
dates provided on 10/19/15. 
 
The actual 1-year survival of patients who received Vigil (11/15) was 73% vs. those who did not 
receive Vigil of 23% (3/13).  One Vigil and one control patient remained alive but had not yet 
achieved actual 1-year survival time point.  These results were recently published by Ghisoli et 
al (Ghisoli, Barve et al. 2016).  Retrospective comparison of patient groups to two prognostic 
risk factor scores (the Wheler score for phase I patients (Wheler, Tsimberidou et al. 2012) and 
the Ewing’s sarcoma prognostic scores (ESPS) (Cotterill, Ahrens et al. 2000, Bacci, Longhi et 
al. 2006, Rodriguez-Galindo, Liu et al. 2007, Ladenstein, Pötschger et al. 2009, Jain and 
Kapoor 2010, Gaspar, Le Teuff et al. 2011, 2016)) suggest similar risk profiles of Vigil and 
control patients (Table 4) thereby supporting evidence of efficacy related to Vigil. 
 
 


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Table 4. Prognostic Indicator Studies of Ewing's sarcoma 
Ewing’s Sarcoma Prognostic Scores (ESPS)° 
 
RISK OF LOW SURVIVAL 
 
Group 
High 
Intermediate 
Low 
Grand Total 
Matched 
Control 
6 
8 
0 
14 
Vigil 
6 
10 
0 
16 
 
High: 16-21 pts 
Intermediate: 11-15 pts 
Low: 0-10 pts 
 
°males higher risk of death 
 
Phase 1 Prognostic Scores* 
 
RISK OF LOW SURVIVAL 
 
Group 
High 
High-
intermediate 
Intermediate 
Low-
intermediate 
Low 
Grand Total 
Matched 
Control 
0 
1 
8 
5 
0 
14 
Vigil 
1 
6 
3 
6 
0 
16 
 
High: 4-5 
pts 
High-intermediate: 
3 pts 
Intermediate: 
2 pts 
Low-intermediate: 
1 pts 
Low: 
0 pts 
 
* Phase 1 Prognostic scores include factors like LDH, number of metastatic sites, albumin, etc. 
 
2.4 
Vigil Phase II Trial: Ewing’s sarcoma (CL-PTL-121, Part 1) 
 
A Phase llb randomized open label trial (CL-PTL-121) comparing Vigil versus standard of care 
chemotherapy (gemcitabine/taxol) in metastatic, refractory or recurrent (≥2 prior systemic 
treatment lines) Ewing’s sarcoma patients was initiated and enrolled the first patient 13 months 
ago.  Part 1 of the trial was open to enrollment at 10 sites (5 in development) across the USA 
but only 13 patients registered into the study.  Eight were randomized to control and received 
gemcitabine/taxol and 5 received Vigil.  No Grade 3 or greater toxicity to Vigil has been 
observed.  Grade 3 or greater toxic events related to chemotherapy were observed in 2 Ewing’s 
patients (hematologic, compromise, edema/facial blisters) and significant chemotherapy related 
toxicity of Grade 1 and 2 involving fever, fatigue, erythroderma, pruritus, neuropathy, nausea, 
vomiting was observed as expected.  Early assessment of survival is shown in Figure 10.  
Kaplan Meier survival of 172 days (5.7 months) was observed in the chemotherapy group while 
the Vigil (initial treatment) group has not yet reached survival median.  Mean survival of the Vigil 
group is 271 days (8.9 months).  This is consistent with the results observed with Phase I/II 
results in study CL-PTL-101.  Futility analysis also revealed that the current trend of positive 
survival to Vigil is on track to achieve statistical significant survival advantage to Vigil at a 90% 
power (46 events).  Further assessment of all advanced Ewing’s patients who received Vigil in 
studies CL-PTL-101 and CL-PTL-121 (21 patients) vs. matched (n=14) and randomized controls 


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(total n=22) provide additional support of potential benefit to Vigil in third-line Ewing’s sarcoma 
(Figure 11).  A Kaplan Meier survival of 757 (24.9 months) days was observed with Vigil. 
 
 
Group 
N 
No. of 
Deaths 
Mean OS 
(mo.) 
Median OS 
(mo.) 
p-value 
Randomized 
Control 
8 
3 
5.7 
5.7 
0.395 
Vigil 
5 
1 
8.9 
Not reached 
 
Figure 10. Overall Survival Since Procurement on Study CL-PTL-121 Preliminary Results.  
Two cross-over patients censored.  Data updated as of 01/19/17. 
 


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Figure 11. Study CL-PTL-101 and -121 Combination Data of Ewing’s sarcoma.  Overall 
Survival Since Procurement.  Median PFS is 6 months.  Expected median PFS is 1.5-2 
months (Juergens, Daw et al. 2011; Choy, Butrynski et al. 2014; Yoon, Kwon et al. 2014).  
Dataset updated 01/23/17.  *MC/RC = Matched Control/Randomized Control 
 
2.5 
Vigil Phase II Trial: Ewing’s sarcoma (CL-PTL-121, Part 2) 
 
Part 2 of CL-PTL-121 was initiated to study the safety of the combination of irinotecan, 
temozolomide and Vigil in patients with refractory or recurrent (≥1 line of prior treatment line(s)) 
Ewing’s sarcoma. 
 
Ten third-line (received ≥ 2 prior systemic chemotherapy regimens) advanced disease Ewing’s 
Sarcoma patients were entered into Part 2 of CL-PTL-121 study.  One patient did not receive 
any protocol treatment, another patient received only 1 dose of temozolomide/irinotecan and did 
not receive Vigil before rapid disease progression.  These two patients were not evaluable for 
efficacy.  One patient is still being treated on study without progression but is too early for 
efficacy evaluation (< 6 months SD, stable disease).  All other 7 currently evaluable patients for 
response have received a mean of 8 cycles of Vigil and temozolomide/irinotecan (range 3 to 
12).  Dose and schedule involved Vigil (1x 10e6 or 1x 10e7 cells/dose on Day 15 every 3 
weeks), irinotecan (50 mg/m2/d Days 1-5 orally every 3 weeks) and temozolomide (100 
mg/m2/d Days 1-5 orally every 3 weeks).  Notably all 7 of these patients had progressed 
following prior temozolomide/irinotecan before entry into CL-PTL-121. 
 
Group 
N 
Mean 
Survival (mo.) 
Median 
Survival 
(mo.) 
P- value 
MC/RC* 
22 
12.8 
6.8 
0.041 
Vigil 
21 
24.9 
24.0 
 
 


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Demographics are shown in Table 5.  No Grade 3 Vigil related toxic effect was observed at 
dose levels of 1x10e6 (n=5) or 1x10e7 (n=5) cells/injection.  No unexpected toxic effect known 
to be related to irinotecan/temozolomide combination was observed.  Table 6 depicts the 
≥Grade 3 adverse events reported to date (out of 175 events).  Seven (EW-167-3010, EW-167-
3012, EW-167-3003, EW-167-3006, EW-167-3011, EW-167-3013, EW-002-3005) of the 10 
patients were available to assess durable (≥ 6 months) progression free response.  One (EW-
167-3014) is alive and doing well but has not yet reached 6-month evaluation.  Additionally, the 
comparison of 1 x 106 cell/ml (n=3) vs 1 x 107 cells/ml (n=4) did not reveal any differences in 
safety or efficacy, measured as OS from time of treatment start of mean 169 days (1 x 106 
cells/ml) vs. 185 days (1 x 107 cells/ml).  
 
Table 5. Demographics of subjects enrolled into Part 2 of CL-PTL-121. 
Intent to treat 
population 
Evaluable patients 
for response 
(≥6mo PFS) 
Number of patients 
10 
7 
Age 
 
 
Mean 
25 
24 
Range 
12 to 46 
12 to 46 
Gender  
 
 
Female 
5 
4 
Male 
5 
3 
Ethnicity 
 
 
Caucasian 
9 
6 
Hispanic/Latino 
1 
1 
Other 
0 
0 
Lines of prior systemic Treatment 
 
 
Mean 
4 
5 
Range 
1 to 13 
1 to 13 
 


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Table 6. Grade 3 or greater Adverse Events reported, CL-PTL-121, Part 2. 
Event Term 
CTC 
Grade 
Relationship 
to VIGIL 
Relationship to 
Temozolomide 
Relationship 
to Irinotecan 
Number 
of 
Subjects 
Number 
of 
Events 
Platelet Count 
Decreased 
3 
Not Related 
Probably 
Related 
Probably 
Related 
1 
1 
Anemia 
3 
Not Related 
Unlikely Related 
Unlikely 
Related 
1 
2 
Chest Wall Pain 
3 
Not Related 
Not Related 
Not Related 
1 
1 
Febrile Neutropenia 
3 
Not Related 
Probably 
Related 
Probably 
Related 
1 
1 
Hypokalemia 
3 
Not Related 
Not Related 
Not Related 
1 
4 
Neutrophil count 
decreased 
3 
Not Related 
Possibly Related 
Possibly 
Related 
2 
2 
  
3 
Not Related 
Probably 
Related 
Probably 
Related 
1 
2 
  
4 
Not Related 
Probably 
Related 
Probably 
Related 
2 
2 
Platelet Count 
Decreased 
3 
Not Related 
Probably 
Related 
Probably 
Related 
2 
2 
  
4 
Not Related 
Not Related 
Not Related 
1 
1 
  
4 
Not Related 
Probably 
Related 
Probably 
Related 
1 
1 
Pneumonitis 
4 
Not Related 
Not Related 
Not Related 
1 
1 
Respiratory Failure 
4 
Not Related 
Not Related 
Not Related 
1 
1 
Respiratory, 
thoracic and 
mediastinal 
disorders - Other, 
Empyema Lt. 
Thorax Infection 
4 
Not Related 
Not Related 
Not Related 
1 
1 
Back Pain 
5 
Not Related 
Not Related 
Not Related 
1 
1 
Musculoskeletal 
and connective 
tissue disorder - 
Other, Progressive 
Ewing’s Sarcoma 
5 
Not Related 
Not Related 
Not Related 
1 
1 
 
An objective response by RECIST (partial response, 61% shrinkage of target lesions) was 
achieved in one patient (EW-167-3006), in addition CT scan abnormalities at baseline cancer 
sites which demonstrated prior PET avid uptake of FDG of this patient no longer demonstrated 
PET uptake after Vigil (see Figure 12 and Figure 13).  This patient underwent biopsy of 
pulmonary and pelvic lesions which were still visible by CT scan but no longer PET avid.  
Results demonstrated no viable tumor presence.  Furthermore, entirely necrotic, hyalinize tissue 
of the pulmonary sample indicates response to on-study therapy.  Another patient EW-167-3003 
similarly underwent PET scan evaluation and showed change from PET avid at baseline to PET 
negative 7 months after treatment initiation.  This patient later after completion of Vigil 
developed an infection of the left lung cavity (status post left pneumectomy) and passed away at 
Month 8 related to progressive infectious pneumonia.  Autopsy was performed and the PET 
negative disease in the posterior chest wall seen on CT scan revealed no viable tumor cells at 
that site consistent with complete histologic disease response.  Additionally, no other sites of 
microscopic cancer were found at autopsy.  Patient EW-167-3010 had durable SD ≥6 months 


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and remains in good health.  Consideration for second resection and assessment of detection of 
viable tumor at CT detectable disease sites is underway. Previously, we also reported complete 
histologic response in a prior Vigil monotherapy treatment patient (F-062), who was not RECIST 
evaluable (Ghisoli, Rutledge et al. 2017). This patient underwent a second harvest and second 
Vigil treatment with persistent detectable disease following first Vigil treatment and remains alive 
without recurrence 4 years/months following Vigil start. It was suggested that neoantigen 
patterns at different sites of cancer growth may have some variation and Vigil activity in part 
may relate to the neoantigen pattern contained on the autologous tissue harvested. As such, 
patients with delayed regression or mixed response could be considered for second Vigil 
harvest and treatment if other more optimal therapies are not available.  
 
 
Figure 12. EW-167-3006 Left Upper Lung posterior target lesion PET scan from baseline, 
Month 3, and Month 6 
 
 
Figure 13. EW-167-3006 Left diaphragm soft tissue target lesion PET scan from baseline, 
Month 3, and Month 6. 
 


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Patient EW-167-3012 had all recurrent disease harvested for Vigil construction and remains 
without disease recurrence ≥ 6 months.  Patients EW-167-3013, EW-167-3011 and EW-002-
3005 had progression in < 6 months despite treatment with Vigil, temozolomide and irinotecan. 
Summary of all participants responses are shown in Figure 14.  
 
Overall, historical disease progression would be expected within 2 months of ≥ third-line disease 
progression. 
 
 
Figure 14. Patient summary of PFS of all 10 intent to treat patients, Part 2 of CL-PTL-121. 
Fifty-seven percent (57%, 4/7) with a durable clinical response rate (for ≥ 6 months). 
 
3.0 
STUDY RATIONALE 
 
The results from the Phase I Vigil trial show product safety (both short-term and long-term 
through 3+ years), confirm effective transgene expression (GMCSF) and RNAi [furin] silencing, 
and support the rationale of the immunotherapeutic triad, i.e., autologous tumor cell therapy with 


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increased GMCSF expression and decreased TGFβ production shown to effectively modulate 
immunogenicity as evidenced by the correlation of IFNγ-ELISPOT responsiveness with overall 
survival and the elicitation of the CD4+ mediated circulating antibody responses against CT47 in 
two of the Phase 1 Ewing’s sarcoma patients.  The preliminary evidence of safety, immune 
stimulation and clinical benefit from Vigil in patients with Ewing’s sarcoma treated in the Phase I 
setting supports evaluation of this therapy in a larger and randomized study and at earlier stage 
of recurrence (first recurrence).  A lower tumor burden is typically associated with greater delay 
in progression with other immunotherapies.  Mathematical modeling supports the concept of 
better control of disease progression with the combined use of effective chemotherapy (e.g., 
temozolomide/irinotecan as second line treatment in Ewing’s sarcoma) and immunotherapy 
(i.e., Vigil) in first relapse patient population (de Pillis, Gu et al. 2006).  The utilization of the 
standard of care temozolomide/irinotecan regimen at investigator consensus defined dose and 
schedule appears to be minimally immunosuppressive and thus a reasonable option for 
combination with Vigil.  Moreover, the temozolomide and irinotecan regimen at effective dose 
levels has low myelosuppressive toxicities (irinotecan 50mg/m2/dose and temozolomide 
100mg/m2/dose) and may increase the tumor antigen release and surface exposure of 
calreticulin (part of the specific danger-signaling system) as well as reduce the frequency of 
CD4+ CD25+ regulatory T-cells (Kim, Kim et al. 2010).  In addition, given that Ewing’s sarcoma 
expression levels of type-1 associated chemokine ligands CXCL9, CXCL10, and CCL5 correlate 
with TIL expression chemokine receptors (CXCR3 and CCR5) (Berghuis, Santos et al. 2011) it 
is notable that temozolomide has been shown to enhance tumor matrix presentation of CXCL9 
and CXCL10 in certain tumor models (Tan, Evrard et al. 2015).  Temozolomide and irinotecan in 
combination have shown benign safety profiles without any Grade 3 treatment-related adverse 
events in seven combination treated Ewing’s Sarcoma patients of Part 2 of CL-PTL-121. 
Furthermore, clinical antitumor activity was demonstrated in 4 of the 7 combination treated 
patients, all of whom had previously failed temozolomide and irinotecan, as evidenced by 2 
partial responses, one histological CR (autopsy) and one prolonged SD currently near 1 year in 
duration. Thus, the combination of low dose Vigil, irinotecan and temozolomide may not only 
increase anti-tumor responses but also prolong time to progression and improve the of quality of 
life in the treatment of Ewing’s sarcoma patients. 
 
4.0 
OBJECTIVES 
 
4.1 
Primary objective 
 
• 
To compare the progression free survival of subjects dosed with Vigil immunotherapy in 
combination with irinotecan and temozolomide vs. irinotecan and temozolomide. 
 
4.2 
Secondary objective(s) 
 
• 
To determine and compare the overall survival of subjects with relapsed or refractory 
Ewing’s sarcoma dosed with Vigil immunotherapy in combination with irinotecan and 
temozolomide. 
• 
To determine the objective response rate (RECIST 1.1) of patients with metastatic 
Ewing’s sarcoma refractory or intolerant to 1 prior line of systemic chemotherapy treated 


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with Vigil immunotherapy dosed with Vigil immunotherapy in combination with irinotecan 
and temozolomide 
• 
To determine the Vigil manufacturing success rate by gathering data from tumor tissue 
collection, Vigil construction, and test procedures. 
 
5.0 
STUDY DESIGN 
 
This is a multicenter, 1:1 randomized Phase III study of intradermal autologous Vigil 
immunotherapy (1.0 x 106 cells/injection; minimum of 4 to a maximum of 12 administrations) in 
combination with irinotecan and temozolomide in subjects with metastatic Ewing’s Sarcoma 
Family of Tumors (ESFT) refractory/intolerant or recurrent to 1 prior line of chemotherapy.  
Participants undergoing a standard surgical procedure (e.g., tumor biopsy or palliative 
resection) may have tumor tissue harvested for manufacture of investigational product 
 
Subjects meeting eligibility criteria including manufacture of a minimum of 4 immunotherapy 
doses of Vigil will be randomized to receive either: 
 
Group A 
(i) 
oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), 
(ii) 
oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle), and 
(iii) 
Vigil 1.0 x 106 cells/injection, intradermally on Day 15. 
 
OR 
Group B 
(i) 
oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), and 
(ii) 
oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle). 
 
If chemotherapy study drugs cannot be administered orally (i.e. size of capsule, nausea, etc.) it 
is the physician’s discretion to request intravenous administration of temozolomide at 100 
mg/m2 daily and/or irinotecan 20 mg/m2 daily intravenously. 
 
One cycle = 21 days.  Screening for the main portion of the study may occur as early as one 
week but no later than 8 weeks following tumor procurement.  Vigil is typically released 
approximately 3 weeks after the completion of the two-day manufacturing process.  At the time 
of data analysis the subjects will be stratified by initial response to frontline treatment (recurrent 
versus refractory (progressive)). 
 
Participants will be managed in an outpatient setting.  Hematologic function, liver enzymes, 
renal function and electrolytes will be monitored.  Blood for immune function analyses including 
IFNγ-ELISPOT analysis of cytotoxic T cell response to autologous tumor antigens will be 
collected at tissue procurement, post-procurement screening and Day 1 (prior to chemotherapy 
administration) at Cycles 2, 4, and 6, end of treatment (EOT), 3 months after EOT, and every 6 


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months thereafter.  Blood for exploratory ctDNA analysis will be collected at tissue procurement, 
prior to chemotherapy administration at baseline, prior to chemotherapy administration on Day 1 
of Cycles 2, 3, 4, and 6, and EOT. 
 
6.0 
STUDY POPULATION 
 
6.1 
Sample Size 
 
Approximately 140 subjects will have tissue procured so that 114 evaluable subjects will be 
registered for enrollment into the study. 
 
6.2 Tissue Procurement Inclusion Criteria 
 
Subjects will be eligible for tissue procurement for the Vigil manufacturing process, if they meet 
all of the following criteria: 
 
1. Histologically confirmed Ewing’s Sarcoma Family of Tumors (ESFT). 
2. Age ≥ 2 years. 
3. Estimated survival ≥ 6 months. 
4. Evidence of EWS translocation by FISH or RT-PCR or Next Generation Sequencing 
(NGS).  If available, NGS sequencing report should be submitted to Gradalis. 
5. Recurrence or refractory to 1 line of systemic chemotherapy including but not limited to 
doxorubicin, vincristine and ifosfamide. 
6. Planned standard of care surgical procedure (e.g., tumor biopsy or palliative resection or 
thoracentesis) and expected availability of a cumulative soft-tissue mass of ~10-30 
grams tissue (“grape” to “golf-ball” size / approximate 2 cm total diameter on imaging) 
and/or pleural fluid estimated volume ≥ 500mL (from a primary or secondary 
thoracentesis, yielding in a high volume of tumor cells) for immunotherapy manufacture. 
7. Tumor intended for immunotherapy manufacture is not embedded in bone and does not 
contain luminal tissue (e.g. bowel, ureter, bile duct). 
8. Ability to understand and the willingness to sign a written protocol specific informed 
consent for tissue harvest or a parental/guardian informed consent and pediatric assent 
when appropriate. 
 
6.3 Tissue Procurement Exclusion Criteria 
 
Subjects meeting any of the following criteria are NOT eligible for tissue procurement for the 
Vigil manufacturing: 
 
1. Medical condition requiring any form of chronic systemic immunosuppressive therapy 
(steroid or other) except physiologic replacement doses of hydrocortisone or equivalent 


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(no more than 30 mg hydrocortisone or 10 mg prednisone equivalent daily) for < 30 days 
duration. 
2. Known history of other malignancy unless having undergone curative intent therapy 
without evidence of that disease for ≥ 3 years except cutaneous squamous cell and 
basal cell skin cancer, superficial bladder cancer, in situ cervical cancer or other in situ 
cancers are allowed if definitively resected. 
3. Brain metastases unless treated with curative intent (gamma knife or surgical resection) 
and without evidence of progression for ≥ 2 months. 
4. Any documented history of autoimmune disease with exception of Type 1 diabetes on 
stable insulin regimen, hypothyroidism on stable dose of replacement thyroid 
medication, vitiligo, or asthma not requiring systemic steroids. 
5. Known HIV or chronic Hepatitis B or C infection. 
6. Known hypersensitivity to any temozolomide component or to dacarbazine (DTIC). 
7. Known hypersensitivity to irinotecan or its excipients. 
8. Known history of allergies or sensitivities to gentamicin. 
9. History of or current evidence of any condition (including medical, psychiatric or 
substance abuse disorder), therapy, or laboratory abnormality that might confound the 
results of the study, interfere with the patient’s participation for the full duration of the 
study, or is not in the best interest of the patient to participate, in the opinion of the 
treating Investigator. 
 
6.4 Study Enrollment Inclusion Criteria 
 
Subjects will be eligible for registration if they meet the tissue procurement inclusion criteria and 
all of the following study enrollment inclusion criteria: 
 
1. Completed manufacture of at least 4 vials of Vigil. 
2. Karnofsky performance status (KPS) / Lansky performance status (LS) ≥80%. 
3. Normal organ and marrow function as defined below: 
Absolute granulocyte count 
≥1,000/mm3 
Absolute lymphocyte count 
≥400/mm3 
Platelets 
≥75,000/mm3 
Hemoglobin 
≥8.0 mg/dL 
Total bilirubin 
≤ institutional upper limit of normal* 
AST(SGOT)/ALT(SGPT) 
≤2x institutional upper limit of normal 
Creatinine 
<1.5 mg/dL 
*documented Gilbert’s syndrome may be considered after medical monitor review 
4. Subject has recovered to CTCAE Grade 1 (except for parameters noted in Item 3, 
above) or better from all adverse events associated with prior therapy or surgery.  Pre-
existing motor, sensory neurologic pathology or symptoms, or dermatologic must be 
recovered to CTCAE Grade 2 or better. 
5. If female of childbearing potential, has a negative urine or serum pregnancy test.  If the 
urine test is positive or cannot be confirmed as negative, a negative serum test will be 
required for study entry. 
6. Ability to understand and the willingness to sign a written informed protocol specific 
consent or a parental/guardian informed consent and pediatric assent when appropriate. 
 
 


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6.5 Study Enrollment Exclusion Criteria 
 
In addition to the procurement exclusion criteria, subjects will NOT be eligible for study 
registration and randomization if meeting any of the following additional criteria: 
 
1. Any anti-neoplastic therapy between tissue procurement for Vigil manufacture and start 
of study therapy. 
2. Live vaccine used for the prevention of infectious disease administered < 30 days prior 
to the start of study therapy. 
3. Post-surgery complication that in the opinion of the treating investigator would interfere 
with the patient’s study participation or make it not in the best interest of the patient to 
participate. 
 
7.0 
WITHDRAWAL 
 
7.1 
Discontinuation from Study Treatment 
 
Treatment will be continued until progressive disease by RECIST 1.1.  However, insofar as 
some patients in our early vaccine trials had increased survival despite initial, short-term volume 
increase. There is accumulating evidence that some subjects treated with immune system 
stimulating agents may appear to demonstrate progression of disease by conventional response 
criteria before demonstrating clinical objective responses and/or stable disease and ultimately 
survival benefit. It may be that enhanced inflammation within tumors initially leads to an 
increase in tumor size or newly visible small lesions. Over time, both the malignant and 
inflammatory portions of the mass may then decrease leading to overt signs of clinical 
improvement.  
 
Therefore, selected subjects may be allowed to continue study therapy after investigator-
assessed RECIST 1.1 progression and with agreement by the Gradalis medical monitor if the 
subject is determined to be deriving clinical benefit, tolerating study drug and meeting the 
following criteria: 
 
1. Absence of symptoms and signs indicating clinically significant PD indicating disease 
progression. 
2. No decline in ECOG performance status. 
3. Absence of rapid progression of disease or of progressive tumor at critical anatomical 
sites (e.g., cord compression) requiring urgent alternative medical intervention. 
 
If subsequent imaging shows an objective response or stable disease relative to baseline, 
treatment with study agents will continue.  If subsequent imaging again shows PD, patients will 
be discontinued from study therapy. 


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Subjects will discontinue study treatment if any of the following occur: 
 
1. Disease progression by RECIST 1.1 criteria. 
2. The patient experiences unacceptable (≥Grade 3) toxicity felt to be related to treatment 
with the Vigil immunotherapy that persists for >3 weeks.  Subjects would be discontinued 
from both Vigil and chemotherapy. 
3. The subject experiences unacceptable (≥Grade 3) toxicity felt to be related to treatment 
with irinotecan/temozolomide that persists for >3 weeks. Vigil therapy may be continued 
in monotherapy. 
4. Persisting Grade 3 or 4 toxicity unrelated to treatment, defined as failing to normalize 
within 3 weeks. 
5. Any Grade ≥ 3 allergic reactions related to Vigil. 
6. Grade 2 autoimmune reactions unless there is evidence of clinical benefit. 
7. An intercurrent illness, which would in the judgment of the investigator, affects 
assessments of clinical status to a significant degree or requires discontinuation of study 
treatment. 
8. Cancer therapy other than protocol treatment. 
9. Non-compliant with protocol or treatment. 
10. Subject refuses to continue treatment. 
 
The date of and reason for discontinuation must be noted in the electronic Case Report Form 
(eCRF).  Every effort should be made to complete the appropriate assessments. 
 
7.2 
Treatment Delay 
 
• 
Treatment may be delayed no more than 3 weeks to allow recovery from toxicity. 
• 
If ≥ one 2-week delay due to disease or infection occurs, subject status must be 
reviewed by sponsor. 
• 
If subjects miss doses, the doses will be made up the following week and continue on a 
revised every 3-week schedule thereafter. 
 
If Vigil administration is delayed, then irinotecan and temozolomide should concurrently be 
delayed. 
 
8.0 
INVESTIGATIONAL PLAN 
 
8.1 
Subject Screening and Registration 
 
Written documentation of full, non-contingent IRB approval of the protocol and consent 
document must be on file before a subject can be registered.  Study participation begins once 
written informed consent is obtained.  Subjects will be assigned a study identification number 
upon scheduling of tissue procurement for Vigil manufacture. 


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No later than the date of surgery, a pre-registration packet will be submitted by the site to 
Vigil@gradalisinc.com for medical monitor review. 
 
One to eight weeks after tissue procurement has occurred eligibility will be reconfirmed by the 
study site.  The Site will register the subject by sending the registration packet to 
Vigil@gradalisinc.com for medical monitor review. 
 
Please allow 48 hours for subject registration as the medical monitor may review the subject’s 
source documents to ensure they meet the eligibility criteria.  Once confirmed, the site will be 
notified of the eligibility and randomization result. 
 
8.2 
Tumor Procurement 
 
Refer to the Study Reference Manual for instructions.  Surgery for tissue procurement for Vigil 
manufacture must be scheduled and approved in advance. 
 
The cumulative equivalent of a “grape” to “golf ball size” mass (~10-30 gm of prime tumor tissue 
/ approximate 2 cm total diameter on imaging) is optimal for Vigil manufacturing.  If surgeons 
have the option of collecting more tissue, more doses of Vigil may be prepared (up to 12 doses).  
Vigil manufacturing is rarely successful with small (<10 gm) tumor masses.  Lesions extending 
into lumen (e.g., bowel) or tumor embedded in bone cannot be processed. 
 
Once the procured tissue is received at Gradalis, Inc. samples will be processed for autologous 
Vigil manufacture. 
 
8.3 
Vigil Immunotherapy Manufacturing 
 
Gradalis, Inc. will manufacture vaccine from the procured subject tumors.  Gradalis, Inc. will 
release Vigil once all release criteria have been met and eligibility has been confirmed. 
 
No tissue or Vigil will be given to the participant or site apart from the outlined clinical protocol. 
 
Any excess tumor tissue, not used for Vigil manufacture will be used towards Vigil research and 
process development assays. 
 
8.4 
Study Treatment Administration 
 
Treatment will be administered on an outpatient basis. 


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8.4.1 Schedule, Dose and Administration 
 
Group A 
Drug 
Dose 
Schedule 
Temozolomide 
100 mg/m2 daily, oral 
Days 1 – 5, every 21 days 
Irinotecan 
50 mg/m2 daily, oral 
Days 1 – 5, every 21 days 
Vigil 
1.0 x 106 cells/injection, 
intradermal 
Day 15, every 21 days 
 
Cross-Over 
Subjects 
Drug 
Dose 
Schedule 
Vigil 
1.0 x 106 cells/injection, 
intradermal 
every 21 days 
 
Subjects will receive Vigil at 1.0 x 106 cells via intradermal injection every 3 weeks for a 
minimum of 4 administrations to a maximum of 12 administrations depending on quantity of 
Vigil manufactured from surgical specimens and so long as the patient is clinically stable and 
without disease progression. 
 
The sites of injection for Vigil will be rotated between the right and left upper arms.  If the 
ipsilateral axillary lymph nodes were radiated or surgically removed during prior therapy, 
alternative sites may be used. 
 
Group B 
Drug 
Dose 
Schedule 
Temozolomide 
100 mg/m2 daily, oral 
Days 1 – 5, every 21 days 
Irinotecan 
50 mg/m2 daily, oral 
Days 1 – 5, every 21 days 
 
Please contact the sponsor with any dose adjustment considerations. 
 
Irinotecan Preparation 
 
Order the injectable formulation of irinotecan from central supplier, Clinigen.  Draw up 1 cycle of 
(five doses) into provided oral syringes and dispense to the subject with instructions to 
refrigerate until administration.  Irinotecan may be mixed with cranberry-grape juice before 
administration to mask the bitter flavor and administered once daily on Days 1 through 5 of each 
3-week cycle.   
 
 


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Temozolomide Preparation 
 
Temozolomide capsules for 1 cycle will be obtained by the patient with administration 
instructions.  Temozolomide may be opened and mixed in apple sauce or juice if unable to 
swallow whole capsules.  Temozolomide is administered on Days 1 through 5 of each 3-week 
course and given at least 1 hour before irinotecan. 
 
Subjects should be instructed to complete a dosing diary for the administration of oral irinotecan 
and oral temozolomide. 
 
Premedications and post medications 
 
Vigil: EMLA® may be utilized at the injection site prior to Vigil administration.  Analgesics may 
be employed as necessary.  As noted above, systemic steroids or other immunosuppressants 
should be avoided due to immune inhibition activity. 
 
Temozolomide / irinotecan: Per package inserts and institutional standards.  A home supply of 
anti-emetic (e.g. ondansetron) is highly recommended to take before temozolomide and as 
needed.  Also, a patient supply of an anti-diarrheal medication (e.g. loperamide) should be 
available after irinotecan for use as needed.  
 
The patient should be educated with a study calendar indicating days to get chemotherapy 
and/or Vigil as well as supportive care medications and when Vigil is given.  
 
Vigil Immunotherapy Transfer 
 
All manufactured Vigil will be stored in the vapor phase of liquid nitrogen until ready for use.  
The site will contact Gradalis, Inc. when the study agent is needed for subject administration. 
 
Gradalis will complete a Drug Transfer and Administration Form to release the subject vaccine.  
The clinic will sign off on the form upon receipt of the administration. 
 
Please reference the Study Reference Manual for preparation and handling information. 
 
 


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Dose Modification for Vigil Toxicity 
 
If > Grade 2 toxicity by NCI Common Toxicity Criteria (excluding Grade 3 injection site 
reactions) develops related to study treatment the Vigil dose will be reduced by 50% and 
continued. 
 
Dose Modifications for temozolomide and irinotecan 
 
Dose modifications for temozolomide, and irinotecan may be necessary for subject safety and 
should be carried out in accordance with the Investigator’s standard of practice, the package 
inserts, and institutional standards.  Please contact the sponsor regarding any dose 
modifications. 
 
If irinotecan + temozolomide is administered beyond 12 cycles, it will be administered off study 
and no longer provided by Gradalis. 
 
If irinotecan + temozolomide is discontinued, Vigil may be administered every 3 weeks until all 
manufactured product is exhausted or when disease progression is noted. 
 
Study Regimen Delay 
 
8.5 
Concomitant Medications and Supportive Care 
 
The following medications and interventions, unless otherwise specified, are prohibited from the 
time of study screening until the End of Treatment visit:  
 
• 
Anti-cancer therapy, including chemotherapy, radiotherapy, or endocrine therapy other 
than those required per protocol.  
• 
Any investigational drug or device other than Vigil. 
• 
Systemic—oral, IV, injectable—corticosteroids (e.g., dexamethasone) should be avoided 
in subjects who are administered Vigil.  If deemed by the investigator to be necessary, 
short term (<30 days) systemic steroids ≤ 0.25 mg/kg (max 10mg) prednisone-equivalent 
per day and inhaled steroids are permitted while on protocol. Other steroid regimens 
and/or immunosuppressives are generally prohibited.  Contact the Gradalis medical 
monitor if other steroidal regimens are clinically needed. 
• 
Localized radiotherapy is permitted for palliation of painful lesions at the investigator’s 
discretion. However, medical management in place of radiation therapy should be used, 
if clinically appropriate.Inactivated vaccines are allowed on study, if deemed appropriate 
by the investigator. Live vaccines are prohibited while on study.  
• 
 Valproic Acid (due to temozolomide) 
• 
Strong CYP34A inducers and inhibitors (due to irinotecan) 


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Subjects should be provided with full supportive care measures, as clinically indicated, and in 
accordance with institutional standards. Such care includes medication for pain control and 
symptom management, antibiotics, bisphosphonates, antiemetics, colony stimulating factors, 
and transfusions of blood or blood products.  Prophylactic GCSF is allowed for both treatment 
arms, if indicated. Treatment for drug-related adverse events should be administered at the 
discretion of the investigator. 
 
Peg-filgrastim may be administered after completion of Day 5 temozolomide to facilitate ANC 
recovery. 
 
8.6 
Contraception 
 
Female subjects of childbearing potential must consent to use a medically acceptable method of 
highly effective contraception (oral hormonal contraceptive, condom plus spermicide, or 
hormone implants) throughout the study period and for 28 days after their final autologous Vigil 
administration. A method of contraception must be employed by all subjects (male and female). 
 
8.7 
Toxicity 
 
Toxicities will be graded and reported according to the NCI Common Toxicity Criteria for 
Adverse Events (CTCAE) Version 5. This document can also be downloaded from the Cancer 
Therapy Evaluation Program (CTEP) home page <http://ctep.cancer.gov>. 
 
Adverse events will be summarized using the MedDRA coding system or higher.  The NCI-
CTCAE will be used for AE grading.  All AEs, regardless of severity, will be followed by the 
Treating Physician until resolution is satisfactory. 
 
9.0 
SCHEDULE OF ASSESSMENTS 
 
Pre-study Assessments / Surgery Pre-Screening 
 
The following evaluations will be performed within 4 weeks of tumor procurement (unless 
otherwise specified): 
 
1. A signed Informed Consent Form for tissue harvest must be obtained. 
2. It has been confirmed that the subject meets all tissue procurement inclusion criteria and 
none of the exclusion criteria. 
3. A complete medical history must be obtained. 
4. A physical examination must be obtained. 


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5. A complete blood count (CBC) with differential and platelet count must be performed.  
(HIV and hepatitis testing is not required if the subject has no medical history of HIV). 
6. Routine pre-operative serum chemistries (including, but not limited to creatinine, total 
bilirubin, alkaline phosphatase, and aspartate transaminase (AST) and/or alanine 
transaminase (ALT). 
7. Blood collection for immune function analysis will be obtained at tissue harvest.  (≤1 
week of tissue procurement) 
8. Plasma collection for immune function analysis (ctDNA) (≤1 week of tissue procurement) 
 
Screening Assessments / Protocol Screening 
 
The following evaluations will be performed on all subjects within 2 weeks of Cycle 1 (unless 
otherwise specified): 
 
1. A signed protocol specific Informed Consent Form must be obtained. 
2. It has been confirmed that the subject meets all inclusion criteria and none of the 
exclusion criteria. 
3. A physical examination (including vital signs, height, temperature and body weight) must 
be obtained. 
4. Assessment of concomitant medications 
5. Assessment of PS on the Karnofsky or Lansky scale (see Appendix A) must be 
obtained. 
6. Radiological assessment of disease status with computed tomography (CT) chest 
/abdomen/pelvis (magnetic resonance imaging (MRI) abd/pelvis may be substituted for 
CT abd/pelvis) within 4 weeks of Cycle 1. The methods used for prestudy assessments 
(e.g., CT or MRI) should be used throughout the study. If possible, the same equipment 
should be used each time. Radiographic assessments must be from after the tissue 
harvest for vaccine manufacture to ensure true baseline disease status is captured.  
Images must be uploaded into WorldPro or submitted to WorldCare. 
7. A complete blood count (CBC) with differential and platelet count must be performed.  
(HIV and hepatitis testing is not required if the subject has no medical history of HIV). 
8. Serum chemistries (creatinine, glucose, total protein, blood urea nitrogen (BUN), total 
carbon dioxide (CO2), albumin, total bilirubin, alkaline phosphatase, and aspartate 
transaminase (AST) and/or alanine transaminase (ALT)) and electrolytes (total calcium, 
chloride, potassium, sodium) must be performed. 
9. Blood collection for immune function analysis (ELISPOT). 
10. Plasma collection for immune function analysis (ctDNA). 
11. Pregnancy test for those of childbearing potential. 
12. Submission to Gradalis, Inc. of preferably 20 unstained tumor slides or tissue block for 
correlative immunohistochemistry assay.  These slides should correlate with tumor 
procured for vaccine manufacture. 
 
 


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Assessments During Treatment 
 
The following evaluations will be performed on Day 1 of each cycle (1 Cycle = 21 days ±3 days) 
(unless otherwise specified): 
 
1. *A physical examination, including vital signs and body weight. 
2. *A toxicity assessment (CTCAE v 5). 
3. *Assessment of concomitant medications. 
4. Radiological assessment of disease status with CT chest /abd/pelvis (MRI abd/pelvis 
may be substituted for CT abd/pelvis) every 6 weeks ± 7 days from Cycle 1 regardless of 
treatment arm assignment or treatment delay.  The methods used for prestudy 
assessments (e.g., CT or MRI) should be used throughout the study. If possible, the 
same equipment should be used each time. 
5. *A CBC with differential and platelet count. 
6. *Serum chemistries (creatinine, glucose, total protein, blood urea nitrogen (BUN), total 
carbon dioxide (CO2), albumin, total bilirubin, alkaline phosphatase, and aspartate 
transaminase (AST) and/or alanine transaminase (ALT)) and electrolytes (total calcium, 
chloride, potassium, sodium). 
7. Blood collection for immune function analysis (ELISPOT) on Day 1 (prior to 
chemotherapy administration) at Cycles 2, 4, and 6. 
8. Plasma collection for immune function analysis (ctDNA) on Day 1 prior to chemotherapy 
administration at Cycles 2, 3, 4 and 6. 
9. *Assessment of Karnofsky Performance Status or LS. 
10. Group A and B participants: oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 
500 mg/m2/cycle) 
11. Group A and B participants: oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 
250mg/m2/cycle) 
12. Group A participants: Vigil 1.0 x 106 cells/injection, intradermally on Day 15. 
13. Cross-over Subjects: Vigil 1.0 x 106 cells/injection, intradermally every 21 days 
14. For those randomized to Group A or in cross-over, Vigil administration.  Day 16 post 
Vigil administration assessment of injection site.  Instruct the subject on Day 15 to 
observe the injection site 24 hours after the product administration.  (This may be 
conducted at home).  Have the subject follow up with the clinic if an injection site 
reaction is present.  Please note if the subject reports of any redness, swelling or other 
response.  If the subject identifies any redness, and /or swelling, please ask the subject 
if it is feasible to take a photograph of the injection site.  If photographing the injection 
site is possible, please instruct the subject to place a measuring tool (bulk supplies 
provided to the site) next to the injection site reaction in the picture, if feasible.  The 
photograph may be provided to the clinic upon the next visit as a hard copy or electronic 
jpg image.  Please instruct the participant to avoid capturing images that would identify 
the subject (i.e. face and head). 
15. Submission of Next Generation Sequencing data, if available in medical records. 
 
Subjects who are stable at the end of Cycle 4, with prior approval by the Sponsor may conduct 
evaluations denoted with an asterisk* on Day 15 of each cycle (1 Cycle = 21 days ±3 days) 
(unless otherwise specified). 


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Subjects who become child-bearing while on study will have a pregnancy test performed prior to 
their next dose.  See Section 8.6. 
 
End of Treatment Assessments 
 
The following evaluations will be performed within 30 days after completion of the study regimen 
and / or within 30 days of disease progression (whichever event occurs first) (unless otherwise 
specified): 
 
1. A physical examination, including vital signs and body weight. 
2. Toxicity assessment (adverse events). 
3. Assessment of concomitant medications taken. 
4. Assessment of Karnofsky Performance Status/Lansky Performance Status 
5. Radiological assessment of disease status with CT chest /abd/pelvis (MRI abd/pelvis 
may be substituted for CT abd/pelvis) within 45 days of the last injection or disease 
recurrence or progression 
6. A CBC with differential and platelet count. 
7. Serum chemistries (creatinine, glucose, total protein, blood urea nitrogen (BUN), total 
carbon dioxide (CO2), albumin, total bilirubin, alkaline phosphatase, and aspartate 
transaminase (AST) and/or alanine transaminase (ALT)) and electrolytes (total calcium, 
chloride, potassium, sodium). 
8. Blood (PBMC) for immune function analysis (ELISPOT). 
9. Plasma for immune function analysis (ctDNA). 
 
Response Follow Up Assessments 
 
If the study agent is discontinued (for reasons such as completion of all available doses of 
vaccine, intolerable toxicity, treatment interruption of more than 4 weeks, intercurrent illness, 
protocol deviation, at PI’s discretion), the subject will be followed quarterly after the end of study 
visit until progression. 
 
The following evaluations will be performed quarterly (every 3 months ±7 days) (unless 
otherwise specified): 
 
1. A physical examination, including vital signs and body weight. 
2. Assessment of concomitant medications taken. 
3. Radiological assessment of disease status with CT chest /abd/pelvis (MRI abd/pelvis 
may be substituted for CT abd/pelvis) must be collected.  The methods used for 
prestudy assessments (e.g., CT or MRI) should be used throughout the study. If 
possible, the same equipment should be used each time. 
4. A CBC with differential and platelet count. 


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5. Serum chemistries (creatinine, glucose, total protein, blood urea nitrogen (BUN), total 
carbon dioxide (CO2), albumin, total bilirubin, alkaline phosphatase, and aspartate 
transaminase (AST) and/or alanine transaminase (ALT)) and electrolytes (total calcium, 
chloride, potassium, sodium). 
6. Blood collection for immune function analysis (ELISPOT) 3 months ±7 days from EOT 
and every 6 months ±7 days thereafter. 
7. Assessment of Performance Status. 
 
Long Term Follow Up 
 
After progression, subjects and their physicians will be contacted quarterly for documentation of 
post study therapies and survival status. 
 
As the intent-to-treat population includes all randomized patients, should a subject be 
randomized and choose to not receive any treatment on this study, the subject should be asked 
if he or she is willing to allow for survival follow up through phone call and/or physician contact 
quarterly. This follow up would include any anti-cancer therapies received and survival status 
information.  
 
Based on findings during the study or during the follow up portion of the trial, Gradalis may 
request for additional blood and / or tissue samples from the research participant.  Collection of 
whole blood (40ml) and / or tissue samples (via biopsy or clinically indicated surgical removal) 
will be optional and used to study the effects of the study agent (included, but not limited to 
testing of biomarkers, predictors or biological responses, toxicity, relationship between genotype 
and study agent responses). 
 
Should Gradalis request for additional blood or tissue and the subject consented to the 
collection, the clinical site will present the option of the procurement to the participant. 
 
10.0 
CROSS-OVER 
 
Subjects randomized to Group B may cross-over to Group A after discussion with, and approval 
from the Gradalis medical monitor or authorized designee. 
 
Within 6 weeks of second relapse or progression and if alternative therapy has not been given 
since EOT, subjects randomized to Group B, will be allowed to cross-over to receive single 
agent Vigil every 21 days following End of Treatment assessment.  Subjects who cross-over 
may receive up to 12 doses of Vigil depending upon the quantity of Vigil manufactured.  After 
the Group B EOT assessment, the subject will start “cross-over” from Assessments During 
Treatment onward.  Vigil and all assessments will occur on Day 1 of each cycle. 
 


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Cross-over must occur within 2 years of End of Treatment assessments of Group B enrollment.  
Subjects who progress ≥ 8 weeks post EOT assessment must re-screen and if they qualify will 
start “cross-over” from Assessments During Treatment onward.  Eligibility criteria for cross-over 
subjects are listed below. 
 
10.1 Inclusion Criteria for Cross-Over 
 
Subjects will be eligible for cross-over, if they meet all of the following criteria: 
 
1. Evidence of radiologic disease progression by RECIST 1.1 after enrollment into Group 
B. 
2. Successful manufacturing of at least 4 vials of Vigil. 
3. Karnofsky performance status (KPS) / Lansky performance status (LS) ≥70%. 
4. Normal organ and marrow function as defined below: 
Absolute granulocyte count 
≥1,000/mm3 
Absolute lymphocyte count 
≥400/mm3 
Platelets 
≥75,000/mm3 
Total bilirubin 
≤ institutional upper limit of normal 
AST(SGOT)/ALT(SGPT) 
≤2x institutional upper limit of normal 
Creatinine 
<1.5 mg/dL 
*documented Gilbert’s syndrome may be considered after medical monitor review 
5. Subject has recovered to CTCAE Grade 1 (except for parameters noted in Item 4, 
above) or better from all adverse events associated with prior therapy or surgery.  Pre-
existing motor, sensory neurologic pathology or symptoms, or dermatologic toxicities 
must be recovered to CTCAE Grade 2 or better. 
6. If female of childbearing potential, has a negative urine or serum pregnancy test.  If the 
urine test is positive or cannot be confirmed as negative, a negative serum test will be 
required for study entry. 
7. Ability to understand and the willingness to sign a written informed protocol specific 
consent or a parental/guardian informed consent and pediatric assent when appropriate. 
 
10.2 Exclusion Criteria for Cross-Over 
 
In addition to the Procurement and Study Enrollment exclusion criteria, Subjects will NOT be 
eligible for cross-over if meeting any of the following criteria: 
 
1. With the exception of irinotecan and temozolomide while on study, any anti-neoplastic 
therapy between tissue procurement for Vigil manufacture and start of study therapy. 
2. Live vaccine used for the prevention of infectious disease administered < 30 days prior 
to the start of study therapy. 
 
 


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11.0 
CONDUCT OF THE STUDY 
 
11.1 Ethics and Regulatory Considerations 
 
This study must have the approval of a properly constituted Institutional Review Board (IRB) or 
Independent Ethics Committee (IEC).  Before the investigational drug is shipped to the 
investigator, the investigator will provide Gradalis, Inc. with a copy of the IRB or IEC approval 
letter stating that the study protocol and informed consent form have been reviewed and 
approved. 
 
11.2 IRB 
 
This trial can be undertaken only after review and full approval of the protocol and an Informed 
Consent Form has been obtained from a properly constituted IRB.  This written approval must 
be dated and it must clearly identify the protocol, any amendments, the Informed Consent Form, 
and any applicable recruiting materials and subject compensation programs approved. 
 
The decision concerning the conduct of the study will be made in writing to the sponsor.  Copies 
of this decision and of all IRB correspondence will be kept on file at the study site; copies will be 
provided to the Sponsor Office. 
 
During the trial, the PI is required to send various documents to the IRB for review: 
 
• 
All protocol amendments and Informed Consent Form revisions. 
• 
Reports of Serious Adverse Events. 
 
The PI provides Gradalis, Inc. with the necessary assurance that an IRB is responsible for the 
initial and continuing review and approval of the proposed clinical study in accordance with 21 
CFR 312.60.  At least once a year, the IRB will be asked to review and re-approve the clinical 
trial protocol; the request must be documented in writing.  At the end of the trial, the PI will notify 
the IRB that the trial has been completed. 
 
11.3 Written Informed Consent 
 
The informed consent document should meet the requirements of the latest version of the 
Declaration of Helsinki and any applicable regulations and guidelines.  It must be approved by 
an IRB or IEC. 
 


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Prior to entry into the trial and before any protocol-required procedures are performed, the 
Investigator must explain the nature of the trial, its intended purpose, and the implications of 
participation to potential subjects or to their legal representatives.  They will be told about the 
possible risks and benefits, and the possible adverse experiences.  They will be informed that 
subjects’ participation is voluntary, and that they may withdraw consent to participate at any 
time.  They will also be informed that if subjects choose not to participate in the trial alternative 
treatments are available; such refusal will not prejudice further treatment of their disease.  
Potential subjects or their legal representatives must be given the opportunity to ask questions 
about the trial protocol and the procedures involved. 
 
Finally, each subject will be told that his or her records may be accessed by authorized 
personnel of Gradalis, Inc. and other authorized individuals without violating the subject’s 
confidentiality, to the extent permitted by the applicable laws and/or regulations.  By signing the 
written Informed Consent Form, the subject or his or her legal representative is authorizing such 
access.  Following this explanation and prior to entry into the trial, the written, dated, and signed 
Informed Consent Form must be obtained from each subject or his or her legal representative; a 
copy will be given to the person signing the form. 
 
11.4 Confidentiality of Records 
 
The Investigator is required to retain, in a confidential manner, sufficient information on each 
subject (i.e., full name, current address, and social security number) so that the subject may be 
contacted by the FDA, Gradalis, Inc., or by their affiliates should the need arise. 
 
11.5 Modification of Protocol 
 
Any changes to this protocol that affect study objectives, study design, study procedures, 
patient population, or significant administrative procedures will require a formal amendment to 
the protocol.  Any proposed protocol amendments must be sent in writing to the applicable IRB.  
Prior to implementation, an amendment must be approved by the Gradalis, Inc., and approved 
by the applicable IRB or IEC. 
 
General administrative changes to the protocol are minor corrections and/or clarifications that 
do not affect the manner in which the study is to be conducted. Such administrative changes will 
be agreed upon by the Gradalis, Inc., and will be documented in a memorandum.  The 
applicable IRB or IEC will be notified of administrative changes according to applicable IRB 
guidelines. 
 
11.6 Protocol Questions and Deviations 
 
When evaluating a potential patient or while a patient is on study, protocol questions can be 
directed to Gradalis via email or phone using the contact information provided in the Contact 
Information section of the Study Reference Manual. 


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11.7 Data Safety Monitoring Board 
 
An independent data monitoring committee (IDMC) or Data Safety Monitoring Board (DSMB) 
will be established for this trial.  Membership will include physicians with appropriate areas of 
expertise in the therapeutic areas of most interest.  A charter will be developed to document 
their operational methods and timing of safety reviews. 
 
12.0 
EVALUATION OF TUMORS 
 
12.1 Tumor Measurements and Response (RECIST 1.1) 
 
Response and progression will also be evaluated in this study using the Response Evaluation 
Criteria in Solid Tumors (RECIST) Version 1.1.  The guidelines are available online at: 
European Organization for Research and Treatment of Cancer (EORTC) RECIST Web page, 
https://www.eortc.be/Recist/documents/RECISTGuidelines.pdf. 
 
Radiological images should be submitted to WorldCare upon receipt from the imaging facility for 
third party review. 
 
13.0 
DRUG INFORMATION 
 
13.1 Vigil Investigational Product 
 
Vigil is made up of irradiated autologous tumor cells which have been electroporated ex vivo 
with the Vigil plasmid designed to suppress expression of both the TGFβ1 and TGFβ2 proteins 
while simultaneously expressing rhGMCSF protein. 
 
Vigil Production 
 
The Vigil manufacturing process is identical to prior Vigil manufacturing (BB-IND 14205) 
(Maples 2010).  Surgically excised tumor is collected in the surgical field and placed in 
0.04mg/ml of gentamicin and sterile saline then packaged for transport to the manufacturing 
facility.  The tumor is mechanically and enzymatically dissociated into a single cell suspension.  
The cells are counted and then transfected with the Vigil plasmid.  The cells are incubated 
overnight to allow transcription of the Vigil plasmid.  The following morning the cells are 
harvested, washed, and then irradiated at 10,000cGy in a standard Blood Bank irradiator.  The 
irradiated cell suspension is then enumerated, aliquoted and frozen (1 x 106 cells).  The freeze 
media consists of 10% DMSO (dimethyl sulfoxide; Cryoserv USP; Bionichepharma US), 1% 
Human Serum Albumin (ABO Pharmaceuticals) in Plasma-Lyte A at pH 7.4 (Baxter).  After 


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freezing the cells are stored in the vapor phase of liquid nitrogen until all release testing is 
completed, all necessary approvals are obtained and the patient is ready for treatment. 
 
Safety Analysis 
 
Vigil plasmid employed in the generation of this product has been tested for identity, sterility, 
purity and strength. 
 
Irradiated Gene Modified Tumor Cells 
 
To ensure safety, all gene-modified tumor cells to be used in Vigil administrations must 
be irradiated 10,000 cGy prior to freezing.  This is the same irradiation process as for 
the TAG vaccine, BB-IND 13650 and prior vaccines (Belagenpumatucel-L and GVAX® 
published trial results and BB-IND 13401 and BB-IND 12118) (Kumar 2009, Maples PB 
2009, Maples PB 2009).  The selection of this radiation dose is based on the desire to 
utilize the lowest possible radiation dose for the transfected cells to optimize the level 
and duration of bifunctional shRNAfurin transcription and GMCSF protein production and 
maximize the safety of vaccine cell injections at the same time.  In addition, 
investigators have demonstrated that irradiating cultured tumor cells of different 
histologic origins at 10,000 cGy completely arrests tumor colony formation. 
 
Preparation 
 
Reference the Study Reference Manual for preparation and handling information. 
 
Vigil concentrate:  
1.0 x 106 cells per injection in a volume of 1mL. 
 
Route of administration:  
Intradermal injection 
 
Storage and Shipping 
 
Frozen, unopened vials are stored in the vapor phase of Liquid Nitrogen below -150° C at 
Gradalis.  Each Vigil concentrate will be shipped individually in a portable liquid nitrogen tank.  
This shipping container will be able to sustain temperature fluctuations for up to 7 days.  This 
will enable sufficient time to reach the different clinical site pharmacies. 
 
Study medications are not expected to pose significant occupational safety risks to 
investigational staff under normal conditions of use and administration.  However, precautions 


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should be taken to avoid direct contact with study medication.  Biosafety Level 1 practices shall 
be employed with this study medication.  Reference the Study Reference Manual. 
 
14.0 ADVERSE EVENTS 
 
14.1 Adverse Event and Serious Adverse Event Definitions 
 
Adverse Event 
 
An AE is any untoward medical occurrence in a patient or clinical investigation subject 
administered a pharmaceutical product and that does not necessarily have a causal relationship 
with this treatment.  An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the use of a 
medicinal (investigational) product, whether or not related to the medicinal (investigational) 
product. 
 
Serious Adverse Event 
 
An AE (experience) or reaction occurring at any dose should be classified as a serious adverse 
event (SAE) if any of the following occur: 
 
• 
Initial or prolonged hospitalization (≥ 24 hours). This does not include hospitalizations 
which are part of routine medical practice.  
• 
A life-threatening condition (i.e. an event in which the patient was at risk of death at the 
time of the event; it does not refer to an event which hypothetically might have caused 
death if it was more severe) 
• 
Significant disability/incapacity (i.e. the AE resulted in a substantial disruption of the 
subject’s ability to carry out normal life functions) 
• 
Congenital anomaly/birth defect 
• 
It does not meet any of the above serious criteria, but may jeopardize the subject or may 
require surgical or medical intervention to prevent one of the outcomes listed above. 
• 
Death 
 
Unexpected Adverse Event 
 
An unexpected event is any AE that is not identified in nature, severity or frequency in the 
clinical Investigator’s brochure or the drug package insert. 
 
 


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Grading Adverse Events 
 
Adverse events (AEs) will be recorded throughout the trial.  Toxicities and AEs will be graded 
and reported using the Common Toxicity Criteria for Adverse Events (CTCAE) Version 5.  All 
AEs, regardless of severity, will be followed by the Treating Physician until resolution is 
satisfactory. 
 
14.2 Attribution of Causality 
 
The relationship of each event to treatment will be assessed by the Treating Physician and 
recorded on the CRF in the EDC. 
 
14.3 Vigil Expected Side Effects 
 
Tumor cell vaccines have been previously administered to patients with cancer.  Side effects 
were minimal, the most frequent of which included local reactions at the site of injection.  
Potential adverse events are listed below. 
 
Local skin reactions at the site of injection: 
 
Erythema, tenderness, induration, urticaria/rash, pruritus. 
 
Other expected adverse events: 
 
Fever, myalgias/arthralgias, chills/rigors, nausea, fatigue, headache, thrombocytopenia and 
other cytopenias, hyperglycemia, vomiting, hypotension, infection at the immunization site. 
 
In addition, there may also be a risk of autoimmune disease development, although to date no 
evidence of this has been seen in any vaccination study.  There may also be worsening of 
tumor related symptoms secondary to immune mediated attack on patient’s tumor. 
 
14.4 Recording of an Adverse Event 
 
Adverse events will be recorded for the duration of a subject’s time on study: following 
procurement of the subject’s tissue for tissue manufacture and for up to 30 days following the 
last administration of Vigil.  All AEs, regardless of causal relationship are to be recorded in the 
eCRF and source documentation.  Additional information about each event, such as treatment 


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required, eventual outcome, and whether or not therapy had to be interrupted or dosages 
reduced, will also be recorded on the eCRF. 
 
Pre-existing conditions will be recorded at baseline on the Medical History Form. If a pre-
existing condition does not change, it does not have to be reported as an AE on subsequent 
cycles. 
 
14.5 Serious Adverse Event Reporting 
 
All SAEs will be reported to Gradalis, Inc. within 24 hours of notification by the site through 
email or facsimile.  This includes any death from any cause following procurement of the 
subject’s tissue for tissue manufacture and for up to 30 days following the last dose of the 
protocol study agent (Vigil). 
 
The site will supply as much information as is available at the time of the initial notification 
(study number, patient initials, patient study number, onset date, relationship, patient 
demographics, event, dosing regimen of study agent) to: 
 
Gradalis, Inc. 
2545 Golden Bear Drive, Suite 110 
Carrollton, TX  75006 
Vigil@gradalisinc.com 
Direct: (214) 442-8124 
Fax: (214) 442-8101 
 
Gradalis, Inc. will report adverse events to the FDA in compliance with 21 CFR 312.32. 
 
15.0 
PATIENT COMPLETION AND WITHDRAWAL 
 
15.1 Indication for Taking Patients Off Study 
 
The Investigator must notify the sponsor at any time following discontinuation of a patient on 
study for the occurrence of a serious or unexpected AE associated with the use of the study 
medication. 
 
 


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16.0 
STATISTICAL CONSIDERATIONS 
 
16.1 Sample Size Justification 
 
This is an open label randomized controlled Phase III clinical trial.  Information concerning the 
predicted survival of the control group is limited, based on rarity of disease and few published 
reports.  However expert advisors in the EWS field estimate a conservative one-year survival 
rate of 25% in the chemotherapy control group.  The one-year survival rate of 60% in the Vigil 
treated group is estimated from EWS patients treated on the Vigil phase 1 protocol.  These 
estimates correspond to a hazard ratio (HR) of 0.383 favoring Vigil over control. 
 
Assuming 1:1 randomization and the use of a two-sided log rank test at the alpha=0.0476 level 
of significance, 67 events will provide 90% power to detect an PFS HR of 0.45.  Assuming a 
Group B (control group) median PFS of 6 months, a one-year accrual period, and a six-month 
follow-up period after randomization of the last subject, it is estimated that the total sample size 
(number of subjects) required to achieve 67 events is 114 (57 patients in each arm). 
 
An unblinded interim analysis will be conducted by an independent data monitoring committee 
(IDMC) when 40 PFS events have occurred.  Based on the results of this interim analysis, the 
planned sample size may be increased to 120 events.  If the sample size is increased to 120 
events, then the study will have approximately 90% power to detect a true HR of 0.55.  Note 
that if the sample size is increased to 120 events, then the accrual period will be extended to 24 
months, with a 6-month follow-up period after randomization of the last patient.  Under these 
assumptions, it is estimated that approximately 164 patients will need to be randomized in order 
to achieve 120 events. Section 16.6 provides further details. 
 
16.2 Definition of Progression Free Survival (PFS) 
 
• 
Progression free survival (PFS) is defined as the time from randomization to the event of 
disease recurrence/progression or death due to any cause.  
• 
Radiographic disease recurrence/progression will be assessed using the RECIST 1.1 
criteria.  If the disease recurrence/progression assessment involves more than one date, 
the earliest date will be used as the event date.  
• 
Patients who are alive and recurrence free at the time of analysis data cut-off, or who 
terminate the study for reasons other than disease progression or death, or who receive 
non-protocol treatment for Ewing’s Sarcoma, will be censored on the last assessment 
date at which a RECIST 1.1 evaluation of disease status was made.  
• 
Patients missing baseline disease assessment will be censored at date of 
randomization. 
• 
For equivocal findings of recurrence (e.g., very small and uncertain new lesions; cystic 
changes or necrosis in existing lesions), treatment may continue until the next scheduled 
assessment.  If recurrence is confirmed at the next scheduled assessment, the date of 
recurrence should be the earlier date when recurrence was suspected. 


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16.3 Analysis Populations 
 
The intent-to-treat (ITT) population will include all randomized subjects.  All efficacy analyses 
will be completed in the ITT population. 
 
The Safety population will include all patients who receive study treatment and patients will be 
analyzed according to actual treatment received.  All safety analyses will be completed in the 
Safety population. 
 
16.4 Efficacy Analyses 
 
16.4.1 Primary Efficacy Analysis 
 
The primary endpoint of PFS is the time from randomization to progression according to 
RECIST version 1.1 or death.  The distributions of PFS in the two arms will be compared using 
a two-sided log-rank test at the alpha=0.0476 level of significance 
 
16.4.2 Secondary Efficacy Analyses 
 
A fixed sequence testing procedure will be used to control the overall level of significance for the 
analysis of the secondary endpoints of OS and ORR.  If the primary analysis of PFS is 
statistically significant (p<0.0476), then OS will be analyzed using a two-sided test at the 
alpha=0.0476 level of significance.  In addition, if the analysis of OS is statistically significant 
(p<00476), then ORR will be analyzed using a two-sided test at the alpha=0.0476 level of 
significance.  However, if an earlier analysis is not statistically significant, the remaining 
analyses will be exploratory rather than confirmatory. 
 
The secondary endpoint of OS is defined as time from randomization to death or to the date of 
last follow-up.  The date of last follow-up confirming survival will be used as the censoring date 
for subjects who are alive and/or do not have a known date of death.  The analysis of OS will be 
conducted using the two-sided log-rank test.  Kaplan-Meier OS curves will be displayed by 
treatment arm.  Median OS and percent OS at fixed time points will be estimated.  
 
The secondary endpoint of ORR will be analyzed using Pearson’s chi-square test.  Overall 
response rate (ORR) is defined as the proportion of patients who have a partial or complete 
response to therapy according to RECIST 1.1.  ORR will be reviewed 6 months after treatment 
with Vigil. 
 
The analyses of all other secondary endpoints will be conducted using two-sided tests at the 
alpha=0.05 level of significance. 


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16.4.3 Sensitivity Analyses of the Primary Endpoint 
 
Five sensitivity analyses of the primary endpoint will be conducted using each of the following 
modifications to the definition of PFS: 
 
• 
If the disease recurrence/progression assessment involves more than one date, the 
latest date will be used as the event date. 
• 
If disease recurrence or death occurs right after missing data for a scheduled 
radiographic disease assessment (including missing the assessment or assessment 
results in an unevaluable status for overall response per RECIST 1.1), the patient will be 
censored at the date of the last radiographic disease assessment. 
• 
If a patient receives non-protocol treatment for Ewing’s Sarcoma, the patient will be 
treated as having progression at the date of the last radiographic disease assessment. 
• 
Patients missing baseline disease assessment will be treating as had having 
progression at the date of randomization. 
• 
For equivocal findings of recurrence (e.g., very small and uncertain new lesions; cystic 
changes or necrosis in existing lesions) for whom treatment continues until the next 
scheduled assessment, and if recurrence is confirmed at the next scheduled 
assessment, the date of recurrence should be the date when recurrence was confirmed. 
 
16.4.4 Sensitivity Analysis of Overall Survival 
 
A sensitivity analysis of OS will be conducted in which subjects who are crossed over to Vigil 
therapy will be censored at the time of crossover. 
 
16.4.5 Subgroup Analyses of the Primary (PFS) and Key Secondary Endpoints 
(OS, ORR) 
 
Subgroup analyses will be conducted in the following subsets of the ITT population: 
 
• 
Race / ethnicity 
• 
Study site / region 
• 
Age group (<15 years (low), ≥15 years (high)) 
• 
Gender 
• 
Number of doses received (≤median number of doses administered, >median number of 
doses administered) 
• 
Number of lines of prior treatments including chemotherapy and radiation 
• 
At time of diagnosis: localized vs. metastatic disease 
• 
At time of diagnosis: skeletal vs. extraskeletal 
• 
At time of diagnosis: tumor volume/burden (<100ml or 8cm vs >100ml or 8cm) 
• 
Response to frontline therapy (CR vs. PR vs. SD vs. progressive disease) 
• 
Time to first recurrence: <2 years, > 2 years 
 


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16.5 Safety Analyses 
 
Safety endpoints include all adverse events (CTCAE 5), laboratory safety assessments, and 
physical examination findings. 
 
16.6 Interim Analysis 
 
An interim analysis for efficacy, futility, and potential sample size re-estimation will be conducted 
by an independent data monitoring committee (IDMC) when 40 PFS events have been achieved 
(60% information fraction).  Based on the Lan-DeMets method for group sequential trials using 
O’Brien-Fleming boundaries (Reboussin, DeMets et al. 2000), the levels of significance for the 
interim and final analyses are alpha=0.00762 and alpha=0.0476, respectively. 
 
Based on the results of the interim analysis, the IDMC will make one of the following four 
recommendations to the sponsor: 
 
• 
Terminate the study for efficacy (p<0.00762 at the interim analysis); 
• 
Continue the study as planned; 
• 
Increase the sample size to 120 events; 
• 
Terminate the study for futility. 
 
Provided that the IDMC does not recommend terminating the study early for efficacy, the 
conditional power of the planned final analysis at 67 events will be computed using the 
methodology of (Jennison and Turnbull 2000) under the assumption that the observed hazard 
ratio at the interim analysis represents the true effect.  In terms of the HR observed at the 
interim analysis, these rules can be translated (approximately) as follows: 
 
• 
If the HR is less than 0.43, terminate for efficacy 
• 
If the HR is in the range from 0.43 to 0.51, continue as planned. 
• 
If the HR is in the range from 0.51 to 0.61 (i.e., conditional power in the range from 50% 
to 80%), increase the sample size to 120 events. 
• 
If the HR is greater than 0.61 (i.e., conditional power less than 50%), terminate for 
futility. 
 
17.0 
DOCUMENTATION 
 
A log of all patients evaluated for this protocol must be maintained at each site.  Patients 
excluded from admission will be provided with a clear explanation of the specific reasons why 
they have been excluded from the study.  Patients who are included will be assigned a patient 
identification number. 


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For each patient treated with the study drug(s), the Investigator or their designee is required to 
prepare and maintain case histories that include all observations and other data pertinent to the 
investigation.  This will include all source documents needed to verify the accuracy of all 
observations and other data contained in the eCRFs on each study patient. 
 
The Investigator or his/her designee is required to retain the records related to the trial for a 
period of 2 years following the date a marketing application is approved for the indication being 
investigated.  If no application is to be filed or if the application is not approved for such 
indication, the records must be retained until 2 years after the investigation is discontinued and 
the regulatory agencies are notified. 
 
The Investigator shall retain study drug disposition records and source documents for the 
maximum period required by the country and institution in which the study will be conducted, or 
for the period specified by Gradalis, whichever is longer.  The Investigator must contact 
Gradalis, Inc. prior to destroying any records associated with the study. 
 
If the Investigator withdraws from the study (e.g., relocation, retirement), the records shall be 
transferred to a mutually agreed upon designee (e.g., another investigator, IRB).  Notice of such 
transfer will be given in writing to Gradalis, Inc. 
 
17.1 Case Report Form (CRF) Procedures 
 
Data for this study will be captured in the EDC.  The investigator or his/her designee is 
responsible for recording all data relating to the trial on the eCRFs in accordance with the site’s 
contract with Gradalis.  The investigator must verify that all data entries on the eCRFs are 
accurate and correct. 
 
 
 


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APPENDIX A 
KARNOFSKY AND LANSKY PERFORMANCE SCALES 
 
Karnofsky 
Lansky 
Score 
Description 
Score 
Description 
100% 
No symptoms. 
100% 
fully active, normal 
90% 
Able to carry on normal activity; 
minor signs or symptoms of 
disease. 
90% 
minor restrictions in strenuous 
physical activity 
80% 
Able to carry on normal activity with 
effort; some signs or symptoms of 
disease. 
80% 
active, but tired more quickly 
70% 
Cares for self, unable to carry on 
normal activity or do active work. 
70% 
greater restriction of play and less 
time spent in play activity 
60% 
Requires occasional assistance but 
is able to care for most of own 
needs. 
60% 
up and around, but active play 
minimal; keeps busy by being 
involved in quieter activities 
50% 
Requires considerable assistance 
and frequent medical care. 
50% 
lying around much of the day, but 
gets dressed; no active playing 
participates in all quiet play and 
activities 
40% 
Disabled; requires special care and 
assistance. 
40% 
mainly in bed; participates in quiet 
activities 
30% 
Severely disabled; hospitalization 
indicated, although death not 
imminent. 
30% 
bed-bound; needing assistance even 
for quiet play 
20% 
Very ill; hospitalization necessary; 
active supportive treatment 
required. 
20% 
sleeping often; play entirely limited 
to very passive activities 
10% 
Moribund, fatal processes 
progressing rapidly. 
10% 
doesn't play; does not get out of bed 
0 
Patient expired. 
0 
unresponsive 
 
 


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APPENDIX B 
SCHEDULE OF ASSESSMENTS 
Procedure 
Prestudy / 
Surgery 
Pre-
Screening 
Protocol 
Screening 
Day 1 of each Cycle1 
(unless otherwise noted) 
End of 
TX 
Response 
Follow-Up 
(q 3mo±7 days) 
Until 
Progression 
Informed consent 
X 
X 
 
 
 
Medical History 
X 
 
 
 
 
Physical Examination 
X 
X 
X 
X 
X 
Toxicity (adverse 
events) 
 
from time of 
procurement 
X 
X 
 
Concomitant 
medications 
 
X 
X 
X 
X 
Performance Status 
 
X 
X 
X 
X 
Radiological Tumor 
Assessment 
(chest/abdomen/pelvis) 
 
within 4 weeks 
(must be post-
procurement) 
every 6 weeks ± 7 days 
from Cycle 1 
Within 45 
days 
X 
CBC with differential 
X2 
X 
X 
X 
X 
HIV testing, if applicable 
 
X 
 
 
 
Hepatitis testing, if 
applicable 
 
X 
 
 
 
Serum Chemistry 
X3 
X 
X 
X 
X 
PBMC collection for 
Immune Function 
Analysis 
≤ 1 week of 
tumor 
procurement 
X 
Cycles 2, 4, and 6 Day 1 
prior to chemotherapy 
administration 
X 
3 months post 
EOT and q 6 
months 
thereafter 
Plasma collection for 
ctDNA 
≤ 1 week of 
tumor 
procurement 
X 
Cycles 2, 3, 4, and 6 Day 1 
prior to chemotherapy 
administration 
X 
 
Pregnancy Test (if 
applicable) 
 
X 
 
 
 
Tissue Procurement 
X 
 
 
 
 
Vigil administration 
depending on randomization 
 
 
Day 15 q 21±3 days 4 
 
 
Injection Site 
Assessment 
 
 
Day 16 only (may be 
conducted at home)5 
 
 
Temozolomide 
 
 
Days 1 – 5, q21 days 
 
 
Irinotecan 
 
 
Days 1 – 5, q21 days 
 
 
Survival Status 
X 
 
 
 
Long Term 
Follow Up6 
Immunohistochemistry  
 
X7 
 
 
 
 
                                                
1 Subjects who are stable at the end of Cycle 4, with prior approval by the Sponsor may conduct evaluations on Day 15 of each cycle (1 Cycle = 
21 days ±3 days) (See Section 9.0). 
2 Obtain from medical records from standard preoperative hematology and chemistry panels. 
3 Obtain from medical records from standard preoperative hematology and chemistry panels. 
4 Cross-over subjects will receive Vigil on Day 1. 
5 Cross-over subjects will have assessment on Day 2. 
6 After progression, subjects will be contacted by phone quarterly for documentation of post study therapies and survival status. 
7 These slides should correlate with tumor procured for vaccine manufacture. 


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sarcoma family of tumors: review of St. Jude Children's Research Hospital studies." 
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56. Saylors, R. L., 3rd, K. C. Stine, J. Sullivan, J. L. Kepner, D. A. Wall, M. L. Bernstein, M. 
B. Harris, R. Hayashi and T. J. Vietti (2001). "Cyclophosphamide plus topotecan in 
children with recurrent or refractory solid tumors: a Pediatric Oncology Group phase II 
study." J Clin Oncol 19(15): 3463-3469. 
57. Senzer, N., M. Barve, J. Kuhn, A. Melnyk, P. Beitsch, M. Lazar, S. Lifshitz, M. Magee, J. 
Oh, S. W. Mill, C. Bedell, C. Higgs, P. Kumar, Y. Yu, F. Norvell, C. Phalon, N. Taquet, D. 
D. Rao, Z. Wang, C. M. Jay, B. O. Pappen, G. Wallraven, F. C. Brunicardi, D. M. 
Shanahan, P. B. Maples and J. Nemunaitis (2012). "Phase I trial of "bi-
shRNAi(furin)/GMCSF DNA/autologous tumor cell" vaccine (FANG) in advanced 
cancer." Mol Ther 20(3): 679-686. 
58. Senzer, N., M. Barve, J. Nemunaitis, J. Kuhn, A. Melnyk, P. Beitsch, M. Magee, J. Oh, 
C. Bedell, P. Kumar, D. D. Rao, B. O. Pappen, G. Wallraven, F. C. Brunicardi, P. B. 
Maples and J. Nemunaitis (2013). "Long Term Follow Up:  Phase I Trial of “bi-shRNA 
furin/GMCSF DNA/Autologous Tumor Cell” Immunotherapy (FANG™) in Advanced 
Cancer." Journal of Vaccines and Vaccination 4(8): 209. 
59. Shankar, A. G., S. Ashley, A. W. Craft and C. R. Pinkerton (2003). "Outcome after 
relapse in an unselected cohort of children and adolescents with Ewing sarcoma." Med 
Pediatr Oncol 40(3): 141-147. 
60. Soepenberg, O., H. Dumez, J. Verweij, D. Semiond, M. J. deJonge, F. A. Eskens, J. ter 
Steeg, J. Selleslach, S. Assadourian, G. J. Sanderink, A. Sparreboom and A. T. van 
Oosterom (2005). "Phase I and pharmacokinetic study of oral irinotecan given once daily 
for 5 days every 3 weeks in combination with capecitabine in patients with solid tumors." 
J Clin Oncol 23(4): 889-898. 
61. Stahl, M., A. Ranft, M. Paulussen, T. Bolling, V. Vieth, S. Bielack, I. Gortitz, G. Braun-
Munzinger, J. Hardes, H. Jurgens and U. Dirksen (2011). "Risk of recurrence and 
survival after relapse in patients with Ewing sarcoma." Pediatr Blood Cancer 57(4): 549-
553. 
62. Tan, K. W., M. Evrard, M. Tham, M. Hong, C. Huang, M. Kato, A. Prevost-Blondel, E. 
Donnadieu, L. G. Ng and J. P. Abastado (2015). "Tumor stroma and chemokines control 
T-cell migration into melanoma following Temozolomide treatment." Oncoimmunology 
4(2): e978709. 
63. Wagner, L., B. Turpin, R. Nagarajan, B. Weiss, T. Cripe and J. Geller (2013). "Pilot study 
of vincristine, oral irinotecan, and temozolomide (VOIT regimen) combined with 
bevacizumab in pediatric patients with recurrent solid tumors or brain tumors." Pediatr 
Blood Cancer 60(9): 1447-1451. 
64. Wagner, L. M. (2010). "Oral irinotecan for treatment of pediatric solid tumors: ready for 
prime time?" Pediatr Blood Cancer 54(5): 661-662. 
65. Wagner, L. M. (2015). "Fifteen years of irinotecan therapy for pediatric sarcoma: where 
to next?" Clin Sarcoma Res 5: 20. 
66. Wagner, L. M., K. R. Crews, L. C. Iacono, P. J. Houghton, C. E. Fuller, M. B. McCarville, 
R. E. Goldsby, K. Albritton, C. F. Stewart and V. M. Santana (2004). "Phase I trial of 
temozolomide and protracted irinotecan in pediatric patients with refractory solid 
tumors." Clin Cancer Res 10(3): 840-848. 
67. Wagner, L. M., N. McAllister, R. E. Goldsby, A. R. Rausen, R. Y. McNall-Knapp, M. B. 
McCarville and K. Albritton (2007). "Temozolomide and intravenous irinotecan for 
treatment of advanced Ewing sarcoma." Pediatr Blood Cancer 48(2): 132-139. 
68. Wagner, L. M., J. P. Perentesis, J. M. Reid, M. M. Ames, S. L. Safgren, M. D. Nelson, 
Jr., A. M. Ingle, S. M. Blaney and P. C. Adamson (2010). "Phase I trial of two schedules 
of vincristine, oral irinotecan, and temozolomide (VOIT) for children with relapsed or 


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refractory solid tumors: a Children's Oncology Group phase I consortium study." Pediatr 
Blood Cancer 54(4): 538-545. 
69. Wheler, J., A. M. Tsimberidou, D. Hong, A. Naing, G. Falchook, S. Piha-Paul, S. Fu, S. 
Moulder, B. Stephen, S. Wen and R. Kurzrock (2012). "Survival of 1,181 patients in a 
phase I clinic: the MD Anderson Clinical Center for targeted therapy experience." Clin 
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recurrent or refractory ewing sarcoma family tumours: a 13-years retrospective study in 
Turkey." In Vivo 28(3): 403-409. 

SAP_001.pdf: 
CL-PTL-130 
 
 
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COVER PAGE 
OFFICIAL PROTOCOL TITLE: 
A Multi-Center Phase III, Randomized, 
Open-Label Trial of Vigil (bi-shRNAfurin 
and GMCSF Augmented Autologous 
Tumor Cell Immunotherapy) in 
combination with Irinotecan and 
Temozolomide as a Second-Line Regimen 
for Ewing’s Sarcoma  
 
 
PROTOCOL NUMBER:  
CL-PTL-130 
 
 
IND NUMBER: 
014205 
 
 
NCT NUMBER: 
NCT03495921 
 
 
STATISTICAL ANALYSIS PLAN 
VERSION  DATE:  
December 5, 2018 
 
 
 
 
 
 
 


CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of 
Gradalis, Inc.  No person is authorized to make it public without written permission of Gradalis, Inc.  These restrictions on disclosure 
will apply equally to all future information supplied to you, which is indicated as privileged or confidential.  This material may be 
disclosed to and used by your staff and associates as may be necessary to conduct this clinical study. 
 
 
 
STATISTICAL ANALYSIS PLAN 
PROTOCOL TITLE: 
A Multi-Center Phase III, Randomized, Open-Label Trial of 
Vigil (bi-shRNAfurin and GMCSF Augmented Autologous 
Tumor Cell Immunotherapy) in combination with Irinotecan 
and Temozolomide as a Second-Line Regimen for Ewing’s 
Sarcoma 
PROTOCOL NUMBER: 
CL-PTL-130 
STUDY AGENT(S): 
Vigil 
bi-shRNAfurin and GMCSF Augmented Autologous Tumor 
Cell Immunotherapy 
SPONSOR: 
Gradalis, Inc. 
2545 Golden Bear Drive, Suite 110 
Carrollton, TX 75006 
Phone: (214) 442-8124 
Fax: (214) 442-8101 
 
 
 


Statistical Analysis Plan 
IND 014205 
 
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CL-SAP-00001 
 
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TABLE OF CONTENTS 
TABLE OF CONTENTS ............................................................................................................... 2 
ABBREVIATIONS ........................................................................................................................ 4 
 
INTRODUCTION ............................................................................................................ 5 
 
STUDY RATIONALE ..................................................................................................... 5 
 
OBJECTIVES ................................................................................................................. 6 
 
Primary objective(s) .................................................................................................... 6 
 
Secondary objective(s) ............................................................................................... 6 
 
END POINTS .................................................................................................................. 6 
 
Primary Endpoint ........................................................................................................ 6 
 
Secondary Endpoint .................................................................................................... 6 
 
STUDY DESIGN ............................................................................................................. 7 
 
SCHEDULE OF ASSESSMENTS .................................................................................. 8 
 
SAMPLE SIZE JUSTIFICATION ................................................................................... 9 
 
Definition of Progression Free Survival (PFS) ............................................................ 9 
 
ANALYSIS POPULATIONS ........................................................................................ 10 
 
Efficacy Analyses ...................................................................................................... 10 
8.1.1 
Primary Efficacy Analysis .................................................................................. 10 
8.1.2 
Secondary Efficacy Analyses ............................................................................ 10 
8.1.3 
Sensitivity Analyses of the Primary Endpoint..................................................... 11 
8.1.4 
Sensitivity Analysis of Overall Survival .............................................................. 11 
8.1.5 
Subgroup Analyses of the Primary (PFS) and Key Secondary Endpoints (OS, 
ORR) 
11 
 
Safety Analyses ........................................................................................................ 12 
 
Interim Analysis ......................................................................................................... 12 
 
CONSIDERATIONS FOR DATA ANALYSIS .............................................................. 13 
 
Programming Environment ....................................................................................... 13 
 
Statistical Notation and Methodology ........................................................................ 13 
 
DATA HANDLING METHODS ..................................................................................... 13 
 
Missing Data ............................................................................................................. 13 
10.1.1 
Date Values ....................................................................................................... 13 
10.1.2 
Non-Date Values ............................................................................................... 14 
 
Visit Windows ............................................................................................................ 14 
 
Data Derivations and Definitions ............................................................................... 14 
 
STUDY POPULATION ................................................................................................. 15 
 
Subject Disposition ................................................................................................... 15 


Statistical Analysis Plan 
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Demographic and Baseline Characteristics .............................................................. 15 
 
Prior Disease medications/therapy and Concomitant Medications ........................... 15 
 
Concomitant procedures ........................................................................................... 15 
 
Medical and Surgery History ..................................................................................... 16 
 
Inclusion/Exclusion Criteria ....................................................................................... 16 
 
Protocol Violations/Deviations .................................................................................. 16 
 
EFFICACY ANALYSIS ................................................................................................ 16 
 
Tumor Assessment ................................................................................................... 16 
12.1.1 
Radiology Tumor Assessment ........................................................................... 16 
 
SAFETY ....................................................................................................................... 17 
 
Exposure to Study Drug ............................................................................................ 17 
 
Adverse Events ......................................................................................................... 17 
 
Serious Adverse Events and Death .......................................................................... 18 
 
Laboratory Evaluations ............................................................................................. 18 
 
Vital Signs ................................................................................................................. 18 
 
Physical Examinations .............................................................................................. 18 
REFERENCES ........................................................................................................................... 19 
 
 
 


Statistical Analysis Plan 
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ABBREVIATIONS 
Abbreviation 
Term 
ATC 
Anatomical Therapeutic Chemical 
BMI 
Body Mass Index 
CBC 
Complete blood count 
CD 
Cluster of differentiation 
CRF 
Case report form 
CT 
computerized tomography 
CTCAE 
Common Toxicity Criteria for Adverse Events 
ctDNA 
Circulating tumor DNA 
CTM 
Clinical Trial Material 
ELISPOT 
Enzyme-Linked ImmunoSorbent Spot 
ESFT 
Ewing’s Sarcoma Family of Tumors 
EWS 
Ewing’s Sarcoma 
GMCSF 
Granulocyte Macrophage-Colony Stimulating Factor 
HR 
Hazard Ratio 
IDMC 
Independent Data Monitoring Committee 
IFN 
Interferon 
ITT 
Intent to Treat 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRI 
Magnetic resonance imaging 
ORR 
Objective Response Rate 
OS 
Overall Survival 
PBMC 
Peripheral Blood Mononuclear Cells 
PFS 
Progression Free Survival 
RECIST 
Response Evaluation Criteria in Solid Tumors 
SAE 
Serious Adverse Event 
STD 
Standard Deviation 
TEAE 
treatment-emergent adverse event 
WHO 
World Health Organization 
 
 
 


Statistical Analysis Plan 
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INTRODUCTION 
 
This document describes the statistical methods and data presentations to be used in the 
summary and analysis of safety and efficacy data from Gradalis Protocol CL-PTL-130.  
Background information is provided for the overall study design and objectives.  The reader is 
referred to the study protocol and case report forms (CRFs) for details of study conduct and 
data collection. 
 
 
STUDY RATIONALE 
 
The results from the Phase I Vigil trial show product safety (both short-term and long-term 
through 3+ years), confirm effective transgene expression (GMCSF) and RNAi [furin] silencing, 
and support the rationale of the immunotherapeutic triad, i.e., autologous tumor cell therapy with 
increased GMCSF expression and decreased TGFβ production shown to effectively modulate 
immunogenicity as evidenced by the correlation of IFNγ-ELISPOT responsiveness with overall 
survival and the elicitation of the CD4+ mediated circulating antibody responses against CT47 in 
two of the Phase 1 Ewing’s sarcoma patients.  The preliminary evidence of safety, immune 
stimulation and clinical benefit from Vigil in patients with Ewing’s sarcoma treated in the Phase I 
setting supports evaluation of this therapy in a larger and randomized study and at earlier stage 
of recurrence (first recurrence).  A lower tumor burden is typically associated with greater delay 
in progression with other immunotherapies.  Mathematical modeling supports the concept of 
better control of disease progression with the combined use of effective chemotherapy (e.g., 
temozolomide/irinotecan as second line treatment in Ewing’s sarcoma) and immunotherapy 
(i.e., Vigil) in first relapse patient population (de Pillis, Gu et al. 2006).  The utilization of the 
standard of care temozolomide/irinotecan regimen at investigator consensus defined dose and 
schedule appears to be minimally immunosuppressive and thus a reasonable option for 
combination with Vigil.  Moreover, the temozolomide and irinotecan regimen at effective dose 
levels has low myelosuppressive toxicities (irinotecan 50mg/m2/dose and temozolomide 
100mg/m2/dose) and may increase the tumor antigen release and surface exposure of 
calreticulin (part of the specific danger-signaling system) as well as reduce the frequency of 
CD4+ CD25+ regulatory T-cells (Kim, Kim et al. 2010).  In addition, given that Ewing’s sarcoma 
expression levels of type-1 associated chemokine ligands CXCL9, CXCL10, and CCL5 correlate 
with TIL expression chemokine receptors (CXCR3 and CCR5) (Berghuis, Santos et al. 2011) it 
is notable that temozolomide has been shown to enhance tumor matrix presentation of CXCL9 
and CXCL10 in certain tumor models (Tan, Evrard et al. 2015).  Temozolomide and irinotecan in 
combination have shown benign safety profiles without any Grade 3 treatment-related adverse 
events in seven combination treated Ewing’s Sarcoma patients of Part 2 of CL-PTL-121. 
Furthermore, clinical antitumor activity was demonstrated in 4 of the 7 combination treated 
patients, all of whom had previously failed temozolomide and irinotecan, as evidenced by 2 
partial responses, one histological CR (autopsy) and one prolonged SD currently near 1 year in 
duration. Thus, the combination of Vigil, irinotecan and temozolomide may not only increase 
anti-tumor responses but also prolong time to progression and improvement of quality of life in 
Ewing’s sarcoma patients. 
 


Statistical Analysis Plan 
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OBJECTIVES 
 
 Primary objective(s) 
 
• 
To compare the progression free survival of subjects dosed with Vigil immunotherapy in 
combination with irinotecan and temozolomide vs. irinotecan and temozolomide. 
 
 Secondary objective(s) 
 
• 
To determine and compare the overall survival of subjects with relapsed or refractory 
Ewing’s sarcoma dosed with Vigil immunotherapy in combination with irinotecan and 
temozolomide. 
• 
To determine the objective response rate (RECIST 1.1) of patients with metastatic 
Ewing’s sarcoma refractory or intolerant to 1 prior line of systemic chemotherapy treated 
with Vigil immunotherapy or dosed with Vigil immunotherapy in combination with 
irinotecan and temozolomide. 
• 
To determine the Vigil manufacturing success rate by gathering data from tumor tissue 
collection, Vigil construction, and test procedures. 
 
 
END POINTS 
 
 Primary Endpoint 
 
The primary endpoint of PFS is the time from randomization to progression according to 
RECIST version 1.1 or death.  Incomplete data resulting from patients who terminate the study 
for reasons other than disease progression or death will be censored in time-to-event analyses 
on the last assessment date at which a RECIST 1.1 evaluation of disease status was made.  
The distributions of PFS in the two arms will be compared using a two-sided log-rank test. 
 
 Secondary Endpoint 
 
The secondary endpoint of OS is defined as time from randomization to death or to the date of 
last follow-up. The date of last follow-up confirming survival will be used as the censoring date 
for subjects who are alive and/or do not have a known date of death.  The analysis of OS will be 
conducted using the two-sided log-rank test.  Kaplan-Meier OS curves will be displayed by 
treatment arm.  Median OS and percent OS at fixed time points will be estimated.  
 
The secondary endpoint of ORR will be analyzed using Pearson’s chi-square test. 
 
All secondary analyses will be conducted using two-sided tests at the alpha=0.05 level of 
significance. 


Statistical Analysis Plan 
IND 014205 
 
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STUDY DESIGN 
 
This is a multicenter, 1:1 randomized Phase III study of intradermal autologous Vigil 
immunotherapy (1.0 x 106 cells/injection cells/injection; minimum of 4 to a maximum of 12 
administrations) in combination with irinotecan and temozolomide in subjects with metastatic 
Ewing’s sarcoma Family of Tumors (ESFT) refractory/intolerant or recurrent to 1 prior line of 
chemotherapy.  Participants undergoing a standard surgical procedure (e.g., tumor biopsy or 
palliative resection) may have tumor tissue harvested for manufacture of investigational product.   
 
Subjects meeting eligibility criteria including manufacture of a minimum of 4 immunotherapy 
doses of Vigil will receive: 
 
Group A 
(i) 
oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), 
(ii) 
oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle), and 
(iii) 
Vigil 1.0 x 106 cells/injection, intradermally on Day 15. 
 
OR 
Group B 
(i) 
oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), and 
(ii) 
oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle) 
 
If chemotherapy study drugs cannot be administered orally (i.e. size of capsule, nausea, etc.) it 
is physician’s discretion to request iv administration of temozolomide at 100 mg/m2 daily and/or 
irinotecan 20 mg/m2 daily intravenously.  
 
One cycle = 21 days.  Screening for the main portion of the study may occur as early as one 
week but no later than 8 weeks following tumor procurement.  Vigil is typically released 
approximately 3 weeks after the completion of the two-day manufacturing process.  At the time 
of analysis the subjects will be stratified by initial response to frontline treatment (recurrent 
versus refractory (progressive)). 
 
Participants will be managed in an outpatient setting.  Hematologic function, liver enzymes, 
renal function and electrolytes will be monitored.  Blood for immune function analyses including 
IFNγ-ELISPOT analysis of cytotoxic T cell response to autologous tumor antigens will be 
collected at tissue procurement, post-procurement screening and prior to chemotherapy 
administration on Day 1 of Cycles 2, 4, and 6, end of treatment (EOT), 3 months after EOT and 
every 6 months thereafter.  Blood for exploratory ctDNA analysis will be collected at tissue 
procurement, prior to chemotherapy administration at baseline, prior to chemotherapy 
administration on Day 1 of Cycles 2, 3, 4, and 6, and EOT. 
 


Statistical Analysis Plan 
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SCHEDULE OF ASSESSMENTS 
 
Procedure 
Prestudy 
Screening 
Day 1 of each Cycle 1 
(unless otherwise noted) 
End of 
TX 
Response 
Follow-Up 
(q 3mo±7 
days) Until 
Progression 
Informed consent 
X 
X 
 
 
 
Medical History 
X 
 
 
 
 
Physical Examination 
X 
X 
X 
X 
X 
Toxicity (adverse 
events) 
 
from time of 
procurement 
X 
X 
 
Concomitant 
medications 
 
X 
X 
X 
X 
Performance Status 
 
X 
X 
X 
X 
Radiological Tumor 
Assessment 
(chest/abdomen/pelvis) 
 
within 4 weeks 
(must be post-
procurement) 
every 6 weeks ± 7 days 
from Cycle 1 
Within 45 
days 
X 
CBC with differential 
X 2 
X 
X 
X 
X 
HIV testing, if applicable 
 
X 
 
 
 
Hepatitis testing, if 
applicable 
 
X 
 
 
 
Serum Chemistry 
X 3 
X 
X 
X 
X 
PBMC collection for 
Immune Function 
Analysis 
≤ 1 week of 
tumor 
procurement 
X 
Cycles 2, 4, and 6 Day 
1prior to chemotherapy 
administration 
X 
3 months post 
EOT and q 6 
months 
thereafter 
Plasma collection for 
ctDNA 
≤ 1 week of 
tumor 
procurement 
X 
(Cycles 2, 3, 4, and 6 Day 1 
prior to chemotherapy 
administration 
X 
 
Pregnancy Test (if 
applicable) 
 
X 
 
 
 
Vigil administration 
depending on randomization 
 
 
Day 15 q 21±3 days 4  
 
 
Injection Site 
Assessment 
 
 
Day 16 only (may be 
conducted at home) 5 
 
 
Temozolomide 
 
 
Days 1 – 5, q21 days 
 
 
Irinotecan 
 
 
Days 1 – 5, q21 days 
 
 
                                                
1 Subjects who are stable at the end of Cycle 4, with prior approval by the Sponsor may conduct evaluations on Day 15 of each cycle (1 Cycle = 
21 days ±3 days) 
2 Obtain from medical records from standard preoperative hematology and chemistry panels. 
3 Obtain from medical records from standard preoperative hematology and chemistry panels. 
4 Cross-over subjects will receive Vigil on Day 1 
5 Cross-over subjects will have assessment on Day 2 


Statistical Analysis Plan 
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Procedure 
Prestudy 
Screening 
Day 1 of each Cycle 1 
(unless otherwise noted) 
End of 
TX 
Response 
Follow-Up 
(q 3mo±7 
days) Until 
Progression 
Survival Status 
X 
 
 
 
Long Term 
Follow Up 6 
Immunohistochemistry 
 
X 7 
 
 
 
 
 
SAMPLE SIZE JUSTIFICATION 
 
This is an open label randomized controlled Phase III clinical trial.  Information concerning the 
predicted survival of the control group is limited, based on rarity of disease and few published 
reports.  However expert advisors in the EWS field estimate a conservative one-year survival 
rate of 25% in the chemotherapy control group.  The one-year survival rate of 60% in the Vigil 
treated group is estimated from EWS patients treated on the Vigil phase 1 protocol.  These 
estimates correspond to a hazard ratio (HR) of 0.383 favoring Vigil over control. 
 
Assuming 1:1 randomization and the use of a two-sided log rank test at the alpha=0.0476 level 
of significance, 67 events will provide 90% power to detect an PFS HR of 0.45.  Assuming a 
Group B (control group) median PFS of 6 months, a one-year accrual period, and a six-month 
follow-up period after randomization of the last subject, it is estimated that the total sample size 
(number of subjects) required to achieve 67 events is 114 (57 patients in each arm). 
 
An unblinded interim analysis will be conducted by an independent data monitoring committee 
(IDMC) when 40 PFS events have occurred.  Based on the results of this interim analysis, the 
planned sample size may be increased to 120 events.  If the sample size is increased to 120 
events, then the study will have approximately 90% power to detect a true HR of 0.55.  Note 
that if the sample size is increased to 120 events, then the accrual period will be extended to 24 
months, with a 6-month follow-up period after randomization of the last patient.  Under these 
assumptions, it is estimated that approximately 164 patients will need to be randomized in order 
to achieve 120 events.  Section 16.6 provides further details. 
 
 Definition of Progression Free Survival (PFS) 
 
• 
Progression free survival (PFS) is defined as the time from randomization to the event of 
disease recurrence/progression or death due to any cause. 
• 
Radiographic disease recurrence/progression will be assessed using the RECIST 1.1 
criteria.  If the disease recurrence/progression assessment involves more than one date, 
the earliest date will be used as the event date.  
                                                
6 After progression, subjects will be contacted by phone quarterly for documentation of survival status. 
7 These slides should correlate with tumor procured for vaccine manufacture. 


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• 
Patients who are alive and recurrence free at the time of analysis data cut-off, or who 
terminate the study for reasons other than disease progression or death, or who receive 
non-protocol treatment for Ewing’s Sarcoma, will be censored on the last assessment 
date at which a RECIST 1.1 evaluation of disease status was made. 
• 
Patients missing baseline disease assessment will be censored at date of 
randomization. 
• 
For equivocal findings of recurrence (e.g., very small and uncertain new lesions; cystic 
changes or necrosis in existing lesions), treatment may continue until the next scheduled 
assessment.  If recurrence is confirmed at the next scheduled assessment, the date of 
recurrence should be the earlier date when recurrence was suspected. 
 
 
ANALYSIS POPULATIONS 
 
The intent-to-treat (ITT) population will include all randomized subjects.  All efficacy analyses 
will be completed in the ITT population. 
 
The Safety population will include all patients who receive study treatment and patients will be 
analyzed according to actual treatment received.  All safety analyses will be completed in the 
Safety population. 
 
 Efficacy Analyses 
 
8.1.1 
Primary Efficacy Analysis 
 
The primary endpoint of PFS is the time from randomization to progression according to 
RECIST version 1.1 or death.  The distributions of PFS in the two arms will be compared using 
a two-sided log-rank test at the alpha=0.0476 level of significance. 
 
8.1.2 
Secondary Efficacy Analyses 
 
A fixed sequence testing procedure will be used to control the overall level of significance for the 
analysis of the secondary endpoints of OS and ORR.  If the primary analysis of PFS is 
statistically significant (p<0.0476), then OS will be analyzed using a two-sided test at the 
alpha=0.0476 level of significance.  In addition, if the analysis of OS is statistically significant 
(p<00476), then ORR will be analyzed using a two-sided test at the alpha=0.0476 level of 
significance.  However, if an earlier analysis is not statistically significant, the remaining 
analyses will be exploratory rather than confirmatory. 
 
The secondary endpoint of OS is defined as time from randomization to death or to the date of 
last follow-up. The date of last follow-up confirming survival will be used as the censoring date 
for subjects who are alive and/or do not have a known date of death.  The analysis of OS will be 
conducted using the log rank test.  Kaplan-Meier OS curves will be displayed by treatment arm.  
Median OS and percent OS at fixed time points will be estimated.  


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The secondary endpoint of ORR will be analyzed using Pearson’s chi-square test.  Overall 
response rate (ORR) is defined as the proportion of patients who have prolonged stable disease 
or a partial or complete response to therapy according to RECIST 1.1.  ORR will be reviewed 6 
months after treatment with Vigil. 
 
The secondary analysis of all secondary endpoints will be conducted using two-sided tests at 
the alpha=0.05 level of significance. 
 
8.1.3 
Sensitivity Analyses of the Primary Endpoint 
 
Five sensitivity analyses of the primary endpoint will be conducted using each of the following 
modifications to the definition of PFS: 
 
• 
If the disease recurrence/progression assessment involves more than one date, the 
latest date will be used as the event date. 
• 
If disease recurrence or death occurs right after missing data for a scheduled 
radiographic disease assessment (including missing the assessment or assessment 
results in an unevaluable status for overall response per RECIST 1.1), the patient will be 
censored at the date of the last radiographic disease assessment. 
• 
If a patient receives non-protocol treatment for Ewing’s Sarcoma, the patient will be 
treated as having progression at the date of the last radiographic disease assessment. 
• 
Patients missing baseline disease assessment will be treated as having progression at 
the date of randomization. 
• 
For equivocal findings of recurrence (e.g., very small and uncertain new lesions; cystic 
changes or necrosis in existing lesions) for whom treatment continues until the next 
scheduled assessment, and if recurrence is confirmed at the next scheduled 
assessment, the date of recurrence should be the earlier date when recurrence was 
suspected. 
 
8.1.4 
Sensitivity Analysis of Overall Survival 
 
A sensitivity analysis of OS will be conducted in which subjects who are crossed over to Vigil 
therapy will be censored at the time of crossover. 
 
8.1.5 
Subgroup Analyses of the Primary (PFS) and Key Secondary 
Endpoints (OS, ORR) 
 
Subgroup analyses will be conducted in the following subsets of the ITT population: 
 
• 
Race / ethnicity 
• 
Study site / region 


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• 
Age group (<15 years (low), ≥15 years (high)) 
• 
Gender 
• 
Number of doses received (≤median number of doses administered, >median number of 
doses administered) 
• 
Number of lines of prior treatments including chemotherapy and radiation 
• 
At time of diagnosis: localized vs. metastatic disease 
• 
At time of diagnosis: skeletal vs. extraskeletal 
• 
At time of diagnosis: tumor volume/burden (<100ml or 8cm vs >100ml or 8cm) 
• 
Response to frontline therapy (CR vs. PR vs. SD vs. progressive disease) 
• 
Time to first recurrence: <2 years, > 2 years 
 
 Safety Analyses 
 
Safety endpoints include all adverse events (CTCAE version 5.0), laboratory safety 
assessments, and physical examination findings. 
 
 Interim Analysis 
 
An interim analysis for efficacy, futility, and potential sample size re-estimation will be conducted 
by an independent data monitoring committee (IDMC) when 40 PFS events have been achieved 
(60% information fraction).  Based on the Lan-DeMets method for group sequential trials using 
O’Brien-Fleming boundaries (Reboussin, DeMets et al. 2000), the levels of significance for the 
interim and final analyses are alpha=0.00762 and alpha=0.0476, respectively. 
 
Based on the results of the interim analysis, the IDMC will make one of the following four 
recommendations to the sponsor: 
• 
Terminate the study for efficacy (p<0.00762 at the interim analysis); 
• 
Continue the study as planned; 
• 
Increase the sample size to 120 events; 
• 
Terminate the study for futility. 
 
Provided that the IDMC does not recommend terminating the study early for efficacy, the 
conditional power of the planned final analysis at 67 events will be computed using the 
methodology of (Harrington 2001) (Jennison and Turnbull 2000), under the assumption that the 
observed hazard ratio at the interim analysis represents the true effect.  In terms of the HR 
observed at the interim analysis, these rules can be translated (approximately) as follows: 
• 
If the HR is less than 0.43, terminate for efficacy 
• 
If the HR is in the range from 0.43 to 0.51, continue as planned. 
• 
If the HR is in the range from 0.51 to 0.61 (i.e., conditional power in the range from 50% 
to 80%), increase the sample size to 120 events. 
• 
If the HR is greater than 0.61 (i.e., conditional power less than 50%), terminate for 
futility. 
 


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CONSIDERATIONS FOR DATA ANALYSIS 
 
 Programming Environment 
 
All analyses will be conducted using SAS® version 9.0. 
 
 Statistical Notation and Methodology 
 
Unless stated otherwise, the term “descriptive statistics” refers to the number of subjects (n), 
mean, median, standard deviation (STD), minimum (min), and maximum (max) for continuous 
data and frequencies and percentages for categorical data.  Min and max values will be 
rounded to the precision of the original value, means and medians will be rounded to 1 decimal 
place greater than the precision of the original value, and STDs will be rounded to 2 decimal 
places greater than the precision of the original value.  Percentages will be rounded to the 
nearest whole number (zeros are not displayed) with values of “< 1%” and “> 99%” shown as 
necessary for values falling near the boundaries.  P-values will be presented with 3 decimal 
places and values less than 0.001 will be presented as < 0.001. 
 
Unless otherwise noted, all data collected during the study will be included in data listings and 
will be sorted by cohort, subject number and then by date/time for each subject number. 
 
 
DATA HANDLING METHODS 
 
 Missing Data 
 
10.1.1 Date Values 
 
In cases of incomplete dates (e.g., pertaining to AE, concomitant medication, medical history, 
etc.), the missing component(s) will be assumed as the most conservative value(s) possible.  
For example, if the start date has a missing day value, the first day of the month will be imputed 
for study day computations (i.e., treatment-emergent status, etc.).  If day is missing for an end 
date, the last day of the month will be imputed.  Similar logic will be assumed for missing month 
and year components.   
 
Date imputation will only be used for computational purposes e.g., treatment-emergent status, 
etc.  Actual data values as they appear in the original CRFs will be shown in the data listings. 
 
 


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10.1.2 Non-Date Values 
 
Every effort will be made to obtain the protocol-required data for all study assessments that are 
scheduled for each scheduled visit for all subjects who have been enrolled.  The primary 
endpoint is progression free survival, survival models (Kaplan-Meier estimates and Cox model) 
which take into account censored observations will be used. There will be no imputation for 
missing data for the primary endpoint.  
 
 Visit Windows 
 
Study visit periods will be windowed as applicable as shown in Section 6.0. 
 
Values will be presented for all scheduled study visits according to the nominal visit obtained 
from the CRF.  If an unscheduled visit falls in a visit window with an existing nominal visit 
assessment, the nominal assessment will be used for summary presentation.  If no nominal visit 
assessment exists for a visit window with unscheduled visit(s), then the latest unscheduled visit 
within the visit window will be used.  If multiple nominal assessments are collected within the 
same visit, the latest value and corresponding date will be used for summary presentation.   
 
All values will be included in the data listings. 
 
 Data Derivations and Definitions 
 
The following definitions and derivations will be used for this study: 
Baseline for all assessments will be considered as the last non-missing assessment prior to the 
first administration of clinical trial material (CTM).  
 
Change from baseline will be calculated as:  
Change = observed value – baseline value. 
 
Study days on or after the initial dose of CTM will be computed as: 
Study Day = Date – Study Drug Start Date + 1. 
For pre-dosing dates, Study Day = Date – Study Drug Start Date. 
 
Age will be calculated from the date of birth to informed consent date.   
 


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Treatment-emergent events will be considered as any event occurring after the first dose of 
treatment drug and prior to a subject’s termination, withdrawal or completion of the study.  
 
Prior medications will be those ended before the first study treatment. 
 
Concomitant medications are those used on or after the date of first study treatment. 
 
 
STUDY POPULATION 
 
Unless otherwise stated, all study population analyses will be performed on the Safety 
Population. 
 
 Subject Disposition 
 
Subject disposition will be presented for all subjects.  The composition of the analysis 
populations and those who enrolled, completed or discontinued from the study will be 
summarized by dose cohort and overall with descriptive statistics.  Reasons for early 
discontinuation will be presented with frequencies and percentages for all categories. 
 
 Demographic and Baseline Characteristics 
 
Demographics and baseline characteristics listing will include date of birth, age, gender, 
ethnicity, race, and height and weight and BMI at screening.  Each of these variables except 
date of birth will also be summarized with descriptive statistics. 
 
 Prior Disease medications/therapy and Concomitant Medications 
 
Concomitant medications will be coded with the World Health Organization (WHO) drug 
dictionary.  Concomitant medications will be summarized with descriptive statistics by drug 
Anatomical Therapeutic Chemical (ATC) Classification System (level one), generic name, and 
cohort.  A data listing will be included that shows all medications by generic name and verbatim 
name.  Prior medications/therapy will be excluded from the summary but be included in the 
listing. 
 
 Concomitant procedures 
 
Concomitant procedures will be summarized by indication type, treatment arm, and time point 
and also will be included in the listing. 


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 Medical and Surgery History 
 
Medical history will be summarized with descriptive statistics by medical history code and 
treatment group.  A comprehensive data listing will also be included. 
 
 Inclusion/Exclusion Criteria 
 
Inclusion and exclusion criteria failures will be included in a data listing. 
 
 Protocol Violations/Deviations 
 
Protocol violations will be summarized with descriptive statistics by category. A listing of all 
events will also be included. 
 
Major protocol deviations include, but are not limited to the following: 
• 
Non-fulfillment of all inclusion criteria or fulfillment of at least one exclusion criteria; 
• 
No consent form found; 
• 
Other relevant deviations, to be judged on an individual basis. 
 
Minor protocol deviations include, but are not limited to: 
• 
Measurements obtained outside the visit window to a limited degree; 
• 
Other deviations will be judged on an individual basis. 
 
 
EFFICACY ANALYSIS 
 
 Tumor Assessment 
 
Disease recurrence will be evaluated in this study using the Response Evaluation Criteria in 
Solid Tumors (RECIST) Version 1.1 (Eisenhauer, Therasse et al. 009). RECIST response will 
be summarized with descriptive statistics. 
 
Local and central radiology tumor assessment will be performed in this study, summary of the 
tumor assessment will be presented. 
 
12.1.1 Radiology Tumor Assessment 
 
Radiology tumor assessment will be performed within 4 weeks of the registration. 


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If the study agent is completed or discontinued before disease recurrence the subject will be 
followed for recurrence on the following schedule. Radiological assessment of disease status 
until disease recurrence (i.e., chest X-ray, (chest CT or MRI is acceptable), pelvic/abdominal CT 
or MRI) performed quarterly (every 3 months ±7 days). The methods used for prestudy 
assessments (e.g., CT or MRI) should be used throughout the study and if possible, the same 
equipment should be used each time. 
 
Radiology tumor assessment will be summarized with descriptive statistics in a summary table. 
A detailed listing will be provided. 
 
 
SAFETY 
 
The safety population will be used for all the analyses of safety data. 
 
 Exposure to Study Drug 
 
Study treatment dosing compliance will be summarized with descriptive statistics by treatment 
group.  All exposure and compliance data will be provided in data listings. 
 
 Adverse Events 
 
All reported terms (investigator descriptions) for AEs will be coded using the most recent version 
of the Medical Dictionary for Regulatory Activities (MedDRA) and summarized with frequencies 
and percentages by treatment group, system organ classification, and preferred term. 
 
A treatment-emergent adverse event (TEAE) is an event that emerges during treatment, having 
been absent pretreatment, or worsens relative to the pretreatment state. 
 
The following summaries will be presented with frequencies and percentage by treatment group, 
system organ classification, and preferred term: 
 
• 
All AEs 
• 
All TEAEs 
• 
TEAEs leading to discontinuation from the study 
• 
TEAEs by relation to study treatment 
• 
TEAEs by CTCAE severity 
• 
All serious TEAEs  
• 
Serious TEAEs by relation to study drug 
• 
TEAEs with fatal outcome 


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For all AE summaries, events will be counted only once for a given subject by primary SOC and 
preferred term.  When an adverse event occurs more than once for a subject, the maximum 
severity and causality will be used. 
 
All AEs will be included in comprehensive data listings.  A separate listing will be included for 
AEs leading to discontinuation from study and AEs with fatal outcome. 
 
 Serious Adverse Events and Death 
 
Separate data listings and summaries will be presented for all SAEs and deaths. 
 
 Laboratory Evaluations 
 
Laboratory assessments and change from baseline will be summarized with descriptive 
statistics by panel, test, treatment group, and time point.  Additionally, abnormal results will be 
summarized with frequencies and percentages by clinical significance, panel, test, treatment 
group, and time point. 
 
A data listing will display all laboratory test results and findings. 
 
 Vital Signs 
 
The results of change from baseline of vital signs will be summarized with descriptive statistics 
by treatment group and time point.  A data listing of all data will also be included. 
 
 Physical Examinations 
 
The results of physical examinations will be summarized with frequencies and percentages by 
category, treatment group, and time point.  A data listing of all results will also be included. 
 
 


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REFERENCES 
 
1. Berghuis, D., S. J. Santos, H. J. Baelde, A. H. Taminiau, R. M. Egeler, M. W. Schilham, 
P. C. Hogendoorn and A. C. Lankester (2011). "Pro-inflammatory chemokine-chemokine 
receptor interactions within the Ewing sarcoma microenvironment determine CD8(+) T-
lymphocyte infiltration and affect tumour progression." J Pathol 223(3): 347-357. 
2. de Pillis, L. G., W. Gu and A. E. Radunskaya (2006). "Mixed immunotherapy and 
chemotherapy of tumors: modeling, applications and biological interpretations." J Theor 
Biol 238(4): 841-862. 
3. Harrington, D. (2001). "Group Sequential Methods with Applications to Clinical Trials. 
Christopher Jennison and Bruce W. Turnbull, CRC/Chapman & Hall, U.K., 2000. No. of 
pages: xviii +390. Price: £ 39.00. ISBN 0-849-30316-8." Statistics in Medicine 20(14): 
2210-2211. 
4. Jennison, C. and B. Turnbull (2000). Group Sequential Methods with Applications to 
Clinical Trials. Chapman and Hall/CRC, Boca Raton, FL. 
5. Kim, T. G., C. H. Kim, J. S. Park, S. D. Park, C. K. Kim, D. S. Chung and Y. K. Hong 
(2010). "Immunological factors relating to the antitumor effect of temozolomide 
chemoimmunotherapy in a murine glioma model." Clin Vaccine Immunol 17(1): 143-153. 
6. Reboussin, D. M., D. L. DeMets, K. M. Kim and K. K. Lan (2000). "Computations for 
group sequential boundaries using the Lan-DeMets spending function method." Control 
Clin Trials 21(3): 190-207. 
7. Tan, K. W., M. Evrard, M. Tham, M. Hong, C. Huang, M. Kato, A. Prevost-Blondel, E. 
Donnadieu, L. G. Ng and J. P. Abastado (2015). "Tumor stroma and chemokines control 
T-cell migration into melanoma following Temozolomide treatment." Oncoimmunology 
4(2): e978709.