Open-label Phase II Trial of Adjuvant bishRNAfurin and GMCSF Augmented Autologous Tumor Cell Vaccine (FANG™) for High Risk Stage III/IV Ovarian Cancer
Phase: PHASE2
Conditions: Ovarian Cancer
Sponsor: Gradalis, Inc.

 
 
 
 
 
Protocol #:  
 
CL-PTL 105 
 
PROTOCOL TITLE:  
Open-label Phase II Trial of Adjuvant bishRNAfurin and GMCSF 
Augmented Autologous Tumor Cell Vaccine (FANG™) for High-Risk Stage 
III/IV Ovarian Cancer 
 
NCT 
 
 
01309230 
 
PROTOCOL DATE:  
September 25, 2015 

 CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of 
Gradalis, Inc. No person is authorized to make it public without written permission of Gradalis, Inc. These restrictions on disclosure 
will apply equally to all future information supplied to you, which is indicated as privileged or confidential. This material may be 
disclosed to and used by your staff and associates as may be necessary to conduct this clinical study. 
 
 
PROTOCOL TITLE: 
Open-label Phase II Trial of Adjuvant bi-
shRNAfurin and GMCSF Augmented Autologous 
Tumor Cell Vaccine (FANG™) for High Risk Stage 
III/IV Ovarian Cancer 
Protocol #: 
CL-PTL 105 
STUDY AGENT: 
Vigil™ formerly known as FANG™ (bi-shRNAfurin 
and GMCSF Autologous Tumor Cell Vaccine) 
SPONSOR: 
Gradalis, Inc. 
2545 Golden Bear Drive, Suite 110 
Carrollton, TX 75006 
Phone: (214) 442-8124 
Fax: (214) 442-8101 
PROTOCOL DATE: 
Amendment #9 dated September 25, 2015 
Amendment #8 dated June 19, 2014 
Amendment #7 dated January 9, 2014 
Amendment #6 dated April 15, 2013 
Amendment #5 dated January 31, 2013 
Amendment #4 dated May 1, 2012 
Amendment #3 dated October 5, 2011 
Amendment #2 dated June 23, 2011 
Amendment #1 dated February 18, 2011 
Initial Protocol dated November 16, 2010 
 
 
 
 

Protocol # CL-PTL 105 
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INVESTIGATOR PROTOCOL SIGNATURE PAGE 
 
 
Protocol Title: 
Open-label Phase II Trial of Adjuvant bi-
shRNAfurin and GMCSF Augmented 
Autologous Tumor Cell Vaccine (FANG™) 
for High Risk Stage III/IV Ovarian Cancer  
Protocol #: 
CL-PTL 105 
SPONSOR: 
Gradalis, Inc. 
2545 Golden Bear Drive, Suite 110 
Carrollton, TX 75006 
Phone: (214) 442-8124 
Fax: (214) 442-8101 
Protocol Date 
Amendment #9 dated September 25, 2015 
Amendment #8 dated June 19, 2014 
Amendment #7 dated January 9, 2014 
Amendment #6 dated April 15, 2013 
Amendment #5 dated January 31, 2013 
Amendment #4 dated March 29, 2012 
Amendment #3 dated October 5, 2011 
Amendment #2 dated June 23, 2011 
Amendment #1 dated February 18, 2011 
Initial Protocol dated November 16, 2010 
 
 
 
I have read and understand the contents of the indicated clinical protocol and will 
adhere to the trial requirements as presented, including all statements regarding 
confidentiality. In addition, should I choose to participate as an investigator, I and 
my sub-investigator(s) agree to conduct the study as outlined herein, in 
accordance with Good Clinical Practices (GCPs), the Declaration of Helsinki, in 
compliance with the obligations and requirements of clinical investigators and all 
other requirements listed in Title 21 Code of Federal Regulations (CFR) Part 312. 
 
_______________________________  
 
 
 
 
 
 
Name of Investigator (please print) 
 
Signature of Investigator 
 
_______________________________ 
Date 
 

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TABLE OF CONTENTS 
INVESTIGATOR PROTOCOL SIGNATURE PAGE ....................................................... 2 
ABBREVIATIONS........................................................................................................... 5 
SYNOPSIS 
............................................................................................................ 7 
1.0 
INTRODUCTION ................................................................................................ 11 
1.1 
Immune Therapy in Cancer ..................................................................... 11 
1.2 
Immune Function Capacity in Cancer Patients ........................................ 12 
1.3 
Immune Function Capacity in Ovarian Cancer Patients........................... 13 
1.4 
Background on Transforming Growth Factors beta (TGFβ) ..................... 14 
1.5 
Background on GMCSF ........................................................................... 16 
1.6 
Background on Gene Vaccines in Cancer ............................................... 17 
1.7 
Blockade of multiple TFGβ isoforms is needed to reverse 
immunosuppression ................................................................................. 19 
1.8 
Background on TAG (TGF2 antisense and GMCSF vaccine) ................ 20 
1.9 
Background on Furin................................................................................ 26 
1.10 
Phase I Results ........................................................................................ 35 
2.0 
STUDY RATIONALE ......................................................................................... 43 
3.0 
OBJECTIVES  ................................................................................................. 45 
3.1 
Primary objective(s) ................................................................................. 45 
3.2 
Secondary objective(s) ............................................................................ 45 
4.0 
STUDY DESIGN ................................................................................................. 45 
4.1 
Design ...................................................................................................... 45 
5.0 
STUDY POPULATION ....................................................................................... 46 
5.1 
Sample Size ............................................................................................. 46 
5.2 
Inclusion Criteria ...................................................................................... 46 
5.3 
Exclusion Criteria ..................................................................................... 47 
5.4 
Withdrawal ............................................................................................... 48 
6.0 
INVESTIGATIONAL PLAN ................................................................................ 49 
6.1 
Patient Registration and Enrollment ........................................................ 49 
6.2 
Tumor Procurement ................................................................................. 50 
6.3 
Vaccine Manufacturing ............................................................................ 50 
6.4 
Study Treatment Administration ............................................................... 51 
6.5 
Toxicity ..................................................................................................... 53 
6.6 
Schedule of Assessments ........................................................................ 53 
7.0 
CONDUCT OF THE STUDY .............................................................................. 57 
7.1 
Ethics and Regulatory Considerations ..................................................... 57 
7.2 
IRB and IBC ............................................................................................. 57 
7.3 
Written Informed Consent ........................................................................ 58 
7.4 
Confidentiality of Records ........................................................................ 59 

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7.5 
Modification of Protocol ............................................................................ 59 
7.6 
Protocol Questions and Deviations .......................................................... 59 
8.0 
EVALUATION OF TUMORS .............................................................................. 60 
8.1 
Disease Evaluation .................................................................................. 60 
9.0 
DRUG INFORMATION ....................................................................................... 60 
9.1 
Investigational Product............................................................................. 60 
9.2 
Safety Analysis ........................................................................................ 61 
9.3 
Concomitant Therapy............................................................................... 62 
10.0 
OCCUPATIONAL SAFETY................................................................................ 62 
11.0 
ADVERSE EVENTS ........................................................................................... 63 
11.1 
Adverse Event and Serious Adverse Event Definitions............................ 63 
11.2 
Attribution of Causality ............................................................................. 64 
11.3 
Expected Side Effects .............................................................................. 64 
11.4 
Recording of an Adverse Event ............................................................... 65 
11.5 
Serious Adverse Event Reporting ............................................................ 65 
12.0 
PATIENT COMPLETION AND WITHDRAWAL ................................................. 66 
12.1 
Indication for Taking Patients Off Study ................................................... 66 
13.0 
DATA EVALUATION ......................................................................................... 66 
13.1 
Statistics and Estimated Sample Size ..................................................... 66 
13.2 
Definition of Evaluable Patients ............................................................... 67 
13.3 
Time to Recurrence of Disease (TTR) ..................................................... 68 
13.4 
Time to Treatment Failure ........................................................................ 68 
13.5 
Survival .................................................................................................... 69 
14.0 
STUDY RECORDS ............................................................................................ 69 
14.1 
Documentation ......................................................................................... 69 
14.2 
Case Report Form (CRF) Procedures ..................................................... 70 
APPENDIX A 
.......................................................................................................... 71 
ECOG Performance Status Scale ................................................................... 71 
APPENDIX B 
.......................................................................................................... 72 
Schedule Of Assessments .............................................................................. 72 
APPENDIX C 
.......................................................................................................... 73 
NCI Common Toxicity Criteria For Adverse Events (CTCAE), Version 3.0 ..... 73 
APPENDIX D 
.......................................................................................................... 74 
FACT-O, Version 4 ......................................................................................... 74 
REFERENCES 
.......................................................................................................... 77 
 

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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  
CR 
Complete response 
CRF 
Case report form 
CTCAE 
Common Toxicity Criteria for Adverse Events 
CTL 
Cytotoxic T lymphocyte 
DC 
Dendritic cell(s) 
DTH 
Delayed-type hypersensitivity 
ECOG PS 
Eastern Cooperative Oncology Group Performance 
Score 
ELISA 
Enzyme-Linked ImmunoSorbent Assay 
ELISPOT 
Enzyme-Linked ImmunoSorbent Spot 
ER 
Endoplasmic reticulum 
FANG™ 
bi-shRNAfurin and GMCSF Augmented Autologous 
Tumor Cell Vaccine 
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 
IBC 
Institutional Biosafety Committee 
IEC 
Independent Ethics Committee 
IL 
Infiltrating lymphocytes 
IRB 
Institutional Review Board 
LAK 
Lymphokine-activated killer 

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Abbreviation 
Term 
LD 
Longest diameter 
LLC 
Large latent complex 
LPI 
Lead Principal Investigator 
MARY CROWLEY 
Mary Crowley Cancer Research Centers 
MHC 
Major histocompatability complex 
MLR 
Mixed lymphocyte reaction 
MR 
Mannose receptor 
NCI 
National Cancer Institute 
NED 
No evidence of disease 
NK 
Natural Killer 
NKT 
Natural Killer T cell(s) 
NSCLC 
Non small cell lung cancer 
PCR 
Polymerase chain reaction 
PD 
Progressive disease 
PI 
Principal Investigator 
PR 
Partial response 
PS 
Performance Status 
RECIST 
Response Evaluation Criteria in Solid Tumors 
SCLC 
Small cell lung cancer 
SD 
Stable disease 
SLC 
Small latent complex 
STMN1 
Stathmin 1 
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 

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SYNOPSIS 
 
Summary:  Despite a gradual improvement in their overall survival over the past 
decade, approximately 75% of women with Stage IIIC ovarian cancer who 
achieve a complete clinical response will relapse as will 50% of those achieving 
pathologic complete response at a median time of 18-24 months.  Phase III 
studies of both maintenance and consolidation therapeutic interventions have not 
translated into an overall survival advantage.  Preliminary studies of 
immunotherapy in patients with ovarian cancer suggest target accessibility 
(potential immunogenicity) to immune mediated approaches.  In an effort to 
overcome limitations of 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 block 
proprotein conversion to active TGF1 and 2) to 1) address the inability to fully 
identify cancer 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™ vaccine in 33 advanced solid tumor patients (1 
of them being a pediatric patient 15 years of age) receiving ≥1 vaccination (at a 
dose of 1.0 x 107 or 2.5 x 107 cells/injection/month for a maximum of 12 
vaccinations) demonstrated safety of the Vigil™ vaccine.  Furthermore, proof of 
principle was established in the manufactured vaccines with increased mean 
GMCSF expression post-transfection to 1135 pg/106 cells/ml and knockdown of 
furin, TGF1 and TGF2 at 78%, 93%, and 95%, respectively).  In addition, 
although a Phase I study, the data suggested an overall survival benefit. 
 
Given the preliminary Phase II evidence of prolongation of time to relapse in 
randomized Vigil™ vs. No Vigil™ treated patients (p=0.005), as of this 
amendment all patients screening for enrollment into the main portion of the trial 
(including those who previously had tumor tissue harvested), will receive Vigil™ 
vaccine and will no longer be randomized.  An extensive Phase II/III protocol is 
designed to more definitively assess benefit.  Additional safety data in ovarian 
cancer patients receiving 1x107 cells/ID injection on a monthly basis (maximum 
12) is required. 
 
This is a Phase II open-label  study of maintenance intradermal autologous 
Vigil™ cancer vaccine administered  to women with Stage III/IV epithelial ovarian 
cancer who attain a clinical complete response (including a post-treatment, 
prevaccination baseline serum CA-125 level of ≤ 20 units/ml) after surgical 
cytoreduction and a total of six courses of front-line (pre- and post- or post-
surgical) doublet chemotherapy.  Patients meeting eligibility criteria will receive at 
least 5 but no more than 6 cycles of NCCN Category 1 chemotherapy.  
Thereafter, patients will receive VigilTM vaccine at a dose of 1.0 x 107 
cells/injection for minimum of 4 to a maximum of 12 vaccinations (based on 
number of vaccine doses manufactured and patient eligibility.  Trial endpoints 

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include time to recurrence (TTR), documentation of immune responses, and 
correlation of immune response and clinical effect. 
 
Objective(s): 
Primary Objectives: 
 To assess time to recurrence (TTR) following the administration of bi-
shRNAfurin and GMCSF autologous tumor cell (Vigil™) vaccine. 
 
Secondary Objectives: 
 To identify and determine the effect of Vigil™ autologous tumor cell 
vaccine on immune surrogate markers. 
 To assess the predictive potential of initial tumor infiltrating lymphocyte 
(TIL) and tumor associated macrophage (TAM) phenotypes. 
 To enlarge the safety database of Vigil™ autologous tumor cell vaccine in 
patients with minimal disease. 
 
Methodology: 
This is a Phase II open-label trial of Vigil™ autologous tumor cell vaccine.  Tumor 
will be harvested at the time of surgical debulking (standard of medical care). 
 
Patients achieving clinical complete response (CR) following primary surgical 
debulking and doublet chemotherapy will be stratified for i) surgical stage (Stage 
IV or suboptimal debulking (>1 cm residual) Stage III disease versus Stage III 
patients with optimal debulking (<1 cm residual)) and ii) post-operative 
chemotherapy, pre-vaccine CA-125 of greater than 10 but less than or equal to 
20 U/mL versus 0≤10 U/ml then randomized 2:1 (Note: patients with Stage IIIc 
ovarian cancer will be additionally evaluated as a subset using descriptive 
statistical endpoint only).  Patients will receive 1.0 x 107 cells / intradermal 
injection of gene transfected autologous tumor cells, Vigil™, once a month for up 
to 12 doses as long as sufficient material is available.  A minimum harvest aliquot 
to produce 4 monthly injections will be required for entry into the study.  These 
patients will be managed in an outpatient setting.  Hematologic function, liver 
enzymes, renal function and electrolytes will be monitored monthly.  Immune 
function analysis including ELISPOT analysis of cytotoxic T cell function to 
autologous tumor antigens will be monitored at (≤24 hours before) tissue harvest, 
≤24 hours before the first cycle chemotherapy (post debulking), ≤24 hours before 
the third cycle chemotherapy (post debulking), baseline (screening); prior to 
Vigil™ injection at Months 2, 4, 6 and EOT.  CA-125 will be monitored at 
baseline, every month for the first year, every 3 months ± 2 weeks for the second 
and third year. 
 
Number of Patients:  Approximately fifty (50) patients with high risk Stage III/IV 
ovarian cancer. 

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Inclusion Criteria: 
1. Histologically confirmed Stage III/IV papillary serous or endometrioid 
ovarian cancer. 
2. Per Amendment #8, treatment naïve, high risk ovarian cancer will no 
longer be stratified, but the following information will be collected: 
a. Stage IV or suboptimal (>1 cm residual) Stage III disease versus 
Stage III patients with optimal (≤1 cm residual) disease, 
b. CA-125 ≤10 U/ml versus CA-125 greater than 10 to 20 U/ml 
c. IP chemotherapy versus IV chemotherapy 
3. Clinically defined CR (no cancer related symptoms, normal physical 
examination and CT scan abdomen/pelvis and chest x-ray, and CA-125 ≤ 
20 U/ml) following completion of surgical debulking.  Patients enrolled 
must complete at least 5 but no more than 6 cycles platinum/taxane 
adjuvant or interval debulking + chemotherapy (or chemotherapy as per 
recommendations of NCCN guidelines, category 1 (IP chemotherapy 
included)). (Patients who complete surgery/chemotherapy with a CA-125 
>20 U/mL pre-randomization have the option of being followed up to 2 
months if serial CA-125 values continue to decrease at a rate of ≥50% per 
month. 
4. Availability of “golf-ball” size ~10-30 grams tissue at time of primary 
surgical debulking. 
5. Successful manufacturing of 4 vials of Vigil™ vaccine. 
6. Recovered from all clinically relevant toxicities related to prior protocol 
specific therapy (including neuropathy ≤Grade 2). 
7. ECOG performance status (PS) 0-2 prior to tumor debulking laparotomy. 
8. ECOG PS 0-1 prior to Vigil™ vaccine administration. 
9. Normal organ and marrow function as defined below: 
Absolute granulocyte count 
≥ 1,500/mm3 
Absolute lymphocyte count 
≥ 200/mm3 
Platelets 
≥ 75,000/mm3 
Total bilirubin 
≤ 2 mg/dL 
AST(SGOT)/ALT(SGPT) 
≤ 2x institutional upper limit of normal 
Creatinine 
< 1.5 mg/dL 
10. Patients must be off all “statin” drugs for ≥ 2 weeks prior to initiation of 
therapy.  Ability to understand and the willingness to sign a written 
informed consent document for tissue harvesting. 
11. Ability to understand and the willingness to sign a written informed 
protocol specific consent. 
Exclusion Criteria: 
1. Surgery involving general anesthesia, radiotherapy, or immunotherapy 
within 4 weeks prior to randomization.  Chemotherapy within 3 weeks prior 
to Vigil™ vaccine administration.  Steroid therapy within 1 week prior to 
vaccine administration. 
2. Patients must not have received any other investigational agents within 4 
weeks of Vigil™ vaccine administration. 

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3. Patients with history of brain metastases. 
4. Patients with compromised pulmonary disease. 
5. Short term (<30 days) concurrent systemic steroids ≤ 0.25 mg/kg 
prednisone per day (maximum 7.5 mg/day) and bronchodilators (inhaled 
steroids) are permitted; other steroid regimens and/or 
immunosuppressives are excluded. 
6. Prior splenectomy. 
7. Prior malignancy (excluding nonmelanoma carcinomas of the skin and 
carcinoma in situ cervix) unless in remission for ≥ 2 years. 
8. Kaposi’s Sarcoma. 
9. Uncontrolled intercurrent illness including, but not limited to ongoing or 
active infection, symptomatic congestive heart failure, unstable angina 
pectoris, cardiac arrhythmia, or psychiatric illness/social situations that 
would limit compliance with study requirements. 
10. Patients with known HIV. 
11. Patients with chronic Hepatitis B and C infection. 
12. Patients with uncontrolled autoimmune diseases. 
Medication and Doses:  Autologous Vigil™ vaccine will be supplied by Gradalis, 
Inc. 
 
Patients will receive 1.0 x 107 cells via intradermal injection one day each month, 
≥3 weeks following completion of doublet chemotherapy (no longer than 2.5 
months post chemotherapy), for a maximum of 12 doses as long as sufficient 
material is available and subject is clinically stable. 
Duration:  If sufficient material is available, the patient may continue therapy up 
to 12 doses, unless disease progression is documented (see Section 4.0, Study 
Design).  If the patient experiences ≥ Grade 2 toxicity (excluding Grade 3 
injection site reaction), they may continue on treatment with doses delayed or 
reduced if, in the opinion of the PI, this is considered to be in the best interest of 
the patient. 
Efficacy Assessments: 
 Time to disease recurrence 
 Immune surrogate markers 
 Quality of Life (FACT-O, Version 4) 
 
Safety Assessments: 
 Physical examination, performance status, height, weight, temperature, 
blood pressure, and pulse 
 Toxicity:  CTCAE v 3.0 

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 INTRODUCTION 
1.0
 
 Immune Therapy in Cancer 
1.1
 
For decades immune-based therapies have been investigated in many types of 
cancer, including melanoma, prostate, renal cell, non-Hodgkin’s lymphoma, 
bladder cancer, and renal cell carcinoma (Quan, Dean et al. 1997; Alexandroff, 
Jackson et al. 1999; Eton, Legha et al. 2002; Kwak 2003; Kaufman, Wang et al. 
2004; Coppin, Porzsolt et al. 2005).  In a majority of these trials, induction of 
tumor specific cellular immune responses have been documented. However, in 
only a minority has there been translation into clinical effectiveness (Walden 
2007).  There have been several hypotheses to explain this demonstrable lack of 
anticancer immune activity in solid tumors.  These include ineffective priming of 
tumor-specific T cells, lack of high-avidity of primed tumor-specific T cells, and 
physical or functional disabling of primed tumor-specific T cells by the primary 
host and or tumor-related mechanism.  For example, in NSCLC a high proportion 
of the tumor-infiltrating lymphocytes are immunosuppressive T regulatory cells 
(CD4+ CD25+) that secrete transforming growth factor-β (TGFβ) and express a 
high level of cytotoxic T lymphocyte (CTL) antigen-4 (Woo, Yeh et al. 2002).  
These cells have been shown to impede immune activation by facilitating T cell 
tolerance to tumor associated antigens rather than cross-priming CD8+ T cells, 
resulting in the inhibition of killer T cells that recognize the tumor without 
attacking it (Dohadwala, Luo et al. 2001; Neuner, Schindel et al. 2002; Woo, Yeh 
et al. 2002).  Elevated levels of immune inhibitors such as IL-10 and TGFβ are 
found in the circulation in patients with advanced cancer, and animal models 
have shown immune suppression is mediated by these cytokines serving both as 
a barrier to antigen recognition and T-cell activation, as well as a defense against 
the body’s immune effector system (Rook, Kehrl et al. 1986; Tsunawaki, Sporn et 
al. 1988; Fontana, Frei et al. 1989). 
 
Recently, however, several advances have been made with respect to 
mechanisms by which the immune system can actually be manipulated to 
facilitate tumor antigen recognition and enhance anticancer immune effector 
activity in cancer.  Two independent approaches involving gene-based vaccines, 
specifically, Belagenpumatucel-L and GVAX®, have demonstrated, in Mary 
Crowley Cancer Research Centers and other institutional studies, remarkable 
safety and significant benefit with respect to response and survival benefits in 
advanced cancer patients (Nemunaitis ; Eager, Harle et al. 2007; Nemunaitis and 
Nemunaitis 2007; Murray N 2010).  Moreover, enhancement of tumor antigen 
recognition following treatment was correlated with patient benefit. 
 

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 Immune Function Capacity in Cancer Patients 
1.2
 
Tumor cells often continue to express normal cellular antigens in addition to neo-
antigens that are recognized by the immune system of the tumor-bearing host.  
The production of tumor specific antibodies by B cells can be elicited by the 
immunization of animals with tumor cells, and tumor-associated determinants 
have been defined by their ability to stimulate an antibody response in 
association with their presence on tumor cells.  Detectable titers of antibodies 
that bind to tumor-associated antigens have been demonstrated in cancer 
patients, but there is no compelling evidence that administration of exogenous 
antibodies to tumor-associated determinants cause solid tumors to regress in 
humans.  However, there is an accumulating body of evidence to suggest that 
many tumors in experimental model systems and from cancer patients express 
molecules that are targeted by the T cell arm of the immune system. 
 
Rejection of antigen-expressing tumor cells has been shown to be mediated by 
specific host cytolytic T cells (Prien RT 1957; Kripke 1974).  Tumor infiltrating 
lymphocytes, or TIL, have been shown to mediate durable regression of 
established tumors in mice with advanced tumor burdens (Yang, Perry-Lalley et 
al. 1990; Barth RJ 1991).  In patients bearing metastatic tumors, a number of 
groups have demonstrated the existence of anti-tumor CTL responses.  
Peripheral blood mononuclear cells as well as TIL contain populations of cells 
and individual clones that demonstrate tumor specificity; they lyse autologous 
tumor cells, but not natural killer targets, allogeneic tumors cells, or autologous 
fibroblasts (Anichini, Mazzocchi et al. 1989; Darrow, Slingluff et al. 1989; Knuth, 
Wolfel et al. 1989; Topalian, Solomon et al. 1989; Van den Eynde B 1989).  
Therefore, tumor specific antigens exist on metastatic human tumors, and are 
capable of stimulating a specific T cell response that can be expanded ex vivo to 
achieve clinical objective responses in tumor bearing patients. 
 
Endogenously synthesized antigens of virtually all mammalian cells are 
processed in an endoplasmic reticulum compartment and converted to small 
epitope peptides that are subsequently displayed on the cell surface in 
association with Class I MHC molecules to stimulate cytotoxic CD8+ cells 
(Townsend, Gotch et al. 1985; Maryanski, Pala et al. 1986; Townsend, Rothbard 
et al. 1986).  However, although exogenous (extracellular) antigens are 
processed by MHC II molecules to activate CD4+ cells, a subgroup of specialized 
antigen processing cells (dendritic cells) are capable of presenting very small 
numbers of exogenous peptides in association with Class I MHC molecules to 
stimulate T cell recognition (Rock, Gamble et al. 1990; Christinck, Luscher et al. 
1991) in a process designated “cross-presentation” (Hirschowitz, Foody et al. 
2004). 
 
The role of dendritic cells (DCs) in cell-mediated immunity has been extensively 
investigated (Gilboa, Nair et al. 1998; Timmerman and Levy 1999; Keilholz, 

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Weber et al. 2002; Conrad and Nestle 2003; Cranmer, Trevor et al. 2004).  DCs 
have been found to play a central role in the induction of anti-tumor immunity in 
tumor-bearing hosts via the process of antigenic cross-presentation (Hirschowitz, 
Foody et al. 2004).  Recent data suggest a distinct mannose receptor (MR) in 
DCs through which endocytosed antigen can be diverted from the classical MHC 
II restricted presentation pathway to the MHC I pathway (Burgdorf, Kautz et al. 
2007). As a result, by way of differential receptor-dependent uptake, DCs either 
present antigens through the MHC II pathway activating of CD4+ cells or cross-
present through the MHC I pathway activating CD8+ T cells.  CD4+ cells further 
augment the activity of natural killer cells and macrophages, in addition to 
amplifying antigen-specific immunity by local secretion of cytokines (Germain 
1994; McAdam, Schweitzer et al. 1998; Pulendran, Smith et al. 1999; 
Banchereau, Briere et al. 2000; Akbari, DeKruyff et al. 2001).  Recent studies in 
animal models have shown that immunization with DCs loaded with defined 
tumor antigens in the form of peptides, proteins, or RNA are capable of priming 
CTL responses and inducing tumor immunity (Flamand, Sornasse et al. 1994; 
Mayordomo, Zorina et al. 1995; Porgador and Gilboa 1995; Boczkowski, Nair et 
al. 1996).  DCs mixed in vivo with irradiated tumor cells have been shown to 
produce a protective immune response against a challenge with autologous 
tumor cells (Coveney, Wheatley et al. 1997).  Tumor-specific CTL response was 
also detectable.  Clinically, infusion of DCs has shown evidence of antitumor 
activity in patients with solid tumors (Hirschowitz, Foody et al. 2004; Ribas 2005; 
Small, Sacks et al. 2007).  These attributes make DCs a pivotal component in 
therapeutic strategies of many current immune-based therapies in cancer. 
 
 Immune Function Capacity in Ovarian Cancer Patients 
1.3
 
The majority of women diagnosed with cancer of the ovary present in advanced 
stage (Jemal, Siegel et al. 2009) with 5-year survival rates of 46.7% for Stage 
IIIa, 41.5% for Stage IIIb, and 32.5% for Stage IIIc (Heintz, Odicino et al. 2006).  
Despite a gradual improvement in overall survival over the past decade, 
approximately 75% of the women with Stage III/IV ovarian cancer who achieve a 
complete clinical response will relapse, as will 50% of those achieving pathologic 
complete response at a median time of 16-24 months depending on risk factors 
(Gadducci, Sartori et al. 1998; Markman, Liu et al. 2003; Gadducci, Cosio et al. 
2005).  Phase III studies of both maintenance and consolidation therapeutic 
interventions have not had an impact on overall survival (Mei, Chen et al. ; 
Foster, Brown et al. 2009; McGuire 2009; Pecorelli, Favalli et al. 2009).  It is 
therefore pertinent that expression of all three immunosuppressive TGF 
isoforms is increased in ovarian tumor as compared with normal ovarian tissue 
(Henriksen, Gobl et al. 1995; Bristow, Baldwin et al. 1999) with significant 
increases in TGF1 in both primary (2.9 fold; p ≤ 0.002) and recurrent (4.4 fold; p 
≤ 0.002) ovarian cancer (Bristow, Baldwin et al. 1999).  Secreted TGFβ from 
ovarian cancer cells generate immunosuppressive Treg cells (CD4+CD25+) from 
peripheral CD4+CD25- cells (Li, Ye et al. 2007).  Both Treg tumor infiltration as 

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well as the granzyme B+/FOX3p+ ratio are associated with outcome in those 
patients treated with neoadjuvant chemotherapy (Polcher, Braun et al.).  The 
contribution of optimal surgical cytoreductive surgery to prognosis may be due, in 
part, to the associated reduction in circulating Tregs (Napoletano, Bellati et al. 
2009).  In a recently described transgenic LSL-K-rasG12D/+p53loxP/loxP murine 
model of induced metastatic ovarian cancer in the setting of a mature immune 
system (Scarlett, Rutkowski et al.), a shift in dendritic cell phenotype from 
immunostimulatory to immunosuppressive was shown to be due to tumor-cell 
derived TGFβ1 and PGE2 both of which were found to be secreted at high levels 
in advanced tumors (Scarlett, Rutkowski et al.). Further, TGF has been linked to 
the emergence of cisplatin resistance (Li, Balch et al. 2009). 
 
Furin, an upstream regulator of TGFβ activity, is a member of the subtilisin-like 
proprotein convertase family. Proteolytic cleavage by furin is required for TGFβ 
convertase activation (i.e. pro-TGFβ TGFβ).  All three of the TGFβ isoforms 
contain the RXXR motif at their cleavage site albeit with different amino acid 
sequences (Kusakabe, Cheong et al. 2008).  High levels of furin mRNA and furin 
are widely expressed in human tumors and, specifically, in ovarian tumors (Page, 
Klein-Szanto et al. 2007).  The presence of furin in tumor cells likely contributes 
significantly to the maintenance of tumor directed, TGFβ-1 peripheral immune 
tolerance (Pesu, Watford et al. 2008). 
 
According to the serial immune function analyses by Coleman (Coleman, Clayton 
et al. 2005), five of nine patients with Stages III-IV ovarian adenocarcinoma who 
achieved post-chemotherapy remission retained the capacity of CD8+ T-cell 
responses to a panel of 11 viral peptides restricted by at least six common HLA 
class I alleles, whereas four patients with disease progression displayed low or 
reduced responses at different stages of treatment.  Chemotherapy produced no 
apparent effect on naïve (CD45RA+CCR7+), central memory (CD45RA-CCR7+), 
or effector memory (CD45-CCR7-) T-cells. 
 
 Background on Transforming Growth Factors beta (TGFβ) 
1.4
 
Transforming growth factors beta (TGFβ) are a family of multi-functional proteins 
that regulate the growth and function of many normal and neoplastic cell types 
(Sporn, Roberts et al. 1986; Massague 1987; Border and Ruoslahti 1992; 
Jachimczak, Bogdahn et al. 1993). TGFβ is synthesized as a prepro-TGFβ 
precursor, processed in the Golgi apparatus with removal of the propeptide and 
secreted as either the small latent complex (SLC) or with latent TGFβ-binding 
protein (LTBP) as the large latent complex (LLC) (Li, Wan et al. 2006). Following 
dissociation from the matrix associated latent complexes, the dimeric TGFβ 
activates a tetrameric TGFβ receptor complex comprised of TGFβRII and 
TGFβRI (ALK5) resulting in the phosphorylation of Smad2 and Smad 3 which 
translocates to the nucleus complexed with Smad4 where a number of 

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transcription factors are engaged. TGFβ2 signal transduction has been found to 
affect the expression of more than 20 different genes (Baker and Harland 1997; 
Heldin, Miyazono et al. 1997; Stiles, Ostrow et al. 1997; Yingling, Datto et al. 
1997). TGFβ exerts a wide range of effects on a variety of cell types and has 
been shown to stimulate or inhibit cell growth, induce apoptosis and increase 
angiogenesis (Jennings, Kaariainen et al. 1994; Merzak, McCrea et al. 1994; 
Ashley, Kong et al. 1998; Ashley, Sampson et al. 1998; Jennings and Pietenpol 
1998). Although TGFβ has been shown to be an effective tumor suppressor in 
epithelial cells and in the early phases of tumorigenesis, once the tumor escapes 
its growth regulatory effects, likely as the result of genetic instability, TGFβ 
appears to function as a tumor promoter (Bierie and Moses 2006; Pardali and 
Moustakas 2007) by virtue of its involvement in all six of the essential hallmark 
cancer-related processes as defined by Hanahan and Weinberg (Hanahan and 
Weinberg 2000). The three known TGFβ ligands (TGFβ1-3) are ubiquitous and 
expressed in the majority of tumors (Arteaga 2006).  These isoforms are highly 
homogenous in their mature regions with greater heterogeneity between their 
propeptide forms (Graycar, Miller et al. 1989).  Therefore, isoform specificity is 
likely controlled through propeptide sequences resulting in highly dynamic and 
spatially regulated expression.  In addition to misregulation of the ligand, there 
are a variety of reported mutations and downregulation by hypermethylation of 
CpG islands in the receptors and Smads (Kim, Im et al. 2000; Levy and Hill 
2006). Many tumors, including breast, colon, esophageal, gastric, hepatocellular, 
pancreatic, SCLC and NSCLC produce high levels of active TGFβ isoforms 
(Constam, Philipp et al. 1992; Eastham, Truong et al. 1995; Friedman, Gold et al. 
1995; Jakowlew, Mathias et al. 1995; Kong, Anscher et al. 1995; Yamada, Kato 
et al. 1995; Eder, Stenzl et al. 1996; Bierie and Moses 2006; Levy and Hill 2006). 
Furthermore, overexpression of TGFβ has been correlated with tumor 
progression and poor prognosis (Bierie and Moses 2006; Levy and Hill 2006). 
Elevated TGFβ2 levels have also been linked with immunosuppression in both 
afferent and efferent limbs (Sporn, Roberts et al. 1986; Massague 1987; Bodmer, 
Strommer et al. 1989; Border and Ruoslahti 1992; Chen, Hinton et al. 1997; 
Bierie and Moses 2006; Li, Wan et al. 2006). Tumor-derived TGFβ1 and PGE2 
induce the upregulation of PD-L1 in immunocompetent splenic dendritic cells and 
are causally related to the shift in dendritic cell phenotype from 
immunostimulatory to immunosuppressive transgenic in the LSL-K-
rasG12D/+p53loxP/loxP murine model of induced metastatic ovarian cancer(Scarlett, 
Rutkowski et al.). TGFβ2 inhibits T cell activation in response to antigen 
stimulation as well as targeting cytotoxic T cell cytolytic pathways (Thomas and 
Massague 2005).  Additionally, TGFβ2 has antagonistic effects on the Natural 
Killer (NK) cells as well as the induction and proliferation of the lymphokine-
activated killer (LAK) cells (Rook, Kehrl et al. 1986; Kasid, Bell et al. 1988; 
Tsunawaki, Sporn et al. 1988; Hirte and Clark 1991; Ruffini, Rivoltini et al. 1993; 
Naganuma, Sasaki et al. 1996). 
 
The immune suppressor functions of TGFβ are likely to play a major role in 
modulating the effectiveness of cancer cell vaccines.  TGFβ  inhibits GMCSF 

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induced maturation of bone marrow derived dendritic cells (DCs) (Yamaguchi, 
Tsumura et al. 1997) as well as expression of MHC Class II and co-stimulatory 
molecules (Geissmann, Revy et al. 1999).  It has been shown that antigen 
presentation by immature DCs result in T cell unresponsiveness (Steinman, 
Hawiger et al. 2003).  TGFβ also inhibits activated macrophages (Ashcroft 1999) 
including their antigen presenting function (Du and Sriram 1998; Takeuchi, Alard 
et al. 1998).  Therefore, both the ubiquitous expression of the TGFβ isoforms as 
well as the inhibitory effects of these isoforms on GMCSF immune modulatory 
function (see below), support a broad based tumor target range for the 
application of a TGFβ suppressed / GMCSF expressing immune enhancing 
therapeutic. 
 
 Background on GMCSF 
1.5
 
One factor that may limit the immunogenicity of tumor cells is the influence of 
cytokines in the tumor microenvironment (Mach and Dranoff 2000).  There is 
evidence that cytokines have a role in tumor formation and it has been 
demonstrated that manipulation of the cytokine balance can be exploited for 
cancer therapy.  Forni et al. (Forni, Giovarelli et al. 1985) showed that modifying 
intracellular cytokine levels could alter the outcome of the host response. 
Protective immunity against later tumor challenge was also reported in some 
instances (Forni, Giovarelli et al. 1985). 
 
More specifically, tumor cells genetically modified to secrete GMCSF have 
consistently demonstrated the most potent induction of anti-tumor immunity 
compared to other cytokines (Dranoff, Jaffee et al. 1993).  Results suggest that 
treatment with GMCSF protein may translate into clinical advantage, perhaps 
through immune stimulation (Anderson, Markovic et al. 1999; Spitler, Grossbard 
et al. 2000; Rini, Weinberg et al. 2003).  Interestingly, systemic cytokine 
administration has not induced direct anticancer response in randomized 
controlled trials, possibly due to a failure of the approach to recreate accurately 
the paracrine function of cytokines in tissue microenvironments (Dranoff 2004). 
 
Increasing evidence suggests that GMCSF is involved in the augmentation of 
tumor antigen presentation (Dranoff, Jaffee et al. 1993; Huang, Golumbek et al. 
1994).  DCs mediate a crucial role in priming antigen-specific immune responses 
(Zeng, Wang et al. 2001).  DCs express diverse receptors that allow for 
recognition and capture of antigens in peripheral tissues, process this material 
efficiently albeit by different routes into the MHC Class I and II presentation 
pathways, upregulate costimulatory molecules upon maturation, and migrate to 
secondary lymphoid tissues (Banchereau, Briere et al. 2000).  It was observed 
following injection of GVAX® in patients that an intense local reaction consisting 
of dendritic cells, macrophages, and granulocytes occurs (Dranoff, Jaffee et al. 
1993; Mach and Dranoff 2000). 

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Dendritic cells have proven to be incredibly versatile with regards to their 
response to inflammatory conditions and pathogens, which has made it difficult 
for researchers to determine the appropriate functional characteristics DCs must 
attain for cancer vaccination (Banchereau, Briere et al. 2000).  In one study B16 
melanoma cells engineered to secrete either GMCSF or Flt-3-Ligand (FL) and 
their immunologic effects were reported (Mach and Dranoff 2000).  Although both 
cytokines provoke a marked expansion of DCs locally and systemically, GMCSF 
stimulated greater levels of protective immunity.  Three profound differences 
have been described, which may account for the disparity in response.  First, 
GMCSF induces a subset of DCs that are superior for the phagocytosis of 
apoptotic tumor cells (Young and Inaba 1996; Pulendran, Lingappa et al. 1997; 
Shen, Reznikoff et al. 1997).  Second, compared to FL, GMCSF evoked higher 
levels of costimulatory molecules, which is characteristic of greater functional 
maturation.  This enhanced activity results in more efficient T cell stimulation, 
thereby broadening the arsenal of induced lymphocyte effector mechanisms 
(Murtaza, Kuchroo et al. 1999).  Third, GMCSF promoted uniformly high levels of 
CD1d on DCs, in contrast to FL, which triggered a more heterogeneous 
expression (Kawano, Cui et al. 1997).  CD1d is a nonclassical MHC Class I 
molecule that presents lipid antigens (Yamaguchi, Furukawa et al. 1990).  The 
CD1d lipid complex activates NKT cells, a population of lymphocytes that display 
a restricted Class I MHC-like receptor (Bendelac, Rivera et al. 1997).  
Importantly, natural killer T cells (NKT) cells may play pivotal roles in both 
endogenous and therapeutic responses to tumors (Smyth, Crowe et al. 2002). 
 
 Background on Gene Vaccines in Cancer 
1.6
 
Gene therapy has received considerable attention in recent years.  Vaccination 
with tumor cells designed to augment tumor antigen presentation and induce 
specific anti-tumor immunity has yielded promising but limited results (Holladay, 
Heitz et al. 1992).  Advances in our understanding of cancer biology and 
developments in vector technologies are advancing the therapeutic potential of 
tumor vaccine approaches.  It is now possible to genetically modify tumor cells 
for vaccination to express specific tumor suppressor genes, immune modulators, 
drug sensitive genes and antisense gene fragments (Huber, Richards et al. 1991; 
Culver, Ram et al. 1992; Trojan, Blossey et al. 1992; Dranoff, Jaffee et al. 1993; 
Ram, Culver et al. 1993; Trojan, Johnson et al. 1993; Fakhrai, Dorigo et al. 1996; 
Swisher, Roth et al. 1999).  Extensive experiences by others, as well, 
demonstrate safety with the use of GMCSF gene based vaccines as a potential 
vaccine therapeutic in cancer patients (Table 1). 
 

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Table 1:  Summary of clinical trials testing GMCSF gene-transduced vaccines 
 
Trial 
Cancer (stage) 
Vaccine 
Vector 
n 
Response 
Dose (number of 
irradiated tumor 
cells) 
GM-CSF 
production 
(ng/106 cells 
every 24 h) 
Concurrent 
treatment 
Simons 
(Simons, 
Jaffee et al. 
1997) 
Renal cell (IV) 
Autologous 
Retrovirus 
16 
1 PR (7 mo) 
4 x 106 – 4 x 108 
17-149 
None 
 
Chang 
(Chang, Li et 
al. 2000) 
Melanoma (IV) 
Autologous 
Retrovirus 
5 
1 CR (>36 mo) 
1 x 107
56-100 
RhIL-2  
3.6 x 10 /kg 
x 15 doses* 
Soiffer 
(Soiffer, 
Lynch et al. 
1998) 
Melanoma 
Autologous 
Retrovirus 
29 
1 PR, 3 MR,  
1 mixR, 3 pts  DFS 
s/p surg/XRT, >36, 
>20 mo 
ND 
84-965 
None 
 
Jaffee 
(Jaffee, 
Hruban et al. 
2001) 
Pancreatic 
cancer 
(I, II, III) 
Allogeneic 
Plasmid 
14 
3 pts DFS  
4-5 years 
1 x 107 – 5 x 108 
ND 
Surgery to 
disease-free 
adjuvant 
XRT/chemo 
Simons 
(Simons, 
Mikhak et al. 
1999) 
Prostate 
Autologous 
Adenovirus 
8 
No responses 
immune 
activation 
observed 
1 x 107 or 5 x 107 
143-1403 
None 
 
Salgia 
(Salgia, 
Lynch et al. 
2003) 
NSCL (IV) 
Autologous 
Adenovirus 
35 
2 pts DFS  
>3 years s/p 
surgery 
1 x 106 – 1 x 107 
Mean 233 
Surgery 
 
Kusumoto 
(Kusumoto, 
Umeda et al. 
2001) 
Melanoma (IV) 
Autologous 
Adenovirus 
9 
No clinical 
response 
2 x 106 – 1 x 107 
80-424 
None 
 
Mastrangelo 
(Mastrangel
o, Maguire et 
al. 1999) 
Melanoma (IV) 
Intratumoral 
injection 
Vaccinia 
virus 
7 
1 PR, 1 CR 
(injected lesion) 
NA 
NA 
None 
 
Hu (Hu, 
McNeish et 
al. 2003) 
Solid tumor 
Intratumoral 
injection 
Herpes 
simplex type 
1 
15 
1 PR  
(injected lesion) 
NA 
NA 
None 
 
Soiffer 
(Soiffer, 
Hodi et al. 
2003) 
Melanoma (IV) 
Autologous 
Adenovirus 
35 
1 CR, 1 PR,  
3-year  
follow-up 10 
patients alive, 4 
DFS 
1 x 106 – 1 x 107 
745 
None 
 
Simons 
(Simons, 
Nelson et al. 
2002) 
Prostate 
Allogeneic 
Adenovirus 
34 
Survival  
31 mo with 
3 x 108 cells vs. 22 
mo with 1 x 108  
cells 
1 x 108 – 3 x 108 
 
None 
 
Simons 
(Simons, 
Higaro et al. 
2003) 
Prostate 
Allogeneic 
Adenovirus 
65 
33 evaluable  
(1 PSA PR,  
2 PSA MR) 
1 x 108 – 3 x 108 
 
None 
 
Nemunaitis 
(Nemunaitis, 
Sterman et 
al. 2004) 
NSCLC (IIIB/IV) 
Autologous 
Adenovirus 
63 
3/33 CR 
Dose-related 
survival 
advantage 
(p <0.05) 
5-100 x 106
44-236 
None 
 
Nemunaitis 
(Nemunaitis, 
Sterman et 
al. 2004) 
NSCLC (IB/II) 
Autologous 
Adenovirus 
20 
7/10 DFS at 
16 mo 
5-100 x 106 
 
None 
 
Tani (Tani, 
Azuma et al. 
2004) 
Renal (IV) 
Autologous 
Retrovirus 
4 
2 patients  
alive >40  
and >58 mo 
1.4-3.7 x 10 8
 
None 
 
Simons 
(Simons, 
Carducci et 
al. 2006) 
Prostate 
(hormone 
naive) 
Allogeneic 
Adenovirus 
21 
76% decrease 
PSA velocity 
1.2x108 
ND 
None 
Small 
(Small, 
Sacks et al. 
2007) 
Prostate 
(hormone 
refractory) 
Allogeneic 
Adenovirus 
55 
Median survival 
metastatic group 
n=(34) 
Low dose-24 m 
High dose-34.9 m 
Prime: 5x108 
Boost: 1x108 or 
3x108 
ND 
None 
Urba (Urba, 
Nemunaitis 
Prostate 
(hormone 
Allogeneic 
Adenovirus 
19 
PSA doubling ↑ 29 
→ 57 wks 
1-5 x 108 
ND 
None 

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Trial 
Cancer (stage) 
Vaccine 
Vector 
n 
Response 
Dose (number of 
irradiated tumor 
cells) 
GM-CSF 
production 
(ng/106 cells 
every 24 h) 
Concurrent 
treatment 
et al. 2008) 
naïve) 
Nemunaitis 
(Nemunaitis, 
Jahan et al. 
2006) 
NSCL IIIB/IV 
Autologous 
and 
transfected 
K562 
Adenovirus 
49 
None 
5 x 106 auto 
5 x 108 K562 
2624 
None 
Laheru 
(Laheru, 
Lutz et al. 
2008) 
Pancreatic 
Allogeneic 
Adenovirus 
50 
Survival 4.3 mo 
vs. 2.3 mo when 
combined with 
cytoxan 
5 x 108 
ND 
Cohort 13 
and 
cyclophosph
omide 
Senzer 
(Senzer 
2009) 
Melanoma 
IIIC/IV 
Intratumoral 
injection 
Herpes 
simplex 1 
virus 
50 
61% 1-year 
survival 
1 x 1061 x 108 
pfu/ml 
ND 
None 
Hofbauer 
(Hofbauer, 
Baur et al. 
2008) 
Melanoma, 
leiomyosarcom
a 
Intratumoral 
injection 
Canarypox 
(ALVAC) 
__ 
SD 
__ 
ND 
None 
Cornelio (G. 
H. Cornelio 
2008) 
Miscellaneous 
Reximmune-
C 
Retrovirus 
7 
Median OS 5 mo 
2.5x109 or 
5.0x1010 pfu 
ND 
None 
Higano 
(Higano, 
Corman et 
al. 2008) 
Prostate 
(hormone 
refractory) 
Allogeneic 
Adenovirus 
80 
Median survival 
Hi 35 mo. 
Int 20 mo. 
Lo 23.1 mo. 
Hi 100x106 or 
200x106 (q28dx6) 
Int 200x106 
(q14dx12) 
Lo 500x106 
x1300x106 
(q14dx11) 
Serum levels 
only 
None 
Ho (Ho, 
Vanneman 
et al. 2009) 
AML 
Autologous 
 
10 
9/10 durable CR 
 
 
Allogeneic, 
nonmyeloabl
ative HSCT 
Emens 
(Emens, 
Asquith et 
al. 2009) 
Breast 
Allogeneic 
Plasmid 
28 
Not reported 
5.0x107 or 5.0x108 
cells 
16.829.4 
pg/mL (5x107) 
153.636.3 
pg/mL (5x10*) 
Cyclophosp
hamide, 
doxorubicin 
Additional studies involving the use of adenoviral vectors to transfer the GMCSF® gene have been approved by the National Institutes of Health in 
patient with prostate, lung, breast, colon and head and nec k cancer, as well as in melanoma patients. 
*Following vaccine injection, four patients received ex vivo expanded lymphocytes (CD3 negative) from harvested vaccine-primed lymph nodes. 
chemo:  Chemotherapy; CR: Complete response; DFS:  Disease-free survival; GMCSF®:  Granulocyte-macrophage colony-stimulating factor; NA: Not 
available; mixR:  Mixed response; mo: Months; MR:  Minor response; ND:  Not described; NSCLC:  Non-small cell lung cancer; PR:  Partial response; 
PSA:  Prostate-specific antigen; pts:  Patients; RhIL: Recombinant human interleukin; s/p: Status post; surg: Surgery; XRT: Radiation therapy. 
 
 Blockade of multiple TFGβ isoforms is needed to reverse 
1.7
immunosuppression 
 
Overexpression of two or more of the TGFβ isoforms has been demonstrated in 
melanoma, gliomas, prostate, gastric, colorectal, ovarian and gastric cancers 
(Tsamandas, Kardamakis et al. 2004; Dallas, Zhao et al. 2005; Polak, Borthwick 
et al. 2007; Vagenas, Spyropoulos et al. 2007).  It was previously noted that 
expression of all three immunosuppressive TGF isoforms is increased in 
ovarian tumor as compared with normal ovarian tissue (Henriksen et al, 1995; 
Bristow et al, 1999) with significant increases in TGF1 in both primary (2.9 fold; 
p ≤ 0.002) and recurrent (4.4 fold; p ≤ 0.002) ovarian cancer (Bristow, Baldwin et 
al. 1999).  TGFβ1 and -β2 both bind to TGFβ receptor 1 and suppress DC and 
helper T cell function through Smad and MAP kinases (Larmonier, Marron et al. 
2007; Park, Letterio et al. 2007).  The recent correlative study by Polak showed 
that tumor infiltrating tolerogenic DCs and suppressor T cell lymphocytes in 
malignant melanoma can be correlated with immunosuppressive TGF-β1, -β2 
and IL-10 expression (Polak, Borthwick et al. 2007).  This mechanism of tumor-

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associated immunosuppression is likely to contribute to tumor escape to immune 
response. 
 
The genetic modification with a TGFβ2 antisense-encoding plasmid represents 
one of many currently examined approaches to inhibit local TGFβ activity.  
Others include the use of neutralizing antibodies, soluble receptors, receptor 
kinase antagonist drugs, antisense reagents, and a number of less specific drugs 
such as angiotensin II antagonist and tranilast (Prud'homme 2007).  Systemic 
TGFβ blockade may result in severe inflammatory disease, although this has not 
been observed, presumably because the neutralization is only partial.  To attain 
localized TGFβ blockade as is applicable to achieve optimal immunostimulation 
with a GM-CSF-modified cancer cell vaccine, the desired outcome may be 
accomplished expediently through the incorporation of a de novo gene modifying 
moiety into the vaccinating cancer cells ex vivo, thereby bypassing 
pharmacokinetic and toxicity concerns from systemic introduction of anti-TGFβ 
neutralizing antibodies, soluble receptors, or receptor kinase antagonists.  In 
view of the overlapping immunosuppressive activities of the various TGFβ 
isoforms, it would be particular advantageous if such a gene modifying moiety 
can globally attenuate the activity of TGFβ1, -β2, and -β3. 
 
 Background on TAG (TGF2 antisense and GMCSF vaccine) 
1.8
 
Based on the hypothesis that the combination of GMCSF and TGFβ2 antisense 
genes will enhance potency of immune stimulation against cancer specific 
antigens, a combined plasmid, TAG was constructed (Gradalis, Inc. Carrollton, 
TX) (Kumar 2009).  Components of this plasmid are the clinically utilized 
pUMVC3 vector backbone (University of Michigan), a bacterial origin of 
replication, a kanamycin resistance gene, a CMV IE promoter / enhancer and 
intron A driving the hGMCSF cDNA followed by a 2A linker sequence and a 930 
base pair fragment of the hTGFβ2 cDNA molecule in antisense orientation 
followed by a rabbit hemoglobin poly-A tail (Kumar 2009; Maples PB 2009). 
 
The TAG expression vector was electroporated into the autologous cells ex vivo. 
Therefore, only the cells present at the time of electroporation would incorporate 
the transfected DNA. 
 
The vector utilized is expected to remain extra-chromosomal. Amplification of the 
insert by PCR suggested that the vector is non-rearranged in the cells. Our 
previous human and animal vaccination studies with TGFβ2 antisense vaccine 
and GVAX® vaccine (GMCSF) had not demonstrated deleterious effects due to 
unwanted gene expression. It is theoretically possible that the structure or 
expression of a gene near an integration site (if the vector was incorporated into 
a chromosome) may be affected by insertion of the added DNA. However, all 

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cells used for vaccinations were irradiated with 10,000 cGy to block their growth 
potential. 
 
GMCSF expression in the TAG product despite being significant is lower with the 
electroporation processing (1ng per 106 tumor cells per 24 hours).  Thus, 
although survival in one GVAX® study (Nemunaitis, Sterman et al. 2004) 
correlated with GMCSF expression ≥40ng/106 cells/24 hours, in a second study 
there was no correlation (see below).  Further, in addition to documented 
variability in level of GMCSF expression between manufacturing processes, the 
levels of expression achieved with the TAG vaccine are deemed clinically 
relevant as 1) use of a plasmid rather than a viral vector obviates the obfuscating 
effects of elicited anti-viral neutralizing antibodies, 2) use of a plasmid likewise 
prevents the development of elicited antibodies interfering with long-term gene 
expression, and 3) concurrent suppression of TGFβ2 would minimize tumor 
associated inhibition of GMCSF induced dendritic cell maturation  (Yamaguchi, 
Tsumura et al. 1997), which we hypothesize would improve product activity and 
theoretically lower minimal toxic effect related to GMCSF and or adenoviral 
presence. In addition, our experience with the bystander GVAX® vaccine with a 
25-fold higher GMCSF secretion than the autologous vaccine in which no 
objective tumor responses were seen suggests that levels of GMCSF protein 
expression from the manufactured vaccine is not an exclusive or possibly even 
necessary factor in vaccine activity. 
 
Thirty-eight patients (BB-IND 13650) underwent harvest for TAG vaccine.  Thirty-
two were successfully manufactured under GMP conditions (Maples PB 2009).  
GMCSF expression and TGFβ2 knockdown met product release criteria.  Three 
(all gastrointestinal tumors with luminal access) had bacterial contaminants and 
could not be released.  Three had insufficient cells.  Twenty-two advanced 
refractory cancer patients have been treated (Maples, Kumar et al. 2009; Maples 
PB 2009; Nemunaitis 2009).  No significant toxic effect was observed.  No Grade 
3/4 toxicity was demonstrated to TAG (Tables 2 and 3).  Significant increase in 
GMCSF expression and knockdown of TGFβ2 but not TGF-β1 were 
demonstrated as components of product release criteria.  In preliminary results 
we have observed 17 of 22 (77%) evaluable patients with stable disease or 
better of at least 3 months.  Two withdrew early for personal reasons with stable 
disease after 1 cycle; two had progressive disease at the 2 month assessment; 
and one withdrew consent at month 3 with progressive disease (Tables 4 and 5).  
One patient achieved CR, confirmed by imaging studies (Figure 1) (melanoma).  
Three patients remain alive under observation following successful administration 
of all manufactured vaccines (008, 013, and 023).  Two additional patients are 
continuing on trial (037 and 041).  Eight of 18 evaluable patients survived > 1 
year following initiation of treatment.  Thus the TAG vaccine appears to be safe 
and has evidence of clinical efficacy (Olivares J 2011). 
 
 

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Table 2: Frequency of Adverse Events Possibly, Probably, or Definitely Related 
to the TAG vaccine 
 
Preferred Term 
Grade 
Relation to 
TAG 
Number of 
Events 
Number of 
Subjects 
Edema 
1 
Definitely 
1 
1 
Fatigue 
1 
Possibly 
1 
1 
Fatigue 
2 
Possibly 
1 
1 
General Pain 
(NOS) 
1 
Definitely 
4 
1 
Injection Site 
Reaction 
1 
Definitely 
3 
3 
Injection Site 
Reaction 
1 
Probably 
2 
2 
Injection Site 
Reaction 
2 
Probably 
1 
1 
Rash 
1 
Possibly 
1 
1 
Rash 
2 
Possibly 
1 
1 
Fatigue 
2 
Possibly 
1 
1 
Left Arm 
Soreness 
1 
Probably 
1 
1 
Left Arm 
Weakness 
1 
Probably 
1 
1 
 
Table 3: Serious Adverse Experiences reported on TAG vaccine 
 
ID# 
Reported Term 
Grade 
Drug Related 
Assessment 
Unexpected 
007 
Small Bowel Obstruction 
3 
Not Related 
Yes 
007 
Dehydration 
3 
Not Related 
No 
010 
Seizure 
3 
Not Related 
Yes 
010 
GI Bleed 
3 
Not Related 
Yes 
010 
Brain Metastasis 
3 
Not Related 
Yes 
017 
Pneumonia (resulting in death) 
5 
Not Related 
Yes 
031 
Pericardial effusion 
3 
Not Related 
Yes 
032 
Weight Loss 
2 
Not Related 
No 
032 
Vomiting 
3 
Not Related 
No 
032 
Aspiration Pneumonia 
3 
Not Related 
No 
 
 
 

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Table 4: Demographics of Subjects on TAG 
 
Age (years) 
16-64 
24 
 
≥65 
14 
Gender 
Male 
17 
 
Female 
21 
Cancer 
Adrenocortical 
2 
 
Bladder Cancer 
2 
 
Breast Cancer 
4 
 
Colon Cancer 
4 
 
Colorectal 
1 
 
Duodenal Cancer 
1 
 
Gastric 
1 
 
Leiomyosarcoma 
1 
 
Melanoma 
5 
 
Neuroendocrine 
2 
 
NSCLC 
7 
 
Ovarian 
1 
 
Rectal Cancer 
1 
 
Renal Cancer 
1 
 
Urachal Adenocarcinoma 
1 
 
Prostate 
1 
 
Hemangiopericytoma 
1 
 
Esophageal 
1 
 
Cervical Cancer 
1 
 
Table 5: Response status of subjects on TAG 
 
 
2.5 x 107 cells / 
injection 
1.0 x 107 cells / 
injection 
Stable disease ≥ 3 months after 
vaccine received 
10 
6 
Unevaluable 
2 
0 
Progressive disease 
3 
0 
Complete response 
0 
1 
Active on treatment < 3 months 
4 
0 
 
 

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Figure 1: Patient 013 had diffusely metastatic melanoma and had previously 
failed multiple standard therapies.  PET CT 11 cycles after TAG demonstrated 
significant response on 7/7/09 in comparison to baseline on 7/7/08.  New 2,3 
FDG uptake however in upper paratracheal nodes in association with a viral 
syndrome confused demonstration of CR.  Re-scan 3 months later with viral 
syndrome resolved revealed CR.  Residual uptake at L 2 was followed up with a 
MRI scan and biopsy which revealed no malignancy. 
 
Harvested malignant tissue was processed to a single cell suspension, and cells 
were transfected with the TAG expression vector via electroporation.  All 
processed TAG vaccines demonstrated GMCSF secretion and low to 
undetectable levels of TGFβ2, as shown in Figures 2A and 2B. 
 

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Figure 2A: GM-CSF expression in TAG vaccines. 
 
GMCSF Expression in Pre and Post TAG Plasmid 
Transfected Autologous Tumor Cells
1
10
100
1000
10000
Pre 
Post
p g /m l
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Breast
Breast
Breast
Breast
Breast
Colon
Colon
Colon
Colon
Rectal
Gastric
Duodenal
Melanoma
Melanoma
Melanoma
Melanoma
Adrenocortical
Adrenocortical
Liver
Ovarian
Neuroendocrine
Renal
Leiomyosarcoma
Bladder
Bladder
Prostate
Hemagiopericytoma
Esophageal
Cervical
Urachal

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Figure 2B: TGFβ-2 expression in TAG vaccines. 
 
 Background on Furin 
1.9
 
Furin, an upstream regulator of TGFβ activity, is a member of the subtilisin-like 
proprotein convertase family. The members of this family are proprotein 
convertases that process latent precursor proteins into their biologically active 
products. This encoded protein is a calcium-dependent serine endoprotease that 
can efficiently cleave precursor proteins at their paired basic amino acid 
processing sites by the consensus sequence –Arg-X-K/Arg-Arg (RXK/RR), with -
RXXR- constituting the minimal cleavage site (Thomas 2002).  Furin is 
synthesized as an inactive zymogen and translocated into the endoplasmic 
reticulum (ER), where the propeptide is cleaved autocatalytically.  Furin is 
activated when it exits the ER and transits to the mildly acidic trans Golgi 
apparatus, where it is further cleaved by the resident, activated furin or by an 
TGF2 Expression in Pre & Post TAG Plasmid 
Transfected Autologous Tumor Cells n=38
1
10
100
1000
10000
100000
Pre
Post
pg/ml
Lung 1
Lung 2
Lung 3
Lung 4
Lung 5
Lung 6
Lung 7
Lung 8
Breast 1
Breast 2
Breast 3
Breast 4
Breast 5 
Colon 1
Colon 2
Colon 3
Colon 4
Rectal 5
Gastric 6
Duodenal 7
Adrenocortical 1
Adrenocortical 2
Adrenocortical 3
Melanoma 1
Melanoma 2
Melanoma 3
Melanoma 4
Bladder 1
Bladder 2
Liver (H)
Ovarian (O)
Neuroendocrine (N)
Renal ®
Leiomyosarcoma (S)
Prostate (P)
Hemagiopericytoma (H)
Esophageal (E)
Cervical ©

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associated molecule in trans.  Its dissociation from the catalytic domain leads to 
its activation.  Following release and intracellular trafficking, furin can be tethered 
at the cell membrane by the cytoskeletal protein filamin.  Proteolytic cleavage by 
furin is required for TGFβ convertase activation (i.e. pro-TGFβ TGFβ).  All 
three of the TGFβ isoforms contain the RXXR motif at their cleavage site albeit 
with different amino acid sequences (Kusakabe, Cheong et al. 2008). Data 
suggest that the unique structure of the TGFβ2 latency associated peptide (LAP) 
region might attenuate furin mediated cleavage but do not exclude a role for furin 
in its processing. 
 
Furin is the primary proconvertase in TGFβ processing events. It is worth noting 
that there appears to be a disparity between the complete cleavage of TGFβ in 
vivo versus the partial cleavage in vitro (Dubois, Laprise et al. 1995). TGFβ in 
turn appears to amplify furin gene transcription through an amplification loop 
(Blanchette, Day et al. 1997; McMahon, Laprise et al. 2003). 
 
High levels of furin mRNA and furin protein are widely expressed in human 
tumors and, specifically, in ovarian cancer (Schalken, Roebroek et al. 1987; 
Cheng, Watson et al. 1997; Mbikay, Sirois et al. 1997; Bassi, Mahloogi et al. 
2000; Bassi, Mahloogi et al. 2001; Bassi, Fu et al. 2005).  There is evidence that 
furin may be an important target in the treatment of prostate cancer (Uchida, 
Chaudhary et al. 2003), colorectal cancer (Scamuffa, Basak et al. 2008; 
Scamuffa, Siegfried et al. 2008), and breast cancer (Lapierre, Siegfried et al. 
2007). In addition, furin plays an important role in immune regulation (Pesu, Muul 
et al. 2006; Pesu, Watford et al. 2008).  In APCs, cytotoxic T lymphocyte-
sensitive epitopes in the trans-Golgi compartment were processed by furin and 
the less frequented TAP independent pathway (Lu, Wettstein et al. 2001).  The 
conditional deletion of endogenous-expressing furin in T lymphocytes allowed for 
normal T-cell development, but impaired the function of regulatory and effector T 
cells, which produced less TGFβ1. Furin-deficient Tregs were less protective in a 
T-cell transfer colitis model and failed to induce Foxp3 in normal T cells. 
Additionally, furin-deficient effector cells were inherently over-active and were 
resistant to suppressive activity of wild-type Treg cells. Thus, furin expression by 
T cells appears to be indispensable in maintaining peripheral tolerance, which is 
due, at least in part, to its non-redundant, essential function in regulating TGFβ1 
production (Pesu, Watford et al. 2008). 
 
We and others have found that up to a 10-fold higher level of TGFβ1 may be 
produced by human colorectal, lung cancer, melanoma, and ovarian cancer cells, 
and likely impact the immune tolerance state by a higher magnitude 
(Bommireddy and Doetschman 2007; Fogel-Petrovic, Long et al. 2007; Polak, 
Borthwick et al. 2007). The presence of furin in tumor cells likely contributes 
significantly to the maintenance of tumor directed, TGFβ1 peripheral immune 
tolerance (Pesu, Watford et al. 2008). Hence, furin knockdown represents a 
novel and attractive approach for optimizing immunosensitization. Initial studies 

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to validate the effect of furin blockade on TGFβ expression was carried out with 
the furin inhibitor decanoyl-Arg-Val-Lys-Arg-CMK (Dec-RVKR-CMK), a peptidyl 
chloromethylketone that binds irreversible to the catalytic site of furin and blocks 
its activity ((Henrich, Cameron et al. 2003)).  Treatment with Dec-RVKR-CMK 
either completely or partially reduces the activity of furin substrates BASE (β-site 
APP-cleaving enzyme), MT5-MMP, and Boc-RVRR-AMC (Pearton, Nirunsuksiri 
et al. 2001). 
 
TGFβ1 and TGFβ2 specific immunoassays (R&D Systems Quantikine ELISA) 
were used to quantify the effect of furin blockade on secreted cytokine levels of 
the human colorectal line CCL-247.  Dec-RVKR-CMK (30 nM) reduced TGF1 
level from 250 pg/ml to below detectable levels, and inhibited TGFβ2 expression 
by approximately 50% (from 35 to 18 pg/ml).   Dec-RVKR-CMK was similarly 
effective in reducing TGFβ1 and TGFβ2 production in the human melanoma line 
CRL-1585 (reductions of 100% and 82%, respectively) and the Human NSCLC 
line H460 (reductions of 100% and 93%, respectively).  These findings indicate 
that TGFβ isoform expression can be effectively reduced through furin blockade. 
 
To consider the applicability of furin knockdown for inhibiting TGFβ isoform 
expression, similar assessments were performed following transfection with furin-
specific siRNAs.  Prospective siRNA targeting sites in the furin mRNA were 
determined by the published recommendations of  Tuschl and colleagues and 
the additional selection parameters that integrates BLAST searches of the 
human and mouse genome databases (http:jura.wi.mit.edu/bioc/siRNAext).  
siRNAs targeting eligible translated and 3’UTRs were purchased from Ambion 
(A-1, -2,-3) and Integrated DNA Design Technologies/IDT (I-1,-2,-3; Figure 3).  
Proprietary siRNAs from Ambion were designed with an additional algorithm that 
utilized a proprietary classification approach support vector machine (SVM) 
approach for enhanced specificity and activity.  siRNAs from IDT utilizes a 
proprietary rational design algorithm that integrates both traditional 21-mer siRNA 
design rules as well as new 27-mer Dicer-substrate specific design criteria. 
 

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Figure 3:  siRNA targeted regions of furin mRNA. 
Prospective siRNA targeting regions in 3’-UTR and encoding regions of furin 
mRNA and the targeted sequence by each siRNA construct is shown. 
 
The transfection of CCL-247 cells was carried out with Lipofectamine 2000 with 
10nM -60nM siRNA and 1x105 cells/ml.  Treatment with each of the 6 siRNA 
constructs resulted in marked reduced levels of TGFβ1 and TGFβ2 in the culture 
supernatants.  Depletion ranged from 60-72% for TGF1 and comparable levels 
(57-77%) for TGFβ2 (Figure 4).  By comparison, the inhibitory effects of 
scrambled siRNA treatment were less profound (<20%).  Further, siRNAs that 
targeted the encoding (A-1, A-2, I-1, I-2) or the 3’-UTR (A-3, I-3) of furin miRNA 
appeared to be equally effective for TGFβ1 and β2 depletion.  Similar levels of 
cytokine reduction were observed over the dose range tested (10-60 nM) for 
each siRNA.  
 
The viability of untreated CCL-247 cells was 84% at 24 hours post-culture, based 
on trypan blue dye exclusion viability analysis.  By comparison, 70% of cells were 
viable after treatment with the fuin inhibitor Dec-RVKR-CMK.  siRNAfurin treated 
cultures displayed a viability of > 70% for all siRNA tested at the same time point, 
as compared to 80% following control siRNA treatment.  There was no 
remarkable survival advantage following treatment with any of the 6 siRNA 
tested.  These findings indicate that the observed TGFβ reductions cannot be 
explained by the marginal cytotoxicity of Dec-RVKR-CMK or siRNAfurin 
transfection.  The relatively robust viability of transfected cultures implies that 
immunosensitization through transgene expression is likely to remain active over 
an extended time period, (i.e. > 48 hrs). 
 
siRNAfurin treatment similarly reduced TGF levels the human melanoma line CRL-
1585 (37-59% reduction of TGF-β1, 25-51% reduction of TGF-β2) and the 
NSCLC line H460 (78-94% reduction of TGF-β1, 72-79% reduction of TGF-β2) 

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(Figure 4).  Our findings indicate that siRNA-mediated furin knockdown is 
effective for the depletion of TGF-β isoforms. 
 
a 
B 
 
Figure 4:  TGF-β 1 (Figure 4a) and TGF-β2 (Figure 4b) expression in human 
cancer lines following siRNAfurin knockdown. 
 
Currently, it is unclear why TGFβ reduction in CRL-1585 was comparatively 
modest.  All three lines displayed similar transfection efficiencies (75-85%) by 
assessment with a GFP expression plasmid.  Followup studies are underway to 
quantify furin reduction at the mRNA and protein levels, in order to correlate 
knockdown outcome with TGF reduction outcomes. 
 
Gradalis, Inc., is at the forefront in the design of “bifunctional” vectors that embed 
both siRNA and miRNA functional components in a miR 30 scaffold for optimized 
gene knockdown (Rao 2009; Rao D 2010).  The siRNA component is encoded 
as a hairpin composed of complete matching sequences of the passenger and 
guide strands.  Following cleavage of the passenger strand by Ago 2, an 
endonuclease with RNase H like activity, the guide strand is incorporated into the 
RNA-induced silencing complex (RISC), which binds to and cleaves 
complementary target mRNA (cleavage dependent process).  In distinction, the 
miRNA component of the “bifunctional” vector incorporates mismatches between 
the passenger and guide strands within the encoding shRNA hairpin in order to 
achieve lower thermodynamic stability.  This configuration allows the passenger 
strand to dissociate from RISC without cleavage (cleavage independent process) 
(Matranga, Tomari et al. 2005; Leuschner, Ameres et al. 2006), and the RISC 
incorporated guide strand acts both through translational repression and 
sequestration of the target mRNA in the cytoplasmic processing bodies (P-body). 
 
We have previously demonstrated the markedly enhanced effectiveness of bi-
functional shRNASTMN1 in knockdown of stathmin (oncoprotein 18) which encodes 
a protein that regulates microtubule remodeling of the cytoskeleton and shown to 
be upregulated in a high proportion of patients with solid cancers (Rana, Maples 

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et al. 2008).  The bifunctional shRNA construct achieved effective knockdown 
against stathmin 1, with a 5 log dose enhancement in potency of tumor cell killing 
compared with the identically targeted siRNA (Rao 2009; Rao D 2010). 
 
A similarly designed bifunctional shRNA was designed to effect furin knockdown.  
The bi-shRNAfurin consists of two stem-loop structures with miR-30a backbone; 
the first stem-loop structure has complete complementary guiding strand and 
passenger strand, while the second stem-loop structure has two bp mismatches 
at positions 11 and 12 of the passenger strand.  Our strategy is to use a single 
targeted site for both cleavage and sequestration.  By the use of a proprietary 
algorithm, the encoding shRNAs are proposed to accommodate mature shRNA 
to be loaded onto more than one types of RISC (Azuma-Mukai, Oguri et al. 
2008).  Our reason for focusing on a single site is that multi-site targeting may 
double the chance for “seed sequence” induced off-target effect (Jackson, 
Koduvayur et al. 2006). 
 
Figure 5:  Plasmid construct of FANG™. 
 
 
 
Construction of the FANG™ expression vector is based on the TAG plasmid 
(Kumar 2009) with the only difference being the replacement of the TGFβ2 
antisense DNA sequence with the bi-shRNAfurin DNA sequence (Figure 5).  The 
two stem-loop double stranded DNA sequence was assembled with 10 pieces of 
synthetic complementing and interconnecting oligonucleotides through DNA 
Or
i
ce
CM
V
Pr
om
ot
er
HI
Ba
m
HI
Ba
m
20
00
30
00
50
00
I
2A
po
ly
A
sh
-F
ur
in
5139 bp

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ligation.  The completed 241 base pairs DNA with Bam HI sites at both ends was 
inserted into the Bam HI site of the TAG expression vector in place of the TGFβ2 
antisense sequence.  Otherwise the two expression vectors (TAG and FANG™) 
are identical (confirmed by sequencing).  Orientation of the inserted DNA was 
screened by PCR primer pairs designed to screen for the shRNA insert and 
orientation.  The FANG™ construct has a single mammalian promoter (CMV) 
that drives the entire cassette, with an intervening 2A ribosomal skip peptide 
between the GMCSF and the furin bifunctional shRNA transcript, followed by a 
rabbit polyA tail.  There is a stop codon at the end of the GMCSF transcript.  
Insertion of picornaviral 2A sequences into mRNAs causes ribosomes to skip 
formation of a peptide bond at the junction of the 2A and downstream 
sequences, leading to the production of two proteins from a single open reading 
frame (Funston, Kallioinen et al. 2008).  We have found that the 2A linker to be 
effective for generating approximately equal levels of GMCSF and anti-TGFβ 
transcripts with the TAG vaccine, and elected to use the same design for Vigil™. 
 
Electroporation of GMP FANG™ plasmid into patient tumor cells (the cGMP 
vaccine manufacturing process) demonstrated GMCSF protein production and 
concomitantly TGFβ1 and –β2 knockdown as predicted.  Figure 6 depicts a 
FANG™-transfected NSCLC tumor’s expression profile over 14 days (FANG-
004).  A summary of the 14 day expression (after manufacturing) profiles of the 
23 tumors processed to date is depicted in Figure 7.  Of note is the comparison 
of the TAG-038 and FANG-004 vaccines produced from the same patient’s 
NSCLC tumor.  The TAG vaccine has similar GMCSF expression and TGF-β2 
knockdown but TAG did not effect the TGF-β1 expression (which is about 10X 
that of pre-transfection TGF-β2) (Figure 8). 
 
 

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Figure 6: Summary of TGFβ1, -β2, and GMCSF expression in FANG-004 tumor 
cells pre and post FANG™ cGMP plasmid transfection.  Cells were incubated for 
14 days and media was periodically sampled for cytokine analysis. 
TGF beta1 Expression by FANG-004 (NSCLC) pre and post 
transfection/post irradiated cells
0
500
1000
1500
2000
2500
3000
3500
4000
Day0
Day1
Day2
Day3
Day4
Day7
Day10
Day14
Days in culture
pg/ml
Pre
Post
TGFb2 Expression by FANG-004 (NSCLC) pre and post transfection/post 
irradiated cells
0
200
400
600
800
1000
Day0
Day1
Day2
Day3
Day4
Day7
Day10
Day14
Days in culture
pg/ml
Pre
Post
GMCSF Expression by FANG-004 (NSCLC) pre and post 
transfected/post irradiated cells
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
Day0
Day1
Day2
Day3
Day4
Day7
Day10
Day14
ng/ml
Pre
Post

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Figure 7:  Summary of TGFβ1, -β2, and GMCSF protein production pre and post 
FANG™ plasmid transfection.  ELISA values from Day 4 of the 14-day 
determinations of cytokine production in manufactured autologous cancer cells.  
TGF1 Expression (Day 7) Pre and Post 
FANG Transfected Autologous Tumor Cells 
1
10
100
1000
10000
Pre
Post
pg/ml
TGF2 Expression (Day 7) Pre and Post 
FANG Transfected Autologous Tumor Cells
1
10
100
1000
10000
Pre
Post
pg/ml
GMCSF Expression (Day 7) Pre and Post 
FANG Transfected Autologous Tumor Cells
1
10
100
1000
10000
Pre
Post
pg/ml
FANG- 0 0 1 M elanoma
FANG- 0 0 2  M elanoma
FANG- 0 0 5 M elanoma
FANG- 0 0 6  M elanoma
FANG- 0 0 7 Colon
FANG- 0 0 8  C olorect al
FANG- 0 0 3  Gallbladder
FANG- 0 0 4  N SCLC
FANG- 0 0 9  B reast  C an
FANG- 0 10  Colorect al
FANG- 0 11 SCLC
FANG- 0 12  M elanoma
FANG- 0 13  Breast  Can
FANG- 0 14  M elanoma
FANG- 0 15 NSCLC
FANG- 0 16  Colon
FANG- 0 17 NSCLC
FANG- 0 18  Colon
FANG- 0 19  M elanoma
FANG- 0 2 0  M elanoma
FANG- 0 2 1 Colorect al
FANG- 0 2 2  M elanoma
FANG- 0 2 3  Pit uit ary

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Data represents autologous vaccines independently generated from 23 patients 
who underwent Vigil™ processing (FANG 001 – 023). 
 
 
Figure 8:  Manufactured consecutively from the same patient tumor.  Values 
represent ELISA determinations of cytokine production at Day 7 post transfection 
in harvested autologous cancer cells transfected with either Vigil™ (blue) or TAG 
plasmid (red).  The TGFβ2 post transfection values are the same for TAG 
(ACTV-038) and Vigil™ (FANG-004), hence the black line. 
 
 Phase I Results 
1.10
 
The Vigil™ vaccine is comprised of autologous tumor cells as a source of TAA’s 
(tumor associated antigens) and two genetic modifications in order to optimize a 
“triad” functionality--patient tumor-specific antigen presentation, dendritic cell 
activation (GMCSF), and tolerance escape (blocking TGF1, 2 activation) 
(Maples PB 2009; Nemunaitis 2011).  To construct Vigil™, autologous cancer 
cells are transfected with a multiple component expression vector encoding 
GMCSF for recruitment and differentiation of antigen presenting dendritic cells 
(DCs) 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. 
 
We recently completed a Phase I trial with Vigil™ vaccine in which 56 cancer 
patients underwent successful vaccine construction and 33 patients (1 of them 
being a pediatric patient 15 years of age) received ≥1 vaccination.  No significant 
toxic effect was observed.  A marked elevation of interferon gamma producing T 
cells was observed in a subset of patients which correlated with survival. 
 
The capacity of Vigil™ to deplete TGF1 and TGF2 isoforms is hypothesized to 
1) effect silencing of a well documented, primarily endogenous family of 
immunosuppressive cytokines and 2) specifically inhibit TGF-suppression of 
GMCSF induced maturation of DCs, expression of MHC Class II and co-
stimulatory molecules. 
TGF1 Expression
0
500
1000
1500
2000
2500
Pre
Post
Day 7
pg/m l
TGF2 Expression
0
100
200
300
400
500
Pre
Post
Day 7
pg/ml
GMCSF Expression
0
500
1000
1500
2000
Pre
Post
Day 7
pg /m l

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Product transgene expression of GMCSF and downregulation of expression of 
TGF1, -2, and Furin are shown in Figure 9.  Mean post-transfection GMCSF 
expression value was 1135 pg/106 cells/ml (Day 4 of QC assay), the GMCSF 
release specification of ≥30pg GMCSF/106 cells/ml was met for each vaccine 
manufactured).  Mean post-transfection TGF1 and 2 knockdown were 93% 
and 95%, respectively (Day 4 of QC assay).  Furin knockdown was 78%. 
 
There was no difference in the rate of adverse events across the 2 dose cohorts.  
Two possibly related Grade 3 treatment-related events were observed: 
abdominal pain and neutropenia.  There were no treatment-related serious 
adverse events.  The most common Grade 1, 2 adverse event occurring at ≥5% 
frequency related to study medication was injection site erythema.  No 
relationship between dose and adverse event frequency or severity was 
observed. 
 
Of the 33 patients who have received ≥1 vaccine dose, 31 were evaluable for 
tumor response (1 adult and 1 pediatric patient progressed following first 
injection).  Mean and median survival of all Vigil™ treated adult patients (n=32) 
from time of procurement was 584 days and 562 days, respectively.  Their mean 
and median survival from time of treatment was 477 days and 490 days, 
respectively.  Factors such as age, sex, dose level, pre-treatment expression 
levels of TGF1 and 2 and Furin and vaccine transgene expression or 
knockdown did not correlate with survival.  However, breakdown of survival 
comparing Vigil™ treated adult patients receiving ≥4 vaccines (n=24) vs. <4 
vaccines (n=8) was significantly different (p<0.001) as shown in Figure 10. 
Although not randomized, comparison to patients fulfilling the same inclusion 
criteria who did not receive Vigil™ formerly known as FANG™ from time of 
procurement (no FANG™) is shown in Figure 11.  Difference in survival (median 
122 days for no FANG™, n=29 and 562 days for FANG™, n=32) achieved 
statistical significance (p<0.00001).  Conservative assessment of only patients 
who survived 4 or more months since procurement revealed a median survival of 
251 days for the no FANG™ patients (n=11) and 572 days for FANG™ patients 
(n=30) (p=0.005).  Interestingly, all four patients with advanced metastatic 
melanoma achieved ≥1 year of survival following treatment with Vigil™ 
(specifically, 967, 835, 560, and 490 days each). 

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a 
 
 
b 
 

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c 
 
 
d 
 
 
Figure 9.  Protein data results for GMCSF gene expression (a), TGFβ1 
knockdown (b), TGFβ2 knockdown (c), and Furin knockdown (d) from 56 
successful vaccines and 4 vaccines not meeting the minimum dose requirement 
but for which protein data were collected.  Failed vaccines (n=5) did not have 
protein data collected.   

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Figure 10.  Survival comparison of treated adult patients who received <4 
FANG™ vaccinations (red) vs. ≥4 vaccinations (blue), n=32, p<0.001.  (Data as 
of 01/08/13) 
 
 
 
Figure 11.  Survival comparison of adult patients receiving FANG™ (blue) to 
those not receiving FANG™ (red), n=61, p<0.00001. (Data as of 01/08/13) 
 
Sequential ELISPOT analysis at baseline and prior to the 4th Vigil™ vaccine was 
performed in 24 patients.  Results are shown in Figure 12.  Twenty-two had no 

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detectable autologous tumor specific cytotoxic CD8+ T-cell activity at baseline.  
Two patients showed ELISPOT reaction at baseline (>10 spots), but while one 
showed reactivity >2 times that of baseline at the Month 3 assessment, the other 
did not have a >2 times increase in ELISPOT spots, and thus was considered a 
negative responder.  Twelve patients had an increase in immune response from 
a baseline mean of 6 IFN producing T-cell spots to an end of Cycle 3 mean of 
106 IFN producing T-cell spots (p = 0.011).  The other 12 patient showed 
neither reactivity nor enhancement of immune response from a baseline mean of 
3 spots through Cycle 3.  These 2 populations were statistically different from 
each other at the end of Cycle 3 in ELISPOT response (106 spots vs. 3 spot, 
p=0.007).  Comparison of survival between the 12 patients demonstrating 
positive ELISPOT response and those 12 not demonstrating response at 
completion of Cycle 3 demonstrated a statistically significant increase in survival 
from time of procurement (p=0.040) and time of treatment start (p=0.023, Figure 
13a, b) in the former group.  Additionally, patients who received ≥3 doses of 
Vigil™ and had a positive ELISPOT had better survival than patients who did not 
receive Vigil™ (p<0.001). 
 
 
 
 
Figure 12.  IFN- (ELISPOT) in FANG™ vaccine treated patients peripheral 
blood mononuclear cells in response to non-transfected autologous tumor cells.  
Blue lines indicate patients achieving ≥10 IFN- producing lymphocytes (positive 
response) after 3 cycles of FANG™ vaccine.  Red lines indicate patients not 
achieving positive ELISPOT response after 3 cycles of the FANG™ vaccine. 
(Data as of 01/08/13) 
 

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All patients demonstrating positive response after 3 complete cycles of Vigil™ 
vaccine who had long term assessment continued to show positive ELISPOT 
response during vaccine treatment and after discontinuation of vaccine therapy 
(Figure 12).  Two patients who were in the non-responsive group at the end of 
Cycle 3 demonstrated a late positive response after 5 cycles of Vigil™ vaccine.  
One of these patients had advanced metastatic melanoma with survival of 490 
days since treatment start, and the other had hepatocellular cancer with a 
survival of 738 days since treatment start. 

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a 
 
b 
 
 
Figure 13:  Survival comparison of patients achieving positive ELISPOT 
response prior to the 4th injection (blue) vs. those not achieving positive ELISPOT 
response prior to the 4th injection (red) from time of procurement, p=0.40 (a) and 
time of first vaccination, p=0.023 (b) (n=24). 
 
This Phase I study was designed to assess the safety of a RNAi mediated, 
GMCSF expressing autologous tumor cell vaccine, Vigil™, and to evaluate the 
p = 0.040 
p = 0.023 

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triad immunotherapeutic concept (Nemunaitis 2011) of concurrent autologous 
tumor cell antigen provision, immunostimulation and inhibition of autologous 
whole cell component endogenous immunosuppression. 
 
Enhanced effectiveness of the bi-shRNA vis-à-vis downregulation of expression 
as compared with siRNA and shRNA has been previously demonstrated (Liu, 
Rao et al. ; Rao, Maples et al. 2010).  In the context of comparative Vigil™ 
plasmid functionality with the prior generation TAG vaccine, the median GMCSF 
expression was similar but TGF2 knockdown was more effective, i.e., 92.5% as 
compared to slightly more than 54% knockdown with TAG.  However, most 
significantly, TGF1 knockdown, which is not affected in TAG, was 93% with 
Vigil™.  These results validate the rationale and confirm the effectiveness of 
inhibition of expression of immunosuppressive TGF isoforms via a bi-shRNA 
mediated knockdown of the proprotein convertase Furin as well as the feasibility 
of an integrated GMCSF + RNAi moiety.  The high degree of TGF1 and 2 
knockdown herein achieved combined with the clinical and immunological data 
presented justify advancement of the current manufacturing process.  A 3+ year 
follow up of 74 Phase I patients with successful vaccine manufacture, of whom 
35 received Vigil™, confirmed tolerability and safety with no evidence of long-
term toxicity. In addition, survival continues to correlate with ELISPOT response 
(median 836 days vs. 440 days with positive and negative ELISPOT respectively) 
(Senzer N 2013) 
 
Results support the hypothesis that the Vigil™ vaccine is safe and, in addition, 
provide a Phase I database justifying continued clinical evaluation and expansion 
of immune assessment assays. 
 
 STUDY RATIONALE 
2.0
 
This study adopts an alternative approach to immune therapy assessment by 
eschewing the treatment of late stage disease and evaluating adjuvant-based 
disease prevention in patients with minimal disease but at high risk for 
recurrence.  According to the serial immune function analyses by Coleman 
(Coleman, Clayton et al. 2005), five of nine patients with Stages III-IV ovarian 
adenocarcinoma who achieved post-chemotherapy remission retained the 
capacity of CD8+ T-cell responses to a panel of 11 viral peptides restricted by at 
least six common HLA class I alleles.  Chemotherapy produced no apparent 
effect on naïve (CD45RA+CCR7+), central memory (CD45RA-CCR7+), or effector 
memory (CD45-CCR7-) T-cells.  These findings affirm the strategy of vaccine 
deployment following chemotherapy, and justify selection of Stage III/IV epithelial 
ovarian cancer patients who have achieved complete clinical or pathological 
remission following cytoreductive surgery and front-line chemotherapy for this 
Vigil™ vaccine study.  Exploratory analysis of two Phase III studies (D9901 and 
D9902) with the recently FDA approved Provenge® has identified prolonged 

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survival benefits in prostate cancer patients initially treated with Provenge® and 
subsequently received docetaxel (median survival of 34.5 months, compared to 
25.4 months in patients treated with placebo followed by docetaxel) (Petrylak 
2006).  Thus the potential for vaccine mediated immune modulation both 
posterior and anterior to chemotherapy is a clinically attractive strategy worthy of 
exploration. 
 
With regard to the appropriateness of a placebo arm in this group of patients, 
to date, in conjunction with platinum/taxol base regimens, maintenance therapies 
have shown neither a DFS endpoint advantage (Sabbatini, Harter et al. 2013) nor 
an OS (overall survival) endpoint advantage low dose taxol (Markman, Liu et al. 
2009), Abagovomab (Sabbatini, Harter et al. 2013), and bevacizumab (Burger, 
Brady et al. 2011; Perren, Swart et al. 2011) while giving rise to a spectrum of 
extended treatment related side effects and questions of results-related 
economic justification (Lesnock, Farris et al. 2011).  In the two recent randomized 
Phase III studies of bevacizumab, GOG-0218 (15 mg/kg x 16 cycles) and ICON7 
(7.5 mg/kg x 12 cycles), in which there was a RFS endpoint advantage, the 
bevacizumab and control curves converged after discontinuation of bevacizumab 
prior to progression at 24 months and ~22 months, respectively, implying that 
continuation of bevacizumab until progression be considered, consistent with the 
results of the OCEANS trial in women with platinum-sensitive recurrent disease 
(for a significant RFS endpoint albeit there was still no significant difference in 
OS at first interim analysis) (Aghajanian, Blank et al. 2012).  This must be 
weighed against the added exposure to bevacizumab associated adverse 
effects.  As subsequently pointed out, the 3.8 month gain in PFS came at the 
patient cost of “23% risk for developing Grade 2 hypertension, 10% risk for 
Grades 3 to 4 hypertension, and 2.3% risk for Grade 3 or worse GI perforation, 
hemorrhage, or fistula formation”(Hensley 2011).  In addition, based on Quality 
Adjusted Life Year (QALY), the cost of chemotherapy plus just 16 cycles of 
bevacizumab is $326,530/QALY versus $13,402/QALY for the same 
chemotherapy and maintenance low-dose paclitaxel (Lesnock, Farris et al. 
2011).  Likewise in a cost-effectiveness analysis of GOG-0218, the cost 
effectiveness ratio (i.e. the cost per year of PFS) was $247,616, $1,800,000 and 
$5,500,000 for the paclitaxel (P) and carboplatin (C); P+C and bevacizumab (B); 
and P+C+B and maintenance B, respectively (Cohn, Kim et al. 2011).  In the 
absence of a confirmed OS advantage, at least for front-line therapy for ovarian 
cancer, the additional cost is hard to justify (Hensley 2011; Tomao, Tomao et al. 
2013); particularly so without FDA approval.  Similarly Grade ≥2 toxic effects 
including peripheral neuropathy (15.5), infection/fever (19.9%), and dermatologic 
events (70.8%), are observed with maintenance therapy involving taxol (Mannel, 
Brady et al.).  Despite a recurrence-free-interval delay in the SWOGS9761/GOG 
178 study of 12 vs. 3 cycles of maintenance Taxol (22 vs. 14 months), long-term 
follow up revealed no survival advantage (53 vs. 48 months; p=0.34) (Markman, 
Liu et al. 2009).  In addition, a Phase III study of maintenance Taxol x 6 cycles 
vs. observation revealed neither a progression-free survival nor overall survival 
advantage (Conte 2007).  The attendant significant morbidity and lack of survival 

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benefit qualified the National Comprehensive Cancer Network (NCCN) 
commentary on use of Taxol maintenance as 2B with the further proviso 
recommending use in a clinical trial. 
 
 OBJECTIVES 
3.0
 
 Primary objective(s) 
3.1
 
 To assess time to recurrence (TTR) following the administration of bi-
shRNAfurin and GMCSF autologous tumor cell (Vigil™) vaccine. 
 
 Secondary objective(s) 
3.2
 
 To identify and determine the effect of Vigil™ autologous tumor cell 
vaccine on immune surrogate markers in this group of patients. 
 To assess the predictive potential of initial tumor infiltrating lymphocyte 
(TIL) and tumor associated macrophage (TAM) phenotypes. 
 To enlarge the safety database of Vigil™ autologous tumor cell vaccine in 
patients with miminal disease. 
 
 STUDY DESIGN 
4.0
 
 Design 
4.1
 
This is a Phase II open-label trial of maintenance Vigil™ autologous tumor cell 
vaccine.  Tumor will be harvested at the time of surgical debulking (standard of 
medical care).  Subsequently, patients achieving clinical CR following primary 
surgical debulking and doublet chemotherapy will be stratified for i) surgical stage 
(Stage IV or suboptimal debulking (>1 cm residual) Stage III disease versus 
Stage III patients with optimal debulking (<1 cm residual)) and ii) post-op 
chemotherapy, pre-vaccine CA-125 >10 ≤ 20 U/mL versus 0≤10 U/ml, then 
randomized 2:1 (Note: patients with Stage IIIc ovarian cancer will be additionally 
evaluated as a subset using descriptive statistics only).  Patients will receive 1.0 
x 107 cells / intradermal injection of gene transfected autologous tumor cells, 
Vigil™, once a month for up to 12 doses as long as sufficient material is 
available.  Enough harvested tissue to provide a minimum of 4 monthly injections 
will be required for entry into the study.  These patients will be managed in an 
outpatient setting.  Hematologic function, liver enzymes, renal function and 
electrolytes will be monitored monthly.  Immune function analysis including 

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ELISPOT analysis of cytotoxic T cell function to autologous tumor antigens will 
be monitored at (≤24 hours before) tissue harvest, ≤24 hours before the first 
cycle of chemotherapy (post debulking), ≤24 hours before the third cycle of 
chemotherapy (post debulking), baseline (screening), prior to Vigil™ injection 
Months 2, 4, 6 and at EOT.  The dates of the last dose of chemotherapy and the 
administration of Vigil™ vaccine #1 will be recorded. 
 
Treatment will be continued until disease recurrence or exhaustion of the 
patient’s vaccine supply.  If ≥ Grade 2 toxicity by NCI Common Toxicity Criteria 
(excluding Grade 2 fever ≤ 24 hours and Grade 2 and 3 injection site reactions) 
develops related to study treatment the vaccine dose will be reduced by 50% and 
continued on a monthly basis.  If a single patient develops ≥ Grade 3 toxicity 
(other than injection site reaction) related to study treatment the trial will be 
placed on hold for reevaluation of design in discussion with FDA.  During this 
hold, no new subjects will initiate dosing, but subjects already being dosed may 
continue dosing as scheduled if deemed clinically appropriate by the PI. 
 
 STUDY POPULATION 
5.0
 
 Sample Size 
5.1
 
Approximately fifty (50) treatment naïve patients with high risk Stage III/IV 
ovarian cancer who achieve clinical CR following surgical debulking and 
chemotherapy will be randomized in this trial. 
 
 Inclusion Criteria 
5.2
 
Patients will be eligible for tissue procurement for the Vigil™ vaccine 
manufacturing process if they meet all of the following critieria: 
 
1. Presumptive Stage III/IV papillary serous or endometrioid ovarian 
cancer. 
2. Per Amendment #8, treatment naïve, high risk ovarian cancer will no 
longer be stratified, but the following information will be collected: 
a. Stage IV or suboptimal (>1 cm residual) Stage III disease versus 
Stage III patients with optimal (≤1 cm residual) disease, 
b. CA-125 ≤10 U/ml versus CA-125 greater than 10 but less than or 
equal to 20 U/ml 
c. IP chemotherapy versus IV chemotherapy 
3. Availability of “golf-ball” size 10-30 grams tissue at time of primary 
surgical debulking. 

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4. ECOG performance status (PS) 0-2 prior to tumor debulking 
laparotomy. 
5. Ability to understand and the willingness to sign a written informed 
consent document for tissue harvest. 
 
Patients will be registered for inclusion in this study if they meet all of the 
following criteria: 
 
1. Histologically confirmed Stage III/IV papillary serous or endometrioid 
ovarian cancer. 
2. Clinically defined CR (no cancer related symptoms, normal physical 
examination and CT scan abdomen/pelvis and CXR, and CA-125 ≤20 
U/ml) following completion of primary surgical debulking.  Patients 
enrolled must complete at least 5 but no more than 6 cycles 
platinum/taxane adjuvant or interval debulking + chemotherapy (or 
chemotherapy as per recommendations of NCCN guidelines, category 
1 (IP chemotherapy included)).  (Patients who complete 
surgery/chemotherapy with a CA-125 >20 U/mL pre-registration have 
the option of being followed up to 2 months if serial CA-125 values 
continue to decrease at a rate of CA-125 decrease of 50% per 
month.) 
3. Successful manufacturing of 4 vials of Vigil™ vaccine. 
4. Recovered from all clinically relevant toxicities related to prior protocol 
specific therapies (including neuropathy to ≤Grade 2). 
5. ECOG performance status (PS) 0-1. 
6. Normal organ and marrow function as defined below: 
Absolute granulocyte count 
≥ 1,500/mm3 
Absolute lymphocyte count 
≥  200/mm3 
Platelets 
≥ 75,000/mm3 
Total bilirubin 
≤ 2 mg/dL 
AST(SGOT)/ALT(SGPT) 
≤ 2x institutional upper limit of normal
Creatinine 
< 1.5 mg/dL 
7. Patients must be off all “statin” drugs for ≥ 2 weeks prior to initiation of 
therapy.   
8. Ability to understand and the willingness to sign a written informed 
protocol specific consent document. 
 
 Exclusion Criteria 
5.3
 
Patients will be excluded from this study if they meet any of the following criteria: 
 

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1. Surgery involving general anesthesia, radiotherapy, or immunotherapy 
within 4 weeks prior to randomization.  Chemotherapy within 3 weeks 
prior to Vigil™ vaccine administration.  Steroid therapy within 1 week 
prior to vaccine administration. 
2. Patients must not have received any other investigational agents within 
4 weeks prior to Vigil™ vaccine administration. 
3. Patients with history of brain metastases. 
4. Patients with compromised pulmonary disease. 
5. Short term (<30 days) concurrent systemic steroids ≤ 0.25 mg/kg 
prednisone per day (maximum 7.5 mg/day) and bronchodilators 
(inhaled steroids) are permitted; patients requiring other steroid 
regimens and/or immunosuppressives at randomization are excluded.  
6. Prior splenectomy. 
7. Prior malignancy (excluding nonmelanoma carcinomas of the skin and 
carcinoma in situ cervix) unless in remission for ≥ 2 years. 
8. Kaposi’s Sarcoma. 
9. Uncontrolled intercurrent illness including, but not limited to ongoing or 
active infection, symptomatic congestive heart failure, unstable angina 
pectoris, cardiac arrhythmia, or psychiatric illness/social situations that 
would limit compliance with study requirements. 
10. Patients with known HIV. 
11. Patients with chronic Hepatitis B and C infection. 
12. Patients with uncontrolled autoimmune diseases. 
 
 Withdrawal 
5.4
 
Off Study Treatment 
 
Patients will be taken off study treatment if any of the following occur: 
 
1. The patient experiences unacceptable (≥Grade 3) toxicity felt to be 
related to treatment with the tumor cell vaccination that persists for >1 
week.  
2. Persisting Grade 3 or 4 toxicity unrelated to treatment, defined as 
failing to normalize within 4 weeks. 
3. ≥ Grade 2 allergic reactions related to the study agent. 
4. Grade 2 autoimmune reactions unless there is evidence of clinical 
benefit. 
5. ≥ Grade 3 autoimmune reactions. 
6. 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. 

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7. Non-protocol therapy (including, but not limited to, chemotherapy not 
listed as Level 1 Category of Evidence per NCCN Guidelines Version 
3.2012, radiotherapy, hormonal therapy, immunotherapy, or surgery) is 
administered during study treatment.  
8. Non-compliant with protocol or treatment. 
9. Patient refuses to continue treatment. 
 
The date of and reason for discontinuation must be noted in the Case Report 
Form (CRF).  Every effort should be made to complete the appropriate 
assessments. 
 
Stopping Rules 
 
Patients with disease recurrence will not receive further vaccine treatment.  
Withdrawal of patients from the study treatment will be in the event of 
unexpected and unacceptable vaccine-related toxicity, ≥ Grade 2 allergic 
reactions, Grade 2 autoimmune reactions unless there is evidence of clinical 
benefit, ≥ Grade 3 autoimmune reactions related to the study agent, or clinically 
significant disease progression. 
 
 INVESTIGATIONAL PLAN 
6.0
 
 Patient Registration and Enrollment 
6.1
 
Written documentation of full, non-contingent IRB approval of the protocol and 
consent document must be on file before a patient can be registered.  Patients 
will be assigned a tissue procurement number upon harvest and a vaccine 
number / patient number will be assigned upon completion of the manufacturing 
process. 
 
Once eligibility has been confirmed, the site Research Nurse or Clinical 
Research Coordinator will email or fax the Registration Form and applicable de-
identified records to MCCRO@marycrowley.org or 214-658-1990.  If sent by fax, 
and email should be sent to the above email address to alert the CRO of the 
incoming fax. 
 
The received documents will be reviewed for completeness and sent to the 
Medical Monitor for review and randomization (if applicable).  If records are not 
complete or if question arise, the site Research Nurse or Clinical Research 
Coordinator will be contacted for more information. 

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Please allow 48 hours for patient registration / randomization.  Once confirmed, 
the completed Registration Form will be returned to the site indicating the 
assigned patient cohort. 
 
 Tumor Procurement 
6.2
 
The investigative sites will ensure patients are scheduled for surgery for tumor 
procurement.  Gradalis, Inc. will manufacture vaccine from the procured tissue, 
and thus will need to be notified at least 24 hours before the scheduled 
procurement in order to prepare for vaccine manufacturing (when possible).  
Additionally, tumors are to be procured on Mondays, Tuesdays, Wednesdays 
and Thursdays only due to the manufacturing being a 2-day process.  Please 
contact the Sponsor if scheduling adjustments need to be made. Refer to the 
Tissue Procurement Study Reference Manual for instructions. 
 
The equivalent of a “golf ball size” mass (10-30 gm tissue) and/or ascites fluid 
(preferably first paracentesis) is necessary for vaccine manufacturing.  
Radiological scans should confirm the presence of a lesion >3cm.  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 
tumor masses. Lesions extending into bowel lumen cannot be processed. 
 
Once the procured tissue is received at Gradalis, Inc. samples will be processed 
for autologous vaccine manufacture.  No tissue or vaccine will be given to the 
participant or site apart from the outlined clinical protocol. 
 
If available without compromising vaccine manufacture or pathology 
requirements, an aliquot of tumor will be collected for TIL and TAM 
immunophenotyping.  Any excess tumor tissue, not used for vaccine 
manufacture will be used towards Vigil™ vaccine research and process 
development assays. 
 
 Vaccine Manufacturing 
6.3
 
Gradalis, Inc. will manufacture vaccine from the procured patient tumors. 
Gradalis, Inc. will release vaccine once all release criteria have been met and 
eligibility has been confirmed. 
 
 
 

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 Study Treatment Administration 
6.4
 
Treatment will be administered on an outpatient basis.  The site must contact the 
Mary Crowley CRO to obtain participant randomization assignment. 
 
Recommended Chemotherapy 
 
Patients will receive 6 cycles of chemotherapy as either adjuvant or interval 
debulking + chemotherapyschedule.  Recommended regimens are those 
classified as Level 1 Category of Evidence per NCCN Guidelines Version 3.2012.  
Regimens other than these should be discussed with the sponsor. Patients will 
not receive maintenance therapy other than that specified by protocol. 
 
Vaccination Schedule and Dose 
 
Patients will be vaccinated according to the schedule outlined in Appendix B.  
Patients will receive Vigil™ vaccine at 1 x 107 cells via intradermal injection for a 
minimum of 4 doses and a maximum of 12 doses starting ≥3 weeks following 
completion of chemotherapy (no longer than 2.5 months post chemotherapy). 
 
Patients with viable cells in sufficient numbers (i.e. at least 4 doses of 1.0 x 107 
cells / injection) will receive monthly intradermal injections of the tumor cell 
vaccine for up to 12 months as long as sufficient material is available.  Patients 
whose vaccine manufacturing fails to achieve a minimum of 4 doses will not 
undergo treatment. 
 
Administration of Vigil™ vaccine 
 
The sites of injection 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 (e.g., anterior thigh) will be used.  The patient will be 
observed for at least 30 minutes (with a 10% window) following vaccination. 
During this observation period, vital signs will be taken every 10 minutes (with a 
10% window).  If clinically stable, vaccine administration may continue for up to 
12 doses given on a monthly basis as long as sufficient material is available. 
 
 

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Vigil™ Vaccine Transfer 
 
All manufactured vaccine 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 patient vaccine administration. 
 
Gradalis will complete a Drug Transfer and Administration Form to release the 
patient vaccine.  The clinic will sign off on the form upon receipt of the 
vaccination. 
 
Please reference the Pharmacy Reference Manual for vaccine preparation and 
handling information. 
 
Treatment Delay 
 
1. Treatment may be delayed no more than 4 weeks to allow recovery 
from toxicity. 
2. Patients who delay treatment for more than 4 weeks due to toxicities 
will be considered off study treatment (see Section 4.4 Off Study 
Treatment). 
3. Treatment delay not related to toxic events (including subjects unable 
to adhere to monthly injection) will not be delayed for more than three 
days.  Unless a delay due to symptoms related to disease or infection 
in which case up to a 2 week delay is allowed. 
4. If ≥ one 2 week delay due to disease or infection occurs, patient status 
must be reviewed by sponsor. 
5. If patients miss doses due to toxicities, the doses will be made up the 
following week and continue on a revised monthly schedule thereafter. 
 
Dose Modification for Toxicity 
 
If ≥ Grade 2 toxicity by NCI Common Toxicity Criteria (excluding Grades 2 and 3 
injection site reactions) develops related to study treatment the vaccine dose will 
be reduced by 50% and continued on a monthly basis.  If a single patient 
develops ≥ Grade 3 toxicity (other than site reaction) related to study treatment 
the trial will be placed on hold for re-evaluation of design in discussion with FDA. 
 
 

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 Toxicity 
6.5
 
Toxicities will be graded and reported according to the NCI Common Toxicity 
Criteria for Adverse Events (CTCAE) Version 3.0 as linked in Appendix C. This 
document can also be downloaded from the Cancer Therapy Evaluation Program 
(CTEP) home page <http://ctep.info.nih.gov>. 
 
Should a report of an infection (any grade) arise, please identify the bacteria or 
fungus related and report the event via Serious Adverse Event form.  In 
particular, if any organisms are identified as anaerobic, infectious disease should 
be consulted for management using standard of care relevant to anaerobic 
bacterium. 
 
 Schedule of Assessments 
6.6
 
The schedule of assessments for the trial is shown in Appendix B.  If a required 
observation or procedure is missed, documentation is required on the Protocol 
Deviation Form, to explain the reason for this protocol deviation. 
 
Prestudy Assessments 
 
The following evaluations will be performed within 4 weeks of the tumor 
debulking laparotomy (unless otherwise specified): 
 
1. A signed Patient Informed Consent Form for tissue harvest must be 
obtained. 
2. It has been confirmed that the patient meets all tissue procurement 
inclusion criteria and none of the exclusion criteria. 
3. A complete medical history must be obtained. 
4. A physical examination (including vital signs, height, and body weight) 
must be obtained. 
5. Assessment of PS on the ECOG scale (see Appendix A) must be 
obtained. 
6. Radiological assessment of tumors (i.e., chest X-ray, (chest CT or MRI 
only if indicated), pelvic/abdominal CT or MRI) used to stage the extent 
of disease must be performed within 6 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. 

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7. 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). 
8. CA-125 tumor assessment (at initial diagnosis per medical history). 
9. Routine pre-operative serum chemistries (including but not limited to 
creatinine, total bilirubin, alkaline phosphatase, and aspartate 
transaminase (AST) and/or alanine transaminase (ALT). 
10. Immune function analysis consisting of ELISPOT analysis of cytotoxic 
T cell reaction to autologous tumor antigens will be obtained at tissue 
harvest.  (within 24 hours before tissue procurement or on the day of 
harvest) 
 
Following tissue procurement, patients will be contacted every 6 weeks 2 weeks 
to assess interest / eligibility of the main randomized portion of the trial and 
survival status.  Blood for immune function analysis will be collected prior to the 
first and third cycles of (≤24 hours before) chemotherapy post debulking.  
Medical records should be provided to support continued eligibility. 
 
The following evaluations will be performed on all subjects within 2 weeks of 
registration / randomization (unless otherwise specified): 
 
1. A signed protocol specific Patient Informed Consent Form must be 
obtained. 
2. It has been confirmed that the patient 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, oxygen saturation, height, 
temperature and body weight) must be obtained. 
5. Assessment of concomitant medications 
6. Assessment of PS on the ECOG scale (see Appendix A) must be 
obtained. 
7. A tumor clinical assessment of the patient’s disease (i.e., by physical 
examination) must be performed within 4 weeks. 
8. Radiological assessment of disease status (i.e., chest X-ray, (chest CT 
or MRI only if indicated), pelvic/abdominal CT or MRI) used to stage 
the extent of disease must be performed 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. 
9. 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). 
10. CA-125 tumor assessment within 4 weeks following completion of 
surgery chemotherapy. If the CA-125 is >20 U/mL, the patient can 
continue with serial CA-125 evaluations up to 2 months if the levels 

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continue to decrease and there is no objective evidence of disease 
recurrence. 
11. 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. 
12. Immune function analysis consisting of ELISPOT analysis of cytotoxic 
T cell reaction to autologous tumor antigens. 
13. Urinalysis must be performed. 
14. EKG must be performed. 
15. FACT-O, Version 4 assessment 
 
Assessments During Treatment 
 
The following evaluations will be performed monthly (every 28 days ±3 days) 
during the therapy (unless otherwise specified): 
 
1. A physical examination, including vital signs and body weight. 
2. A toxicity assessment (adverse events). 
3. Assessment of concomitant medications taken. 
4. Tumor response by clinical assessment of the patient’s disease (i.e., 
by physical examination). 
5. Radiological assessment of disease status (i.e., chest X-ray, (chest CT 
or MRI only if indicated), pelvic/abdominal CT or MRI) must be 
performed ≤ 1 week prior to Cycle 4) and then at standard of care 
intervals. 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. 
6. A CBC with differential and platelet count. 
7. CA-125 tumor assessment until recurrence.  (monthly for the first year, 
then every 3 months ± 2 weeks for the second and third year.) 
8. Serum chemistry and electrolytes. 
9. Immune function analysis consisting of effector T cell response 
(ELISPOT assay will be monitored - Months 2, 4, 6 (prior to Vigil™ 
administration). 
10. Assessment of PS on the ECOG scale (see Appendix A). 
11. Vaccine administration.  The patient will be observed for at least 30 
minutes (with a 10% window) following vaccination. During this 
observation period, vital signs will be taken every 10 minutes (with a 
10% window). 
12. Day 2 assessment of injection site. 
13. FACT-O, Version 4 assessment at baseline, Month 3, Month 6, Month 
12 and Month 18 (or until recurrence). 

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Radiological assessment of disease status (i.e., chest X-ray, (chest CT or MRI 
only if indicated), pelvic/abdominal CT or MRI) must be collected if CA-125 >35 
U/mL after two consecutive measurements taken one month apart. 
 
Off Treatment Assessments 
 
The following evaluations will be performed within 30 days following the last 
Vigil™ administration and within 30 days of disease recurrence (unless otherwise 
specified): 
 
1. A physical examination, including vital signs and body weight. 
2. Toxicity assessment (adverse events) (Vigil™ administered patients 
only). 
3. Assessment of concomitant medications taken. 
4. Assessment of PS on the ECOG scale (see Appendix A). 
5. A tumor clinical assessment of the patient’s disease (i.e., by physical 
examination). 
6. Radiological assessment of disease status (i.e., chest X-ray, (chest CT 
or MRI only if indicated), pelvic/abdominal CT or MRI) used to stage 
the extent of disease. (within 45 days of the last injection or disease 
recurrence) 
7. A CBC with differential and platelet count. 
8. CA-125 tumor assessment. 
9. Serum chemistry and electrolytes. 
10. Serum for immune function analysis consisting of ELISPOT analysis of 
cytotoxic T cell function to autologous tumor antigens. 
 
Follow Up Assessments 
If the Vigil™ vaccine 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 
patient will be contacted by phone every 3 months after the end of study visit for: 
 
 disease status 
 request of medical records, when applicable (laboratory findings, radiological 
scans, and progress report) 
 documentation of 2nd line therapy 
 recording of response and duration of response to that therapy 
 survival 
 assessment of any additional cancer treatments) 

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Note: Patients who die or withdraw consent are considered off study and no 
further information except survival data will be collected. 
 
Based on findings during the study or during the follow up portion of the trial, 
Gradalis may request for additional blood and / or tissue samples from the 
research participant.  Collection of whole blood (40ml) and / or tissue samples 
(via biopsy or clinically indicated surgical removal) will be optional and used to 
study the effects of the study agent (included, but not limited to testing of 
biomarkers, predictors or biological responses, toxicity, relationship between 
genotype and study agent responses). 
 
Should Gradalis request for additional blood or tissue, the clinical site will present 
the option of the procurement to the participant and obtain written informed 
consent. 
 
 CONDUCT OF THE STUDY 
7.0
 
 Ethics and Regulatory Considerations 
7.1
 
This study must have the approval of a properly constituted Institutional Review 
Board (IRB) or Independent Ethics Committee (IEC), and the Institutional 
Biosafety Committee (IBC).  Before the investigational drug is shipped to the 
investigator, the investigator will provide Gradalis, Inc. with a copy of the IRB or 
IEC and IBC approval letter stating that the study protocol and informed consent 
form have been reviewed and approved. 
 
 IRB and IBC 
7.2
 
This trial can be undertaken only after review and full approval of the protocol 
and a Patient 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 Patient Informed Consent Form, and any 
applicable recruiting materials and subject compensation programs approved. 
 
Since this trial will also be reviewed by a properly constituted IBC, this trial will 
not be undertaken until the protocol has been reviewed and received full 
approval from a properly constituted IBC.  This written approval must be dated 
and it must clearly identify the protocol and any amendments. 
 

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The decision concerning the conduct of the study will be made in writing to the 
sponsor.  Copies of this decision and of all IRB and IBC correspondence will be 
kept on file at the study site; copies will be provided to the Mary Crowley 
Research Office. 
 
During the trial, the PI is required to send various documents to the IRB and IBC 
for review: 
 
1. All protocol amendments and Patient Informed Consent Form 
revisions. 
2. Reports of all Serious Adverse Events. 
 
The PI provides Gradalis, Inc. with the necessary assurance that an IRB and IBC 
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 
and IBC 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 and IBC that the trial has been completed. 
 
 Written Informed Consent 
7.3
 
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. 
 
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 patients 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 patients’ participation 
is voluntary, and that they may withdraw consent to participate at any time.  They 
will also be informed that if patients choose not to participate in the trial 
alternative treatments are available; such refusal will not prejudice further 
treatment of their disease.  Potential patients or their legal representatives must 
be given the opportunity to ask questions about the trial protocol and the 
procedures involved. 
 
Finally, each patient will be told that his or her records may be accessed by 
authorized personnel of Mary Crowley, Gradalis, Inc. and other authorized 
individuals without violating the patient’s confidentiality, to the extent permitted by 
the applicable laws and/or regulations.  By signing the written Patient Informed 
Consent Form, the patient or his or her legal representative is authorizing such 
access.  Following this explanation and prior to entry into the trial, the written, 

Protocol # CL-PTL 105 
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dated, and signed Patient Informed Consent Form must be obtained from each 
patient or his or her legal representative; a copy will be given to the person 
signing the form. 
 
 Confidentiality of Records 
7.4
 
The Investigator is required to retain, in a confidential manner, sufficient 
information on each patient (i.e., full name, current address, and social security 
number) so that the patient may be contacted by the FDA, Gradalis, Inc., or by 
Mary Crowley should the need arise. 
 
 Modification of Protocol 
7.5
 
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 and IBC. 
 
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 and IBC will be 
notified of administrative changes according to applicable IRB guidelines. 
 
 Protocol Questions and Deviations 
7.6
 
When evaluating a potential patient or while a patient is on study, protocol 
questions can be sent to MCCRO@marycrowley.org.  Mary Crowley CRO will 
work with Gradalis in obtaining an answer / clarification.  Please allow 24 hours 
for a response.  For urgent questions affecting patient safety, please contact the 
Gradalis personnel by the phone provided in the Gradalis Contact Information 
section of the Study Reference Manual. 
 
For Planned Protocol Deviation requests, the site will complete the Eligibility 
Inquiry/Planned Protocol Deviation Form and email or fax it to 
MCCRO@marycrowley.org or 214-658-1990.  If sent by fax, an email should be 
sent to the above email address to alert Mary Crowley of the fax.  Mary Crowley 
CRO will work with Gradalis and the Medical Monitor to review the 

Protocol # CL-PTL 105 
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request.  Please allow 48 – 72 hours for review.  Once complete, the form will be 
returned to the site. 
 
 EVALUATION OF TUMORS 
8.0
 
 Disease Evaluation 
8.1
 
Disease recurrence will be evaluated in this study using the Response Evaluation 
Criteria in Solid Tumors Version 1.1 (RECIST) (unidimensional measurement) of 
the tumor lesions are used in the RECIST criteria (Eisenhauer, Therasse et al. 
2009). 
 
Disease recurrence is defined as the appearance of any measurable or 
evaluable lesion or as asymptomatic CA-125 levels greater than 35 U/mL at two 
consecutive measurements, at least one month apart.  The time to recurrence is 
measured from date of first treatment until the first date that recurrence is 
objectively documented whether local, regional, or distant. 
 
 DRUG INFORMATION 
9.0
 
 Investigational Product 
9.1
 
The Vigil™ vaccine 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™ Vaccine Production 
 
The Vigil™ Phase II ovarian vaccine cGMP manufacturing process is identical to 
prior Vigil™ vaccine cGMP manufacturing (BB-IND 14205) (Maples 2010).  
Surgically excised tumor is collected in the surgical field and placed in sterile 
saline and 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 FANG™ plasmid.  The cells are 
incubated overnight to allow transcription of the FANG™ plasmid.  The following 
morning the cells are harvested, washed, and then irradiated at 10,000cGy in a 
standard Blood Bank irradiator.  The irradiated cell suspension is then 
enumerated, aliquoted and frozen (1 x 107 cells).  The freeze media consists of 

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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 
9.2
 
The FANG™ plasmid employed in the generation of this autologous cell vaccine 
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 patient vaccinations 
must be irradiated 10,000 cGy prior to freezing.  This is the same irradiation 
process as for the TAG vaccine, BB-IND 13650 and prior vaccines 
(Belagenpumatucel-L and GVAX® published trial results and BB-IND 13401 and 
BB-IND 12118) (Kumar 2009; Maples PB 2009; Maples PB 2009).  The selection 
of this radiation dose is based on the desire to utilize the lowest possible 
radiation dose for the transfected cells to optimize the level and duration of 
bifunctional shRNAfurin transcription and GMCSF protein production and 
maximize the safety of vaccine cell injections at the same time.  In addition, 
investigators have demonstrated that irradiating cultured tumor cells of different 
histologic origins at 10,000 cGy completely arrests tumor colony formation. 
 
Preparation 
 
Reference the Study Reference Manual for vaccine preparation and handling 
information. 
 
Drug concentrate:  1.0 x 107cells per injection in a volume of 1mL. 
 
Route of administration:  
Intradermal injection 
 
Storage 
 
Frozen, unopened vials are stored in the vapor phase of Liquid Nitrogen. 
 

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Administration of vaccine 
 
Patients with viable cells in sufficient numbers for at least 1.0 x 107 cells / 
injection for 4 doses will receive monthly intradermal injections of the tumor cell 
vaccine (as assigned cohort and available cell yield following tumor harvest and 
processing) as long as sufficient material is available.  Patients who fail to 
achieve successful manufacturing of a minimum of 4 doses will not undergo 
treatment.  The sites of injection will be rotated between the right and left upper 
arms.  If the ipsilateral axillary lymph nodes were radiated or surgically resected 
during prior therapy, alternative sites (e.g., anterior thigh) will be used.  The 
patient will be observed for at least 30 minutes (with a 10% window) following 
vaccination. During this observation period, vital signs will be taken every 10 
minutes (with a 10% window).  If clinically stable vaccine administration may 
continue for up to 12 doses given on a monthly basis as long as sufficient 
material is available. 
 
 Concomitant Therapy 
9.3
 
All concomitant treatments, including blood and blood products, must be reported 
on the source documentation. The following concomitant medications must be 
documented on the Con Meds page of the CRF: 
 
 Prophylactic antibiotics  Antifungals  
 Premedications 
 Antinauseants, antipyretics 
 
Short term (<30 days) concurrent systemic steroids ≤ 0.25 mg/kg prednisone per 
day (maximum 7.5 mg/day) and bronchodilators (inhaled steroids) are permitted; 
other steroid regimens and/or immunosuppressives are excluded. 
 
 OCCUPATIONAL SAFETY 
10.0
 
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 2 practices shall be employed with this study 
medication.  Reference the Study Reference Manual. 
 
 

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 ADVERSE EVENTS 
11.0
 
 Adverse Event and Serious Adverse Event Definitions 
11.1
 
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  
 A life-threatening condition (i.e. an event in which the patient was at risk of 
death at the time of the event; it does not refer to an event which 
hypothetically might have caused death if it was more severe)  
 Significant disability/incapacity (i.e. the AE resulted in a substantial disruption 
of the subject’s ability to carry out normal life functions) 
 Congenital anomaly/birth defect 
 It does not meet any of the above serious criteria, but may jeopardize the 
subject or may require surgical or medical intervention to prevent one of the 
outcomes listed above.  
 Death 
 
Unexpected Adverse Event 
 
An unexpected event is any AE that is not identified in nature, severity or 
frequency in the Clinical Investigator’s brochure or the drug package insert. 
 
 

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Grading Adverse Events 
 
Adverse events (AEs) will be recorded throughout the trial. Toxicities and AEs 
will be graded and reported using the Common Toxicity Criteria for Adverse 
Events (CTCAE) Version 3.0 as linked in Appendix C. All AEs, regardless of 
severity, will be followed by the Treating Physician until resolution is satisfactory. 
 
 Attribution of Causality 
11.2
 
The relationship of each event to treatment will be assessed by the Treating 
Physician and recorded on the CRF. 
 
 Expected Side Effects 
11.3
 
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 
11.4
 
Adverse events will be recorded for the duration of a patient’s study treatment, 
and for up to 30 days following the last study treatment.  All AEs, regardless of 
causal relationship are to be recorded in the CRF 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 CRF. 
 
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 
11.5
 
All SAEs will be reported by email or facsimile upon becoming aware of the event 
within 24 hours to Gradalis, Inc.  This includes any death from any cause while a 
patient is receiving treatment (Vigil™) on this protocol, or  30 days following the 
last dose of protocol treatment (Vigil™). 
 
The site will supply as much information as is available at the time of the initial 
facsimile (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. 
 
 

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 PATIENT COMPLETION AND WITHDRAWAL 
12.0
 
 Indication for Taking Patients Off Study 
12.1
 
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. 
 
 DATA EVALUATION 
13.0
 
 Statistics and Estimated Sample Size 
13.1
 
The primary objective of this study is to investigate whether maintenance Vigil™ 
vaccine after a clinically defined complete response to induction 
platinum/paclitaxel chemotherapy results in prolonged time to recurrence (TTR) 
compared with standard of care observation. TTR was used for the purpose of 
sample-size estimation. Based on prior published experience (Markman, Liu et 
al. 2003), the median RFS after a clinical complete response to induction therapy 
for the control arm was estimated to be approximately 16 months for those with 
stage IV or suboptimal (> 1 cm residual) stage III disease and 24 months for 
stage III patients with optimal (< 1 cm residual) disease. Given an average RFS 
of 24 and 16 months, respectively, a 10% increase in RFS (i.e. TTR) would 
provide adequate statistical power to support expanding the study (Table 6). This 
analysis was performed using a statistical power calculator 
(http://www.dssresearch.com/KnowledgeCenter/toolkitcalculators/statisticalpower
calculators.aspx) to conduct a 1-tail test of a sample average in comparison to 
the historical value, a standard deviation of 1.0, and an alpha error level of 
5%.  In order to expand the protocol treatment base and increase the rate of 
accrual while maintaining the objectives of the protocol, the study, which was 
initially limited to patients with stage IIIc ovarian cancer, is being expanded to 
patients with Stage III/IV disease. In the SEER database: 1988-2001(Kosary 
1988-2001), women with Stage IIIc disease comprised 19.4% of the ovarian 
cancer population; those with Stage III inclusive, 35.3%; and those with Stage IV, 
31.7%. Thus, the eligibility expansion increases the base catchment population 
from 19.4% of women with ovarian cancer to 67% of which only a subset will 
meet protocol criteria.  
 
In the completed Phase I study of Vigil™ vaccine in patients with solid tumors 
experiencing progressive disease following prior therapy, 50% of patients with 
serial assessments (n=18) demonstrated conversion from γIFN ELISPOT 

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negative to positive by Month 4 (Senzer N 2012).  The percent γIFN ELISPOT 
conversions and the timing thereof will be recorded in the current patient 
population of patients with minimal or no residual disease using descriptive 
statistics to assess the hypothesis that there is an inverse relationship between 
tumor volume and immune responsiveness based in part on the quantitative 
relationship between tumor volume and the production of immunosuppressive 
factors.  
 
Although all patients will be evaluatated for RFS, subgroup analyses will be 
separately perfomed on patients with negative ELISPOT at baseline and those 
with positive ELISPOT at baseline. 
 
Table 6: Statistical Power of 2:1 Randomization Trial per Surgical Stage 
Stratification Group (1 tailed t test) 
 
Surgical Stage 
Stratification 
Vigil™ Vaccine 
(TTR in months)
Observation 
Control  
(TTR in months) 
Power  
n 
Stage III patients 
with optimal (< 1 
cm residual) 
26.4 
24 
1.0 
30 
Stage IV or 
suboptimal (> 1 
cm residual) 
stage III disease 
17.6 
16 
1.0 
30 
 
Exploratory analysis of two Phase III studies (D9901 and D9902) with the 
recently FDA approved Provenge documentated prolonged survival benefits in 
Provenge-treated prostate cancer patients (25.9 months vs. 21.4 months and 
25.8 vs. 21.7 months, respectively). Supporting our clinical objective, patients 
who subsequently received docetaxel experienced a median survival of 34.5 
months compared to 25.4 months in patients treated with placebo followed by 
docetaxel.  Descriptive statistics will be employed to format the response rates 
and response durations to 2nd line therapy in patients that relapse in both 
FANG™ and observational groups. 
 
 Definition of Evaluable Patients 
13.2
 
All measurements should be taken and recorded in metric notation using a ruler 
or calipers. All baseline evaluations will be performed as closely as possible to 
the beginning of treatment and never more than 2 weeks before the beginning of 
the treatment. 
 

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The same method of assessment and the same technique should be used to 
characterize each identified and reported lesion at baseline and during follow-up. 
Imaging-based evaluation is preferred to evaluation by clinical examination when 
both methods have been used to assess the antitumor effect of treatment. 
 
Clinical Lesions: Clinical lesions will only be considered measurable when they 
are superficial (e.g., skin nodules, palpable lymph nodes). In the case of skin 
lesions, documentation by color photography including a ruler to estimate the 
size of the lesion is recommended. 
 
Chest X-rays: Lesions on chest X-ray are acceptable as measurable lesions 
when they are clearly defined and surrounded by aerated lung. However, CT is 
preferable. 
 
Conventional CT and MRI: These techniques should be performed with cuts of 
10mm or less in slice thickness contiguously. 
 
Spiral CT: Spiral CT should be performed using a 5mm contiguous 
reconstruction algorithm.  
 
Ultrasound: When the primary endpoint of the study is objective response 
evaluation, ultrasound should not be used to measure tumor lesions. It is, 
however, a possible alternative to clinical measurements of superficial palpable 
lymph nodes, subcutaneous lesions, and thyroid nodules. Ultrasound may also 
be useful to confirm the complete disappearance of superficial lesions usually 
assessed by clinical examination. 
 
 Time to Recurrence of Disease (TTR) 
13.3
 
Time to recurrence will be measured from the treatment start date (date of first 
dose) to either the date the patient is first recorded as having disease recurrence 
(even if the patient went off treatment because of toxicity), or the date of death if 
the patient dies due to any causes before recurrence.  
 
 Time to Treatment Failure 
13.4
 
Time to treatment failure will be measured as the date from when the patient 
started treatment (first dose) to the date the patient is withdrawn due to: AE(s), 
progressive disease/insufficient therapeutic response, death, failure to return, 
refused treatment/did not cooperate/withdrew consent, or started a new 

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treatment. Patients who do not fail treatment will be censored at the date of last 
follow-up. 
 
 Survival 
13.5
 
Survival will be measured, as the time from start of treatment to the date of death 
or the last date the patient was known to be alive. 
 
Long-term survival information will be collected. 
 
 STUDY RECORDS 
14.0
 
 Documentation 
14.1
 
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 Research Coordinator 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 
CRFs on each study patient. 
 
The Investigator or his/her designee is required to retain the records related to 
the trial for a period of 2 years following the date a marketing application is 
approved for the indication being investigated.  If no application is to be filed or if 
the application is not approved for such indication, the records must be retained 
until 2 years after the investigation is discontinued and the regulatory agencies 
are notified. 
 
The Investigator shall retain study drug disposition records and source 
documents for the maximum period required by the country and institution in 
which the study will be conducted, or for the period specified by Gradalis, which 
ever is longer.  The Investigator must contact Gradalis, Inc. prior to destroying 
any records associated with the study. 
 

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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 
14.2
 
CRFs will be supplied by Gradalis, Inc. for recording all data from each patient. 
CRFs must be typewritten or printed legibly using black ballpoint pen or 
completed electronically.  The investigator or his/her designee is responsible for 
recording all data relating to the trial on the CRFs.  The investigator must verify 
that all data entries on the CRFs are accurate and correct. 
 
If an item is not available or is not applicable, it should be documented as such; 
no blank spaces should be left on a CRF. 

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APPENDIX A 
 
ECOG Performance Status Scale 
 
ECOG Performance Status Scale 
Grade 
Description 
0 
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction. 
1 
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of light or 
sedentary nature—(e.g., light 
housework or office work). 
2 
In bed <50% of the time. 
Ambulatory and capable of all 
self care, but unable to carry out 
any work activities; up and about 
> 50% of waking hours. 
3 
In bed >50% of the time.  
Capable only of limited self-care, 
confined to bed or chair >50% of 
waking hours. 
4 
100% bedridden.  Completely 
disabled; cannot carry out any 
self care; totally confined to bed 
chair. 
5 
Dead 
 
 
 

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APPENDIX B 
 
Schedule Of Assessments 
 
Procedure 
Prestudy 
Screening 
Monthly
(q 28±3 days) 
End of TX 
Follow-Up 
 
Informed consent 
X 
X 
 
 
 
Phone Contact 
X1 
 
 
 
 
Medical History 
X 
Interval Medical 
History within 4 
weeks 
 
 
 
Physical Examination 
X 
X 
X 
X 
 
Toxicity (adverse events) 
 
 
X 
X 
 
Concomitant medications 
 
X 
X 
X 
 
Performance Status 
X 
X 
X 
X 
 
Clinical Tumor 
Assessment 
 
X 
X 
X 
 
Radiological Tumor 
Assessment 
(abdomen/pelvis)2 
within 6 weeks 
within 4 weeks 
 
X 
 
CBC with differential 
X 
X 
X 
X 
 
HIV testing, if applicable 
 
X 
 
 
 
CA-125 tumor 
assessment3 
X 
X 
X 
X 
 
Serum Chemistry 
X4 
X 
X 
X 
 
Immune Function Analysis 
At tissue procurement 
and prior to the 1st and 
3rd cycles of 
chemotherapy post 
debulking 
X 
Months 2, 4, 6 
prior to vaccine 
administration 
X 
 
Urinalysis 
 
X 
 
 
 
EKG 
 
X 
 
 
 
Vaccine administration5 
 
 
X 
 
 
Injection Site Assessment 
 
 
Day 2 
only 
 
 
FACT-O6 
 
X 
X 
 
 
Survival Status 
X 
 
 
X 
X7 
 
 
 
                                                 
 
1 Following tissue procurement, patients will be contacted every 6 weeks 2 weeks to assess interest / eligibility of the main randomized portion 
of the trial and survival status. 
2 Radiographic assessments taken at baseline, ≤ 1 week prior to Cycle 4 , at SOC intervals, and when CA-125 >35U/mL. 
3 CA-125 taken at baseline, monthly for the first year, every 3 months ± 2 weeks for the second and third year. 
4 Obtain from medical records from standard preoperative hematology and chemistry panels. 
5 The patient will be observed for at least 30 minutes (with a 10% window) following vaccination. During this observation period, vital signs will 
be taken every 10 minutes (with a 10% window). 
6 FACT-O assessment will be collected at baseline, Month 3, Month 6, Month 12, and Month 18. 
7 After EOT, subjects will be contacted by phone quarterly for disease status, medical records (when applicable), documentation of 2nd line 
therapy and recording of response and duration of response to that therapy, additional cancer treatments as well as survival. 

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APPENDIX C 
 
NCI Common Toxicity Criteria For Adverse Events (CTCAE), Version 3.0 
 
 
Publish Date: MARCH 31, 2003 
 
As of April 02, 2003 NCI has introduced version 3.0 of the Common Toxicity Criteria for
Adverse 
Events. 
These 
may 
be 
obtained 
at 
the 
following 
web 
link
http://ctep.cancer.gov/reporting/ctc.html or on the Mary Crowley CTMS. 
 
DO NOT USE CTC VERSION 2.0 TO GRADE TOXICITIES IN THIS STUDY! 
 
 
 
 

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APPENDIX D 
 
FACT-O, VERSION 4 
 

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