Conditions: Ewing's Sarcoma
Keywords: Ewing's Sarcoma Family of Tumors, ESFT, Sarcoma Soft Tissue Sarcoma
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 22
Healthy volunteers: no

Brief summary:
A two-part trial in patients with metastic Ewing's sarcoma. Participants in Part 1 will be randomized to receive either Vigil immunotherapy or gemcitabine and docetaxel with the objective of comparing the overall survival between the two arms. Participants enrolled in Part 2 will receive Vigil immunotherapy in combination of temozolomide and irinotecan with the objective to determine the safety profile of the combination treatment.

Detailed description:
Part 1 Methodology:

This is a multicenter, 1:1 randomized Phase IIb study of intradermal autologous Vigil immunotherapy (1.0 x 10e7 cells/injection; minimum of 4 to a maximum of 12 administrations) versus gemcitabine / docetaxel in patients with metastatic Ewing's sarcoma Family of Tumors (ESFT) refractory or intolerant to at least 2 prior lines of chemotherapy. Patients undergoing a standard surgical procedure (e.g., tumor biopsy or palliative resection) may have tumor tissue harvested for manufacture of investigational product. Patients meeting eligibility criteria including manufacture of a minimum of 4 immunotherapy doses will be randomized to receive either (1) intradermal Vigil every 28 days for 4-12 administrations, or (2) gemcitabine 675 mg/m2 IV at 10 mg/m2/min D1 and D8 and docetaxel 75 mg/m2 IV D8 every 21 days. The primary trial objective is to determine the overall survival of patients treated with Vigil versus gemcitabine/docetaxel. Randomization may occur as early as vaccine is released (typically 3 - 4 weeks following tumor procurement) but no later than 8 weeks following tumor procurement. Randomization of patients will be stratified by Karnofsky Performance Status (KPS) ≥ 80% vs \< 80%.

Patients will be managed in an outpatient setting. Hematologic function, liver enzymes, renal function and electrolytes will be monitored monthly. Blood for immune function analyses including IFNγ-ELISPOT analysis of cytotoxic T cell response to autologous tumor antigens will be collected at tissue procurement, baseline, and prior to product administration at Cycles 2, 4, end of treatment, and every 6 months thereafter.

Part 2 Methodology:

Based on the limited accrual to Part 1 of this study, Gradalis is opening Part 2 of this clinical protocol to assess the safety of Vigil immunotherapy in combination with irinotecan and temozolomide. Part 2 will be conducted at the same centers as Part 1, studying intradermal autologous Vigil cancer vaccine (1.0 x 10e7 cells/injection; minimum of 4 to a maximum of 12 administrations) in patients with metastatic Ewing's sarcoma Family of Tumors (ESFT) refractory or intolerant to at least 1 prior line of chemotherapy. Patients undergoing a standard surgical procedure (e.g., tumor biopsy or palliative resection) may have tumor tissue harvested for manufacture of investigational product. Patients meeting eligibility criteria including manufacture of a minimum of 4 immunotherapy doses of Vigil will be registered to receive: (i) oral temozolomide 100 mg/m2 daily (Days 1 - 5, total dose 500 mg/m2/cycle), (ii) irinotecan 50 mg/m2 daily (Days 1 - 5, total dose 250mg/m2/cycle), orally or irinotecan 20mg/m2 daily (Days 1 - 5, total dose 100mg/m2/cycle ), intravenously (iii) peg-filgrastim 100μg/kg (Day 6) subcutaneously (optional and may be administered at home), and (iv) Vigil 1.0 x 107 cells/injection, intradermally on Day 15 and every 3 weeks thereafter. One cycle = 21 days. Registration onto Part 2 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.

Patients 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 Day 15 Vigil administration at Cycles 2, 4, end of treatment, and every 6 months thereafter. Blood for ctDNA analysis will be collected prior to chemotherapy administration at baseline, Cycle 2 - Week 1 Day 1, Cycle 4 - Week 1 Day 1, and EOT.

Arms:
arm 1: Vigil immunotherapy 1.0 x 107 cells/injection; minimum of 4 to a maximum of 12 administrations every 28 days arm 2: Gemcitabine 675 mg/m2 IV at 10 mg/m2/min D1 and Docetaxel 75 mg/m2 IV starting on D8 and given every 21 days. arm 3: (i) oral temozolomide 100 mg/m2 daily (Days 1 - 5, total dose 500 mg/m2/cycle), (ii) irinotecan 50 mg/m2 daily (Days 1 - 5, total dose 250mg/m2/cycle), orally or irinotecan 20mg/m2 daily (Days 1 - 5, total dose 100mg/m2/cycle ), intravenously (iii) peg-filgrastim 100μg/kg (Day 6) subcutaneously (optional and may be administered at home), and (iv) Vigil 1.0 x 107 cells/injection, intradermally on Day 15 and every 3 weeks thereafter. One cycle = 21 days.

Interventions: intervention 1: Vigil intervention 2: Temozolomide intervention 3: Irinotecan intervention 4: Gemcitabine intervention 5: Docetaxel
Intervention descriptions: intervention 1: Vigil 1.0 x 10e7 cells/injection, minimum of 4 to a maximum of 12 administrations. intervention 2: oral temozolimidetemozolomide 100 mg/m2 daily (Days 1 - 5, total dose 500 mg/m2/cycle) intervention 3: irinotecan 50 mg/m2 daily (Days 1 - 5, total dose 250mg/m2/cycle), orally or irinotecan 20mg/m2 daily (Days 1 - 5, total dose 100mg/m2/cycle ), intravenously intervention 4: 675 mg/m2 IV at a rate of 10 mg/m2/min on Day 1 and Day 8 every 21 days intervention 5: 75 mg/m2 IV administered on Day 8 and every 21 days

Protocol document:
Prot_SAP_000.pdf: 
CL-PTL-121
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: 
PROTOCOL NUMBER: 
IND NUMBER: 
NCT NUMBER: 
PROTOCOL DATE: 
IRB APPROVAL DATE: 
A two-part Phase IIb Trial of Vigil (bi-
shRNAfurin and GMCSF Augmented 
Autologous Tumor Cell 
Immunotherapy) in Ewing’s Sarcoma 
CL-PTL-121
014205 
NCT02511132 
Amendment 3 dated March 29, 2017 
April 12, 2017 


 
 
PROTOCOL TITLE: 
A two-part Phase IIb Trial of Vigil (bi-shRNAfurin 
and GMCSF Augmented Autologous Tumor Cell 
Immunotherapy) in Ewing’s Sarcoma 
PROTOCOL #: 
CL-PTL-121 
STUDY AGENT: 
Vigil formerly known as FANG (bi-shRNAfurin 
and GMCSF 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 3 dated March 29, 2017 
Amendment 2 dated September 25, 2015 
Amendment 1 dated June 25, 2015 
Initial Protocol dated March 5, 2015 
 
 
 
 


Protocol No. CL-PTL-121 
CONFIDENTIAL 
AM3 03/29/17 
Page 2 
 
INVESTIGATOR PROTOCOL SIGNATURE PAGE 
 
 
PROTOCOL TITLE: 
A two-part Phase IIb Trial of Vigil (bi-
shRNAfurin and GMCSF Augmented 
Autologous Tumor Cell Immunotherapy) in 
Ewing’s Sarcoma 
PROTOCOL #: 
CL-PTL-121 
SPONSOR: 
Gradalis, Inc. 
2545 Golden Bear Drive, Suite 110 
Carrollton, TX 75006 
Phone: (214) 442-8124 
Fax: (214) 442-8101 
PROTOCOL DATE: 
Amendment 3 dated March 29, 2017 
Amendment 2 dated September 25, 2015 
Amendment 1 dated June 25, 2015 
Initial Protocol dated March 5, 2015 
 
 
 
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 
 


Protocol No. CL-PTL-121 
CONFIDENTIAL 
AM3 03/29/17 
Page 3 
 
TABLE OF CONTENTS 
INVESTIGATOR PROTOCOL SIGNATURE PAGE ....................................................... 2 
ABBREVIATIONS........................................................................................................... 5 
SYNOPSIS 
............................................................................................................ 7 
 
INTRODUCTION ................................................................................................ 14 
 
Summary ................................................................................................. 14 
 
Relapsed and Refractory Ewing’s sarcoma ............................................. 14 
 
VIGIL 
 ................................................................................................. 19 
 
Vigil Phase II Trial: Ovarian Cancer ......................................................... 22 
 
Vigil Phase I Trial:  Ewing’s Sarcoma Subset .......................................... 26 
 
Efficacy Evidence of Vigil in Ewing’s Sarcoma ........................................ 27 
 
STUDY RATIONALE ......................................................................................... 33 
 
PART 1 OBJECTIVES ....................................................................................... 33 
 
Primary objective(s) ................................................................................. 33 
 
Secondary objective(s) ............................................................................ 33 
 
PART 2 OBJECTIVES ....................................................................................... 34 
 
Primary objective ..................................................................................... 34 
 
Secondary Objectives .............................................................................. 34 
 
STUDY DESIGN ................................................................................................. 34 
 
STUDY POPULATION ....................................................................................... 36 
 
Sample Size ............................................................................................. 36 
 
Tissue Procurement Inclusion Criteria ..................................................... 36 
 
Tissue Procurement Exclusion Criteria .................................................... 36 
 
Study Enrollment Inclusion Criteria .......................................................... 37 
 
Study Enrollment Exclusion Criteria ......................................................... 38 
 
Withdrawal ............................................................................................... 38 
 
INVESTIGATIONAL PLAN ................................................................................ 39 
 
Patient Registration and Enrollment ........................................................ 39 
 
Tumor Procurement ................................................................................. 39 
 
Vigil Immunotherapy Manufacturing ........................................................ 39 
 
Study Treatment Administration ............................................................... 40 
 
Concomitant Medications and Supportive Care ....................................... 42 
 
Toxicity ..................................................................................................... 43 
 
Schedule of Assessments ........................................................................ 43 
 
CONDUCT OF THE STUDY .............................................................................. 48 
 
Ethics and Regulatory Considerations ..................................................... 48 
 
IRB ........................................................................................................... 48 
 
Written Informed Consent ........................................................................ 48 


Protocol No. CL-PTL-121 
CONFIDENTIAL 
AM3 03/29/17 
Page 4 
 
 
Confidentiality of Records ........................................................................ 49 
 
Modification of Protocol ............................................................................ 49 
 
Protocol Questions and Deviations .......................................................... 50 
 
Data Safety Monitoring Board .................................................................. 50 
 
EVALUATION OF TUMORS .............................................................................. 50 
 
Tumor Measurements and Response (RECIST 1.1) ............................... 50 
 
DRUG INFORMATION ....................................................................................... 50 
 
Vigil Investigational Product ..................................................................... 50 
 
Safety Analysis ........................................................................................ 51 
 
OCCUPATIONAL SAFETY................................................................................ 52 
 
ADVERSE EVENTS ........................................................................................... 52 
 
Attribution of Causality ............................................................................. 53 
 
Expected Side Effects .............................................................................. 53 
 
Recording of an Adverse Event ............................................................... 54 
 
Serious Adverse Event Reporting ............................................................ 54 
 
PATIENT COMPLETION AND WITHDRAWAL ................................................. 54 
 
Indication for Taking Patients Off Study ................................................... 54 
 
PART 1 STATISTICAL CONSIDERATIONS ..................................................... 55 
 
Sample Size Justification ......................................................................... 55 
 
Analysis Populations ................................................................................ 55 
 
Efficacy Analyses ..................................................................................... 55 
15.3.1 
Primary Efficacy Analysis ..................................................................... 55 
15.3.2 
Secondary Efficacy Analyses ............................................................... 56 
 
Safety Analyses ....................................................................................... 56 
 
PART 2 STATISTICAL CONSIDERATIONS ..................................................... 56 
 
DOCUMENTATION ............................................................................................ 56 
 
Case Report Form (CRF) Procedures ..................................................... 57 
APPENDIX A 
.......................................................................................................... 58 
Karnofsky Performance Scale ......................................................................... 58 
APPENDIX B 
.......................................................................................................... 59 
Schedule Of Assessments .............................................................................. 59 
APPENDIX C 
.......................................................................................................... 61 
NCI Common Toxicity Criteria For Adverse Events (CTCAE), Version 4.03 ... 61 
REFERENCES 
.......................................................................................................... 62 
 


Protocol No. CL-PTL-121 
CONFIDENTIAL 
AM3 03/29/17 
Page 5 
 
ABBREVIATIONS 
 
Abbreviation 
Term 
AE 
Adverse event 
ALT 
Alanine transaminase (also referred to as SGPT) 
ANC 
Absolute neutrophil count 
APC 
Antigen Presenting Cells 
AST 
Aspartate transaminase (also referred to as SGOT) 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CD 
Cluster of differentiation 
CMV 
Cytomegalovirus 
CO2 
Total carbon dioxide  
cCR 
Clinically defined Complete Response 
CR 
Complete response 
CRF 
Case report form 
CTCAE 
Common Toxicity Criteria for Adverse Events 
CTL 
Cytotoxic T lymphocyte 
DC 
Dendritic cell(s) 
ESFT 
Ewing’s Sarcoma Family of Tumors 
ELISA 
Enzyme-Linked ImmunoSorbent Assay 
ELISPOT 
Enzyme-Linked ImmunoSorbent Spot 
ER 
Endoplasmic reticulum 
FANG 
bi-shRNAfurin and GMCSF Augmented Autologous 
Tumor Cell Immunotherapy 
FL 
Flt-3-Ligand 
GMCSF 
Granulocyte Macrophage-Colony Stimulating Factor 
GMP 
Good Manufacturing Practice 
GVAX 
GMCSF Secreting autologous or allogenic tumor 
vaccine 
HLA 
Human Leukocyte Antigen 
IEC 
Independent Ethics Committee 
IL 
Infiltrating lymphocytes 
IRB 
Institutional Review Board 
ITT 
Intent to Treat 
KPS 
Karnofsky Performance Score 
LAK 
Lymphokine-activated killer 


Protocol No. CL-PTL-121 
CONFIDENTIAL 
AM3 03/29/17 
Page 6 
 
Abbreviation 
Term 
LD 
Longest diameter 
LLC 
Large latent complex 
LPI 
Lead Principal Investigator 
MHC 
Major histocompatibility complex 
MLR 
Mixed lymphocyte reaction 
MR 
Mannose receptor 
NCI 
National Cancer Institute 
NED 
No evidence of disease 
NGS 
Next Generation Sequencing 
NK 
Natural Killer 
NKT 
Natural Killer T cell(s) 
NSCLC 
Non small cell lung cancer 
OS 
Overall Survival 
PBMC 
Peripheral Blood Mononuclear Cells 
PCR 
Polymerase chain reaction 
PD 
Progressive disease 
PFS 
Progression Free Survival 
PI 
Principal Investigator 
PR 
Partial response 
PS 
Performance Status 
RECIST 
Response Evaluation Criteria in Solid Tumors 
RFS 
Recurrence Free Survival 
SCLC 
Small cell lung cancer 
SLC 
Small latent complex 
TAA 
Tumor Associated Antigens 
TAP 
transporter associated with Ag processing 
TGF 
Transforming growth factor- 
TIL 
Tumor infiltrating lymphocytes 
TNF 
Tumor necrosis factor 
Treg 
Regulatory T cell 
TTR 
Time to recurrence 
ULN 
Upper limits of normal 
WNL 
Within normal limits 


Protocol No. CL-PTL-121 
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AM3 03/29/17 
Page 7 
 
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 with high-risk 
recurrent disease (relapse < 2 years) is generally < 10% at 5 years.  Moreover, of 
patients who progress after second line treatment, eighty percent do not achieve 
a second complete response and of these patients < 10% survive one year.  
Refractory patients to both frontline and second line therapy have even worse 
prognosis. 
 
In an effort to overcome limitations of first-generation immunostimulatory cancer 
vaccines, we designed a novel autologous whole cell vaccine, Vigil formerly 
known as FANG, 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 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 TGF1 and TGF2.  The induced 
immunomodulatory effect in Ewing’s sarcoma was reflected in positive IFNγ 
ELISPOT response to autologous tumor cells in 10/12 assayed patients, one 
RECIST Criteria partial response (PR), a second patient achieving no evidence 
of disease (NED) and a total 1-year survival (Kaplan Meier method) of 73%. 
 
Objective(s): 
 
Part 1 
Primary Objectives: 
 To determine and compare overall survival of patients with metastatic 
Ewing’s sarcoma refractory or intolerant to ≥ 2 prior lines of systemic 
chemotherapy treated with Vigil immunotherapy vs. 
gemcitabine/docetaxel. 
 
Secondary Objectives: 
 To determine the γIFN ELISPOT conversion rate of subjects treated with 
Vigil immunotherapy vs gemcitabine/docetaxel. 


Protocol No. CL-PTL-121 
CONFIDENTIAL 
AM3 03/29/17 
Page 8 
 
 To determine and compare the proportion of patients surviving 1 year 
when treated with Vigil immunotherapy vs gemcitabine/docetaxel. 
 To determine and compare the overall survival of patients with relapsed or 
refractory Ewing’s sarcoma who are IFNγ ELISPOT negative at baseline 
and are treated with Vigil immunotherapy vs. gemcitabine/docetaxel. 
 To determine and compare the progression free survival of patients with 
metastatic Ewing’s sarcoma refractory or intolerant to ≥ 2 prior lines of 
systemic chemotherapy treated with Vigil immunotherapy vs 
gemcitabine/docetaxel. 
 To determine and compare the objective response rate (RECIST 1.1) of 
patients with metastatic Ewing’s sarcoma refractory or intolerant to ≥ 2 
prior lines of systemic chemotherapy treated with Vigil immunotherapy vs 
gemcitabine/docetaxel. 
 To determine and compare the safety profile of Vigil immunotherapy vs 
gemcitabine/docetaxel in patients with relapsed or refractory Ewing’s 
sarcoma. 
 
Part 2 
Primary Objective: 
 To determine safety profile of Vigil immunotherapy in combination with 
irinotecan and temozolomide in patients with metastatic Ewing’s sarcoma 
refractory or intolerant to at least 1 prior line of systemic chemotherapy. 
 
Secondary Objectives: 
 To determine the γIFN ELISPOT conversion rate of patients dosed with 
Vigil immunotherapy in combination with irinotecan and temozolomide. 
 To determine the progression free survival of patients dosed with Vigil 
immunotherapy in combination with irinotecan and temozolomide. 
 To determine the overall survival of patients with relapsed or refractory 
Ewing’s sarcoma who are IFNγ ELISPOT negative at baseline and are 
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 at least 1 prior line 
of systemic chemotherapy treated with Vigil immunotherapy dosed with 
Vigil immunotherapy in combination with irinotecan and temozolomide. 
Part 1 Methodology: 
This is a multicenter, 1:1 randomized Phase IIb study of intradermal autologous 
Vigil immunotherapy (1.0 x 107 cells/injection; minimum of 4 to a maximum of 12 
administrations) versus gemcitabine / docetaxel in patients with metastatic 
Ewing’s sarcoma Family of Tumors (ESFT) refractory or intolerant to at least 2 
prior lines of chemotherapy.  Patients undergoing a standard surgical procedure 
(e.g., tumor biopsy or palliative resection) may have tumor tissue harvested for 
manufacture of investigational product.  Patients meeting eligibility criteria 
including manufacture of a minimum of 4 immunotherapy doses will be 
randomized to receive either (1) intradermal Vigil every 28 days for 4-12 


Protocol No. CL-PTL-121 
CONFIDENTIAL 
AM3 03/29/17 
Page 9 
 
administrations, or (2) gemcitabine 675 mg/m2 IV at 10 mg/m2/min D1 and D8 
and docetaxel 75 mg/m2 IV D8 every 21 days.  The primary trial objective is to 
determine the overall survival of patients treated with Vigil versus 
gemcitabine/docetaxel.  Randomization may occur as early as vaccine is 
released (typically 3 - 4 weeks following tumor procurement) but no later than 8 
weeks following tumor procurement.  Randomization of patients will be stratified 
by Karnofsky Performance Status (KPS) ≥ 80% vs < 80%. 
 
Patients will be managed in an outpatient setting.  Hematologic function, liver 
enzymes, renal function and electrolytes will be monitored monthly.  Blood for 
immune function analyses including IFNγ-ELISPOT analysis of cytotoxic T cell 
response to autologous tumor antigens will be collected at tissue procurement, 
baseline, and prior to product administration at Cycles 2, 4, end of treatment, and 
every 6 months thereafter.   
 
Part 2 Methodology: 
Based on the limited accrual to Part 1 of this study, Gradalis is opening Part 2 of 
this clinical protocol to assess the safety of Vigil immunotherapy in combination 
with irinotecan and temozolomide.  Part 2 will be conducted at the same centers 
as Part 1, studying intradermal autologous Vigil cancer vaccine (1.0 x 107 
cells/injection; minimum of 4 to a maximum of 12 administrations) in patients with 
metastatic Ewing’s sarcoma Family of Tumors (ESFT) refractory or intolerant to 
at least 1 prior line of chemotherapy.  Patients undergoing a standard surgical 
procedure (e.g., tumor biopsy or palliative resection) may have tumor tissue 
harvested for manufacture of investigational product.  Patients meeting eligibility 
criteria including manufacture of a minimum of 4 immunotherapy doses of Vigil 
will be registered to receive: (i) oral temozolomide 100 mg/m2 daily (Days 1 – 5, 
total dose 500 mg/m2/cycle), (ii) irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 
250mg/m2/cycle), orally or irinotecan 20mg/m2 daily (Days 1 – 5, total dose 
100mg/m2/cycle ), intravenously (iii) peg-filgrastim 100μg/kg (Day 6) 
subcutaneously (optional and may be administered at home), and (iv) Vigil 1.0 x 
107 cells/injection, intradermally on Day 15 and every 3 weeks thereafter.  One 
cycle = 21 days.  Registration onto Part 2 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. 
 
Patients 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 Day 15 Vigil administration at Cycles 2, 4, 
end of treatment, and every 6 months thereafter.  Blood for ctDNA analysis will 
be collected prior to chemotherapy administration at baseline, Cycle 2 – Week 1 
Day 1, Cycle 4 – Week 1 Day 1, and EOT. 
 


Protocol No. CL-PTL-121 
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Page 10 
 
Number of Patients: 
 
Part 1: Approximately 62 patients will be enrolled. 
Part 2: Approximately 6-9 patients will be enrolled. 
Tissue Procurement Inclusion Criteria: 
 
Patients 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). 
5. Metastatic disease 
6. Refractory or intolerant to ≥ 2 lines of systemic chemotherapy (Part 1) or 
Refractory or intolerant to at least 1 line of systemic chemotherapy (Part 
2). 
7. Planned standard of care surgical procedure (e.g., tumor biopsy or 
palliative resection or thoracentesis) and expected availability of a 
cumulative mass of ~10-30 grams tissue (“golf-ball” size) or pleural fluid 
estimated volume ≥ 500mL (must be primary tap) for immunotherapy 
manufacture. 
8. Tumor intended for immunotherapy manufacture is not embedded in bone 
and does not contain luminal tissue (e.g. bowel, ureter, bile duct). 
9. Ability to understand and the willingness to sign a written informed 
consent document for tissue harvest. 
 
Tissue Procurement Exclusion Criteria: 
 
Patients 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 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. 


Protocol No. CL-PTL-121 
CONFIDENTIAL 
AM3 03/29/17 
Page 11 
 
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 history of allergies or sensitivities to gentamicin. 
6. Known hypersensitivity reactions to docetaxel or to other drugs formulated 
with polysorbate 80 that would preclude treatment with docetaxel (Part 1 
only). 
7. 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. 
8. Known HIV or chronic Hepatitis B or C infection. 
 
 
Study Enrollment Inclusion Criteria: 
 
Patients will be eligible for registration if they meet all of the following inclusion 
criteria: 
 
1. Successful manufacturing of at least 4 vials of Vigil. 
2. Karnofsky performance status (KPS) ≥60% (Part 1) or KPS ≥80% (Part 2). 
3. Estimated survival ≥ 4 months (Part 1) or estimated survival of ≥6 months 
(Part 2). 
4. Normal organ and marrow function as defined below: 
Absolute granulocyte count 
≥1,500/mm3 
Absolute lymphocyte count 
≥400/mm3 
Platelets 
≥100,000/mm3 
Total bilirubin 
≤ institutional upper limit of normal 
AST(SGOT)/ALT(SGPT) 
≤2x institutional upper limit of normal
Creatinine 
<1.5 mg/dL 
5. Subject has recovered to CTCAE Grade 1 or better from all adverse 
events associated with prior therapy or surgery.  Pre-existing motor or 
sensory neurologic pathology or symptoms 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. 
 
 
 
 


Protocol No. CL-PTL-121 
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AM3 03/29/17 
Page 12 
 
Study Enrollment Exclusion Criteria: 
 
In addition to the procurement exclusion criteria, patients will NOT be eligible for 
study registration and randomization if meeting any of the following 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. 
 
Medication and Doses:  Autologous Vigil will be supplied by Gradalis, Inc. 
 
Part 1 
Patients will receive either 1 x 107 cells via intradermal injection every 28 days for 
a 4 - 12 administrations (depending on the quantity of Vigil manufactured from 
surgical specimens), or gemcitabine 675 mg/m2 IV at 10 mg/m2/min D1 and D8 
and docetaxel 75 mg/m2 IV D8 every 21 days. 
 
Part 2 
Patients will receive (i) oral temozolomide 100 mg/m2 daily (Days 1 – 5, total 
dose 500 mg/m2/cycle), (ii) irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 
250mg/m2/cycle), orally or irinotecan 20mg/m2 daily (Days 1 – 5, total dose 
100mg/m2/cycle), intravenously (iii) peg-filgrastim 100μg/kg (Day 6) 
subcutaneously (optional and may be administered at home), and Vigil 1.0 x 107 
cells/injection, intradermally on Day 15 and every 3 weeks thereafter. One cycle 
equals 21 days.   
Duration:  Patients may receive repeat cycles of treatment until disease 
progression, unacceptable toxicity, withdrawal of consent or other criterion is met 
for discontinuation from study.  Patients randomized to Vigil will receive up to 12 
doses depending upon the quantity of Vigil manufactured from the surgical 
specimen.   
Efficacy Assessments: 
 Overall survival (OS) 
 Progression Free Survival (PFS) 
 Response Rate (RECIST 1.1) 
Safety Assessments: 
 Laboratory assessments 
 Physical examination, performance status, height, weight, temperature, 
blood pressure, and pulse 
 Toxicity:  CTCAE v 4.03 


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Exploratory Assessments: 
Circulating 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-L expression and TIL populations. 


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 INTRODUCTION 
 
 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 of 
diagnosis is 14 -15 years old (Cotterill, Ahrens et al. 2000; Stahl, Ranft et al. 2011).  
Molecularly the disease is distinguished by a translocation of the EWSR1 gene on 
chromosome 22q12 with in FLI1 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 (11;22)(q24;12) (Arvand and Denny 
2001).   
 
Patients with initially diagnosed localized disease have a disease-free survival rate 
(EFS) of 60-70% with standard 5-drug chemotherapy regimen (VAC +IE or VIDE), 
while patients with metastatic disease have a EFS 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 patients enrolled in trials from 1979 to 1993, the 5 year 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, the toxicity profile has also increased during the multi-agent, 
front line chemotherapy treatment course (Esiashvili, Goodman et al. 2008).  Second or 
greater line therapy regimen only show temporary benefit with dismal survival outcome. 
 
 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 with 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).  Patients who relapse have a marked reduction in overall 
survival with published results in 122 (good prognostic) patients of only 12%, 5 year 
survival, and 19% 2-year survival (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 within the first 2 years after initial 
diagnosis, which make up 72% of relapses (Stahl, Ranft et al. 2011), the 2-year OS 


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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). 
 
Ewing’s Sarcoma can present with localized or metastatic disease at time of diagnosis. 
Presentation with localized disease is associated with a 6-year event free survival 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 
significantly deteriorates to 28% with OS 29% (Guerney, Swensen et al. 1999; Miser, 
Goldsby et al. 2007; Leavey, Mascarenhas et al. 2008).  Once first recurrent disease 
occurs survival is much worse with a 5 year OS of only 13% (Bacci, Ferrari et al. 2003).  
Recent analysis of 262 first recurrence Ewing’s Sarcoma patients treated on INT0091 
revealed similar results with 12% 5 year OS, and only 19% 2 year survival.  This 
analysis included 122 (good prognostic) patients who had local recurrence manageable 
with only surgery and/or irradiation (Leavey, Mascarenhas et al. 2008).  
 
The NCCN guidelines do not provide standard of care recommendations for second-
line treatment.  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).  However, none of these regimens have been determined to have 
significant advantage in a randomized comparative clinical assessment (Gaspar, 
Hawkins et al. 2015).  The Irinotecan/Temozolomide regimen is the most common 
second-line regimen utilized.  Irinotecan/Temozolomide shows moderate durable 
responses (Casey, Wexler et al. 2009; Raciborska, Bilska et al. 2013; Palmerini, Jones 
et al. 2016).  Although results of progression free survival (PFS) and OS do not 
significantly vary between this and other dose intense regimens (Yildiz, Sen et al. 
2014), PFS in second-line varies 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). 
 
Prognostic markers for clinical benefit in second relapse is the response status to prior 
therapy regimen (i.e. responder 5 year survival 48% vs. non-responder 0% respectively 
to second-line chemotherapy, p=0.0001), (Barker, Pendergrass et al. 2005). Another 
study revealed similar results: of 161 patients treated with second-line chemotherapy, 
had a 5-year survival of 25% in responders (CR/PR)  compared to non-responders 
(SD/PD), (2-year survival 45% vs 10%) (Rasper, Jabar et al. 2014).  Few patients 
respond to second-line achieving a second remission and survival at 5 years is 
severely limited (Table 1) (Rodriguez-Galindo, Billups et al. 2002; Bacci, Ferrari et al. 
2003; Shankar, Ashley et al. 2003; Barker, Pendergrass et al. 2005; McTiernan, 
Cassoni et al. 2006; Leavey and Collier 2008; Stahl, Ranft et al. 2011), particularly in 
patients with high risk disease (time to relapse (TTR) within 2 years of front-line 
treatment).  In one large retrospective analysis of 714 patients in first relapse, the 1-


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year OS was 43%, 5-year OS was 13%, and 10 year OS was 9% (Stahl, Ranft et al. 
2011).   
 
Table 1.  Relapse from Time of Initial Treatment 
PI 
Number 
Patients 
Primary 
Metastatic 
Disease at 
Diagnosis 
5-year 
Overall 
Suvival 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. 
 
Second-line chemotherapy for relapsed Ewing’s sarcoma generally has had limited 
efficacy with only 9% to 13% of patients achieving a second disease free remission 
(Bacci, Ferrari et al. 2003; Barker, Pendergrass et al. 2005; Rasper, Jabar et al. 2014).  
There is no standard of care for third-line treatment.  Multiple groups have evaluated 
the combination of gemcitabine and docetaxel in the treatment of pediatric sarcomas 
with particular attention to Ewing’s sarcoma (Table 2) (Navid, Billups et al. 2008; Mora, 
Cruz et al. 2009; Fox, Patel et al. 2012; Rapkin, Qayed et al. 2012). Both Gemcitabine 
and Docetaxel have been evaluated separately in phase I pediatric solid tumor studies 
establishing MTD and toxicities in pediatric populations (Seibel, Blaney et al. 1999; 
Reid, Qu et al. 2004).  Gemcitabine with Docetaxel showed a 53% response rate in 
adult uterine leiomyosarcoma (Hensley, Maki et al. 2002) and showed superior activity 
in direct comparison to gemcitabine alone (Maki, Wathen et al. 2007).  Further studies 
in adult sarcoma studies showed response rates ranging 16-43% specifically 2 
previously treated Ewing’s patients responded with 1 PR and 1 SD (Leu, Ostruszka et 
al. 2004).  Gemcitabine in combination with docetaxel began to be used in pediatric 
settings and an early report evaluated 14 patients with refractory bone sarcomas and 
showed an ORR of 29% with one SD response out of 2 Ewing’s patients (Navid, Willert 
et al. 2008).  A second report of 10 patients included 6 Ewing’s patients, of which 4 
experienced response (3 CR and 1 PR) and one had stable disease (Mora, Cruz et al. 
2009). Of 14 patients with Ewing’s sarcoma in another study, two achieved PR and six 
SD as best response (Fox, Patel et al. 2012).  Toxicity primarily involves transient 
hematopoietic suppression which has been managed with use of Filgrastim and dose 
reduction. 
 
 


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Table 2. Gemcitabine in Combination with Docetaxel in Refractory Pediatric Sarcomas 
Reference 
Total 
Number 
pts 
Efficacy 
Overall 
Gemcitabine 
Dosing 
Docetaxel 
Dosing 
Number 
Ewings pts 
Ewings 
Respones 
(Fox, Patel 
et al. 
2012) 
53 
9% PR 
675 mg/m2, 90 min, 
D1,8 
75 mg/m2 D8 
14 
2 PR, 6 SD 
(Mora, 
Cruz et al. 
2009) 
10 
50% ORR, 30% 
SD 
1000 mg/m2, 90 min, 
D1,8 
100 mg/m2 D8 (2-
4hr) 
6 
3 CR, 1 PR, 1 
SD 
(Rapkin, 
Qayed et 
al. 2012) 
19 
11% ORR, 39% 
SD 
675 mg/m2, 90 min, 
D1,8 
75 mg/m2 D8 (1hr) 
2 
none 
(Navid, 
Willert et 
al. 2008) 
14 
29% ORR, 14% 
SD 
675 mg/m2, 90 min, 
D1,8 
100 (or 75) mg/m2 
D8 (1hr) 
2 
1 SD 
*all received Neupogen starting on Day 9 till recovery 
Overall Response Rate (ORR); Stable Disease (SD); Partial Response (PR); Complete Response (CR) 
 
Despite the use of different chemotherapy regimen, the majority of relapsed patients 
will not achieve a second or third remission, or show only temporary benefit followed by 
relapse and a grim prognosis.  
 
To date, there is no proven significant benefit of third-line chemotherapy.  Prognostic 
markers for clinical benefit in second relapse is the response status to prior therapy 
regimen (i.e. responder 5 year survival 48% vs. non-responder 0% respectively to 
second-line chemotherapy, p=0.0001), (Barker, Pendergrass et al. 2005). Another 
study revealed similar results: of 161 patients treated with second-line chemotherapy, 
had a 5-year survival of 25% in responders (CR/PR)  compared to non-responders 
(SD/PD), (2-year survival 45% vs 10%) (Rasper, Jabar et al. 2014).  In a relatively 
large retrospective analysis 86% of 195 patients, did not achieve a second remission 
and 97% had died with a median of 11.7 mo, while no patient achieved disease control.  
For the 26 of 195 patients who did go into a second remission, 12 relapsed again 
(10/12 died and 2 were living with uncontrolled disease at 6 and 13 months)(Bacci, 
Ferrari et al. 2003). 
 
This reflects the limitations of conventional cytotoxic chemotherapy underscores the 
desperate need for treatment development in the metastatic Ewing’s sarcoma in high-
risk first relapse patients(TTR within 2 years of front line treatment) or in second 
relapse. 
 
Immune escape mechanisms also appear to play a role in the development and 
metastasis of Ewing’s Sarcoma.  Patients with primary disseminated disease showed 
increased frequency of T-regulatory cells in the bone marrow compared to localized 
disease at presentation (Brinkrolf, Landmeier et al. 2009).  Factors associated with 
survival benefit are increased number of tumor infiltrating CD8+ T-lymphocytes 
(p=0.05) as well as lower tumor volume (<200 ml) at diagnosis (Berghuis, Santos et al. 
2011).  Furthermore, the EWS/FLI-1 fusion protein itself presenting uniquely on EWS 


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tumor cells and absent on normal cells, may be a driver neoantigenic target capable of 
eliciting an effective cytotoxic immune response (Liu, Huang et al. 2012; Peng, Huang 
et al. 2014).  Re-activation of immunogenic antitumor response as a treatment strategy 
for EWS could be exploited either in a monotherapy or in combination with lymphocyte-
sparing chemotherapy agents like irinotecan and temozolomide, which have been 
found with lower rates of lymphopenia in treated patients (Kushner, Kramer et al. 
2006). 
 
Thus, preliminary results in 83% of Ewing’s Sarcoma patients demonstrated actual 
“turn on” of activated T cells in circulation with marked capacity to release γIFN at 
exposure to unmanipulated autologous tumor cells as defined by γIFN ELISPOT assay 
after treatment with Vigil immunotherapy.  ELISPOT activity has been verified by Mt. 
Sinai and plates are third party read, Zellnet, Fort Lee, NJ with a high confidence in this 
functional immune assessment.  ELISPOT results have also demonstrated significant 
correlation with survival and TTR in previously published work involving other solid 
tumor patients (Senzer, Barve et al. 2013; Oh, Barve et al. 2016).  Moreover, these 
results have correlated with tumor responses (1 CR/NED, 1 PR) in 2 Ewing’s sarcoma 
patients.  One who observed complete regression and is described in prior publication 
(Ghisoli, Rutledge et al. 2017).  Notably γIFN ELISPOT level appeared to correlate 
lightly to disease response and progression (Figure 1) in this patient.  
 
 
Figure 1. Case Report 1 (F-062). Relapsed Ewing’s Sarcoma Response to Vigil 
 
 


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 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 
TGF1, 2 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 TGF 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). 
 
Eighty patients in total (54 females, aged 13-84 / 26 males, aged 12-76) have been 
entered into Phase I trial involving 19 solid tumor cancers.  Four hundred sixty-four 
Vigil injections were administered and no ≥ Grade 3 toxic events attributed to Vigil have 
been observed.  Dose variance ranged from 2 x 106 cells/injection to 2.5 x 107 
cells/injection via intradermal injection 1x/month for a minimum of 4 months.  The 
prolonged survival shown in Figure 2 involving predominantly advanced stage, heavily 
pretreated patients suggests further safety support as it is greater than historical 
expectation (Senzer, Barve et al. 2013). 
 


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Figure 2. 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 3). 
 
 


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Figure 3. Correlated survival and immune response in first 30 patients entered into 
Vigil Phase I trial 
 
These results have recently been updated.  Sixty-seven of these Phase I patients have 
now undergone ELISPOT γIFN testing.  Fifty-two demonstrated change from negative 
at baseline (prior to Vigil treatment) to positive γIFN ELIPSOT reactivity after Vigil.  
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 (Figure 4). 
 


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Figure 4. Four-year update of all Phase I patients with γIFN ELISPOT analysis. 
 
 Vigil Phase II Trial: Ovarian Cancer 
 
All ovarian cancer patients who underwent γIFN ELISPOT assay testing underwent a 
sub analysis from the Phase I population to validate the efficacy findings.  Results 
demonstrated correlation with survival advantage in ovarian cancer patients in which 
Vigil induced above threshold levels of circulating γIFN expressive T cells to 
autologous tumor (Figure 5).   
 


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Figure 5. Subset advanced ovarian population analysis of γIFN ELISPOT response to 
survival 
 
A Mary Crowley Cancer Research Center registry database, randomly selected 
matched Phase I trial ovarian cancer patients who did not receive Vigil or TAG.  This 
historical sub-analysis revealed survival advantage of Vigil ELISPOT positive patients 
to the historical control (MCCRC database).  Additionally, ELISPOT negative patients 
had similar survival duration as the matched database (Figure 6).  Thus, 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 (CR) 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.  Crossover was allowed at time of relapse 
for patientsrandomized to standard of care therapy.  Results have been published by 
Oh et al (Oh, Barve et al. 2016). 
 


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Figure 6. Phase I Ovarian versus MCCRC Match Control Ovarian, Overall Survival 
 
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 CTL-PTL-105 was closed to further 
accrual and patients not yet randomized in consolidation chemotherapy section were 
allowed to enter into 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.  Median time to Vigil 
relapse by KM analysis (log rank) was 604 days vs. control standard of care, 377 days 
(p=0.033) 
 


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Figure 7. Correlated survival of Vigil compared to control in Ovarian Cancer (CL-PTL 
105) with immune responses 
 
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 γIFN 
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 γIFN 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).  One year 
follow up analysis of all patients entered into study CL-PTL-105 was recently 
completed.  Results demonstrate continued benefit (Figure 8). 
 


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Figure 8. Comparison of Phase II control ovarian cancer patients to Vigil RFS from 
time of procurement and time of treatment 
 
 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 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 patients received Vigil.  Fourteen 
concurrent patients (control) did not receive Vigil after undergoing similar surgery and 
Vigil construction.  Nine of the latter were unable to have vaccine released (6 
contaminants, 3 insufficient viable tumor cells) and 5 chose other treatment 
management.  All products constructed fulfilled release criteria of GMCSF production 
and TGFβ1, β2 knockdown.  See demographics in Table 3. 
 
Table 3.  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 Tx (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 


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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.  AE’s 
reported are shown in Table 4. 
 
Table 4. Phase 1 (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 
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 AE’s were observed to Vigil.  There were 11 SAEs reported 
involving 7 participants.  None of the SAEs were related to Vigil.   
 
 Efficacy Evidence of Vigil in Ewing’s Sarcoma 
 
Survival advantage, however, was demonstrated to Vigil compared to the matched 
comparator. 
Figure 9 shows the results of the Kaplan-Meier analysis of the survival data. Overall 
survival from time of procurement comparing these 2 concurrent groups (Vigil vs. No 
Vigil) revealed a 17+ month improvement in survival in the Vigil treated patients 
compared to the No Vigil control group (MC) from a mutually comparable time point, 


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time of tissue procurement (for Vigil construction).  Focus on the Vigil group from time 
of treatment revealed a median survival of 689 days. 
 
 
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 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 MC 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 indication risk factor scores (Wheler score (Wheler, 
Tsimberidou et al. 2012) and, 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 5). 
 
 
 


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Table 5.  Prognostic Indicator Studies of Ewing’s Sarcoma 
 
 
A Phase llb randomized open label trial (CL-PTL-121) comparing Vigil vs. standard of 
care chemotherapy (Gemcitabine/Taxol) in metastatic Ewing’s Sarcoma patients was 
initiated and enrolled the first patient 13 months ago.  Patients with refractory or 
recurrent disease failing ≥2 prior systemic treatment lines and undergoing surgical 
disease resection were targeted (for trial CL-PTL-121).  The trial is open to enrollment 
at 10 sites (5 in development) across the USA but only 13 patients have registered into 
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 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.  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 have received Vigil in studies CL-PTL-101 and 
CL-PTL-121 (21 patients) vs. matched controls (n=14) and randomized controls (total 
n=22) revealed further support of potential benefit to Vigil in third-line Ewing’s Sarcoma 
(Figure 11).  A Kaplan Meier survival of 524 (17.2 months) days was observed with 
Vigil. 
 
 


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Figure 10. Overall Survival Since Procurement on Study CL-PTL-121 Preliminary 
Results.  Two crossover patients censored.  Data updated as of 01/19/17.  
*RC=Randomized Control 
 
 
Figure 11. Study CL-PTL-101 and 121 Combination Data of Ewing’s Sarcoma.  Overall 
Survival Since Procurement.  Dataset updated 01/23/17.  *MC/RC = Matched 
Control/Randomized Control 


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Additionally, a long-term follow up of one of the two responding Phase 1 Ewing’s 
patients (Figure 1) (first patient response characterization is shown in Figure 12) 
revealed long term RECIST qualified response to Vigil.  A complete regression related 
to a second course of Vigil therapy (Ghisoli, Rutledge et al. 2017) was recently 
confirmed.  This patient had extensive disease initially and at rapid progression during 
frontline chemotherapy (TTR <2 years) underwent Vigil treatment with disease 
stabilization for > 6 months.  Following recurrence in her lungs, she underwent a 
second Vigil construction and treatment for third-line disease management.  Results 
yielded complete regression by PET scan.  ELISPOT response was shown with both 
Vigil treatments but was shown (in retrospect) to have deteriorated following the first 
Vigil treatment with recovery in immune response following the second Vigil treatment 
using the second recurrent disease sample for construction (Figure 1).  Overall, ten of 
12 (83%) of Ewing’s Sarcoma patients who received Vigil demonstrated marked 
elevation of effector T cells in circulation as demonstrated by γIFN ELISPOT assay 
(Figure 13) (Ghisoli, Barve et al. 2015; Ghisoli, Barve et al. 2016).  The ELISPOT 
assessments have been independently validated by Mt. Sinai Human Immune Core 
Monitoring facility (involving Ewing’s Sarcoma samples) (Figure 14).  Interestingly, very 
low cell loads 15/5 x 103 PBMC’s at a 1:3 ratio to the autologous tumor cell stimulant 
were required as the results were so highly positive that the utilized cell numbers had 
to be significantly diluted from standard ELISPOT routine in order to distinguish 
ELISPOTs on the assay. 
 
 
Figure 12. Case Report 2 (F-089).  Relapsed Ewing’s Sarcoma Response to Vigil 
 


Protocol No. CL-PTL-121 
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Figure 13. Immune Response by γIFN ELISPOT Assay Related to Vigil. 
 
Figure 14. Mt Sinai – Gradalis Lab:  Concurrent Comparison of γIFN ELISPOT 
Response.  Autologous Tumor Cells (pre-transfection) and PBMCs.  *ZellNet 
Consulting, Inc (Fort Lee, NJ) third party spot read out.  Results based on 3:1 ratio 
tumor to PBMC’s (15 x 103/5 x 103 cells) 
 


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 STUDY RATIONALE 
 
The results from the Phase I Vigil trial supported high confidence in safety (both short-
term and long-term through 3+ years), confirmed effective transgene expression (GM-
CSF) and RNAi [furin] silencing, and supported the triad rationale autologous tumor cell 
therapy with increased GMCSF expression and decreased TGFβ production, to 
modulate immunogenicity as shown by the correlation of IFNγ-ELISPOT 
responsiveness with overall survival (Senzer, Barve et al. 2012; Senzer, Barve et al. 
2013).  The preliminary evidence of safety, immune stimulation and clinical benefit from 
Vigil in patients with ESFT 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).  Less tumor burden is typically associated with greater delay in 
progression with other immunotherapies.  Thus, better control of delayed disease 
progression with increased response to chemotherapy (second line treatment involving 
Temozolomide/Irinotecan) in concurrence with Vigil immunotherapy in first relapse 
patient population is suggested.  Median time to progression of the Phase I EWS 
patients (third-line) was approximately 6 months compared to historical experience of 
similar patients of 2 months (Rodriguez-Galindo, Billups et al. 2002; Bacci, Ferrari et al. 
2003; Shankar, Ashley et al. 2003; Barker, Pendergrass et al. 2005; McTiernan, 
Cassoni et al. 2006; Leavey, Mascarenhas et al. 2008; Juergens, Daw et al. 2011; 
Stahl, Ranft et al. 2011; Tap, Demetri et al. 2012; Choy, Butrynski et al. 2014; Rasper, 
Jabar et al. 2014; Ghisoli, Barve et al. 2016). Thus, Part 2 combination of 
Vigil/Temozolomide/Irinotecan is justified. 
 
 PART 1 OBJECTIVES 
 
 Primary objective(s) 
 
 To determine and compare the overall survival of patients with metastatic 
Ewing’s sarcoma refractory or intolerant to ≥ 2 prior lines of systemic 
chemotherapy treated with Vigil immunotherapy vs. gemcitabine/docetaxel. 
 
 Secondary objective(s) 
 
 To determine the γIFN ELISPOT conversion rate of subjects treated with Vigil 
immunotherapy vs gemcitabine/docetaxel. 
 To determine and compare the proportion of patients surviving 1 year when 
treated with Vigil immunotherapy vs gemcitabine/docetaxel. 
 To determine and compare the overall survival of patients with metastatic 
Ewing’s sarcoma refractory or intolerant to ≥ 2 prior lines of systemic 
chemotherapy who are IFNγ ELISPOT negative at baseline and are treated 
with Vigil immunotherapy vs. gemcitabine/docetaxel. 


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 To determine and compare the progression free survival of patients with 
metastatic Ewing’s sarcoma refractory or intolerant to ≥ 2 prior lines of 
systemic chemotherapy treated with Vigil immunotherapy vs 
gemcitabine/docetaxel. 
 To determine and compare the objective response rate (RECIST 1.1) of 
patients with metastatic Ewing’s sarcoma refractory or intolerant to ≥ 2 prior 
lines of systemic chemotherapy treated with Vigil immunotherapy vs 
gemcitabine/docetaxel 
 To determine and compare the safety profile of Vigil immunotherapy vs 
gemcitabine/docetaxel in patients with relapsed or refractory Ewing’s 
sarcoma. 
 
 PART 2 OBJECTIVES 
 
 Primary objective 
 
 To determine safety profile of Vigil immunotherapy in combination with 
irinotecan and temozolomide in patients with metastatic Ewing’s sarcoma 
refractory or intolerant to at least 1 prior line of systemic chemotherapy. 
 
 Secondary Objectives 
 
 To determine the γIFN ELISPOT conversion rate of patients dosed with Vigil 
immunotherapy in combination with irinotecan and temozolomide. 
 To determine the progression free survival of patients dosed with Vigil 
immunotherapy in combination with irinotecan and temozolomide. 
 To determine the overall survival of patients with relapsed or refractory 
Ewing’s sarcoma who are IFNγ ELISPOT negative at baseline and are 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 at least 1 prior line of 
systemic chemotherapy treated with Vigil immunotherapy dosed with Vigil 
immunotherapy in combination with irinotecan and temozolomide. 
 
 STUDY DESIGN 
 
Part 1 
 
This is a multicenter, 1:1 randomized Phase IIb study of intradermal autologous Vigil 
immunotherapy (1.0 x 107 cells/injection; minimum of 4 to a maximum of 12 


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administrations) versus gemcitabine / docetaxel in patients with metastatic Ewing’s 
sarcoma Family of Tumors (ESFT) refractory or intolerant to at least 2 prior lines of 
chemotherapy.  Patients undergoing a standard surgical procedure (e.g., tumor biopsy 
or palliative resection) may have tumor tissue harvested for manufacture of 
investigational product.  Patients meeting eligibility criteria including manufacture of a 
minimum of 4 immunotherapy doses will be randomized to receive either (1) 
intradermal Vigil every 28 days for 4-12 administrations, or (2) gemcitabine 675 mg/m2 
IV at 10 mg/m2/min D1 and D8 and docetaxel 75 mg/m2 IV D8 every 21 days.  The 
primary trial objective is to determine the overall survival of patients treated with Vigil 
versus gemcitabine/docetaxel.  Randomization may occur as early as vaccine is 
released (typically 3 - 4 weeks following tumor procurement) but must occur no later 
than 8 weeks following tumor procurement.  Randomization of patients will be stratified 
by Karnofsky Performance Status (KPS) ≥ 80% vs < 80%. 
 
Patients will be managed in an outpatient setting.  Hematologic function, liver enzymes, 
renal function and electrolytes will be monitored monthly.  Blood for immune function 
analyses including IFNγ-ELISPOT analysis of cytotoxic T cell response to autologous 
tumor antigens will be collected at tissue procurement, baseline and and prior product 
administration at Cycles 2, 4, end of treatment, and every 6 months thereafter.   
 
 
Part 2 
 
Based on the limited accrual to Part 1 of this study, Gradalis is opening Part 2 of this 
clinical protocol to assess the safety of Vigil immunotherapy in combination with 
irinotecan and temozolomide.  Part 2 will be conducted at the same centers as Part 1, 
studying intradermal autologous Vigil cancer vaccine (1.0 x 107 cells/injection; 
minimum of 4 to a maximum of 12 administrations) in patients with metastatic Ewing’s 
sarcoma Family of Tumors (ESFT) refractory or intolerant to at least 1 prior line of 
chemotherapy.  Patients undergoing a standard surgical procedure (e.g., tumor biopsy 
or palliative resection) may have tumor tissue harvested for manufacture of 
investigational product.  Patients meeting eligibility criteria including manufacture of a 
minimum of 4 immunotherapy doses of Vigil will be registered to receive: (i) oral 
temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), (ii) irinotecan 
50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle), orally or irinotecan 20mg/m2 
daily (Days 1 – 5, total dose 100mg/m2/cycle ), intravenously (iii) peg-filgrastim 
100μg/kg (Day 6) subcutaneously (optional and may be administered at home), and 
Vigil 1.0 x 107 cells/injection, intradermally on Day 15 and every 3 weeks thereafter.  
One cycle = 21 days.  Registration onto Part 2 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. 
 
Patients 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, and prior to Day 15 Vigil 


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administration at Cycles 2, 4, end of treatment, and every 6 months thereafter.  Blood 
for ctDNA analysis will be collected prior to chemotherapy administration at baseline, 
Cycle 2 – Week 1 Day 1, Cycle 4 – Week 1 Day 1, and EOT. 
 
 STUDY POPULATION 
 
 Sample Size 
 
Approximately 62 patients will be enrolled into Part 1.  Approximately 6 to 9 patients will 
be enrolled into Part 2. 
 
 Tissue Procurement Inclusion Criteria 
 
Patients 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). 
5. Metastatic disease 
6. Refractory or intolerant to ≥ 2 prior lines of systemic chemotherapy (Part 1) or 
Refractory or intolerant to at least 1 line of systemic chemotherapy (Part 2). 
7. Planned standard of care surgical procedure (e.g., tumor biopsy or palliative 
resection or thoracentesis) and expected availability of a cumulative mass of 
~10-30 grams tissue (“golf-ball” size) for or pleural fluid estimated volume ≥ 
500mL (must be primary tap) immunotherapy manufacture. 
8. Tumor intended for immunotherapy manufacture is not embedded in bone and 
does not contain luminal tissue (e.g. bowel, ureter, bile duct). 
9. Ability to understand and the willingness to sign a written informed consent 
document for tissue harvest. 
 
 Tissue Procurement Exclusion Criteria 
 
Patients 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 


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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 history of allergies or sensitivities to gentamicin. 
6. Known hypersensitivity reactions to docetaxel or to other drugs formulated with 
polysorbate 80 that would preclude treatment with docetaxel (Part 1 only). 
7. 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. 
8. Known HIV or chronic Hepatitis B or C infection. 
 
 Study Enrollment Inclusion Criteria 
 
Patients will be eligible for registration if they meet all of the following inclusion criteria: 
 
1. Successful manufacturing of at least 4 vials of Vigil. 
2. Karnofsky performance status (KPS) ≥60% (Part 1) or KPS ≥80% (Part 2). 
3. Estimated survival ≥ 4 months (Part 1) or estimated survival of ≥6 months (Part 
2). 
4. Normal organ and marrow function as defined below: 
Absolute granulocyte count 
≥1,500/mm3
Absolute lymphocyte count 
≥400/mm3
Platelets 
≥100,000/mm3 
Total bilirubin 
≤ institutional upper limit of normal 
AST(SGOT)/ALT(SGPT) 
≤2x institutional upper limit of normal 
Creatinine 
<1.5 mg/dL 
5. Subject has recovered to CTCAE Grade 1 or better from all adverse events 
associated with prior therapy or surgery.  Pre-existing motor or sensory 
neurologic pathology or symptoms 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 document. 
 


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 Study Enrollment Exclusion Criteria 
 
In addition to the procurement exclusion criteria, patients will NOT be eligible for study 
registration and randomization if meeting any of the following 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. 
 
 Withdrawal 
 
Discontinuation of Study Treatment 
 
Patients will discontinue study treatment if any of the following occur: 
 
1. Disease progression by RECIST 1.1 criteria according to investigator 
assessment. 
2. The patient experiences unacceptable (≥Grade 3) toxicity felt to be related to 
treatment with the Vigil immunotherapy that persists for >2 weeks. (Part 1 or Part 
2) 
3. The patient experiences unacceptable (≥Grade 3) toxicity felt to be related to 
treatment with gemcitabine / docetaxel that persists for >2 weeks. Vigil therapy 
may be continued in monotherapy. (Part 1) 
4. The patient experiences unacceptable (≥Grade 3) toxicity felt to be related to 
treatment with irinotecan/temozolomide that persists for >2 weeks. Vigil therapy 
may be continued in monotherapy. (Part 2) 
5. Persisting Grade 3 or 4 toxicity unrelated to treatment, defined as failing to 
normalize within 4 weeks. 
6. Any Grade ≥ 3 allergic reactions related to the study agent. 
7. Grade 2 autoimmune reactions unless there is evidence of clinical benefit. 
8. 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. 
9. Cancer therapy other than protocol treatment. 
10. Non-compliant with protocol or treatment. 
11. Patient refuses to continue treatment. 
 
The date of and reason for discontinuation must be noted in the electronic Case Report 
Form (CRF).  Every effort should be made to complete the appropriate assessments. 


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 INVESTIGATIONAL PLAN 
 
 Patient Registration and Enrollment 
 
Written documentation of full, non-contingent IRB approval of the protocol and consent 
document must be on file before a patient can be registered.  Study participation 
begins once written informed consent is obtained.  Patients will be assigned a study 
identification number upon completion of the screening process. 
 
Two to eight weeks after tissue procurement has occurred eligibility will be reconfirmed 
by the study site.  The Research Nurse or Clinical Research Coordinator will register 
the patient. 
 
Please allow 48 hours for patient 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 (Part 1 and 2) and randomization (Part 1 only) by 
the Contract Research Organization. 
 
 Tumor Procurement 
 
Refer to the Tissue Procurement Manual for instructions. 
 
The cumulative equivalent of a “golf ball size” mass (~10-30 gm tissue) is optimal for 
vaccine manufacturing.  If surgeons have the option of collecting more tissue, more 
doses of vaccine may be prepared (up to 12 doses).  Vaccine 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 vaccine manufacture. 
 
 Vigil Immunotherapy Manufacturing 
 
Gradalis, Inc. will manufacture vaccine from the procured subject tumors.  Gradalis, 
Inc. will release vaccine once all release criteria have been met and eligibility has been 
confirmed. 
 
No tissue or vaccine will be given to the participant or site apart from the outlined 
clinical protocol. 
 
Any excess tumor tissue, not used for vaccine manufacture will be used towards Vigil 
vaccine research and process development assays. 


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 Study Treatment Administration 
 
Treatment will be administered on an outpatient basis. 
 
Schedule, Dose and Administration 
 
Part 1 
 
Subjects will be randomized to receive Vigil or gemcitabine / docetaxel according to the 
schedule outlined in Appendix B. 
 
Subjects randomized to Vigil will receive 1.0 x 107 cells via intradermal injection every 4 
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 will be used.  Subjects receiving Vigil will be observed for at 
least 30 minutes following administration.  
 
OR 
 
Patients randomized to gemcitabine / docetaxel will receive gemcitabine 675 mg/m2 IV 
at a rate of 10mg/m2/min (e.g., 675 mg/m2 should run over 67.5 minutes) on D1 and 
Day 8, and docetaxel 75 mg/m2 IV D8 every 21 days. 
 
Drug 
Dose 
Schedule 
Gemcitabine 
675 mg/m2 IV at a rate of 
10mg/m2/min 
Day 1 and Day 8 every 21 days 
Docetaxel 
75 mg/m2 IV 
D8 every 21 days 
 
Part 2 
 
Subjects will receive Vigil at 1.0 x 107 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 will be used.  Subjects receiving Vigil will be observed for at 


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least 30 minutes following administration. Vigil will be given in combination with 
Irinotecan and Temozolimide as outlined below. 
 
Drug 
Dose 
Schedule 
Temozolomide 
100 mg/m2 daily, oral 
Days 1 – 5, every 21 days 
Irinotecan 
50 mg/m2 daily, oral 
OR 
20 mg/m2 daily, IV 
Days 1 – 5, every 21 days 
peg-filgrastim (optional) 
100μg/kg, subcutaneous 
injection 
Day 6, every 21 days 
may be administered at 
home 
Vigil 
1.0 x 107 cells/injection, 
intradermal 
Day 15, every 21 days 
 
Temozolomide, irinotecan and peg-filgrastim should be administered and adjusted per 
package insert and institutional standards.  
 
Premedications 
 
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 immunosuppressents should be avoided due to immune inhibition activity. 
 
Gemcitabine / docetaxel: Per package insert and institutional standards.  To reduce 
the frequency and severity of fluid retention, it is recommended to administer 
dexamethasone 8mg PO bid for 3 days starting one day prior to each docetaxel 
administration (minimum of 3 doses).  (Oral suspensions / modifications are 
acceptable). 
 
Temozolomide / irinotecan / peg-filgrastim: Per package inserts and institutional 
standards. 
 
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 Pharmacy 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 vaccine dose will be reduced by 50% 
and continued. 
 
Vigil Treatment Delay 
 
 Treatment may be delayed no more than 4 weeks to allow recovery from 
toxicity. 
 Subjects who delay treatment for more than 4 weeks due to toxicities will 
be considered off study treatment (see Section 5.4 Withdrawal). 
 Treatment delay not related to toxic events (including subjects unable to 
adhere to monthly/3 week cycle injection) may not extend for more than 
three days unless due to symptoms related to disease or infection in 
which case up to a 2 week delay is allowed. 
 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 monthly/ every 3 week schedule thereafter. 
 
Dose Modifications for gemcitabine, docetaxel, temozolomide, irinotecan, 
and peg-filgrastim 
 
Dose modifications for gemcitabine, docetaxel, temozolomide, irinotecan, and peg-
filgrastim may be necessary for subject safety and should be carried out in accordance 
with the Investigator’s standard of practice and the package inserts and institutional 
standards. 
 
If irinotecan + temozolomide is administered beyond 12 months, it will be administered 
off study.  If irinotecan + temozolomide is discontinued, Vigil may be administered 
every 3 weeks until all manufactured product is exhausted or disease progression is 
noted. 
 
 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:  
 


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Systemic anti-cancer therapy, including chemotherapy, radiotherapy, or endocrine 
therapy other than those required per protocol are prohibited from the time of study 
screening until the End of Treatment visit.  
 
Any investigational drug or device other than Vigil is prohibited from the time of study 
screening until the End of Treatment visit.  
 
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 prohibited. 
 
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 the chemotherapy arm. Treatment for drug-related adverse events should 
be administered at the discretion of the investigator.  
 
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 vaccinations are permitted 2 weeks post administration of investigational 
product.  Nasal flu vaccine is prohibited. 
 
 Toxicity 
 
Toxicities will be graded and reported according to the NCI Common Toxicity Criteria 
for Adverse Events (CTCAE) Version 4.03 as linked in Appendix C. 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. 
 
 Schedule of Assessments 
 
The schedule of assessments for the trial is shown in Appendix B.  If a required 
observation or procedure is missed, documentation is required to explain the reason for 
this protocol deviation. 
 


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Prestudy Assessments 
 
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. 
3. A complete medical history must be obtained. 
4. A physical examination must be obtained. 
5. A complete blood count (CBC) with differential and platelet count must be 
performed.  (HIV 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. PBMC collection for immune function analysis will be obtained at tissue harvest.  
(≤24 hours of tissue procurement) 
 
The following evaluations will be performed on all subjects within 2 weeks of 
randomization (in Part 1) or within 2 weeks of registration (in Part 2) (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. An interval medical history must be obtained within 4 weeks. 
4. A physical examination (including vital signs, height, temperature and body 
weight) must be obtained. 
5. Assessment of concomitant medications 
6. Assessment of PS on the Karnofsky scale (see Appendix A) must be obtained. 
7. Radiological assessment of disease status with CT chest /abd/pelvis (MRI 
abd/pelvis may be substituted for CT abd/pelvis) within 4 weeks. 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. 
8. A complete blood count (CBC) with differential and platelet count must be 
performed.  (HIV testing is not required if the subject has no medical history of 
HIV). 
9. 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. 


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10. PBMC collection for immune function analysis. 
11. Plasma collection for immune function analysis (ctDNA) (Part 2). 
12. Pregnancy test for those of childbearing potential. 
13. 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).  
14. Submission to Gradalis, Inc of 20 unstained tumor slides or tissue block for 
correlative immunohistochemistry assay.  These slides should correlate with 
tumor procured for vaccine manufacture. 
 
Assessments During Treatment 
 
The following evaluations will be performed once at the beginning of each cycle (1 
Cycle = 28 days ±3 days for Vigil treated subjects and 1 Cycle = 21 days ±3 for 
gemcitabine/docetaxel treated patients in Part 1); (1 Cycle = 21 days ±3 days for Vigil + 
temozolomide + irinotecan treated subjects in Part 2) (unless otherwise specified): 
 
1. A physical examination, including vital signs and body weight. 
2. A toxicity assessment (CTCAE v 4.03). 
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 12 weeks ± 7 days.  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. PBMC collection for immune function analysis prior to Vigil or chemotherapy 
administration at Cycles 2, and 4. 
8. PBMC collection for immune function analysis prior to Day 15 Vigil administration 
at Cycles 2, and 4 (Part 2). 
9. Plasma collection for immune function analysis (ctDNA) prior to chemotherapy 
administration at Cycle 2 – Week 1 Day 1 and Cycle 4 – Week 1 Day 1 (Part 2). 
10. Assessment of Karnofsky Performance Status. 
11. For those randomized to Vigil, Vigil administration.  The subject will be observed 
for at least 30 minutes. Day 2 post Vigil administration assessment of injection 
site.  Instruct the subject on Day 1 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 


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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).  This is applicable to those 
who receive Vigil in either Part 1 or Part 2 of the protocol. 
12. For those randomized to chemo in Part 1: gemcitabine 675 mg/m2 IV 
administered at a rate of 10mg/m2/min on D1 and D8 and docetaxel 75 mg/m2 IV 
D8 every 21 days. 
13. For those registered into Part 2 of the study: (i) (i) oral temozolomide 100 mg/m2 
daily (Days 1 – 5, total dose 500 mg/m2/cycle), (ii) irinotecan 50 mg/m2 daily 
(Days 1 – 5, total dose 250mg/m2/cycle), orally OR irinotecan 20mg/m2 daily 
(Days 1 – 5, total dose 100mg/m2/cycle), intravenously (iii) peg-filgrastim 
100μg/kg (Day 6) subcutaneously (optional and may be administered at home), 
and (iv) Vigil 1.0 x 107 cells/injection, intradermally on Day 15 and every 3 weeks 
thereafter. 
 
End of Treatment Assessments 
 
The following evaluations will be performed within 30 days after completion of the study 
agent or chemotherapy 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 
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. PBMC for immune function analysis. 
9. Plasma for immune function analysis (ctDNA) (Part 2). 
 
 


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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 every 3 
months 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. 
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. PBMCs for immune function analysis every 6 months from EOT. 
7. Assessment of Performance Status. 
 
Long Term Follow Up 
 
After progression, subjects and their physicians will be contacted quarterly for 
documentation of 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). 
 


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Should Gradalis request for additional blood or tissue, the clinical site will present the 
option of the procurement to the participant and obtain written informed consent. 
 
 CONDUCT OF THE STUDY 
 
 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. 
 
 IRB 
 
This trial can be undertaken only after review and full approval of the protocol and a 
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 all 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. 
 
 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. 
 
 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. 
 
 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. 
 
 


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 Protocol Questions and Deviations 
 
When evaluating a potential patient or while a patient is on study, protocol questions 
can be directed to the CRO via email or phone using the contact information provided 
in the Contact Information section of the Study Reference Manual. 
 
 Data Safety Monitoring Board 
 
A 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. 
 
 EVALUATION OF TUMORS 
 
 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 
 
 DRUG INFORMATION 
 
 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.  


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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 107 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 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 Pharmacy Reference Manual for preparation and handling information. 
 
Vigil concentrate:  1.0 x 107cells 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. 
 


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 OCCUPATIONAL SAFETY 
 
Study medications are not expected to pose significant occupational safety risks to 
investigational staff under normal conditions of use and administration.  However, 
precautions should be taken to avoid direct contact with study medication.  Biosafety 
Level 1 practices shall be employed with this study medication.  Reference the 
Pharmacy Reference Manual. 
 
 ADVERSE EVENTS 
 
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 
 
 


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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. 
 
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 4.03 as linked in Appendix C.  All AEs, regardless of severity, will be followed 
by the Treating Physician until resolution is satisfactory. 
 
 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. 
 
 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/arthralagias, 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. 
 


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 Recording of an Adverse Event 
 
Adverse events will be recorded for the duration of a patient’s study treatment 
(following the first dose of the Investigational Product), and for up to 30 days following 
the last study treatment.  All AEs, regardless of causal relationship are to be recorded 
in the eCRF and source documentation.  Additional information about each event, such 
as treatment 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. 
 
 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 while a patient is 
receiving the study agent on this protocol, or  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. 
 
 PATIENT COMPLETION AND WITHDRAWAL 
 
 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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 PART 1 STATISTICAL CONSIDERATIONS 
 
 Sample Size Justification 
 
This is an open label randomized controlled Phase II 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 logrank test at the alpha=0.05 
level of significance, 46 events will provide 90% power to detect an OS HR of 0.383.  
Assuming a one-year accrual period, and a one-year follow-up period after 
randomization of the last subject, it is estimated that the total sample size (number of 
subjects) required to achieve 46 events is 62 (31 patients in each arm). 
 
 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 
 
15.3.1 Primary Efficacy Analysis 
 
The primary endpoint of OS is time from randomization to death.  Kaplan-Meier OS 
curves will be displayed by treatment arm.  Median OS and percent OS at fixed time 
points will be estimated. 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 primary analysis of OS will be conducted using a two-sided log rank test at 
the alpha=0.05 level of significance. 
 
 


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15.3.2 Secondary Efficacy Analyses 
 
The secondary 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. 
 
The secondary endpoint of 1 year survival proportion will be estimated using 
Greenwood’s formula to estimate the standard errors of the Kaplan-Meier survival 
probability at a specified time point. 
 
The secondary endpoint of ORR will be analyzed using Fisher’s exact test. 
 
All secondary analyses will be conducted using two-sided tests at the alpha=0.05 level 
of significance. 
 
 Safety Analyses 
 
Safety endpoints include all adverse events (CTCAE 4.03), laboratory safety 
assessments, and physical examination findings. 
 
 PART 2 STATISTICAL CONSIDERATIONS 
 
Since this part of the protocol is an uncontrolled, non-randomized safety cohort with 
approximately 6-9 subjects studied, there will be no statistical analysis of outcome.  
Descriptive statistics only will be conducted. 
 
 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. 
 
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. 


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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. 
 
 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 PERFORMANCE SCALE 
 
STATUS 
MEANING 
100% 
No symptoms. 
90% 
Able to carry on normal activity; minor signs or symptoms of disease. 
80% 
Able to carry on normal activity with effort; some signs or symptoms of 
disease. 
70% 
Cares for self, unable to carry on normal activity or do active work. 
60% 
Requires occasional assistance but is able to care for most of own needs. 
50% 
Requires considerable assistance and frequent medical care. 
40% 
Disabled; requires special care and assistance. 
30% 
Severely disabled; hospitalization indicated, although death not imminent. 
20% 
Very ill; hospitalization necessary; active supportive treatment required. 
10% 
Moribund, fatal processes progressing rapidly. 
0 
Patient expired. 
 
 
 


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APPENDIX B 
 
SCHEDULE OF ASSESSMENTS 
 
Procedure 
Prestudy 
Screening 
At the beginning 
of each Cycle 
(unless otherwise 
noted) 
End of 
TX 
Response 
Follow-Up 
(q 3mo±7 
days) Until 
Progression
Informed consent 
X 
X 
 
 
 
Medical History 
X 
Interval Medical 
History within 4 
weeks 
 
 
 
Physical Examination 
X 
X 
X 
X 
X 
Toxicity (adverse 
events) 
 
 
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 12 weeks ± 7 
days 
Within 45 
days 
X 
CBC with differential 
X1 
X 
X 
X 
X 
HIV testing, if applicable 
 
X 
 
 
 
Hepatitis testing, if 
applicable 
 
X 
 
 
 
Serum Chemistry 
X2 
X 
X 
X 
X 
PBMC collection for 
Immune Function 
Analysis 
≤ 24 hours 
prior to tumor 
procurement 
X 
(Cycles 2, and 4, prior 
to Vigil or chemo 
administration  
X 
q 6 months 
PBMC collection for 
Immune Function 
Analysis (Part 2) 
≤ 24 hours 
prior to tumor 
procurement 
X 
(Cycles 2, and 4, prior 
to Day 15 Vigil 
administration) 
X 
q 6 months 
Plasma collection for 
ctDNA (Part 2) 
 
 
(Cy 2, W1D1; Cy 4, 
W1D1 prior to 
chemotherapy 
administration
X 
 
Pregnancy Test (if 
applicable) 
 
X 
 
 
 
Vigil administration 
depending on randomization of 
Part 1 and Part 2 participants 
 
 
Part 1 – Day 1 q 28±3 
days 3 
Part 2 – Day 15 q 21±3 
days3  
 
 
Injection Site 
Assessment 
 
 
Day 2 or Day 16 only 
(may be conducted at 
home) 
 
 
Gemcitabine / Docetaxel 
depending on randomization 
 
 
gemcitabine D1 and 8 
docetaxel D8 q 21d 
 
 
Temozolomide / 
Irinotecan 
Part 2 participants 
 
 
(p.o. temozolomide 
100 mg/m2 daily for 5 
days (Days 1 – 5), I.V. 
 
 
                                                 
 
1 Obtain from medical records from standard preoperative hematology and chemistry panels. 
2 Obtain from medical records from standard preoperative hematology and chemistry panels. 
3 If assigned to the Vigil arm, the patient will be observed for at least 30 minutes following Vigil administration. 


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Procedure 
Prestudy 
Screening 
At the beginning 
of each Cycle 
(unless otherwise 
noted) 
End of 
TX 
Response 
Follow-Up 
(q 3mo±7 
days) Until 
Progression
irinotecan 20mg/m2 
daily (Days 1 – 5, total 
dose 100mg/m2/cycle), 
or oral irinotecan 50 
mg/m2 daily for 5 days 
(Days 1 – 5), q21 days 
Peg-filgrastim 
Part 2 participants 
 
 
s.c. peg-filgrastim 
100ug/kg, Day 6 
optional and may be 
administered at home
 
 
Survival Status 
X 
 
 
 
Long Term 
Follow Up4 
 
 
 
                                                 
 
4 After progression, subjects will be contacted by phone quarterly for documentation of survival status. 


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APPENDIX C 
 
NCI COMMON TOXICITY CRITERIA FOR ADVERSE EVENTS (CTCAE), VERSION 
4.03 
 
 
Publish Date: June 14, 2010 
 
As of June 14, 2010 NCI has introduced version 4.03 of the Common Toxicity Criteria for
Adverse Events. These may be obtained at the following web link http://ctep.cancer.gov.
 
DO NOT USE CTC VERSION 3.0 TO GRADE TOXICITIES IN THIS STUDY! 
 
 
 
 


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