Treatment of Patients With Castration Resistant Prostate Cancer Using a Multi-Targeted Recombinant Ad5 PSA/MUC1/Brachyury Based Immunotherapy Vaccines
Phase: PHASE1
Conditions: Prostatic Neoplasms, Prostatic Cancer
Sponsor: National Cancer Institute (NCI)

Abbreviated Title: Ad5-based vaccines in mCRPC 
CC Protocol #: 18C0073, Amendment B 
 
 
 
 
 
IBC #: RD-18-I-06 
Version Date: September 20, 2018 
NCT #: NCT03481816 
 
 
 
 
 
 
 
 
 
Title: Treatment of Patients with Castration Resistant Prostate Cancer using Multi-Targeted 
Recombinant Ad5 PSA/MUC1/Brachyury Based Immunotherapy Vaccines 
 
NCI Principal Investigator: 
Marijo Bilusic, MD, PhD 
Genitourinary Malignancies Branch (GMB), CCR, NCI 
10 Center Drive 
Building 10, Room B2L312 
Bethesda, MD 20892 
Phone: 240-760-6064 
Email: marijo.bilusic@nih.gov 
 
 
 
 
 
 
 
 
 
Investigational Agents: 
Drug Name: 
ETBX-071; adenoviral 
PSA vaccine 
ETBX-061; adenoviral 
MUC1 vaccine 
ETBX-051; adenoviral 
brachyury vaccine 
IND Number: 17811 
17811 
17811 
Sponsor: 
Center 
for 
Cancer 
Research 
Center 
for 
Cancer 
Research 
Center 
for 
Cancer 
Research 
Manufacturer: Etubics 
Etubics 
Etubics 
 
 

Abbreviated Title: Ad5-based vaccines in mCRPC 
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PRÉCIS 
Background: 
x The overall goal of the current project is to expand our immunotherapeutic approach for 
the treatment of prostate cancer employing a multi-targeted approach. 
x Therapeutic cancer vaccines targeting overexpressed proteins offer a potential method to 
activate T cells against tumors. 
x A novel adenovirus based vaccines targeting three (3) human tumor associated antigens 
(TAA), PSA, MUC1, and brachyury, respectively have demonstrated anti-tumor cytolytic 
T cell responses in pre-clinical animal models of cancer. 
 
Objectives: 
x To determine the overall safety and recommended phase 2 dose of a combination of three 
immunotherapeutic vaccines (ETBX-071, ETBX-061, and ETBX-051) when administered 
subcutaneously (SC) to subjects with metastatic castration resistant prostate cancer 
 
Eligibility: 
x Subjects age 18 and older with cytologically or histologically confirmed prostate cancer 
for which no curative standard approved therapy is available. 
x Metastatic Castration Resistant Prostate Cancer (mCRPC) patients with rising PSA or 
progressive disease despite castration levels of testosterone.
x 
x Prior treatment with immunotherapy, hormonal therapy, radiotherapy, chemotherapy, 
and/or other experimental therapy is allowed.
x ECOG performance status < 1 
x Adequate organ and bone marrow function. 
x Subjects with a history of autoimmune disease (active or past) and subjects requiring 
systemic steroids are not eligible (physiologic doses of steroids for steroid replacement as 
well as nasal, topical and inhaled steroids are allowed). Autoimmune-related thyroid 
disease, type I diabetes and vitiligo are permitted if the condition is well controlled. 
 
Design: 
x This is a Phase I trial in subjects with mCRPC. A combination of three therapeutic vaccines 
(ETBX-071, ETBX-061, and ETBX-051) which use the same modified Adenovirus vector 
backbone, separately encoding three well studied TAA,PSA, MUC1, and brachyury, 
respectively) will be assessed. The vaccines will be tested at standard dose levels, with a 
dose de-escalation (if required) design employed. The dose level of each vaccine tested 
will be 5x1011 VP. This dose has been found in a prior phase 1 testing of a similar vaccine 
Ad5 [E1-, E2b-]-CEA(6D) (ETBX-011) to be well tolerated (with no DLT’s or related 
SAE’s), and be optimal for induction of immune responses. 
x Up to six patients will be enrolled at dose level 1. If  ≤ 1 of 6 patients experience a DLT, 
an initiation of the dose expansion phase will occur. If ≥ 2 of 6 experience DLT at dose 
level 1, then dose de-escalation will occur. Up to six patients will be enrolled at the lower 
dose level (-1) (1x1011 VP). If  ≤ 1 of 6 patients experience a DLT, then the maximum 

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tolerated (MTD) will be declared at this dose, and initiation of the dose expansion phase 
will occur. If  ≥ 2 of 6 experience DLT at dose level -1, then a further dose de-escalation 
will occur. Up to six patients will be enrolled at the lower dose level (-2) (5x1010 VP).  If  
≤ 1 of 6 patients experience a DLT, then the maximum tolerated (MTD) will be declared 
at this dose, and initiation of the dose expansion phase will occur. If  ≥ 2 of 6 experience 
DLT at dose level -2, then the study will be stopped. 
x A dose expansion phase of study will be enrolled after the MTD of the  vaccines have been 
determined. An additional 12 subjects will be enrolled in the dose expansion component of 
the trial, for a total of 18 subjects at the MTD. 
x The ETBX-051, ETBX-61 and ETBX-71 vaccines will be administered subcutaneously 
(SC) at separate injection sites (proximal limb, preferably the thigh), will be administered 
SC every 3 weeks for 3 doses (dose de-escalation cohorts) followed by boosts every 8 
weeks for 1 year (only patients enrolled in dose expansion cohort). 
 
 
 

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TABLE OF CONTENTS 
PRÉCIS ............................................................................................................................................2 
TABLE OF CONTENTS .................................................................................................................4 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ............................................9 
1 INTRODUCTION ...................................................................................................................12 
1.1 
Study Objectives and Endpoints ................................................................................. 12 
1.1.1 
Primary Objectives.................................................................................................. 12 
1.1.2 
Secondary Objectives.............................................................................................. 12 
1.1.3 
Exploratory Objectives ........................................................................................... 12 
1.1.4 
Primary Endpoints .................................................................................................. 12 
1.1.5 
Secondary Endpoints .............................................................................................. 12 
1.1.6 
Exploratory Endpoints ............................................................................................ 12 
1.2 
Background and Rationale .......................................................................................... 12 
1.2.1 
Overall Objective .................................................................................................... 12 
1.2.2 
PSA Expression in Prostate Cancer and It’s Use as an Immunotherapy Target ..... 13 
1.2.3 
MUC1 Expression in Cancer and It’s Use as an Immunotherapy Target ............... 14 
1.2.4 
Brachyury Expression in Cancer and It’s Potential as an Immunotherapy Target . 15 
1.2.5 
Adenovirus-Based Vectors ..................................................................................... 16 
1.2.6 
Clinical Experience with an Ad5 [E1-, E2b-]-based Cancer Immunotherapy 
Vaccine ................................................................................................................... 17 
1.2.7 
ETBX-071   Pre-clinical Studies............................................................................. 20 
1.2.8 
ETBX-061 Pre-clinical Studies............................................................................... 24 
1.2.9 
ETBX-051 Pre-clinical Studies............................................................................... 26 
1.2.10 Clinical Experience ................................................................................................. 28 
1.2.11 Rationale ................................................................................................................. 28 
2 ELIGIBILITY ASSESSMENT AND ENROLLMENT ..........................................................28 
2.1 
Eligibility Criteria ....................................................................................................... 28 
2.1.1 
Inclusion Criteria .................................................................................................... 28 
2.1.2 
Exclusion Criteria ................................................................................................... 30 
2.1.3 
Recruitment Strategies ............................................................................................ 31 
2.2 
Screening Evaluation .................................................................................................. 31 
2.3 
Registration Procedures .............................................................................................. 32 

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2.3.1 
Treatment Assignment Procedure ........................................................................... 32 
2.4 
Baseline Evaluation .................................................................................................... 32 
3 STUDY IMPLEMENTATION ...............................................................................................33 
3.1 
Study Design ............................................................................................................... 33 
3.1.1 
Dose Limiting Toxicity ........................................................................................... 34 
3.1.2 
Dose De-Escalation ................................................................................................. 35 
3.2 
ETBX Vaccine Dose Preparation ............................................................................... 38 
3.2.1 
Instructions for Dose Preparation – 5 x 1011 Virus Particles .................................. 38 
3.2.2 
Instructions for Dose Preparation – 1 x 1011 Virus Particles .................................. 38 
3.2.3 
Instructions for Dose Preparation – 5 x 1010 Virus Particles (For Dose De-
escalation) ............................................................................................................... 38 
3.2.4 
Administration ........................................................................................................ 39 
3.2.5 
Other Considerations .............................................................................................. 39 
3.3 
Dose Modifications and Delays .................................................................................. 39 
3.3.1 
Dosing Delay (applies to events occurring outside of the DLT evaluation period) 39 
3.3.2 
Dose Modifications ................................................................................................. 40 
3.4 
Study Calendar ............................................................................................................ 41 
3.5 
Criteria for Removal from Protocol Therapy and Off Study Criteria ......................... 45 
3.5.1 
Criteria for removal from protocol therapy ............................................................ 45 
3.5.2 
Off-Study Criteria ................................................................................................... 45 
3.5.3 
Off Protocol Therapy and Off-Study Procedure ..................................................... 45 
4 CONCOMITANT MEDICATIONS/MEASURES .................................................................45 
4.1 
Concurrent Medications/Interventions........................................................................ 46 
4.1.1 
Anticancer Therapy ................................................................................................. 46 
4.1.2 
Other Medications ................................................................................................... 46 
5 BIOSPECIMEN COLLECTION .............................................................................................46 
5.1 
Correlative Studies for Research/Pharmacokinetic Studies ........................................ 46 
5.1.1 
Immunological Studies ........................................................................................... 46 
5.2 
Sample Collection Summary ...................................................................................... 49 
5.3 
Sample Storage, Tracking, Disposition (Clinical Services Program [CSP]) .............. 49 
5.3.1 
Protocol Completion/Sample Destruction .............................................................. 50 
6 DATA COLLECTION AND EVALUATION ........................................................................51 

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6.1 
Data Collection ........................................................................................................... 51 
6.2 
Data Sharing Plans ...................................................................................................... 51 
6.2.1 
Human Data Sharing Plan ....................................................................................... 51 
6.2.2 
Genomic Data Sharing Plan .................................................................................... 52 
6.3 
Response Criteria ........................................................................................................ 52 
6.3.1 
Disease Parameters ................................................................................................. 52 
6.3.2 
Methods for Evaluation of Measurable Disease ..................................................... 53 
6.3.3 
Response Criteria .................................................................................................... 55 
6.3.4 
Duration of Response .............................................................................................. 57 
6.3.5 
Progression-Free Survival (PFS) ............................................................................ 58 
6.4 
Toxicity Criteria .......................................................................................................... 58 
7 SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN .......................................................................................................................................58 
7.1 
Definitions................................................................................................................... 58 
7.1.1 
Adverse Event ......................................................................................................... 58 
7.1.2 
Suspected adverse reaction ..................................................................................... 58 
7.1.3 
Unexpected adverse reaction .................................................................................. 58 
7.1.4 
Serious..................................................................................................................... 58 
7.1.5 
Serious Adverse Event ............................................................................................ 59 
7.1.6 
Disability ................................................................................................................. 59 
7.1.7 
Life-threatening adverse drug reaction ................................................................... 59 
7.1.8 
Protocol Deviation (NIH Definition) ...................................................................... 59 
7.1.9 
Non-Compliance (NIH Definition) ......................................................................... 59 
7.1.10 Unanticipated Problem............................................................................................ 59 
7.2 
NIH IRB and Clinical Director (CD) Reporting ......................................................... 59 
7.2.1 
NIH IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths
 ................................................................................................................................ 59 
7.2.2 
NIH IRB Requirements for PI Reporting at Continuing Review ........................... 60 
7.2.3 
NIH IRB Reporting of IND Safety Reports ............................................................ 60 
7.3 
IND Sponsor Reporting Criteria ................................................................................. 60 
7.3.1 
Reporting Pregnancy ............................................................................................... 61 
7.4 
Safety Reporting Criteria to the Pharmaceutical Collaborators .................................. 61 
7.4.1 
Etubics Corporation (ETBX-051, ETBX-061, ETBX-071) ................................... 61 

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7.5 
Institutional Biosafety Committee (IBC) Reporting Criteria ...................................... 61 
7.5.1 
Serious Adverse Event Reports to IBC ................................................................... 61 
7.5.2 
Annual Reports to IBC............................................................................................ 62 
7.6 
Data and Safety Monitoring Plan ................................................................................ 63 
7.6.1 
Principal Investigator/Research Team .................................................................... 63 
7.6.2 
Sponsor Monitoring Plan ........................................................................................ 63 
7.6.3 
Safety Monitoring Committee (SMC) .................................................................... 63 
8 STATISTICAL CONSIDERATIONS.....................................................................................63 
8.1 
Statistical Hypotheses ................................................................................................. 64 
8.1.1 
Primary Endpoints .................................................................................................. 64 
8.1.2 
Secondary Efficacy Endpoints ................................................................................ 64 
8.2 
Sample Size Determination......................................................................................... 64 
8.3 
Populations for Analysis ............................................................................................. 65 
8.3.1 
Definitions............................................................................................................... 65 
8.4 
Statistical Analyses ..................................................................................................... 65 
8.4.1 
General Approach ................................................................................................... 65 
8.4.2 
Analysis of the Primary Endpoints ......................................................................... 65 
8.4.3 
Analysis of the Secondary Efficacy Endpoints ....................................................... 65 
8.4.4 
Safety Analyses ....................................................................................................... 66 
8.4.5 
Baseline Descriptive Statistics ................................................................................ 67 
8.4.6 
Planned Interim Analyses and Halting Guidelines ................................................. 67 
8.4.7 
Subgroup Analyses ................................................................................................. 67 
8.4.8 
Tabulation of Individual Participant Data............................................................... 67 
8.4.9 
Exploratory Analyses .............................................................................................. 67 
9 COLLABORATIVE AGREEMENTS ....................................................................................68 
9.1 
Cooperative Research And Development Agreement (CRADA) .............................. 68 
10 HUMAN SUBJECTS PROTECTIONS ..................................................................................68 
10.1 
Rationale for Subject Selection ................................................................................... 68 
10.1.1 Selection Based on Gender, Ethnicity, and Race .................................................... 68 
10.1.2 Strategies/Procedures for Recruitment ................................................................... 68 
10.1.3 Justification for Exclusions ..................................................................................... 68 
10.2 
Participation of Children ............................................................................................. 68 

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10.3 
Participation of Subjects Unable to Give Consent ...................................................... 68 
10.4 
Evaluation of Benefits and Risks/Discomforts ........................................................... 69 
10.5 
Consent Process and Documentation .......................................................................... 69 
10.5.1 Telephone re-consent procedure ............................................................................. 69 
10.5.2 Consent process for non-English speaking subjects ............................................... 70 
11 PHARMACEUTICAL INFORMATION ................................................................................70 
11.1 
Combination ETBX-071, ETBX-061, and ETBX-051 Vaccine Regimen (IND# 
17811) ......................................................................................................................... 70 
11.1.1 Source ..................................................................................................................... 70 
11.1.2 Toxicity ................................................................................................................... 72 
11.1.3 Formulation and Preparation................................................................................... 74 
11.1.4 Stability and Storage ............................................................................................... 74 
11.1.5 Administration Procedures...................................................................................... 74 
11.1.6 Incompatibilities ..................................................................................................... 74 
11.1.7 Other Considerations .............................................................................................. 74 
12 REFERENCES ........................................................................................................................76 
13 APPENDICES .........................................................................................................................82 
13.1 
Appendix A: Performance Status Criteria .................................................................. 82 
13.2 
Appendix B: Injection Site Reactions Diary Card ...................................................... 83 
 

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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation or Specialist Term Explanation 
E-HCG 
E-Human chorionic gonadotropin 
Ad 
Adenovirus 
Ad5 
Adenovirus serotype-5 
Ad5 [E1-] 
Ad5 with deletions in the early 1 (E1) and early 3 (E3) gene 
regions 
Ad5 [E1-, E2b-] 
Ad5 with deletions in the early 1 (E1), early 2b (E2b), and early 
3 (E3) gene regions 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
Brachyury 
A transcription factor within the T-box complex of genes 
BSI 
BioSpecimen Inventory 
BUN 
Blood urea nitrogen 
CAP 
College of American Pathologists 
CBC 
Complete blood count 
CCR 
(NCI) Center for Cancer Research 
CEA 
Carcinoembryonic Antigen 
CEA(6D) 
Modified Carcinoembryonic Antigen 
CLIA 
Clinical Laboratory Improvement Amendments 
CMI 
Cell-mediated immunity 
CMV 
Cytomegalovirus 
CR 
Complete response 
CRADA 
Cooperative Research and Development Agreement 
CRF 
Case report form 
CSC 
Cancer stem cell 
CT 
Computed tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
DFS 
Disease-free survival 
DCR 
Disease control rate 
DLT 
Dose-limiting toxicity 

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Abbreviation or Specialist Term Explanation 
DLTs 
Dose-limiting toxicities 
E1 
Adenovirus early 1 gene 
E2b 
Adenovirus early 2b gene 
E3 
Adenovirus early 3 gene 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
EGFR 
Epidermal growth factor receptor 
ELISA 
Enzyme-linked immunosorbent assay 
EOS 
End of study 
ETBX-011 
Ad5 [E1-, E2b-]-CEA 
ETBX-051 
Ad5 [E1-, E2b-]-brachyury 
ETBX-061 
Ad5 [E1-, E2b-]-MUC1 
ETBX-071 
Ad5 [E1-, E2b-]-PSA 
FDA 
Food and Drug Administration 
FFPE 
Formalin-fixed paraffin-embedded 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practice 
GM-CSF 
Granulocyte-macrophage colony-stimulating factor 
cGMP 
Current Good Manufacturing Practice 
HBV 
Hepatitis B virus 
HCV 
Hepatitis C virus 
HHS 
Health and Human Services 
HIV 
Human immunodeficiency virus 
HPV 
Human papilloma virus 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
IFN 
Interferon 
IHC 
Immunohistochemistry 
IL 
Interleukin 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
ISH 
In situ hybridization 

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Abbreviation or Specialist Term Explanation 
LLD 
Longest lesion diameter 
LLN 
Lower limit of normal 
MRI 
Magnetic resonance imaging 
MTD 
Maximum tolerated dose 
MUC1 
A Transmembrane glycoprotein 
MUC1c 
A modified MUC1 with agonist epitope 
NCI 
National Cancer Institute 
NIH 
National Institutes for Health 
ORR 
Objective response rate 
OS 
Overall survival 
PBMC 
Peripheral blood mononuclear cell 
PD 
Progressive disease 
PFS 
Progression-free survival 
PR 
Partial response 
PSA 
Prostate specific antigen 
PT 
Prothrombin 
PTT 
Partial thromboplastin time 
aPTT 
Activated partial thromboplastin time 
RECIST 
Response Evaluation Criteria in Solid Tumors 
SAE 
Serious adverse event 
SC 
Subcutaneous 
SD 
Stable disease 
SRC 
Safety Review Committee 
SUSAR 
Suspected unexpected serious adverse reactions 
Treg 
Regulatory T cells 
ULN 
Upper limit of normal 
VP 
Virus particles 
 
 
 

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1 
INTRODUCTION 
1.1 
STUDY OBJECTIVES AND ENDPOINTS 
1.1.1 Primary Objectives 
x To determine the overall safety and recommended phase 2 dose of a combination of three 
immunotherapeutic vaccines (ETBX-071, ETBX-061, and ETBX-051) when administered 
subcutaneously (SC) in subjects with Metastatic Castration Resistant Prostate Cancer 
(mCRPC). 
1.1.2 Secondary Objectives 
x To make preliminary assessments of objective response rate (ORR), disease control rate 
(DCR), duration of response, progression-free survival (PFS), overall survival (OS) and 
PSA doubling time (PSADT) in subjects with advanced cancer treated with the ETBX-071, 
ETBX-061, and ETBX-051 vaccines. 
1.1.3 Exploratory Objectives 
x To evaluate the immunogenicity of the ETBX-071, ETBX-061, and ETBX-051 vaccines 
platform 
x To monitor circulating PSA serum/plasma levels. 
x To evaluate CD8 and CD4 immunologic response as measured by an increase in PSA, 
MUC-1, and Brachyury-specific T cells. 
x To evaluate tumors for expression of the target antigens, PSA, MUC-1, and Brachyury 
x Correlations between immunogenicity  and efficacy outcomes will be assessed. 
1.1.4 Primary Endpoints 
x Refer to section 8.1.1 
1.1.5 Secondary Endpoints 
x Refer to section 8.1.2 
1.1.6 Exploratory Endpoints 
x Immunogenicity of the ETBX-071, ETBX-061, and ETBX-051 vaccines by flow 
cytometric analysis of T-cell frequency, activation status, cytokine profiles, antibody levels 
and analysis in sera of sCD27 and sCD40L 
x Evaluate circulating PSA levels and correlations with immunogenicity and/or efficacy. 
1.2 
BACKGROUND AND RATIONALE 
1.2.1 Overall Objective 
As a result of the discovery of new biomarkers associated with tumor development and metastasis, 
many tumor-associated antigens (TAAs) are being utilized in immunotherapeutic modalities 
designed to induce anti-tumor directed cytotoxic immune responses. It is increasingly clear that 
not any one of these TAA is sufficient, as a single entity, by which one can develop a potent 
immunotherapeutic response. Furthermore, the addition of immune checkpoint inhibitors has 
augmented the immunotherapy approach against TAAs. Consequently, efforts are focused on 

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developing multi-targeted immunotherapy approaches. The overall goal of the current project is to 
expand the immunotherapeutic approach for the treatment of mCRPC employing a multi-targeted 
approach. Previous studies have demonstrated safety, dose response, and favorable overall survival 
in metastatic colorectal cancer (mCRC) patients using Ad5 [E1-, E2b-]-based vaccine platform 
containing a modified carcinoembryonic antigen (CEA) (Ad5 [E1-, E2b-[-CEA(6D) referred to as 
ETBX-011.(1) This vaccine was employed as an immunotherapeutic tested as a single agent in a 
clinical setting.(2, 3) 
 
Patients in that study exhibited evidence of a favorable survival probability, with all 25 patients 
treated at least 2 times with vaccine exhibiting a 12-month overall survival probability of 48%, 
with a mean overall survival of 11 months. The phenotypic heterogeneity in terms of expression 
of different TAAs in a given primary or metastatic tumor mass is a well-established phenomenon. 
One can speculate that the use of an immunotherapeutic vaccine regimen targeting three distinct 
TAAs, each of which is widely expressed on the majority of human carcinomas, would be 
potentially therapeutically advantageous over the use of a vaccine targeting only one TAA. With 
the safety and immunogenicity of Ad5 [E1-, E2b-]–CEA established in patients as a single agent, 
a multi-target approach is now being investigated. The objective is to develop a combination 
immunotherapeutic approach designed to induce broad anti-tumor immune responses directed 
against tumors that over express PSA, MUC1, and/or Brachyury.(4, 5)The  objective is to develop 
a combination immunotherapeutic approach designed to induce broad anti-tumor immune 
responses directed against prostate cancer. In proof of concept research, published pre-clinical 
studies indicated that a multi-targeted vaccine based upon the Ad5 [E1-, E2b-] platform and 
containing CEA, MUC1, and brachyury TAA induces immune responses directed against all three 
target CEA, MUC1, and brachyury antigens with minimal to no “antigenic competition” in in vitro 
studies of human dendritic cells(6), or in murine vaccination studies.(6) 
 
We plan to test a combination of multi-targeted PSA, MUC1, and brachyury targeted Ad5 [E1-, 
E2b-]-based vectored vaccines in a phase 1 clinical to test the safety and efficacy  in patients with 
mCRPC. Subsequent trials will involve the use of these vaccines in combination with checkpoint 
inhibitor monoclonal antibodies (MAbs) and other immune modulators. 
1.2.2 PSA Expression in Prostate Cancer and It’s Use as an Immunotherapy Target 
Prostate cancer is an ideal candidate for immunotherapy for several reasons. The slow growing 
nature of cancer within the prostate allows sufficient time to generate an anti-tumor immune 
response following a prime/boost or multiple immunization strategies.(7, 8) The presence of PSA 
in patient serum enables the malignancy to be detected early and, in some cases, before tumors are 
radiologically detectable.(9) This in turn can facilitate earlier treatment.(10) Circulating T cells 
that react with prostate TAAs have previously been detected, which suggests that self-tolerance to 
these antigens can be overcome.(10) The prostate is considered to be a non-essential organ and 
therefore induced immunological responses directed against specific prostate TAAs should not 
cause acute off target toxicity.(9, 10) Most importantly, the first prostate cancer specific 
immunotherapy, Sipuleucel-T (Provenge®, Dendreon Corporation, Seattle, WA), has been 
licensed by the US Food and Drug Administration (FDA) in 2010 for asymptomatic or minimally 
symptomatic mCRPC.(11, 12) Sipuleucel-T consists of autologous peripheral blood mononuclear 
cells with antigen presenting dendritic cells that have been activated ex vivo with a recombinant 

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fusion protein (PA2024) consisting of PAP linked to granulocyte-macrophage colony stimulating 
factor (GM-CSF).(12) In a phase III trial, mCPRC patients receiving Sipuleucel-T exhibited a 22% 
reduction in mortality.(11) The success of the therapeutic Sipuleucel–T has now paved the way 
for other immunotherapeutic prostate cancer vaccines to be granted regulatory approval and enter 
the market. 
ProstvacTM vaccine offers an alternative strategy to Sipuleucel-T and employs genetically altered 
poxviruses to deliver targeting information to immune cells and generate an immune response. 
Administered subcutaneously, the poxviruses deliver the transgenes for the tumor associated 
antigens (PSA) to antigen presenting cells through cellular infection. Once these pox viruses are 
within the cellular cytoplasm, the transgenes are processed. The end result is an antigen presenting 
cell expressing a PSA peptide within the major histocompatibility complex, resulting in PSA-
specific cytolytic T lymphocytes activation. Prostvac was evaluated in a randomized phase 2 trial. 
Subjects with minimally symptomatic mCRPC were randomized 2:1 (85/41) to vaccine therapy 
versus placebo. Treated subjects had prolonged median overall survival (OS) 26 vs. 18 months. 
Prostvac is very well-tolerated, with common side effects of grade 1 injection-site reactions or flu-
like symptoms.(13) This approach has been taken to pivotal randomized phase 3 trial, now fully 
enrolled (N=1297), and awaiting event driven results.(14) 
In addition, a recombinant Ad-5 [E1-, E2b-] PSA vector (Ad-PSA) has now been developed.  PSA 
has been incorporated into a replication incompetent early generation Ad5 [E1-]-based vector 
platform and tested in a phase 1 trial.(15) Sequential cohorts of subjects had increasing doses of a 
single injection of Ad5-PSA. Most subjects developed detectable cellular mediated anti-PSA 
responses 18/32 (67%).  This approach is being evaluated in a phase 2 trial using multiple doses 
(3 injections) at 1 month intervals.(16) Infection of human dendritic cells with Ad-PSA induced 
high levels of expression of PSA.  Moreover, Ad-PSA infected dendritic cells were shown to 
activate a PSA specific human T-cell line to produce >3,000 pg of IFN g in an MHC restricted 
manner, while control Ad5 infected dendritic cells expressed less than 1pg IFN-g. Thus, PSA based 
vaccination approaches have preliminary evidence of clinical activity as well as an ability to induce 
anti-PSA directed cellular immunity.  Improved vectors, such as the new Etubics Ad5 [E1-, E2b-
]-based vector platform, should facilitate clinical development of this targeted approach. Non-
replicating adenoviral vectors should improve the safety of this approach, and the ability to 
circumvent neutralizing anti-viral immune responses would enable sustained boosting to maximize 
immune responses.  These features can be provided by Ad5 [E1-, E2b-]-based vectors. 
1.2.3 MUC1 Expression in Cancer and It’s Use as an Immunotherapy Target 
MUC1 (CD227) is a tumor associated antigen (TAA) that is over-expressed on a majority of 
human carcinomas and several hematologic malignancies.(17-20) The MUC1 glycoprotein is 
normally expressed at the surface of glandular epithelial cells(21) and is over-expressed and 
aberrantly glycosylated in carcinomas.(17, 22) Thus, MUC1 has been recognized as a priority 
TAA that can be utilized as a target for tumor immunotherapy.(23, 24) Several clinical trials have 
been and are being performed to evaluate the use of MUC1 in immunotherapeutic vaccines.(25-
30) Some of these trials have indicated that targeting MUC1 is safe and may provide survival 
benefit .(28-30) 
 
Multiple enhancer agonist epitopes were previously identified, several of which are in the MUC1 
C-terminus region. (31, 32) This is potentially important because studies have demonstrated that 

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15 
 
the C-terminus of MUC1 has oncogenic potential, associates with poor prognosis and drug 
resistance, and induces “stemness” features in a range of human carcinomas. The human T-cell 
lines generated using these MUC1 agonist epitopes were more efficient than those generated with 
the corresponding native epitopes in terms of antigen-specific interferon (IFN)–γ production and 
lysis of tumor cells endogenously expressing native MUC1.(24) Therefore, it is believed that 
MUC1 containing modified agonist epitopes has a greater potential as an immunogenic agent for 
vaccine development. 
1.2.4 Brachyury Expression in Cancer and It’s Potential as an Immunotherapy Target 
Brachyury is an embryonic transcription factor of the T-box family that regulates the formation of 
the posterior mesoderm in the developing embryo, a process that requires the conversion of 
epithelial cell layers into mesenchymal cells.(33) While in the majority of adult normal tissues 
Brachyury is undetectable, with the exception of low levels found in normal testis, thyroid and a 
subset of B cells(34), aberrantly high levels of Brachyury have been observed in the primary and/or 
metastatic sites of non-small cell (NSCLC) and small cell (SCC) lung cancer(35, 36), colon(37), 
hepatocellular(38), prostate(39) and breast carcinomas(40), including triple negative breast cancer 
(TNBC)(41). High levels of Brachyury are also characteristic of the rare tumor type chordoma(42), 
which is thought to originate from remnants of the embryonic notochord where Brachyury is 
normally found. Recent studies have now characterized the role of Brachyury in the biology of 
carcinomas and demonstrated the ability of this transcription factor to drive the phenotypic 
conversion of tumor cells from an epithelial to a mesenchymal-like phenotype (also designated as 
an epithelial-mesenchymal transition, EMT, or carcinoma “mesenchymalization”)(43). Carcinoma 
cells undergoing this phenotypic transition exhibit enhanced motility and invasiveness in vitro, 
propensity to metastasize in vivo, and features of tumor stemness(44), including resistance to a 
range of therapeutics such as chemotherapy, radiation, small molecule therapies and, potentially, 
immunotherapy(45-48). In agreement with a role for Brachyury in the progression of carcinomas, 
multiple studies have now shown that the level of Brachyury in the primary tumor correlates with 
poor patient prognosis in carcinomas of the lung(49), colon(37) breast(40), triple negative 
breast(41) and gastrointestinal stromal tumor (GIST)(50). Brachyury expression has also been 
shown to be correlated with advanced stage prostate cancer(39). 
Transcription factors have been considered “difficult to drug” due to their primary location in the 
nucleus and lack of a hydrophobic groove for drug attachment. Studies have shown, however, that 
Brachyury-specific T cells can be generated both in vitro and in vivo.(33, 51) Utilizing 9-mer 
peptides of the Brachyury protein, for example, Brachyury-specific CD8+ T cells have been 
expanded in vitro from the blood of cancer patients; these Brachyury-specific CD8+ T cells were 
utilized in cytotoxic assays for effective lysis of human tumor cells that endogenously express 
Brachyury.(33, 51) 
 
The inherent immunogenicity of Brachyury was also revealed from the analysis of immune 
responses in cancer patients immunized against CEA or PSA. In addition to generating responses 
against the tumor-associated antigens contained within their respective vaccines, development of 
Brachyury-reactive CD8+ T cells was also observed.(52) This expansion of Brachyury-specific T 
cells may have been the result of cross-presentation of the antigen to the immune system, following 
tumor destruction in response to the vaccine. These studies demonstrated that Brachyury is 
immunogenic, and has the potential to function as a target for anticancer vaccination. In addition, 

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16 
 
two recently completed Phase I clinical studies with a recombinant yeast-Brachyury vaccine(53) 
or an MVA-Brachyury-TRICOM vaccine(54) also demonstrated the generation of Brachyury-
specific T cells as well as safety in humans, thus providing further evidence of 
immunogenicity(55). These combined properties, i.e., tumor-restricted expression, relevant 
function in tumor progression, and immunogenicity, make Brachyury a potential target for 
immunotherapy-mediated approaches against cancer. Preventing or reverting the EMT process in 
carcinomas via the use of Brachyury-based cancer vaccines represents an attractive modality to 
minimize tumor dissemination and the emergence of therapeutic resistance. 
 
1.2.5 Adenovirus-Based Vectors 
Adenoviruses (Ads) have emerged as leading candidate vectors to deliver vaccines designed to 
induce CMI and antibody responses (56-58). Ad vectors infect multiple cell types, including 
dendritic cells which result in priming of a vigorous CMI response. Ads are a family of DNA 
viruses characterized by an icosahedral, non-enveloped capsid containing a linear double-stranded 
genome (57, 58). None of the human Ads are associated with neoplastic disease and only cause 
relatively mild, self-limiting illness in immunocompetent individuals. Ad serotype-5 (Ad5) is the 
most widely used subtype for human vaccines. The wild-type Ad5 genome is approximately 36 
kilobases and encodes genes that are divided into early and late viral functions, depending on 
whether they are expressed before or after DNA replication. Ad5 vectors do not integrate (i.e., 
their genomes remain episomal), so the risk for insertional mutagenesis and/or germ-line 
transmission is extremely low if at all. 
 
1.2.5.1 Early Generation Ad5 Vectors 
Early generation Ad5 vectors (Ad5 [E1-]) contain deletions in the early 1 (E1) gene and early 3 
(E3) gene regions (56-59). The E1 gene is required for DNA synthesis, capsid protein expression, 
and viral replication, and the E3 gene is required for anti-host immunity. Ad5 [E1-] vectors have 
a decreased ability to replicate and cannot produce infectious virus in cells that do not express the 
Ad5 E1 genes. Recombinant Ad5 [E1-] vectors are propagated in human cells (typically human 
embryonic kidney 293 cells), allowing for Ad5 [E1-] vector replication and packaging (57). There 
have been over 300 human clinical trials that utilized Ad5 [E1-] vectors, with more than 2000 
subjects given the virus SC, intramuscularly, or intravenously (58). Ad5 [E1-] vectors have a 
number of positive attributes; one of the most important is their relative ease for scale up and 
current good manufacturing practices (cGMP) production. Furthermore, recombinant Ad5 [E1-] 
vectors have a large carrying capacity that approaches 7 kilobases. However, preclinical and 
clinical studies have demonstrated that pre-existing immunity against Ad5 can be an inhibitory 
factor to the use of Ad5 [E1-] vaccines in practice (57, 59). Most humans have antibodies against 
Ad5, with up to two-thirds having lymphoproliferative responses against Ad5 (60, 61). CMI 
directed against pre-existing or newly synthesized Ad5 cell surface proteins interfere with Ad5 
[E1-] vaccines by eliminating the vaccine and vector-infected cells. This reduces the effectiveness 
of the early generation Ad5 [E1-]-based vaccines. 
 

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1.2.5.2 New Generation Ad5 Vectors (Ad5 [E1-, E2b-]) 
A new and advanced generation of Ad5 vectors have been developed that, in addition to deletions 
in the E1 and E3 gene regions, have deletions in the early 2b (E2b) gene regions (Ad5 [E1-, E2b-
]) (62-65). The E2b genes are required for viral replication and encode viral DNA polymerase as 
well as the preterminal protein. In addition, the deletion of the E2b genes drastically reduces late 
gene expression (capsid type proteins), which decreases anti-vector immune responses and enables 
longer term transgene expression with enhanced immunogenicity. Thus, the Ad5 [E1-, E2b-] 
vector overcomes limitations of early generation vectors, as it permits the immunization of people 
who have been previously exposed to Ad5. In preclinical studies of cancer and infectious disease, 
Ad5 [E1-, E2b-] vector-based vaccines were used in multiple homologous immunization regimens 
and induced immune responses despite the presence of pre-existing Ad5 immunity (1, 3, 6, 65-
73). 
 
1.2.6 Clinical Experience with an Ad5 [E1-, E2b-]-based Cancer Immunotherapy Vaccine 
1.2.6.1 Clinical Safety 
Etubics Corporation has performed a Phase I/II clinical trial (IND#14325) with an Ad5 [E1-, E2b-
]-based vector containing a modified carcinoembryonic antigen (Ad5 [E1-, E2b-]-CEA(6D)) for 
the immunotherapy of CEA expressing cancer.(2, 3) The Phase I/II study consisted of a dose-
escalation study of four dosage levels (1x109, 1x1010, 1x1011, 5x1011 VP/dose) of ETBX-011 
(Phase I component), and the maximally tolerated dose of ETBX-011 (Phase II and 5x1011 
VP/dose components). Ad5 [E1-, E2b-]-CEA(6D) was administered by SC injection every 3 
weeks. Thirty-two patients with metastatic colorectal cancer (mCRC), median age 57.5 (range 38–
77) who had failed a median of three prior chemotherapeutic regimens (range 2–5), had a 
Karnofsky performance status of 90 (range 70–100), and had three sites of metastatic disease 
(range 1–4), were enrolled. The majority of patients were able to receive all three immunizations. 
Four patients who stopped immunizations early did so due to significant disease progression. A 
total of 94 immunization treatments was administered to all patients. There was no dose-limiting 
toxicity and no serious adverse effects (SAE) that resulted in treatment discontinuation at any 
vaccine dose level. The most common toxicity was a self-limited, injection site reaction. Other 
reactions occurred with less than a 10% incidence of all adverse effects (AE) reported and included 
fever, flu-like symptoms, anorexia, chills, nausea, and headache. These symptoms were also self-
limiting and did not require intervention other than symptomatic measures such as 
acetaminophen.(2, 3) 
 
1.2.6.2 Clinical Immune Response 
A secondary objective of the study listed above was to evaluate CEA specific immune responses 
following immunization treatments with the product candidate. As determined by an ELISA 
technique,(2, 3) no antibody activity directed against CEA was observed. CMI responses were 
assessed in colorectal cancer patients treated in cohort 1, cohort 2, cohort 3/Phase II, and cohort 5. 
PBMCs were isolated prior to immunotherapy treatment and after all treatments as well as three 
weeks following the last treatment from patients. CEA specific ELISpot assays were performed 
on PBMC as previously described(1-3) to determine the numbers of interferon gamma (IFN-J) 
secreting lymphocytes (SFC) after exposure to CEA peptides in vitro. The highest CMI responses 
were determined during immunizations, regardless of time point (weeks 3, 6, or 9) in the patients 

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18 
 
treated in cohort 1, cohort 2, cohort 3/Phase II, and cohort 5. This analysis revealed a dose response 
to increasing levels of product (Figure 1). The highest CMI levels occurred in patients that 
received the highest dose of 5x1011 VP (Cohort 5). 
 
 
 
 
 
Pre
Post
Pre
Post
Pre
Post
Pre
Post
0
100
200
300
400
500
Cohort 1
Cohort 2
Cohort 3/Phase II
Cohort 5
IFNJ SFC per 106 PBMC
 
Figure 1. CMI responses in treated patients. CMI (IFN-J secretion) was assessed at baseline (Pre) and 
after administrations of Ad5 [E1-, E2b-]-CEA(6D) (Post). The highest CMI responses (regardless of 
time point) observed in the patients after treatment revealed a dose response. The highest CMI levels 
occurred in patients that received the highest dose of 5x1011 VP (Cohort 5). The CMI responses for 
Cohort 3/Phase II and Cohort 5 were significantly elevated (P=0.0002 and P=0.0317, respectively; 
Mann-Whitney test) as compared to their baseline (Pre) values. Specificity of the responses was 
demonstrated by the lack of reactivity with the irrelevant antigens E-galactosidase and HIV-gag (data 
not shown). For positive controls, PBMCs were exposed to concanavalin A (data not shown).  SFC: 
spot forming cells. Values = Mean r SEM for each Cohort. 

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19 
 
 
 
In a preliminary study,(2) a population of polyfunctional CD8+ T cells was observed (those that 
secrete more than 1 cytokine when activated) after immunizations that secreted multiple cytokines, 
a sign of greater functionality of T cells induced by the vaccine. In further follow-up analysis(2) 
of a few patient blood samples, a decrease in CEA directed immune responses was noted after 
immunotherapy immunizations was stopped. This observation supports a rationale for booster 
immunizations to maintain immune responses. 
 
Ad5 NAb and CMI were measured against Ad5 and correlated with CEA-specific CMI. Each 
patient had their serum and PBMC sample tested at baseline (prior to treatment) and at 9 weeks 
after completion of 3 treatments. Nineteen of 31 colorectal cancer patients (61%) tested in this 
study had Ad5 neutralizing activity in serum samples prior to the onset of treatment with Ad5 [E1-
, E2b-]-CEA(6D). The mean pre-treatment Ad5 NAb titer value obtained among all patients was 
1:189 r1:71 SEM and the mean pre-treatment Ad5 NAb titer among seropositive patients was 
1:308 r 1:108. Analysis of serum samples from patients who received 3 immunizations revealed 
Ad5 NAb titers that were significantly increased (P<0.0001, Mann-Whitney test) by week 9 (mean 
1:4767 r 1:1225 SEM) when compared with their respective baseline values. Analysis of PBMC 
for CMI responses to Ad5 also revealed a significant increase (P<0.01, Mann-Whitney test) in Ad5 
directed CMI responses after immunizations with Ad5 [E1-, E2b-]-CEA(6D) (22.6 r 9.3 SEM 
IFN-J secreting SFC at week 0 versus 191.1 r 83.7 IFN-J SFC at week 9). 
 
1.2.6.3 Clinical Evidence of Activity 
The Ad5 [E1-, E2b-]-CEA(6D) treated colorectal cancer patients (total = 32) were followed for 
survival and Kaplan-Meier plots and survival proportions performed (PRISM software).(2, 3) 
0
20
40
60
80
100
0
3
   6
9
26
56
Weeks
Patient  005
IFNJ SFC per 106 PBMC
0
3
9
26
0
100
200
300
400
500
Weeks
Patient 024
IFNJ SFC per 106 PBMC
0
3
6
9
26
0
150
300
450
Weeks
Patient 031
IFNJ SFC per 106 PBMC
0
3
6
9
26
39
0
10
20
30
40
50
60
Weeks
Patient 501
IFNJ SFC per 106 PBMC
0
500
1000
1500
0
3
6
9
26
Weeks
Patient 507
39
IFNJ SFC per 106 PBMC
 
Figure 2. Profiles of CEA directed CMI responses in 5 mCRC patients during and after the course of 
immunotherapy. Note the decrease in CMI responses from their peak values after immunizations ended. 
Values are Mean r SEM.  

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Events were determined by information from the social security death index (SSDI) database, 
clinical charts and telephone calls (Figure 3). 
The seven patients in cohorts 1 and 2 experienced a 12-month survival proportion of 29%. The 21 
patients in cohort 3 and Phase II experienced a 12-month survival proportion of 48%. The six 
patients in cohort 5 experienced a 12-month survival proportion of 50%. Twenty-nine-month 
overall survival of the intent-to-treat population (32 patients) was 20% (Figure 3a) with a median 
survival time of 11 months from informed consent/first injection. For the subset of 28 patients that 
received all 3 immunizations, the 29-month survival was 23% (Figure 3b) with a median survival 
time of 13 months. For the 22 patients optimally dosed with the two highest doses of vaccine (1 
and 5 x 1011) and receiving all 3 immunizations, the 28-month overall survival was 19% (Figure 
3c). Median overall survival was 13 months in the optimally treated patients. Since there was no 
active control group in the study, comparisons for significance in survival time cannot be made. 
There were 3 stable disease events observed immediately after completion of treatment 
(immunizations). 
1.2.7 ETBX-071   Pre-clinical Studies 
Pre-clinical studies were performed in mice based upon immunization and treatment schedules 
used in earlier murine model studies. In a pre-clinical study with the Ad5 [E1-, E2b-]-HER2/neu 
immunotherapy vaccine to treat HER2/neu expressing tumors(74), a dose response study was 
performed using doses of 108, 109, or 1010 VP injected 3 times subcutaneously (SC) at weekly 
intervals. A dose response effect was observed on the induced CMI responses with the highest 
CMI response level being observed after immunizations with 1010 VP of vaccine. In addition, a 
study was performed to assess CMI induction following a single or multiple homologous 
immunizations with Ad5 [E1-, E2b-]-HER2/neu, groups of Ad5 naive BALB/c mice (n=5/group) 
were immunized once, twice, or three times at weekly intervals with 1010 VP of Ad5 [E1-, E2b-]-
HER2/neu. Two weeks following the last immunization, splenocytes were exposed to HER2/neu 
peptides or irrelevant antigens and analyzed by ELISpot for the number of IFN-and IL-2 secreting 
0
4
8
12
16
20
24
28
0
20
40
60
80
100
a
Months
Percent Survival Proportion
0
4
8
12
16
20
24
28
0
20
40
60
80
100
b
Months
Percent Survival Proportion
0
4
8
12
16
20
24
28
0
20
40
60
80
100
c
Months
Percent Survival Proportion
  
Figure 3. Kaplan-Meier survival plots on long-term overall survival of treated mCRC patients. Panel a 
represents all treated patients. Panel b represents patients that received all 3 treatments. Panel c 
represents patients immunized 3 times with the 2 highest doses of vaccine. There were 23 events. 

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21 
 
T-cells. Significantly elevated numbers of IFN-γ and IL-2 secreting cells were observed in mice 
after one, two, or three immunizations with Ad5 [E1-, E2b-]-HER2/neu. The numbers of IFN-γ 
and IL-2 secreting cells observed after two or three immunizations were significantly higher than 
those observed after one immunization (p<0.02 and p<0.002, respectively). However, the 
differences were not statistically significant between two and three immunizations. This provided 
us with a rationale for using a dose of 1010 VP injected 3 times SC in order to induce significant 
tumor associated antigen (TAA) directed immune responses. In cancer immunotherapy studies 
with Ad5 [E1-, E2b-]-based cancer vaccines directed against CEA, HER2/neu, or HPV-E6/E7 to 
treat CEA, HER2/neu, or HPV-E6/E7 expressing tumors, respectively, the induced immune 
responses resulted in anti-tumor activity as evidence by inhibition of tumor growth in treated mice 
as compared to control mice(1, 71, 72, 74). For the CEA murine tumor model, immunotherapy 
treatment had to be initiated within one day of tumor implantation due to the fast-growing 
aggressive nature of the tumor. For the HER2/neu and HPV-E6/E7 murine tumor models, the 
tumors were not as fast growing and allowed for establishment of implanted tumor cells before 
tumor immunotherapy was initiated. The antitumor activity observed in all 3 tumor models 
indicated that the vaccines could exhibit activity against small or palpable (larger) tumors. The 
pre-clinical studies using ETBX-071 (Ad5 [E1- E2b-]-PSA) are described below. 
 
ETBX-071 is a PSA targeting vaccine comprising the Ad5 [E1-, E2b-] vector and a PSA gene 
insert (Ad5 [E1-, E2b-]-PSA). Studies were performed to assess the use of Ad5 [E1-, E2b-]-PSA 
as a cancer vaccine in a BALB/c mouse model. Ad5 [E1-, E2b-]-PSA induced potent CMI against 
PSA in mice. Studies were also performed to show anti-tumor activity of the vaccine in a murine 
model of PSA expressing cancer. These data indicate that in vivo delivery of Ad5 [E1-, E2b-]-
PSA can induce PSA directed anti-tumor immunity against PSA expressing cancers. 
1.2.7.1 Immunogenicity 
To assess immune responses generated following multiple homologous immunizations with Ad5 
[E1-, E2b-]-PSA (ETBX-071), initial studies were performed using a BALB/c mouse model. 
Groups of BALB/c mice (n = 5/group) were immunized three times SC at 1-week intervals with 
1010 VP of Ad5 [E1-, E2b-]-PSA. Control mice were injected with buffer solution only. Two weeks 
following the last immunization, splenocytes were harvested and exposed to PSA protein and 
assessed for CMI responses by ELISpot for IFN-γ or IL-2 secreting splenocytes. As shown in 
Figure 4, PSA directed CMI responses were induced in vaccinated but not control mice. 
Specificity of the CMI responses was demonstrated by a lack of reactivity against irrelevant HIV-
gag or cytomegalovirus virus (CMV) antigens in ELISpot assays. 
Antibody responses were also tested using a previously described quantitative assay(74) and PSA 
directed antibody responses were detected in immunized but not control mice (Figure 5). 
 

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To determine if infected human DC could stimulate human antigen-specific T cell lines to secrete 
IFN-γ, infected dendritic cells (DC) were incubated with antigen-specific T cell lines and tested 
for IFN-γ secreting activity as a measure of stimulation. Human DC were infected with Ad5 vector, 
incubated for 48 hours, washed, and used for stimulation of human antigen-specific T cells. As 
shown in Table 1 below, infection of human dendritic cells (from a HLA-A2 donor) with 
recombinant Ad5-PSA vectors encoding transgenes can activate PSA-specific T cell lines to 
produce IFN-γ. 
Additional studies were performed to assess immune responses generated by the Ad5 [E1-, E2b-
]-PSA (ETBX-071) component of a multi-targeted PSA, MUC1, brachyury vaccine regimen. In 
these studies, groups (n = 5) of C57Bl/6 mice were used. The mice were injected SC 3 times at 2-
week intervals with 3X1010 VP containing a 1/1/1 mixture of Ad5 [E1-, E2b-]-PSA, Ad5 [E1-, 
E2b-]-MUC1, and Ad5 [E1-, E2b-]-brachyury (tri-immunization). Two weeks after the last 
immunization CMI activity was determined employing ELISpot assays for IFN-J secreting cells 
(SFC) after exposure of splenocytes to PSA peptide pools, as previously described.(1-3, 6, 65, 67-
C o n tro l A d 5 [E 1 -, E 2 b -]-P S A
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
IFN J S F C p e r 1 0
6 s p le n o c y te s
C o n tro l A d 5 [E 1 -, E 2 b -]-P S A
0
2 0 0
4 0 0
6 0 0
8 0 0
IL -2 S F C /1 0 6 sp len o cytes
Figure 4. PSA induced CMI 
responses 
as 
assessed 
by 
ELISpot assays for IFN-J or IL-
2 secreting splenocytes (spot 
forming 
cells; 
SFC) 
were 
detected in vaccine immunized 
mice but not in control mice. 
Specificity of responses was 
confirmed by lack of reactivity 
to irrelevant HIV-gag or CMV 
peptide pools. A positive control 
included 
cells 
exposed 
to 
concanavalin A (Con A, data not 
shown). Values = Mean r SEM. 
A n ti-P S A A b R e s p o n s e
C o n tro ls
A d 5 [E 1 -, E 2 b -]-P S A
0
2
4
6
8
1 0
n g E q u iv a le n ts Ig G B o u n d
 
Figure 5. Antibody responses. 
BALB/c mice were immunized 
three times with either injection 
buffer (control group) or 10
10 VP 
of Ad5 [E1-, E2b-]-PSA at 7 day 
intervals. Sera from individual 
mice were assessed 14 days after 
the final immunization for PSA 
directed antibody responses using 
a quantitative ELISA. Values = 
Mean ± SEM. 

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70, 73, 74) As shown in Figure 6, significant CMI responses to were detected in immunized mice. 
Flow cytometry intracellular cytokine staining was performed as previously described(70, 72) on 
spleen cells after exposure to PSA peptides to assess the quantity of activated CD8+ T cells. As 
shown in Figure 7, IFN-J and IFN-J/TNF-D expressing polyfunctional CD8+ cells were detected 
in immunized but not control splenocytes. These above results demonstrate that the Ad5 [E1-, E2b-
]-PSA vaccine component in the multi-targeted vaccine is effective at inducing PSA directed 
immune responses. 
 
 
 
 
1.2.7.2 Anti-tumor Activity 
The anti-tumor activity of the Ad5 [E1-, E2b-]-PSA vaccine was tested in a murine model of PSA 
expressing cancer. BALB/c mice were immunized three times SC at weekly intervals with 1X1010 
VP of Ad5 [E1-, E2b-]-null (empty vector controls) or 1X1010 VP of Ad5 [E1-, E2b-]-PSA 
vaccine. Two weeks after the last immunization (vaccination), mice were implanted with 1x106 
PSA expressing murine tumor cells. All mice were monitored for tumor growth and tumor volumes 
m e d ia
S IV n e f p e p tid e s
P S A p e p tid e s
0
1
2
3
IFN J
+
(% C D 8 E
+ S p le n o c y te s)
m e d ia
S IV n e f p e p tid e s
P S A p e p tid e s
0 .0
0 .5
1 .0
1 .5
IFN J
+ T N F D
+
(% C D 8 E
+ S p le n o c y te s)
Figure 
7. 
PSA 
induced 
stimulation of CD8+ cells 
expressing IFN-J (Top) and 
IFN-J /TNF-D (Bottom) in mice 
immunized with vaccine (grey 
bars) but not in controls injected 
with Ad5-null (white bars). 
Specificity of the responses was 
confirmed by lack of reactivity 
to media alone or irrelevant 
SIV-nef peptides. Values = 
Mean r SEM.  
 
0
4
7
1 1 1 4 1 8 2 1 2 4 2 8 3 2 3 7
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
1 1 0 0
D a y s p o s t tu m o r in n o c u la tio n
T u m o r v o lu m e m m
3
A d 5 [E 1 -, E 2b -]-n u ll
A d 5 [E 1 -, E 2 b -]-P S A
*
p = 0 .0 2
Figure 
8. 
Anti-tumor 
responses. BALB/c mice were 
immunized three times with 
either 10
10 VP of Ad5 [E1-, 
E2b-]-null (control group) or 
10
10 VP of Ad5 [E1-, E2b-]-
PSA at 7 day intervals. Mice 
were implanted 
with PSA 
expressing tumors cells and 
monitored for tumor growth. 
Note the significant inhibition 
of 
tumor 
growth 
in 
the 
immunized mice as compared 
to controls. Values = Mean ± 
SEM. 
Figure 6. PSA induced CMI 
responses as assessed by ELISpot 
assays 
for 
IFN-J 
secreting 
splenocytes (spot forming cells; 
SFC) were detected in vaccine 
immunized (grey bars) mice but not 
in 
control 
(white 
bars) 
mice 
(injected with Ad5-null empty 
vector). Specificity of the responses 
was confirmed by lack of reactivity 
to irrelevant SIV-nef or SIV-vif 
peptides. 
A 
positive 
control 
included 
cells 
exposed 
to 
concanavalin A (Con A, data not 
shown). Values = Mean r SEM. 

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24 
 
were calculated to determine if pre-immunization with Ad5 [E1-, E2b-]-PSA inhibited growth of 
tumors in immunized but not control mice. As shown in Figure 8, mice immunized with Ad5 [E1-
, E2b-]-PSA experienced slower tumor growth as compared to control mice injected with Ad5-
null (empty vector). These studies indicate that the Ad5 [E1-, E2b-]-PSA component in the multi-
targeted vaccine regimen can induce PSA directed anti-tumor responses in immunized animals. 
 
Table 1 
Dendritic cells infected with 
Antigen Specific T Cells 
 
T-PSA-(HLA-A2) 
T-CEA-(HLA-A2) 
Ad5 [E1-, E2b-]-PSA (20,000 MOI) >3,000 
<0.732 
Ad5 [E1-, E2b-]-PSA (10,000 MOI) >3,000 
0.84 
Ad5 [E1-, E2b-]-Null (20,000 MOI) 0.9 
0.89 
DCs 
4.24 
0.78 
No DCs (PSA T Cells only) 
<0.732 
ND 
No DCs (CEA T Cells only) 
ND 
<0.732 
Results are expressed in picograms of IFN-γ per 5X105 T cells/mL. DC only = <0.732. MOI: 
multiplicity of infection. 
1.2.8 ETBX-061 Pre-clinical Studies 
ETBX-061 is a MUC1 targeting vaccine comprising the Ad5 [E1-, E2b-] vector and a modified 
MUC1 (MUC1c) gene insert. The best MUC1c modified for immune enhancer capability has been 
selected and incorporated into the recombinant Ad5 [E1-, E2b-] platform to produce a new and 
more potent immunotherapeutic vaccine (Ad5 [E1-, E2b-]-MUC1) for treating MUC1 expressing 
cancers. 
1.2.8.1 Immunogenicity 
Studies were performed to assess immune responses generated by the Ad5 [E1-, E2b-]-MUC1 
(ETBX-061) component of a multi-targeted PSA, MUC1 and brachyury vaccine regimen. In these 
studies, groups (n = 5) of C57Bl/6 mice were used. The mice were injected SC 3 times at 2-week 
intervals with 3X1010 VP containing a 1/1/1 mixture of Ad5 [E1-, E2b-]-PSA, Ad5 [E1-, E2b-]-
MUC1, and Ad5 [E1-, E2b-]-brachyury (tri-immunization). Two weeks after the last immunization 
CMI activity was determined employing ELISpot assays for IFN-J secreting cells (SFC) after 
exposure of splenocytes to MUC1 peptide pools, as previously described.(1-3, 6, 65, 67-70, 73, 
74) As shown in Figure 9, significant CMI responses to were detected in immunized mice. Flow 
cytometry intracellular cytokine staining was performed as previously described (70, 72) on spleen 
cells after exposure to MUC1 peptides to assess the quantity of activated CD8+ T cells. As shown 
in Figure 10, IFN-J and IFN-J/TNF-D expressing polyfunctional CD8+ cells were detected in 
immunized but not control splenocytes. Testing was also performed to determine if immunization 
with the Ad5 [E1-, E2b-]-MUC1 vaccine induced MUC1 directed antibody. Using a previously 
described quantitative ELISA assay(74) with purified MUC1 glycoprotein, antibody responses to 
MUC1 were detected in sera from immunized but not control mice. (Figure 11). These studies 
indicate that the Ad5 [E1-, E2b-]-MUC1 component in the multi-targeted vaccine can induce 
MUC1 directed immune responses in immunized animals. 
 

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
25 
 
 
 
 
 
1.2.8.2 Anti-tumor Activity 
A study was conducted to test the anti-tumor capability of the Ad5 [E1-, E2b-]-MUC1 (ETBX-
061) component of the multi-targeted vaccine regimen. Immunotherapy studies were performed 
in mice with established MUC1 expressing tumors. Groups (n = 7) of C57Bl/6 mice were injected 
SC in the right flank with 5X105 MC38-MUC1 expressing murine tumor cells. After palpable 
tumors were detected, mice were treated by 3 SC injections with 1X1010 VP of Ad5 [E1-, E2b-]-
null (no transgene empty vector) or Ad5 [E1-, E2b-]-MUC1, respectively. Tumor volumes were 
calculated and tumor growth curves were plotted as previously described.(1, 6, 74) As published 
previously, numbers of 7-10 mice/group are sufficient for statistical evaluation of treatment.(1, 6, 
74) Significant anti-tumor activity was observed in MUC1 expressing tumor-bearing mice treated 
by immunotherapy with Ad5 [E1-, E2b-]-MUC1 but not in control mice injected with Ad5-null 
(empty vector). Figure 12 shows the significant anti-tumor activity and increased survival 
observed in MUC1 expressing tumor-bearing mice treated by immunotherapy. This study indicates 
 
Figure 9. MUC1 induced CMI 
responses as assessed by ELISpot 
assays for IFN-J secreting 
splenocytes (spot forming cells; 
SFC) were detected in vaccine 
immunized (grey bars) mice but 
not in control (white bars) mice 
(injected with Ad5-null empty 
vector). Specificity of the ELISpot 
assay responses was confirmed by 
lack of reactivity to irrelevant SIV-
nef or SIV-vif peptides. A positive 
control included cells exposed to 
concanavalin A (Con A, data not 
shown). Values = Mean r SEM. 
 
m e d ia
S IV n e f p o o l
M U C 1
p e p tid e p o o l
0 .0
0 .2
0 .4
0 .6
IFN J
+
(% C D 8 E
+ S p len o cytes)
m e d ia
S IV n e f p o o l
M U C 1
p e p tid e p o o l
0 .0
0 .1
0 .2
0 .3
IFN J
+ T N F D
+
(% C D 8 E
+ S p len o cytes)
 
Figure 
10. 
MUC1 
induced 
stimulation 
of 
CD8+ 
cells 
expressing IFN-J (Top) and IFN-J 
/TNF-D 
(Bottom) 
in 
mice 
immunized with vaccine (grey 
bars) but not in controls injected 
with 
Ad5-null 
(white 
bars). 
Specificity of the responses was 
confirmed by lack of reactivity to 
media alone or irrelevant SIV-nef 
peptides. Values = Mean r SEM.  
 
A d 5 [E 1 -,E 2 b -]-N u ll
A d 5 [E 1 -,E 2 b -]-M U C 1 c
0
1 0
2 0
3 0
4 0
M U C 1 A n tib o d y
n g e q u iv a le n ts o f Ig G
  
Figure 11. MUC1 directed 
antibody responses induced 
in vaccine immunized mice 
but not in control mice. 
Values = Mean r SEM.   

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
26 
 
that the Ad5 [E1-, E2b-]-MUC1 component of the vaccine regimen can induce anti-tumor activity 
against MUC1 expressing tumor cells. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.2.9 ETBX-051 Pre-clinical Studies 
ETBX-051 is a is a brachyury targeting vaccine comprising the Ad5 [E1-, E2b-] vector and a 
modified brachyury gene insert. A modified brachyury gene was incorporated into the recombinant 
Ad5 [E1-, E2b-] platform to produce an immunotherapeutic vaccine (Ad5 [E1-, E2b-]-brachyury) 
for treating brachyury expressing cancers. 
 
1.2.9.1 Immunogenicity 
Studies were performed to assess immune responses generated by the Ad5 [E1-, E2b-]-brachyury 
(ETBX-051) vaccine component of a multi-targeted PSA, MUC1, brachyury vaccine regimen. 
Groups (n = 5) of C57Bl/6 mice were used. The mice were injected SC 3 times at 2-week intervals 
 
Figure 13. Brachyury induced 
CMI responses as assessed by 
ELISpot 
assays 
for 
IFN-J 
secreting 
splenocytes 
(spot 
forming 
cells; 
SFC) 
were 
detected in immunized (grey 
bars) mice but not in control 
(white bars) mice (injected with 
Ad5-null 
empty 
vector). 
Specificity of the ELISpot assay 
responses was confirmed by lack 
of reactivity against irrelevant 
SIV-nef or SIV-vif peptides. A 
positive control included cells 
exposed to concanavalin A (Con 
A, data not shown). Values = 
Mean r SEM. 
0
5
1 0
1 5
2 0
2 5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
A d 5 [E 1 -E 2 b -]-n u ll
A d 5 [E 1 -E 2 b -]-M U C 1 c
*
*
D a y s
T u m o r v o lu m e (m m
3 )
0
5
1 0
1 5
2 0
2 5
3 0
3 5
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
A d 5 [E 1 -E 2 b -]-n u ll
A d 5 [E 1 -E 2 b -]-M U C 1 c
P < 0 .0 1
D a y s
P e rc e n t s u rv iv a l
  
Figure 12. C57Bl/6 mice were 
inoculated SC with MC38-MUC1 
expressing tumor cells in the left 
flank and administered 1010 VP of 
Ad5-null (empty vector) or 1010 VP 
of Ad5 [E1-, E2b-]-MUC1 in the 
right flank on days 0, 7, 14.  Mice 
treated with Ad5 [E1-, E2b-]-MUC1 
had significantly (P<0.05) smaller 
tumors on days 15 and 18 as 
compared to controls (top) and 
significantly 
(P<0.01) 
longer 
survival (bottom). Values = Mean r 
SEM. Experiment was terminated 
on day 36. 

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
27 
 
with 3X1010 VP containing a 1/1/1 mixture of Ad5 [E1-, E2b-]-PSA, Ad5 [E1-, E2b-]-MUC1, and 
Ad5 [E1-, E2b-]-brachyury (tri-immunization) or with 3X1010 VP Ad5 [E1-, E2b-]-null (empty 
vector controls). Two weeks after the last immunization CMI activity was determined employing 
ELISpot assays for IFN-γ secreting cells (SFC) after exposure of splenocytes to a brachyury 
peptide pool as previously described.(6) As shown in Figure 13, significant CMI responses to 
were detected in immunized mice. Flow cytometry intracellular cytokine staining was performed 
as previously described(70, 72) on spleen cells after exposure to brachyury peptides to assess the 
quantity of activated CD8+ T cells. As shown in Figure 14, IFN-γ and IFN-γ/TNF-α expressing 
polyfunctional CD8+ cells were detected in splenocytes from immunized but not control mice. 
These studies indicate that the Ad5 [E1-, E2b-]-brachyury component in the multi-targeted vaccine 
regimen can induce brachyury directed immune responses in immunized animals. 
 
 
 
 
 
1.2.9.2 Anti-tumor Activity 
A preliminary study was conducted to test the anti-tumor capability of the Ad5 [E1-, E2b-]-
brachyury (ETBX-051) component of the multi-targeted vaccine regimen. Groups (n = 7) of 
m e d ia
S IV n e f p o o l
B ra c h y u ry
p e p tid e p o o l
0 .0
0 .1
0 .2
0 .3
IFN J
+
(% C D 8 E
+ S p le n o c y te s)
m e d ia
S IV n e f p o o l
B ra c h y u ry
p e p tid e p o o l
0 .0 0
0 .0 5
0 .1 0
IFN J
+ T N F D
+
(% C D 8 E
+ S p le n o c y te s)
Figure 14. Brachyury induced 
stimulation of CD8+ cells 
expressing IFN-J (Top) and IFN-J/ 
TNF-D  (Bottom) in vaccine 
immunized (grey bars) mice but 
not in control mice injected with 
Ad5-null (white bars). Specificity 
of the responses was confirmed by 
lack of reactivity to media alone 
or irrelevant SIV-nef peptides. 
Values = Mean r SEM.  
0
5
10
15
20
25
0
200
400
600
800
1000
Ad5 [E1-,E2b-]-null
Ad5 [E1-,E2b-]-Brachyury
Days
Tumor volume mm3
Figure 15. C57Bl/6 mice were 
inoculated with MC38-brachyury 
expressing tumor cells sc in the 
left flank and administered 1010 
VP of Ad5-Null (empty vector) 
(N=4) or 1010 VP of Ad5 [E1-, 
E2b-]-brachyury (N=5) in the 
right flank on days 5, 11, and 17. 
Tumors were smaller in treated 
mice on days 15, 19, and 22. 
Values = Mean r SEM  
  

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
28 
 
C57Bl/6 mice were injected SC in the right flank with 5X105 brachyury expressing murine tumor 
cells. After palpable tumors were detected, mice were treated by 3 SC injections with 1X1010 VP 
of Ad5 [E1-, E2b-]-brachyury or with Ad5 [E1-, E2b-]-null. Tumor volumes were calculated and 
tumor growth curves were plotted as previously described.(6) Data in Figure 15 shows that 
immunotherapy of mice with Ad5 [E1-, E2b-]-brachyury resulted in smaller tumors as compared 
to control mice injected with Ad5 [E1-, E2b-]-null. This study indicates that the Ad5 [E1-, E2b-]-
brachyury component of the vaccine regimen can induce anti-tumor activity against brachyury 
expressing tumor cells. 
1.2.10 Clinical Experience 
The combination of 3 different vaccines: ETBX-071, ETBX-061, and ETBX-051 have not been 
tested in humans. This Phase I study is the first clinical study of this vaccines combination in 
subjects with mCRPC. 
1.2.11 Rationale 
The preclinical data merit a Phase I study to evaluate the safety, preliminary efficacy, and 
immunogenicity of the combination ETBX-071, ETBX-061, and ETBX-051 vaccines. Subsequent 
trials will involve the use of this vaccine in combination with checkpoint inhibitor monoclonal 
antibodies (MAbs) and other immune modulators. 
 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT 
2.1 
ELIGIBILITY CRITERIA 
2.1.1 Inclusion Criteria 
2.1.1.1 Age ≥ 18 years (male). 
2.1.1.2 Ability to understand and provide signed informed consent that fulfills Institutional 
Review Board (IRB)’s guidelines. 
2.1.1.3 Cytologically or histologically confirmed prostate cancer for which no curative standard 
approved therapy is available by either the Laboratory of Pathology at the NIH Clinical 
Center or Walter Reed National Military Medical Center at Bethesda prior to starting this 
study. If no pathologic specimen is available, patients may enroll with a pathologist’s 
report showing a histological diagnosis of prostate cancer and a clinical course consistent 
with the disease. 
2.1.1.4 Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1. 
2.1.1.5 Subjects who have received prior PSA, MUC1, and/or brachyury-targeted 
immunotherapy (e.g. vaccine) are eligible for this trial if this treatment was discontinued 
at least 3 months prior to enrollment. 
2.1.1.6 Resolution of all toxic side effects of prior chemotherapy, radiotherapy, or surgical 
procedures to NCI CTCAE Grade ≤ 1. 
2.1.1.7 Adequate hematologic function at screening, as follows: 
x Absolute neutrophil count (ANC) ≥1.0 x 109/L 
x Hemoglobin ≥ 9 g/dL 
x Platelets ≥ 75,000/microliter. 

Abbreviated Title: Ad5-based vaccines in mCRPC 
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29 
 
x Prothrombin (PT)-international normalized ratio (INR) < 1.5. 
x Partial thromboplastin time (PTT) < 1.5 x upper limit of normal (ULN). 
2.1.1.8 Adequate renal and hepatic function at screening, as follows: 
x Serum creatinine ≤ 1.5 x upper limit of normal (ULN) OR creatinine clearance (CrCl) ≥ 40 
mL/min (if using the Cockcroft-Gault formula below): 
a) Female CrCl = [(140 - age in years) x weight in kg x 0.85] / [72 x serum 
creatinine in mg/dL] 
b) Male CrCl = [(140 - age in years) x weight in kg x1.00] / [72 x serum creatinine 
in mg/dL] 
x Total bilirubin ≤ 1.5 x ULN OR in subjects with Gilbert’s syndrome, a total bilirubin ≤ 3.0 
x ULN 
x Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 2.5 x ULN, 
unless liver metastases are present, then values must be ≤ 5 x ULN) 
2.1.1.9 The effects of ETBX-051, ETBX-061 and ETBX-071 vaccines on the developing human 
fetus are unknown.  For this reason subjects must agree to use a condom and acceptable 
contraceptive method with their partner during the study and for one month after the last 
dose of vaccines. 
2.1.1.10 Ability to attend required study visits and return for adequate follow up, as required by 
this protocol. 
2.1.1.11 Castrate testosterone level (<50ng/dl or 1.7nmol /L) 
2.1.1.12 Metastatic disease documented by at least one of the following: 
x Metastatic bone disease on an imaging study, or 
x Soft tissue disease documented by CT/MRI 
2.1.1.13 Progressive disease at study entry defined as one or more of the following criteria 
occurring in the setting of castrate levels of testosterone: 
x Radiographic progression defined as any new or enlarging bone lesions or growing lymph 
node disease, consistent with prostate cancer 
OR 
x PSA progression defined by sequence of rising values separated by >1 week (2 separate 
increasing values over a minimum of 2ng/ml (PCWG2 PSA eligibility criteria). If patients 
had been on flutamide, PSA progression is documented 4 weeks or more after withdrawal. 
For patients on bicalutamide or nilutamide disease progression is documented 6 or more 
weeks after withdrawal. The requirement for a 4-6 week withdrawal period following 
discontinuation of flutamide, nilutamide or bicalutamide only applies to patients who have 
been on these drugs for at least the prior 6 months. For all other patients, they must stop 
bicalutamide, nilutamide or flutamide the day prior to enrollment. 
2.1.1.14 Patients must agree to continue to continuation of androgen deprivation therapy (ADT) 
with a gonadotropin-releasing hormone analogue/antagonist or bilateral orchiectomy. 
2.1.1.15 Prior treatment with immunotherapy, hormonal therapy, radium 223, chemotherapy 

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
30 
 
and/or other experimental therapy is allowed. 
2.1.2 Exclusion Criteria 
2.1.2.1 Treatment with an investigational drug study within 28 days of before starting on study 
treatment. 
2.1.2.2 Subjects with concurrent cytotoxic chemotherapy or radiation therapy. There must be at 
least 28 days between any other prior chemotherapy (or radiotherapy) and study 
treatment. Prior antibody therapy must be discontinued 8 weeks prior to start of study 
treatment. Prior hormonal therapy can be discontinued 24 hours prior to start of study 
treatment. 
2.1.2.3 Any prior PSA, MUC1, and/or brachyury-targeted immunotherapy (e.g., vaccine) must 
have been discontinued at least 12 weeks before initiation of study treatment. Subjects 
must have recovered from all acute toxicities from prior treatment prior to screening for 
this study. 
2.1.2.4 Prior treatment with Adenovirus-Based vectors immunotherapy 
2.1.2.5 Known active brain or central nervous system metastasis, or seizures requiring 
anticonvulsant treatment, cerebrovascular accident, or transient ischemic attack (< 6 
months prior to enrollment). 
2.1.2.6 Subjects with a history of autoimmune disease (active or past), such as but not restricted 
to inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, 
scleroderma, or multiple sclerosis. Autoimmune-related thyroid disease, type I diabetes 
and vitiligo are permitted if the condition is well controlled. 
2.1.2.7 Subjects with serious intercurrent chronic or acute illness, such as cardiac or pulmonary 
disease, hepatic disease, or other illness considered by the Investigator as high risk for 
investigational drug treatment. 
2.1.2.8 Subjects with a history of heart disease, such as congestive heart failure (class II, III, or 
IV defined by the New York Heart Association functional classification), history of 
unstable or poorly controlled angina, or history (< 1 year prior to enrollment) of 
ventricular arrhythmia. 
2.1.2.9 Subjects with a medical or psychological impediment that would impair the ability of the 
subject to receive therapy per protocol or impact ability to comply with the protocol or 
protocol-required visits and procedures. 
2.1.2.10 Presence of a known active acute or chronic infection, including human 
immunodeficiency virus (HIV, as determined by enzyme-linked immunosorbent assay 
[ELISA] and confirmed by western blot) and hepatitis B and hepatitis C virus 
(HBV/HCV, as determined by HBsAg and hepatitis C serology). 
2.1.2.11 Subjects on systemic intravenous or oral steroid therapy (or other immunosuppressive, 
such as azathioprine or cyclosporin A) are excluded on the basis of potential immune 
suppression. Subjects must have had at least 6 weeks of discontinuation of any steroid 
therapy (except that used as premedication for chemotherapy or contrast-enhanced 
studies) prior to enrollment. Physiologic (replacement) doses od steroids as well as nasal, 
topical or inhaled steroids are allowed. 
2.1.2.12 Subjects with known allergy or hypersensitivity to any component of the investigational 

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
31 
 
product will be excluded. 
2.1.2.13 Subjects with acute or chronic skin disorders that will interfere with injection into the 
skin of the extremities or subsequent assessment of potential skin reactions will be 
excluded. 
2.1.2.14 Subjects vaccinated with a live (attenuated) vaccine (e.g., FluMist®) or a killed 
(inactivated)/subunit vaccine (e.g., PNEUMOVAX®, Fluzone®) within 28 days or 
14 days, respectively, of the first planned dose of ETBX vaccine. 
2.1.2.15 Patients with second malignancy within 3 years of enrollment; Patients curatively treated 
non-melanoma skin cancers or carcinoma in situ of the bladder, are not excluded. 
2.1.2.16 Use of herbal products that may decrease PSA levels (e.g. saw palmetto) 
2.1.2.17 Patients who have received radiation therapy, radionuclide therapy or undergone surgery 
within certain duration (4 weeks) of enrollment 
2.1.3 Recruitment Strategies 
This 
study 
will 
be 
listed 
on 
available 
websites 
(www.clinicaltrials.gov, 
https://ccr.cancer.gov/clinical-trials-search-start) and participants will be recruited from the 
current patient population at NIH. 
2.2 
SCREENING EVALUATION 
Confirmation of diagnosis by NCI Laboratory of Pathology or Walter Reed National Military 
Medical Center (at any time prior to enrollment). If no tissue is available, a pathologist’s report 
documenting histological diagnosis of prostate cancer along with a clinical course consistent with 
the disease may be used. 
 
Studies should be done within 28 days prior to enrollment unless otherwise noted below. 
x History and physical exam including ECOG performance status, height, weight and 
vital signs 
x 12-lead EKG 
x Scans 
o CT CAP (chest abdomen and pelvis) with oral and IV contrast (MRI 
abdomen/pelvis with contrast + chest CT without contrast if IV contrast is 
contraindicated or CT CAP is inadequate) 
o Bone scan 
x Clinical laboratory tests (within 16 days prior to enrollment) 
o Hepatic Panel (Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, 
Direct Bilirubin) 
o Acute Care Panel (Sodium [NA], Potassium [K], Chloride [CL] Total CO2 
[Bicarbonate], Creatinine, Glucose, Urea nitrogen, eGFR) 
o CBC with differential 
o Urinalysis (24-hour collection will be done as needed) 
o Serum PSA 
o Testosterone level 
o PT/INR, PTT 

Abbreviated Title: Ad5-based vaccines in mCRPC 
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32 
 
x HBV (HBsAg), HCV (anti-HCV), HIV (anti-HIV) screening (within 3 months prior to 
enrollment) 
x Appropriate tumor imaging and assessment. All baseline tumor measurements should 
be performed based on the subject’s qualifying scan obtained within 28 days prior to 
the start of treatment. 
2.3 
REGISTRATION PROCEDURES 
Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) 
within 24 hours of signing consent.  A registration Eligibility Checklist from the web site 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and sent via 
encrypted email to:  NCI Central Registration Office ncicentralregistration-l@mail.nih.gov.  After 
confirmation of eligibility at Central Registration Office, CRO staff will call pharmacy to advise 
them of the acceptance of the patient on the protocol prior to the release of any investigational 
agents.  Verification of Registration will be forwarded electronically via e-mail to the research 
team. A recorder is available during non-working hours. 
2.3.1 Treatment Assignment Procedure 
Cohorts 
Number 
Name 
Description 
1 
Dose De-Escalation 
Subjects enrolled to dose de-escalation cohorts 
2 
Dose Expansion 
Subjects enrolled at the MTD after the MTD is established 
Arms 
Number 
Name 
Description 
1 
Dose De-Escalation 
ETBX-071; adenoviral PSA vaccine + ETBX-061; adenoviral 
MUC1 vaccine + ETBX-051; adenoviral brachyury vaccine 
dose de-escalation 
2 
Dose Expansion 
ETBX-071; adenoviral PSA vaccine + ETBX-061; adenoviral 
MUC1 vaccine + ETBX-051; adenoviral brachyury vaccine 
Stratifications 
None 
 
Randomization and Arm Assignment 
Not a randomized study.  Subjects in Cohort 1 will be assigned to Arm 1; subjects in Cohort 2 will 
be assigned to Arm 2. 
2.4 
BASELINE EVALUATION 
All subjects are required to complete baseline evaluations within 1 day prior to the first planned 
dosing of the study drug (Any screening evaluation done on D-1 of treatment can also serve for 

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
33 
 
the baseline evaluation). The following procedures and evaluations will be performed and 
documented in the subject’s source records. 
x History and physical exam, including weight, ECOG status and vital signs 
x Hepatic Panel (Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct 
Bilirubin) 
x Acute Care Panel (Sodium [NA], Potassium [K], Chloride [CL] Total CO2 [Bicarbonate], 
Creatinine, Glucose, Urea nitrogen, eGFR) 
x CBC with differential 
x PSA and testosterone 
If these assessments have been previously performed within 24 hours prior to the first dosing, a 
second assessment at baseline/week 0 can be omitted. 
3 
STUDY IMPLEMENTATION 
3.1 
STUDY DESIGN 
This is a Phase I trial in subjects to test the safety of 3 different adenovirus based vaccines (ETBX-
051, ETBX-61, ETBX-71) administered to patients with metastatic castration resistant prostate 
cancer at the same time. All vaccines utilize the same second generation E1(-), E2 (-) vector. 
 
ETBX-071 is a PSA-targeting vaccine that comprises the Ad5 [E1-, E2b-] vector containing a PSA 
gene insert. The investigational product ETBX-061 is a MUC1-targeting vaccine that comprises 
the Ad5 [E1-, E2b-] vector containing a modified MUC1 (MUC1c) gene insert. The investigational 
product ETBX-051 is a brachyury-targeting vaccine that comprises the Ad5 [E1-, E2b-] vector 
containing a modified brachyury gene insert. 
Table 2: Investigational Products 
Table 2a 
Product Name(s): 
ETBX-071 (Ad5 [E1-, E2b-]-PSA Vaccine) 
 
Dosage Form: 
Suspension for injection 
Dose 
5 x 1011 VP (standard dose), 1 x 1011 VP (DL-1), or 5 x 1010 VP (DL-2). 
Route of Administration 
SC injection 
Physical Description 
ETBX vaccine is supplied as a sterile, clear solution in a 2-mL single-
dose vial. The vaccine is provided at a concentration of 5 x 1011 VP per 
1 mL and contains ARM formulation buffer (20 mM TRIS, 25 mM 
NaCl, 2.5% glycerol, pH 8.0). Each vial contains approximately 1.1 mL 
of the vaccine. The product should be stored at ≤ -20°C. 
Manufacturer 
Etubics 
Table 2b 

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
34 
 
Product Name(s): 
ETBX-061 (Ad5 [E1-, E2b-]-MUC1 Vaccine) 
Dosage Form: 
Suspension for injection 
Dose 
5 x 1011 VP (standard dose), 1 x 1011 VP (DL-1), or 5 x 1010 VP (DL-2). 
Route of Administration 
SC injection 
Physical Description 
ETBX vaccine is supplied as a sterile, clear solution in a 2-mL single-
dose vial. The vaccine is provided at a concentration of 5 x 1011 VP per 
1 mL and contains ARM formulation buffer (20 mM TRIS, 25 mM 
NaCl, 2.5% glycerol, pH 8.0). Each vial contains approximately 1.1 mL 
of the vaccine. The product should be stored at ≤ -20°C. 
Manufacturer 
Etubics 
Table 2c 
Product Name(s): 
ETBX-051 (Ad5 [E1-, E2b-]-Brachyury Vaccine) 
Dosage Form: 
Suspension for injection 
Dose 
5 x 1011 VP (standard dose), 1 x 1011 VP (DL-1), or 5 x 1010 VP (DL-2). 
Route of Administration 
SC injection 
Physical Description 
ETBX vaccine is supplied as a sterile, clear solution in a 2-mL single-
dose vial. The vaccine is provided at a concentration of 5 x 1011 VP per 
1 mL and contains ARM formulation buffer (20 mM TRIS, 25 mM 
NaCl, 2.5% glycerol, pH 8.0). Each vial contains approximately 1.1 mL 
of the vaccine. The product should be stored at ≤ -20°C. 
Manufacturer 
Etubics 
 
 
The vaccines will be administered subcutaneously (SC) in 3 separate injections every 3 weeks for 
3 immunizations (there are no booster injections in dose escalation cohort). It is preferred to 
administer the injections in the anterolateral section of the same thigh separated from each other 
by a minimum of 5 cm. It is also preferred to administer ETBX-071 most proximal, ETBX-061 in 
the middle, and ETBX-051 most distal and the injected thigh should be alternated each treatment 
visit when possible. In the dose expansion cohort, ETBX-051, ETBX-61, ETBX-71 vaccines will 
be administered on Weeks 0, 3, and 6 for a total of three injections followed by booster injections  
every 8 weeks for 1 year. 
3.1.1 Dose Limiting Toxicity 
A dose limiting toxicity (DLT) is defined as occurring within 28 days after the first vaccine 
administration and meeting any of the below criteria: 

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x Any Grade 3 or greater toxicity as defined by National Cancer Institute (NCI) Common 
Terminology Criteria for Adverse Events (CTCAE) Version 5 that is possibly, 
probably, or definitely related to the vacines with the exception of transient (≤ 24 hours) 
Grade 3 flu-like symptoms or fever, which is controlled with medical management or 
transient (≤ 24 hours), Grade 3 fatigue, local skin reactions or rash, headache, nausea, 
emesis that resolves to Grade ≤ 1 or laboratory abnormalities that are not associated 
with organ pathology. 
x Any Grade 2 or higher autoimmune reaction (except endocrine-related immune 
toxicity) or immediate hypersensitivity reaction. Any grade 3 autoimmune endocrine-
related toxicity that has not resolved clinically within 7 days of initiating therapy will 
also be defined as a DLT. 
x Generalized erythroderma or macular or papular rash lasting less than 7 days and not 
associated with desquamation will not be DLTs. 
x For purposes of dose de-escalation the DLT evaluation period will be for 1 week 
following the first dose of drug. 
x Subjects experiencing a DLT will be removed from protocol therapy. 
3.1.2 Dose De-Escalation 
Up to six patients will be enrolled at dose level 1.  If ≤1 of 6 patients experience a DLT, then 
initiation of the dose expansion phase will occur.  If ≥2 of 3 or 6 experience DLT at the initial dose 
level, then dose de-escalation will occur. Up to six patients will be enrolled at the lower dose level 
(-1) (1x1011 VP).  If ≤1 of 6 patients experience a DLT, then the maximum tolerated (MTD) will 
be declared at this dose, and initiation of the dose expansion phase will occur.  If ≥2 of 3 or 6 
experience DLT at dose level -1, then a further dose de-escalation will occur. Up to six patients 
will be enrolled at the lower dose level (-2) (5x1010 VP).  If ≤1 of 6 patients experience a DLT, 
then the maximum tolerated (MTD) will be declared at this dose, and initiation of the dose 
expansion phase will occur.  If ≥2 of 3 or 6 experience DLT dose level -2, then the study will be 
stopped. 
In the second part, dose expansion will occur when the MTD has been determined. An additional 
12 subjects will be enrolled in the dose expansion component of the trial, for a total of 18 subjects 
treated at the MTD. 
A schematic overview of the study is shown in Figure 16. 
In the initial component of the study, 3 to 6 subjects will be sequentially enrolled starting at the 
standard dose at dose level 1. During enrollment at each dose level, there will be a minimum of 7 
days between enrolling successive subjects. DLTs will be monitored continuously. 
Dose levels are shown in Table 3. No intra-patient dose escalations are permitted. 
 

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Table 3 Dose Levels 
Dose level (DL) 
ETBX VP 
Standard (Dose level 1) 5X1011 
-1 
1X1011 
-2 
5X1010 
 
Standard dose DL 1 (5 x 1011 VP): 
x If ≤ 1 of the initial 6 subjects experience a DLT, then dose expansion will commence. 
x If ≥ 2 of the initial 3 subjects or ≥ 2 of the 6 total subjects experience a DLT, enrollment 
at DL -1 will commence. 
 
Dose de-escalation DL -1 (1 x 1011 VP): 
x If ≤ 1 of the initial 6 subjects experience a DLT, this dose level will be defined as the 
MTD and dose expansion will commence. 
x If ≥ 2 of the initial 3 subjects or ≥ 2 of the 6 total subjects experience a DLT, enrollment 
at DL -2 will commence. 
 
Dose de-escalation DL-2 (5 x 1010 VP): 
x If ≤ 1 of 6 subjects experience a DLT, this dose level will be defined as the MTD and 
dose expansion will commence. 
x If ≥ 2  of the initial 3 subjects, or if ≥ 2 of a total 6 subjects at DL -2 experience a DLT, 
dosing will be suspended, and the study will be re-evaluated. 

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Figure 16: Study Design and Treatment Schema 
 
 
 
 
 
 








 
 
 
 
 
 
 
 
Standard 
Dose Level  
N=3-6 
(5x10
11VP) 
If ≥ 2 of initial 3 subjects, or 
≥ 2 of the 6 total subjects at 
standard dose experience 
DLT, de-escalate to dose 
level -1 
If ≤ 1 of 6 subjects at 
standard dose experience 
DLT,  
proceed to dose expansion 
If ≥ 2 of initial 3 subjects, or 
≥ 2 of the 6 total subjects at 
dose level -1 experience 
DLT, de-escalate to dose 
level -2 
If ≥ 2 of initial 3 subjects, or 
≥ 2 of the 6 total subjects at 
dose level -2 experience 
DLT, stop study 
If ≤ 1 of 6 subjects at dose 
level -1 experience DLT,  
proceed to dose expansion 
If ≤ 1 of 6 subjects at dose 
level -2 experience DLT,  
proceed to dose expansion 
X 
Dose   
Level -2  
N=3-6 
(5x10
10VP) 
 
Dose   
Level -1  
N=3-6 
(1x10
11VP) 
 

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3.2 
ETBX VACCINE DOSE PREPARATION 
The dose of each ETBX vaccine to be injected is 5 x 1011 VP per 1 mL.  Dose levels of 1 x 1011 
VP per mL (dose level -1), or 5 x 1010 VP per mL (dose level -2) are available for de-escalation.  
Prior to injection, the appropriate vial should be removed from the freezer and allowed to thaw at 
controlled room temperature (20–25°C, 68-77°F) for at least 20 minutes and not more than 30 
minutes, after which it should be kept at 2-8°C (35–46°F). 
Each vial is sealed with a rubber stopper and has a white flip-off seal. The end user of the product 
will need to flip the white plastic portion of the cap up/off with their thumb to expose the rubber 
stopper and then puncture the stopper with an injection needle to withdraw the liquid. The rubber 
stopper is secured to the vial with an aluminum-crimped seal. 
The thawed vial should be swirled and then, using aseptic technique, the pharmacist or pharmacist 
designee should withdraw the appropriate volume from the appropriate vial using an appropriately 
sized syringe. 
The vaccine dose should be injected as soon as possible using a 1 to 1/2 inch, 20 to 25-gauge 
needle. If the vaccine cannot be injected within 1 hour, the syringe should be returned to the 
pharmacy and properly disposed in accordance with institutional policy and procedure, and 
disposition must be recorded on the investigational product accountability record. 
Note that storage of the vaccine in the vial at 2–8°C (35–46°F) must not exceed 8 hours. Also, 
once the vaccine has been thawed, it must not be refrozen, and, if not used, it should be disposed 
of as described above. 
3.2.1 Instructions for Dose Preparation – 5 x 1011 Virus Particles 
Withdraw 1 mL of contents from the vial, prepare the injection site with alcohol, and administer 
to the subject by SC injection in the thigh without any further manipulation (refer to the vial label 
for a description of the vialed ETBX vaccine concentration). 
3.2.2 Instructions for Dose Preparation – 1 x 1011 Virus Particles 
Withdraw 0.2 mL of contents from the vial, prepare the injection site with alcohol, and administer 
to the subject by SC injection in the thigh without any further manipulation (refer to the vial label 
for a description of the vialed ETBX vaccine concentration). 
3.2.3 Instructions for Dose Preparation – 5 x 1010 Virus Particles (For Dose De-escalation) 
From a 5.0-mL vial of 0.9% sodium chloride for injection, remove 0.5 mL of fluid using a 1.0 mL 
tuberculin syringe, leaving 4.50 mL. Then, using another 1.0 mL tuberculin syringe, remove 0.5 
mL from the vial labeled ETBX-071, ETBX-061, or ETBX-051, respectively, and deliver this 
volume into the 4.50 mL of sterile saline remaining in the 5-mL sterile saline vial. Mix the contents 
by inverting the 5 mL of diluted ETBX vaccine. Then withdraw 1 mL of the diluted ETBX vaccine, 
prepare the injection site with alcohol, and administer to the subject by SC injection in the thigh 
(refer to the vial label for a description of the vialed ETBX vaccine concentration). Alternatively, 
4.5 mL of 0.9% sodium chloride for injection may be added to an appropriately sized sterile empty 
vial (e.g. 10 mL) using an appropriately sized syringe. Then, using a new syringe, remove 0.5 mL 
from the vial labeled ETBX-071, ETBX-061, or ETBX-051, respectively and deliver this volume 
into the sterile vial containing the 4.5 mL 0.9% sodium chloride for injection. Mix the contents by 

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inverting the 5 mL of diluted ETBX vaccine. Then withdraw 1 mL of the diluted ETBX vaccine, 
prepare the injection site with alcohol, and administer to the subject by subcutaneous injection in 
the thigh (refer to the vial label for a description of the vialed ETBX vaccine concentration). 
3.2.4 Administration 
ETBX-051, ETBX-61 and ETBX-71  vaccines will be administered on Weeks 0, 3, and 6 for a 
total of three injections. In dose expansion, ETBX-051, ETBX-61 and ETBX-71 vaccines will be 
administered on Weeks 0, 3, and 6 for a total of three injections followed by booster injections at 
8-week intervals for up to 1 year. All study drug administration treatments should occur within ± 
7 days of the planned visit date. Treatment may be shorter for patients who experience progressive 
disease (unequivocal or confirmed) or unacceptable toxicity, withdraw consent, or if the 
Investigator feels it is no longer in their best interest to continue treatment. 
 
The vaccines will be administered subcutaneously as 3 separate injections preferably in the 
anterolateral section of the same thigh separated from each other by a minimum of 5 cm. It is also 
preferred to administer ETBX-071 most proximal, ETBX-061 in the middle, and ETBX-051 most 
distal and the injected thigh should be alternated each treatment visit when possible. 
3.2.5 Other Considerations 
The Ad5 [E1-, E2b-] vector is non-replicating and its genome does not integrate into the human 
genome. However, since this is a non-replicating recombinant virus, we recommend that it be 
handled under Biosafety Level-2 conditions. Any vialed ETBX vaccine material that has been used 
in the study should be autoclaved or incinerated after use according to institutional policy and 
according to local, state, and federal regulations.  Refer to the Material Safety Data Sheets for 
additional handling instructions. 
3.3 
DOSE MODIFICATIONS AND DELAYS 
3.3.1 Dosing Delay (applies to events occurring outside of the DLT evaluation period) 
Patients must have recovered to ≤ grade 2 for injection site reaction or grade ≤ 1 for  any other 
toxicity related to the vaccines for the parameters used to assess levels of organ function required 
for eligibility (see Section 2) after each vaccination in order to receive a subsequent vaccination 
(see Sections 2.1.1.7 and 2.1.1.8). 
If ≥ grade 3 nonautoimmune toxicity attributable to the vaccines persists for > 42 days, the patient 
will not receive further vaccine inoculations and will be removed from the protocol treatment and 
followed for resolution of toxicity and immune/survival endpoints. 
Patients who develop grade 3 injection site reactions will have their vaccines held until injection 
site reaction resolves to grade 2 or less. Patients will receive a diary card to record injections site 
reactions (see Appendix B). 
Patients who develop ≥ a grade 2 allergic or autoimmune disease that threatens vital organ function 
or any ≥ grade 3 autoimmunity, not related to a therapeutic response, will be removed from the 
protocol treatment and followed for resolution of toxicity and immune/survival endpoints. 
Patients who develop any grade 4 toxicity attributable to the vaccines will be removed from the 
protocol treatment and followed for resolution of toxicity and immune/survival endpoints. 

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For unrelated acute illnesses present at the time of a scheduled vaccination, dosing can be delayed 
until symptoms subside, or the subject may be withdrawn at the discretion of the Investigator; 
delays up to 4 weeks are considered acceptable in this setting. 
If a scheduled vaccine dose is missed due to scheduling or logistical issues, the vaccine may be 
given within 7 days of the appointed time. 
3.3.2 Dose Modifications 
No dose modifications are allowed with these vaccines except for dose cohort de-escalation as 
described in section 3.1.

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3.4 
STUDY CALENDAR 
Assessment 
Screening l 
Treatment 
p 
(Every 3-Week Dosing) 
Bi-monthly Boosters (every 
8 weeks – only expansion 
cohort) 
p 
 
End 
of 
Treatment 
(within 
90 
days after the 
last vaccine) k 
Post 
Study 
Therapy Follow 
Up 
Study Week 
Day 
-28 to -1 
Baseline/
Wk-0 
Wk-3 
Wk-6 
Week 14, 22, 30, 38, 46, 54 
 
 
Clinic Visit 
X 
X 
X 
X 
X 
X 
 
Informed Consent0 
X 
 
 
 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Physical Examination, 
Heighta, Weight, ECOG 
X 
Xb 
X 
X 
X 
X 
 
Medical Historyc 
X 
X 
 
 
 
 
 
Concomitant 
Medications 
X 
X 
X 
X 
X 
X 
 
Vital Signsd 
X 
X 
X 
X 
X 
X 
 
12-Lead ECG 
X 
 
 
 
X 
X 
 
Confirm Contraceptive 
Measurese 
X 
 
 
 
 
 
 
Study Drug Injection/ 
Injection Site Reaction 
Monitoringf 
 
X 
X 
X 
X 
 
 

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Assessment 
Screening l 
Treatment 
p 
(Every 3-Week Dosing) 
Bi-monthly Boosters (every 
8 weeks – only expansion 
cohort) 
p 
 
End 
of 
Treatment 
(within 
90 
days after the 
last vaccine) k 
Post 
Study 
Therapy Follow 
Up 
Study Week 
Day 
-28 to -1 
Baseline/
Wk-0 
Wk-3 
Wk-6 
Week 14, 22, 30, 38, 46, 54 
 
 
Dispensation of Subject 
Diary Cardg 
 
X 
 
 
 
 
 
Review of Subject Diary 
Cardg 
 
 
X 
 
 
 
 
Tumor Imaging/CT scan 
and bone scanh 
X 
 
 
 
X 
 
 
Urinalysis 
Xl 
 
 
 
 
X 
 
Chemistry Panel m 
Xl 
Xb 
X 
X 
X 
X 
 
CBC, Differential, 
Platelets 
Xl 
Xb 
X 
X 
X 
X 
 
Serum PSA and 
testosterone level 
Xl 
X 
X 
X 
X 
X 
 
PT/INR, PTTT 
Xl 
 
 
 
 
X 
 
Serum Virology (HIV, 
HBV, HCV)i 
X 
 
 
 
 
 
 
Adverse Events 
 
X 
X 
X 
X 
X 
 
Exploratory Immune 
Analysis 
 
X 
 
X 
X (only week 14 and 30) 
X 
 
Telephone Follow Upj 
 
 
 
 
 
 
X 

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Assessment 
Screening l 
Treatment 
p 
(Every 3-Week Dosing) 
Bi-monthly Boosters (every 
8 weeks – only expansion 
cohort) 
p 
 
End 
of 
Treatment 
(within 
90 
days after the 
last vaccine) k 
Post 
Study 
Therapy Follow 
Up 
Study Week 
Day 
-28 to -1 
Baseline/
Wk-0 
Wk-3 
Wk-6 
Week 14, 22, 30, 38, 46, 54 
 
 
Confirmation of 
diagnosis (path report) n 
X 
 
 
 
 
 
 
 
0Informed consent may be obtained on D0 after eligibility is confirmed and other screening protocols may be used to obtain screening labs and 
procedures as per NCI’s internal SOP 
a Height will only be assessed at screening. 
b If the assessment was performed within 24 hours prior to the first dosing, a second assessment at baseline/week 0 can be omitted. 
c Complete medical history will be evaluated at screening and includes current and past cardiac and pulmonary history, documentation of diagnosis 
including history of current and prior cancers, prior treatment(s), and prior radiologic studies. Any new events in the medical history will be evaluated 
at baseline. 
d Vital signs include temperature, heart rate, blood pressure, and respiratory rate. Vital sign assessments are to be obtained after the subject has been 
in a seated resting position for at least 5 minutes. For the first injection, vital signs must be assessed 30 and 60 minutes after the injection. Vital signs 
must be assessed 30 minutes after the subsequent injections. 
e Contraceptive measures are described in 2.1.1.9. 
f Injection site reactions will be monitored. 
g Subjects will be given a diary card for the self-evaluation and reporting of injection site reactions, see Section Appendix B. Local injection site 
reactions are expected to occur over several days after vaccination. Erythema and some soreness are possible. Injection site reactions will be 
monitored by clinic staff after each dose prior to discharge and by subject self-evaluation and reporting. Subjects will be given a diary card with 
reporting fields for diameter of erythema and duration (see Appendix B).  In addition, pain at the injection site, fever, and chills will be collected as 
Yes/No and medications (over-the-counter ibuprofen, acetaminophen, aspirin, etc.) taken for injection site pain or discomfort as Yes/No. The site 
staff will review the diary cards with the subject during the next clinic visit and record responses in the case report form. 
h All baseline tumor measurements should be performed based on the subject’s qualifying scan obtained within 28 days prior to the start of treatment. 
Tumor imaging and assessments will be performed as described in Section 6.3. Tumor imaging may be performed +/-5 days from the next treatment 
for logistical and scheduling purposes. 
i Serum virology test for HIV (as determined by ELISA and confirmed by western blot), and HBV and HCV (as determined by HBsAg and hepatitis 
C serology) can be performed up to 12 weeks prior to enrollment. 

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j After the subject completes or withdraws from study therapy, the study team will contact the subject approximately every 3 months for 12 months 
and then approximately every 6 months every 6 months for 24 months and then every 12 months thereafter for another 24 months to collect follow-
up information, including survival status and any current cancer treatment regimen. 
k If the patient cannot return to the Clinical Center within 90 days after the last vaccine, the patient will be contacted by phone to AEs and laboratory 
assessments will be deferred until a later point if at feasible. 
l Should be performed within 16 days prior to enrollment. 
m Chemistry include acute care panel (sodium, potassium, chloride, bicarbonate, creatinine, glucose, BUN, eGFR) and hepatic panel (alkaline 
phosphatase, ALT, AST, Total Bilirubin, Direct Bilirubin. Blood work may be obtained +/-5 days from the next treatment day for logistical and 
scheduling purposes  
n Confirmation of diagnosis by NCI Laboratory of Pathology or Walter Reed National Military Medical Center can be done at any time prior to 
enrollment. 
p Initial cohort: Treatment every 3 weeks (21 days: – 3 days up to + 3 days) intervals for 3 doses. Expansion cohort: Treatment every 3 weeks (21 
days: – 3 days up to + 3 days) intervals for 3 doses then every 8 weeks (56 days: – 3 days up to + 8 days) intervals thereafter (treatment may be 
delayed up to 8 days for special circumstances like severe weather, travel delays or patient event/vacation).

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3.5 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA 
Regardless of reason for removal from study therapy, patients will be asked to have a 90 day follow 
up safety visit after the last vaccine. Patients who refuse to return for this visit will be asked to 
review any safety concerns by phone within this time period. 
3.5.1 Criteria for removal from protocol therapy 
x Completion of protocol therapy. 
x Clinical or radiographic progression of disease (not including PSA progression). Patients 
with serological progression alone will remain on treatment at the discretion of the PI. 
x Unacceptable Toxicity as listed in section 3.3.1 
x Any serious adverse event that is unexpected relative to the known safety profile of the 
investigational agents in the opinion of the investigator 
x Participant requests to be withdrawn from active therapy 
x Investigator discretion 
 
3.5.2 Off-Study Criteria 
x Completion of study follow up period 
x PI decides to end study 
x Participant requests to be withdrawn from study. Reasons for withdrawal will be 
documented. 
x Noncompliance with protocol guidelines (patient removed at discretion of Principal 
Investigator) 
x Lost to follow-up 
x Death 
 
3.5.3 Off Protocol Therapy and Off-Study Procedure 
Authorized staff must notify Central Registration Office (CRO) when a subject is taken off 
protocol therapy and when a subject is taken off-study. A Participant Status Updates Form from 
the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be 
completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-
l@mail.nih.gov. 
4 
CONCOMITANT MEDICATIONS/MEASURES 
Subjects must inform the investigators of the current or planned use of all other medications during 
the study (including prescription medications, vitamins, herbal and nutritional supplements, and 
over-the-counter medications). 
For the administration of the ETBX vaccines, antiemetics, stool softeners, and antidiarrheal agents 
may be administered as required, but are not anticipated to be needed and should not be used 
prophylactically on the first cycle. The selection of the specific antiemetic regimen is at the 
discretion of the treating physician. Antiemetic regimens will not include steroids. 

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Other supportive care with blood components, antibiotics, analgesics, general medical therapy, 
etc., will be delivered as required. Any patients taking antibiotics for infection must complete that 
course of therapy and be free of evidence of further infection before receiving any dose of vaccine. 
Use of prophylactic antibiotics is allowed. 
Concurrent systemic corticosteroid use (daily or every other day for continued use > 14 days) 
should be avoided within 28 days before the first planned dose of ETBX vaccines. Use of inhaled 
steroids, nasal sprays, and topical creams for small body areas is allowed. Use of physiological 
doses of steroids for replacement therapy (up to 10 mg prednisone/day or equivalent) is permitted. 
Use of steroids as premedication for imaging studies is also allowed. 
Symptomatic anemia should be treated with appropriate red blood cell or erythropoietin support. 
Thrombocytopenia should be treated conservatively. In the absence of bleeding or a planned 
invasive procedure, platelet transfusions should be given for a platelet count below 10,000/mm3. 
If invasive procedures are planned or the patient develops bleeding, platelet transfusions should 
be administered in accordance with the standard of practice, usually maintaining a platelet count 
of > 50,000/mm3. 
4.1 
CONCURRENT MEDICATIONS/INTERVENTIONS 
4.1.1 Anticancer Therapy 
If a subject requires additional systemic anticancer treatment, study treatment must be 
discontinued. Local intervention is discouraged unless medically unavoidable. Subjects receiving 
local intervention (e.g., palliative radiation) are allowed to continue to receive study treatment at 
the investigator’s discretion. 
4.1.2 Other Medications 
Subjects must be instructed to inform the investigators of the current or planned use or all other 
medications during the study (including prescription medications, over-the-counter medications, 
vitamins and herbal and nutritional supplements). It is the responsibility of the investigator to 
ensure that details regarding all medications are documented. Bisphosphonates started prior to 
screening activities or initiated during the course of the study to control bone pain may be used 
with caution. 
Colony stimulating factors (e.g., erythropoietin and granulocyte colony-stimulating factors) 
administered as dictated by safety purposes are acceptable while the subject is enrolled on study. 
Pain medications administered as dictated by standard practice are acceptable while the patient is 
enrolled on the study. 
No concurrent investigational agents are permitted. 
 
5 
BIOSPECIMEN COLLECTION 
5.1 
CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES 
5.1.1 Immunological Studies 
Samples from patients enrolled at the Clinical Center will be available for immunologic correlative 
studies as described below. 

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5.1.1.1 Peripheral blood 
Five (10mL) green top sodium heparin tubes for PBMC isolation and 1 (8mL) red top SST tube 
for serum samples will be drawn at baseline (week 0), week 6, week 14, week 30 and at the end of 
study treatment at bi-monthly injection days, and at end of study as described in section 3.4.  The 
following assessments are planned in the Laboratory of Tumor Immunology and Biology (LTIB), 
CCR, NCI for the samples collected.  Please see section 5.1.1.3 for sample processing information. 
 
5.1.1.1.1 Analyses of PBMCs: 
Pre- and post-therapy PBMCs, separated by Ficoll-Hypaque density gradient separation, will be 
analyzed for antigen-specific immune responses using an intracellular cytokine staining assay. 
PBMCs will be stimulated in vitro with overlapping 15-mer peptide pools encoding the tumor-
associated antigens PSA, MUC-1, and Brachyury. Control peptide pools will involve the use of 
human leukocyte antigen (HLA) peptide as a negative control and CEFT peptide mix as a positive 
control. CEFT is a mixture of peptides of CMV, Epstein-Barr virus, influenza, and tetanus toxin. 
Post-stimulation analyses of CD4 and CD8 T cells will involve the production of IFN-γ, IL-2, 
tumor necrosis factor, and CD107a. The absolute number of CD4+ or CD8+ T lymphocytes 
producing cytokine or positive for CD107a will be calculated per 1 × 106 cells plated at the start 
of the IVS. The background signal (obtained with the negative control peptide pool) and any value 
obtained prior to vaccination, will be subtracted from those obtained after vaccination ([post-
TAA − post-HLA] − [pre-TAA − pre-HLA]). An antigen specific immune response will be scored 
as positive if a patient has more than 250 CD4+ or CD8+ T cells that produce IFNg, TNF, IL-2, or 
are positive for CD107a at the end of the stimulation assay per 1x106 cells that are plated at the 
start of the assay.  A detailed description of this assay has been previously reported(55).  If 
sufficient PBMCs are available, assays may also be performed for the development of T cells to 
other tumor-associated antigens. 
 
If sufficient PBMCs are available, PBMCs from selected subjects may be analyzed for changes in 
standard immune cell types (CD4 and CD8 T cells, natural killer [NK] cells, regulatory T cells 
[Tregs], myeloid-derived suppressor cells [MDSCs], and dendritic cells) as well as 123 immune 
cell subsets, as described elsewhere(75), and for function of specific immune cell subsets, 
including CD4 and CD8 T cells, NK cells, Tregs, and MDSCs. 
 
5.1.1.1.2 Analyses of antibodies and soluble factors: 
Ad5 Neutralizing antibody titers will be determined as previously described(2, 3).  Dilutions of 
heat-inactivated test sera will be mixed with Ad5 [E1-]-null viral particles and then added to 
HEK293 cells.  An MTS tetrazolium bioreduction assay will be used to measure Ad5 neutralizing 
antibody titers. 
Sera will be analyzed pre- and post-therapy for the following soluble factors: sCD27 and  sCD40 
ligand. 

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Selected patients may be analyzed for cytokines and antibodies to human tumor antigens, including 
PSA, MUC-1, and Brachyury. 
5.1.1.1.3 Additional assays 
Blood samples may be used for additional research studies, which may include phenotypic and 
functional analysis of immune-cell subsets, T cell clonality, and analyses for cytokines (IFN-γ, IL-
10, IL-12, IL-2, IL-4, etc.), chemokines, antibodies, tumor-associated antigens, and/or other 
markers. Auto-antigen proteomic arrays may also be explored 
 
5.1.1.2 Analyses of Tumor Specimens 
Analyses of Tumor Tissue (IHC) for expression of Target Antigens and Immune Related Markers 
For patients with lesions amenable to biopsy (or in selected patients with archival tissue), study of 
expression of target antigens (including PSA, MUC-1, and Brachyury) will be performed 
(collaboration with Dr. Houssein Sater) 
For patients with lesions amenable to biopsy, immune infiltration within the tumor 
microenvironment pre vs. post treatment may be performed to examine the frequency and 
localization of tumor infiltrated leukocytes (e.g. CD8, CD4 T-cells, Treg, NK cells, macrophage, 
M1/2 profile) by IHC (collaboration with Dr. Houssein Sater). 
Level of PD-L1 expression may be assessed by immunohistochemistry staining (IHC). Of note, 
further techniques to evaluate the expression of PD-L1 and/or marker candidates impacting the 
targeting or contributing to improve its expression may be also investigated if needed. 
Tissue from select patients may be tested for T cell clonality. 
 
5.1.1.3 Sample Processing 
5.1.1.3.1 Blood samples 
Blood samples will be processed at: 
Clinical Services Program 
NCI Frederick Cancer Research and Development Center 
PO Box B 
Frederick, MD 21702 
301-846-1000 
 
On the days that samples are drawn, Jennifer Bangh at CSP should be notified (phone: [301] 846-
5893; fax [301] 846-6222).  She will arrange courier delivery of the specimens to the processing 
lab. Couriers are assigned to CCR and this mechanism is in place. 
 
The weekly patient lists of samples drawn will be emailed to Caroline Jochems at 
jochemscm@mail.nih.gov, 
Jen 
Bangh 
at 
jb478s@nih.gov 
and 
Theresa 
Burks 
(burkst@mail.nih.gov). 
 
After processing, all non-FFPE samples will be transferred for labeling and tracking at: 
 

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Clinical Services Program 
NCI Frederick Cancer Research and Development Center 
PO Box B 
Frederick, MD 21702 
301-846-1000 
 
5.2 
SAMPLE COLLECTION SUMMARY 
Test/assay 
Sample/ 
volume 
blood 
Type 
of 
tube 
Collection point 
(+/- 48hrs) 
Location 
of 
specimen analysis 
PSA-specific T 
cell response 
 
15 mL blood 
Two 10 
mL 
Sodium 
heparin 
Baseline, week 6, 
week 14, week 30 
and end of 
treatment 
Clinical Services 
Program, Frederick/ 
LTIB 
PMBC subset 
correlation 
 
15 mL blood 
Two 10 
mL 
Sodium 
heparin 
Baseline, week 6, 
week 14, week 30 
and end of 
treatment 
Clinical Services 
Program, Frederick/ 
LTIB 
T cell clonality 
 
4 mL blood 
One 10 
mL 
Sodium 
heparin 
Baseline, week 6, 
week 14, week 30 
and end of 
treatment 
Clinical Services 
Program, Frederick/ 
LTIB 
Cytokine 
analysis 
 
8 mL blood 
One SST 
Baseline, week 6, 
week 14, week 30 
and end of 
treatment 
Clinical Services 
Program, Frederick/ 
LTIB 
 
5.3 
SAMPLE STORAGE, TRACKING, DISPOSITION (CLINICAL SERVICES PROGRAM [CSP]) 
Samples will be ordered in CRIS and tracked through a Clinical Trial Data Management system.  
Should a CRIS screen not be available, the CRIS downtime procedures will be followed.    Samples 
will not be sent outside NIH without IRB notification and an executed MTA. 
All data associated with the patient samples is protected by using a secure database.  All samples 
drawn at the NIH Clinical Center will be transported to the NCI Frederick Central Repository by 
couriers. 
Samples will be tracked and managed by the Central Repository database.  All samples will be 
stored in liquid nitrogen.  These freezers are located at NCI Frederick Central Repository in 
Frederick, Maryland. 

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NCI Frederick Central Repositories (managed under a subcontract) store, among other things, 
biological specimens in support of NIH clinical studies. All specimens are stored in secure, limited 
access facilities with sufficient security, back-up and emergency support capability and monitoring 
to ensure long-term integrity of the specimens for research. 
The subcontractor’s role is limited to clinical research databases and repositories containing patient 
specimens. The subcontractor neither conducts nor has any vested interest in research on human 
subjects, but does provide services and supports the efforts of its customers, many of which are 
involved in research on human subjects. 
It is the intent and purpose of the subcontractor to accept only coded, linked samples and sample 
information.  Protected information will not be sent electronically or by hard copy or on vial labels. 
Sample data is stored in the BioSpecimen Inventory (BSI) System II. This inventory tracking 
system is used to manage the storage and retrieval of specimens as well as maintain specimen data. 
BSI is designed for controlled, concurrent access. It provides a real-time, multi-user environment 
for tracking millions of specimens. The system controls how and in what order database updates 
and searches are performed. This control prevents deadlocks and race conditions. For security, BSI 
has user password access, 3 types of user access levels, and 36 user permissions (levels of access) 
that can be set to control access to the system functions. BSI provides audit tracking for processes 
that are done to specimens including shipping, returning to inventory, aliquoting, thawing, 
additives, and other processes. BSI tracks the ancestry of specimens as they are aliquoted, as well 
as discrepancies and discrepancy resolution for specimens received by the repository. If a 
specimen goes out of the inventory, the system maintains data associated with the withdrawal 
request. Vials are labeled with a unique BSI ID which is printed in both eye-readable and bar-
coded format. No patient-specific information is encoded in this ID. 
Investigators are granted view, input and withdrawal authority only for their specimens.  They may 
not view specimen data or access specimens for which they have not been authorized. Access to 
specimen storage is confined to repository staff. Visitors to the repositories are escorted by 
repository staff at all times. 
5.3.1 Protocol Completion/Sample Destruction 
All specimens obtained in the protocol are used as defined in the protocol. Any specimens 
remaining at the completion of the protocol will be stored in the conditions described above. The 
study will remain open as long as sample or data analysis continues. Samples will be stored until 
they are no longer of scientific value or until a subject withdraws consent for their continued use, 
at which time they will be destroyed. Once primary research objectives for the protocol are 
achieved, intramural researchers can request access to remaining samples, provided they have an 
IRB-approved protocol and patient consent or an exemption from OHSRP. 
The PI will report any loss or destruction of samples to the NIH IRB as soon as he/she is made 
aware of such loss. The PI will report destroyed samples to the IRB if samples become 
unsalvageable because of environmental factors such as a broken freezer or lack of dry ice in a 
shipping container, or if a patient withdraws consent. Samples will also be reported as lost if they 
are lost in transit between facilities or misplaced by a researcher. Freezer problems, lost samples, 
or other problems associated with samples will also be reported to the IRB and the NCI Clinical 
Director. 

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6 
DATA COLLECTION AND EVALUATION 
6.1 
DATA COLLECTION 
The PI will be responsible for overseeing entry of data into an in-house password protected 
electronic system (C3D) and ensuring data accuracy, consistency and timeliness. The principal 
investigator, associate investigators/research nurses and/or a contracted data manager will assist 
with the data management efforts. All data obtained during the conduct of the protocol will be kept 
in secure network drives or in approved alternative sites that comply with NIH security standards. 
Primary and final analyzed data will have identifiers so that research data can be attributed to an 
individual human subject participant. 
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH 
Intramural Records Retention Schedule as applicable. 
Loss or destruction of data: Should we become aware that a major breach in our plan to protect 
subject confidentiality and trial data has occurred, the IRB will be notified. 
 
All adverse events, including clinically significant abnormal findings on laboratory evaluations, 
regardless of severity, will be followed until return to baseline or stabilization of event. Patients 
will be followed for adverse events for 90 days after removal from study treatment or until off-
study, whichever comes first. 
 
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following: 
x 
Results in discontinuation from the study 
x 
Is associated with clinical signs or symptoms 
x 
Requires treatment or any other therapeutic intervention 
x 
Is associated with death or another serious adverse event, including hospitalization. 
x 
Is judged by the Investigator to be of significant clinical impact 
x 
If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome. 
6.2 
DATA SHARING PLANS 
6.2.1 Human Data Sharing Plan 
What data will be shared? 
I will share human data generated in this research for future research as follows: 
x Coded, linked data in an NIH-funded or approved public repository. 
x Coded, linked data in BTRIS (automatic for activities in the Clinical Center) 
x Identified or coded, linked data with approved outside collaborators under appropriate 
agreements. 
 
How and where will the data be shared? 
Data will be shared through: 

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x An NIH-funded or approved public repository.  Insert name or names:_clinicaltrials.gov_. 
x BTRIS (automatic for activities in the Clinical Center) 
x Approved outside collaborators under appropriate individual agreements. 
x Publication and/or public presentations. 
 
When will the data be shared? 
x Before publication. 
x At the time of publication or shortly thereafter. 
6.2.2 Genomic Data Sharing Plan 
No large scale genomic data are collected on this study.  The GDS plan does not apply. 
6.3 
RESPONSE CRITERIA 
For the purposes of this study, patients should be re-evaluated for response at week 14 and every 
8 weeks thereafter and at the of study treatment.  In addition to a baseline CT and bone scan, 
confirmatory scans should also be obtained up to 6 (not less than 4) weeks following initial 
documentation of objective response. 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline 
(version 1.1; Eisenhauer 2009).  Changes in the largest diameter (unidimensional measurement) 
of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the 
RECIST criteria. 
6.3.1 Disease Parameters 
Measurable disease:  Measurable lesions are defined as those that can be accurately measured in 
at least one dimension (longest diameter to be recorded) as:  
x By chest x-ray: >20 mm;  
x By CT scan:  
o Scan slice thickness 5 mm or under: as >10 mm 
o Scan slice thickness >5 mm: double the slice thickness  
x With calipers on clinical exam: >10 mm. 
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). 
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a lymph node 
must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended 
to be no greater than 5 mm).  At baseline and in follow-up, only the short axis will be measured 
and followed. 
Non-measurable disease.  All other lesions (or sites of disease), including small lesions (longest 
diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered 
non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial 

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effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not 
followed by CT or MRI), are considered as non-measurable. 
Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts should not be 
considered as malignant lesions (neither measurable nor non-measurable) since they are, by 
definition, simple cysts. 
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, if 
they meet the definition of measurability described above. However, if non-cystic lesions are 
present in the same patient, these are preferred for selection as target lesions. 
Target lesions.  All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in 
total, representative of all involved organs, should be identified as target lesions and recorded and 
measured at baseline.  Target lesions should be selected on the basis of their size (lesions with the 
longest diameter), be representative of all involved organs, but in addition should be those that 
lend themselves to reproducible repeated measurements.  It may be the case that, on occasion, the 
largest lesion does not lend itself to reproducible measurement in which circumstance the next 
largest lesion which can be measured reproducibly should be selected.  A sum of the diameters 
(longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated 
and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then 
only the short axis is added into the sum.  The baseline sum diameters will be used as reference to 
further characterize any objective tumor regression in the measurable dimension of the disease. 
Non-target lesions.  All other lesions (or sites of disease) including any measurable lesions over 
and above the 5 target lesions should be identified as non-target lesions and should also be 
recorded at baseline.  Measurements of these lesions are not required, but the presence, absence, 
or in rare cases unequivocal progression of each should be noted throughout follow-up. 
6.3.2 Methods for Evaluation of Measurable Disease 
All measurements should be taken and recorded in metric notation using a ruler or calipers.  All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 4 weeks before the beginning of the treatment. 
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 unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam. 
Clinical lesions:  Clinical lesions will only be considered measurable when they are superficial 
(e.g., skin nodules and palpable lymph nodes) and t10 mm diameter as assessed using calipers 
(e.g., skin nodules).  In the case of skin lesions, documentation by color photography, including a 
ruler to estimate the size of the lesion, is recommended. 
Chest x-ray:  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:  This guideline has defined measurability of lesions on CT scan based 
on the assumption that CT slice thickness is 5 mm or less.  If CT scans have slice thickness greater 
than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness.  MRI is 
also acceptable in certain situations (e.g. for body scans). 

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Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal resolution; 
however, there are many image acquisition variables involved in MRI, which greatly impact image 
quality, lesion conspicuity, and measurement.  Furthermore, the availability of MRI is variable 
globally.  As with CT, if an MRI is performed, the technical specifications of the scanning 
sequences used should be optimized for the evaluation of the type and site of disease.  Furthermore, 
as with CT, the modality used at follow-up should be the same as was used at baseline and the 
lesions should be measured/assessed on the same pulse sequence.  It is beyond the scope of the 
RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body 
parts, and diseases.  Ideally, the same type of scanner should be used and the image acquisition 
protocol should be followed as closely as possible to prior scans.  Body scans should be performed 
with breath-hold scanning techniques, if possible. 
PET-CT:  At present, the low dose or attenuation correction CT portion of a combined PET-CT is 
not always of optimal diagnostic CT quality for use with RECIST measurements.  However, if the 
site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to 
a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for 
RECIST measurements and can be used interchangeably with conventional CT in accurately 
measuring cancer lesions over time.  Note, however, that the PET portion of the CT introduces 
additional data which may bias an investigator if it is not routinely or serially performed. 
Ultrasound:  Ultrasound is not useful in assessment of lesion size and should not be used as a 
method of measurement.  Ultrasound examinations cannot be reproduced in their entirety for 
independent review at a later date and, because they are operator dependent, it cannot be 
guaranteed that the same technique and measurements will be taken from one assessment to the 
next.  If new lesions are identified by ultrasound in the course of the study, confirmation by CT or 
MRI is advised.  If there is concern about radiation exposure at CT, MRI may be used instead of 
CT in selected instances. 
Endoscopy, Laparoscopy:  The utilization of these techniques for objective tumor evaluation is not 
advised.  However, such techniques may be useful to confirm complete pathological response 
when biopsies are obtained or to determine relapse in trials where recurrence following complete 
response (CR) or surgical resection is an endpoint. 
Tumor markers:  Tumor markers alone cannot be used to assess response.  If markers are initially 
above the upper normal limit, they must normalize for a patient to be considered in complete 
clinical response.  Specific guidelines for both CA-125 response (in recurrent ovarian cancer) and 
PSA response (in recurrent prostate cancer) have been published [JNCI 96:487-488, 2004; J Clin 
Oncol 17, 3461-3467, 1999; J Clin Oncol 26:1148-1159, 2008].  In addition, the Gynecologic 
Cancer Intergroup has developed CA-125 progression criteria which are to be integrated with 
objective tumor assessment for use in first-line trials in ovarian cancer [JNCI 92:1534-1535, 2000]. 
Cytology, Histology:  These techniques can be used to differentiate between partial responses (PR) 
and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germ cell 
tumors, where known residual benign tumors can remain). 
The cytological confirmation of the neoplastic origin of any effusion that appears or worsens 
during treatment when the measurable tumor has met criteria for response or stable disease is 
mandatory to differentiate between response or stable disease (an effusion may be a side effect of 
the treatment) and progressive disease. 

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FDG-PET:  While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in assessment 
of progression (particularly possible 'new' disease).  New lesions on the basis of FDG-PET imaging 
can be identified according to the following algorithm: 
a. Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD 
based on a new lesion. 
b. No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive FDG-
PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD.  If 
the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, 
additional follow-up CT  scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-
PET scan).  If the positive FDG-PET at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic images, this is not 
PD. 
c. FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy 
in cases where a residual radiographic abnormality is thought to represent fibrosis or 
scarring.  The use of FDG-PET in this circumstance should be prospectively described 
in the protocol and supported by disease-specific medical literature for the indication.  
However, it must be acknowledged that both approaches may lead to false positive CR 
due to limitations of FDG-PET and biopsy resolution/sensitivity. 
Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake greater 
than twice that of the surrounding tissue on the attenuation corrected image. 
6.3.3 Response Criteria 
6.3.3.1 Evaluation of Target Lesions 
Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes 
(whether target or non-target) must have reduction in short axis to <10 mm. 
Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking 
as reference the baseline sum of diameters. 
Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, 
taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest 
on study).  In addition to the relative increase of 20%, the sum must also demonstrate an absolute 
increase of at least 5 mm. (Note:  the appearance of one or more new lesions is also considered 
progressions). 
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to 
qualify for PD, taking as reference the smallest sum of diameters while on study. 
 
6.3.3.2 Evaluation of Non-Target Lesions 
Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor 
marker level.  All lymph nodes must be non-pathological in size (<10 mm short axis). 
Note:  If tumor markers are initially above the upper normal limit, they must normalize for a 
patient to be considered in complete clinical response. 

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Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor 
marker level above the normal limits. 
Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal progression 
of existing non-target lesions.  Unequivocal progression should not normally trump target lesion 
status.  It must be representative of overall disease status change, not a single lesion increase. 
Although a clear progression of “non-target” lesions only is exceptional, the opinion of the treating 
physician should prevail in such circumstances, and the progression status should be confirmed at 
a later time by the review panel (or Principal Investigator). 
 
6.3.3.3 Evaluation of Best Overall Response 
The best overall response is the best response recorded from the start of the treatment until disease 
progression/recurrence (taking as reference for progressive disease the smallest measurements 
recorded since the treatment started).  The patient's best response assignment will depend on the 
achievement of both measurement and confirmation criteria. 
 
For Patients with Measurable Disease (i.e., Target Disease) 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
>4 wks. Confirmation** 
CR 
Non-
CR/Non-PD 
No 
PR 
>4 wks. Confirmation** 
CR 
Not 
evaluated 
No 
PR 
PR 
Non-
CR/Non-
PD/not 
evaluated 
No 
PR 
SD 
Non-
CR/Non-
PD/not 
evaluated 
No 
SD 
Documented at least once >4 
wks. from baseline** 
PD 
Any 
Yes or 
No 
PD 
no prior SD, PR or CR 
Any 
PD*** 
Yes or 
No 
PD 

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Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
Any 
Any 
Yes 
PD 
* 
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
** 
Only for non-randomized trials with response as primary endpoint. 
*** 
In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as 
disease progression. 
Note: 
Patients with a global deterioration of health status requiring discontinuation of treatment without 
objective evidence of disease progression at that time should be reported as “symptomatic 
deterioration.”  Every effort should be made to document the objective progression even after 
discontinuation of treatment. 
 
For Patients with Non-Measurable Disease (i.e., Non-Target Disease) 
 
 
 
 
 
 
 
 
 
 
 
 
6.3.4 Duration of Response 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first date that 
recurrent or progressive disease is objectively documented (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started). 
The duration of overall CR is measured from the time measurement criteria are first met for CR 
until the first date that progressive disease is objectively documented. 
Duration of stable disease:  Stable disease is measured from the start of the treatment until the 
criteria for progression are met, taking as reference the smallest measurements recorded since the 
treatment started, including the baseline measurements. 
n-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
*  ‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is increasingly used as 
an endpoint for assessment of efficacy in some trials so to assign this category when no lesions can be 
measured is not advised 

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6.3.5 Progression-Free Survival (PFS) 
PFS is defined as the duration of time from start of treatment to time of progression or death, 
whichever occurs first. 
6.4 
TOXICITY CRITERIA 
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on the study.  The descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 5 will be utilized for AE reporting.  All 
appropriate treatment areas should have access to a copy of the CTCAE version 5.  A copy of the 
CTCAE 
version 
5 
can 
be 
downloaded 
from 
the 
CTEP 
web 
site: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc  
 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN 
7.1 
DEFINITIONS 
7.1.1 Adverse Event 
Any untoward medical occurrence in a human subject, including any abnormal sign (for example, 
abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with 
the subject’s participation in research, whether or not considered related to the subject’s 
participation in the research. 
7.1.2 Suspected adverse reaction 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. For the purposes of IND safety reporting, ‘reasonable 
possibility’ means there is evidence to suggest a causal relationship between the drug and the 
adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug. 
7.1.3 Unexpected adverse reaction 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in the 
investigator brochure or is not listed at the specificity or severity that has been observed; or, if an 
investigator brochure is not required or available, is not consistent with the risk information 
described in the general investigational plan or elsewhere in the current application. "Unexpected” 
also refers to adverse events or suspected adverse reactions that are mentioned in the investigator 
brochure as occurring with a class of drugs or as anticipated from the pharmacological properties 
of the drug, but are not specifically mentioned as occurring with the particular drug under 
investigation. 
7.1.4 Serious 
An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a Serious 
Adverse Event or if it compromises the safety, welfare or rights of subjects or others. 

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7.1.5 Serious Adverse Event 
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following: 
x 
Death, 
x 
A life-threatening adverse drug experience 
x 
Inpatient hospitalization or prolongation of existing hospitalization 
x 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions 
x 
A congenital anomaly/birth defect. 
x 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition. 
7.1.6 Disability 
A substantial disruption of a person’s ability to conduct normal life functions. 
7.1.7 Life-threatening adverse drug reaction 
Any adverse event or suspected adverse reaction that places the patient or subject, in the view of 
the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that had it occurred in a more severe form, might have caused death. 
7.1.8 Protocol Deviation (NIH Definition) 
Any change, divergence, or departure from the IRB-approved research protocol. 
7.1.9 Non-Compliance (NIH Definition) 
The failure to comply with applicable NIH Human Research Protections Program (HRPP) policies, 
IRB requirements, or regulatory requirements for the protection of human research subjects. 
7.1.10 Unanticipated Problem 
Any incident, experience, or outcome that: 
x Is unexpected in terms of nature, severity, or frequency in relation to 
(a) the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study docu-ments, and 
(b) the characteristics of the subject population being studied; AND 
x Is related or possibly related to participation in the research; AND 
x Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
7.2 
NIH IRB AND CLINICAL DIRECTOR (CD) REPORTING 
7.2.1 NIH IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths 
The Protocol PI will report in the NIH Problem Form to the NIH IRB and NCI CD: 
x All deaths, except deaths due to progressive disease 

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x All Protocol Deviations 
x All Unanticipated Problems 
x All non-compliance 
Reports must be received within 7 days of PI awareness via iRIS. 
7.2.2 NIH IRB Requirements for PI Reporting at Continuing Review 
The protocol PI will report to the NIH IRB: 
1. A summary of all protocol deviations in a tabular format to include the date the deviation 
occurred, a brief description of the deviation and any corrective action. 
2. A summary of any instances of non-compliance 
3. A tabular summary of the following adverse events: 
x All Grade 2 unexpected events that are possibly, probably or definitely related to the 
research; 
x All Grade 3 and 4 events that are possibly, probably or definitely related to the research; 
x All Grade 5 events regardless of attribution; 
x All Serious Events regardless of attribution. 
NOTE: Grade 1 events are not required to be reported. 
7.2.3 NIH IRB Reporting of IND Safety Reports 
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NIH IRB. 
 
7.3 
IND SPONSOR REPORTING CRITERIA 
During the first 30 days after the subject receives investigational agent, the investigator must 
immediately report to the sponsor, using the mandatory MedWatch form 3500a or equivalent, any 
serious adverse event, whether or not considered drug related, including those listed in the protocol 
or investigator brochure and must include an assessment of whether there is a reasonable 
possibility that the drug caused the event. For serious adverse events that occur more than 30 days 
after the last administration of investigational agent/intervention, only report those that have an 
attribution of at least possibly related to the agent/intervention. 
Required timing for reporing per the above guideline: 
x Deaths (except death due to progressive disease) must be reported via email within 24 
hours.  A complete report must be submitted within one business day. 
x Other serious adverse events as well as deaths due to progressive disease must be 
reported within one business day 
Events will be submitted to the Center for Cancer Research (CCR) at: CCRsafety@mail.nih.gov 
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7.3.1 Reporting Pregnancy 
7.3.1.1 Paternal exposure 
Male patients should refrain from fathering a child or donating sperm during the study and for one 
month after the last dose of study vaccines. Pregnancy of the patient’s partner is not considered to 
be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective 
termination, ectopic pregnancy, normal birth, or congenital abnormality) occurring from the date 
of the first dose until one month after the last dose should, if possible, be followed up and 
documented. 
 
7.4 
SAFETY REPORTING CRITERIA TO THE PHARMACEUTICAL COLLABORATORS 
7.4.1 Etubics Corporation (ETBX-051, ETBX-061, ETBX-071) 
All events listed below must be reported in the defined timelines to CCRsafety@mail.nih.gov. 
The CCR Office of Regulatory Affairs will send all reports to the manufacturer as described below. 
In the event of any new SAE (of any Grade) occurring during the reporting period (from first dose 
until decision made to discontinue treatment), the investigator must immediately (i.e., within a 
maximum 2 business days after becoming aware of the event) inform the Manufacturer (Etubics 
Corporation) or designee by telephone, by fax, or by email. 
When an event (or follow-up information) is reported by telephone, a written report must be sent 
immediately thereafter by fax or email. 
Reporting procedures and timelines are the same for any new information on a previously reported 
SAE (=follow-up). Any new SAE is reported to: 
Jelena Berglund, PhD 
VP Operations 
401 West Harrison Street, Seattle, WA 98119 
Telephone: 206-838-5110 
Cell: 919-265-7089 (primary number) 
Fax: 206-838-2978 
Email: jelena@etubics.com 
All Medwatch 3500a forms and written reports should be transmitted, which must be completed 
by the investigator following specific completion instructions. Relevant pages from the CRF may 
be provided in parallel (e.g., medical history, concomitant drugs). 
In all cases, the information provided in the SAE Report Form must be consistent with the data on 
the event that is recorded in the corresponding sections of the CRF. 
 
7.5 
INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) REPORTING CRITERIA 
7.5.1 Serious Adverse Event Reports to IBC 
The Principal Investigator (or delegate) will notify IBC of any unexpected fatal or life-threatening 
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possible but in no event later than 7 calendar days of initial receipt of the information. Serious 
adverse events that are unexpected and associated with the use of the ETBX-071, ETBX-061, and 
ETBX-051vaccines, but are not fatal or life-threatening, much be reported to the NIH IBC as soon 
as possible, but not later than 15 calendar days after the investigator’s initial receipt of the 
information. Adverse events may be reported by using the FDA Form 3500a. 
7.5.2 Annual Reports to IBC 
Within 60 days after the one-year anniversary of the date on which the IBC approved the initial 
protocol, and after each subsequent anniversary until the trial is completed, the Principal 
Investigator (or delegate) shall submit the information described below. Alternatively, IRB 
continuing review report can be sent to the IBC in lieu of a separate report. Please include the IBC 
protocol number on the report. 
7.5.2.1 Clinical Trial Information 
A brief summary of the status of the trial in progress or completed during the previous year. The 
summary is required to include the following information: 
x the title and purpose of the trial 
x clinical site 
x the Principal Investigator 
x clinical protocol identifiers; 
x participant population (such as disease indication and general age group, e.g., adult or 
pediatric); 
x the total number of participants planned for inclusion in the trial; the number entered into 
the trial to date whose participation in the trial was completed; and the number who 
dropped out of the trial with a brief description of the reasons 
x the status of the trial, e.g., open to accrual of subjects, closed but data collection ongoing, 
or fully completed, 
x if the trial has been completed, a brief description of any study results. 
7.5.2.2 Progress Report and Data Analysis 
Information obtained during the previous year's clinical and non-clinical investigations, including: 
x a narrative or tabular summary showing the most frequent and most serious adverse 
experiences by body system 
x a summary of all serious adverse events submitted during the past year 
x a summary of serious adverse events that were expected or considered to have causes not 
associated with the use of the gene transfer product such as disease progression or 
concurrent medications 
x if any deaths have occurred, the number of participants who died during participation in 
the investigation and causes of death 
x a brief description of any information obtained that is pertinent to an understanding of the 
gene transfer product’s actions, including, for example, information about dose-response, 
information from controlled trials, and information about bioavailability. 

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7.6 
DATA AND SAFETY MONITORING PLAN 
7.6.1 Principal Investigator/Research Team 
The clinical research team will meet on a regular basis when patients are being actively treated on 
the trial to discuss each patient, enrollment and data management issues. Decisions about dose 
level enrollment and dose de-escalation will be made based on the toxicity data from prior patients. 
All data will be collected in a timely manner and reviewed by the principal investigator. Adverse 
events will be reported as required above. Any safety concerns, new information that might affect 
either the ethical and or scientific conduct of the trial, or protocol deviations will be immediately 
reported to the IRB using iRIS. 
The principal investigator will review adverse event and response data on each patient to ensure 
safety and data accuracy. The principal investigator will personally conduct or supervise the 
investigation and provide appropriate delegation of responsibilities to other members of the 
research staff. 
7.6.2 Sponsor Monitoring Plan 
As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring 
program. The CCR’s program allows for confirmation of: study data, specifically data that could 
affect the interpretation of primary study endpoints; adherence to the protocol, regulations, and 
SOPs; and human subjects protection. This is done through independent verification of study data 
with source documentation focusing on: 
x Informed consent process 
x Eligibility confirmation 
x Drug administration and accountability 
x Adverse events monitoring 
x Response assessment. 
The monitoring program also extends to multi-site research when the CCR is the coordinating 
center. 
This trial will be monitored by personnel employed by an CCR contractor. Monitors are qualified 
by training and experience to monitor the progress of clinical trials. Personnel monitoring this 
study will not be affiliated in any way with the trial conduct. 
7.6.3 Safety Monitoring Committee (SMC) 
This protocol will require oversight from the Safety Monitoring Committee (SMC).  Initial review 
will occur as soon as possible after the annual NIH IRB continuing review date.  Subsequently, 
each protocol will be reviewed as close to annually as the quarterly meeting schedule permits or 
more frequently as may be required by the SMC. For initial and subsequent reviews, protocols will 
not be reviewed if there is no accrual within the review period. Written outcome letters will be 
generated in response to the monitoring activities and submitted to the Principal investigator and 
Clinical Director or Deputy Clinical Director, CCR, NCI. 
 
8 
STATISTICAL CONSIDERATIONS 

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8.1 
STATISTICAL HYPOTHESES 
8.1.1 Primary Endpoints 
x Safety and tolerability of the recommended phase 2 dose of PSA/MUC1/Brachyury 
vaccine as determined by the fraction of patients who experience a DLT. 
x Dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) 
x Treatment-emergent adverse event (AEs) and serious adverse events (SAEs). 
x Clinically significant changes in safety laboratory tests, physical examinations, 
electrocardiograms (ECGs), and vital signs. 
8.1.2 Secondary Efficacy Endpoints 
Preliminary assessments of: 
x objective response rate (ORR) – percentage of subjects who experience partial or complete 
response at any time point during treatment period 
x disease control rate (DCR) –percentage of subjects who experience partial response, 
complete response or stable disease lasting for at least 6 months 
x duration of response – the time from when measurement criteria for PR or CR are met until 
disease recurrence or progression per dose cohort and overall 
x progression-free survival (PFS) - the time from the date of first treatment to the date of 
disease progression or death (any cause) whichever occurs first per dose cohort 
x overall survival (OS) - the time from the date of first treatment to the date of death (any 
cause) per dose cohort and overall 
x PSA doubling time (PSADT) – week 14 and at the end of study treatment in subjects with 
advanced cancer treated with the ETBX-071, ETBX-061, and ETBX-051 vaccines 
8.2 
SAMPLE SIZE DETERMINATION 
Up to six patients will be enrolled in an initial cohort.  If ≤1 of 6 patients experience a DLT, then 
initiation of the dose expansion phase will occur.  If ≥2 of 3 or 6 experience DLT in the initial dose 
level 1, then dose de-escalation will occur. Up to six patients will be enrolled at the lower dose 
level (-1) (1x1011 VP).  If ≤1 of 6 patients experience a DLT, then the maximum tolerated (MTD) 
will be declared at this dose, and initiation of the dose expansion phase will occur.  If ≥2 of 6 (or 
>=2 of the first 3) experience DLT at dose level -1, then a further dose de-escalation will occur. 
Up to six patients will be enrolled at the lower dose level (-2) (5x1010 VP).  If ≤1 of 6 patients 
experience a DLT, then the maximum tolerated (MTD) will be declared at this dose, and initiation 
of the dose expansion phase will occur.  If ≥2 of 6 (or >=2 of the first 3) experience DLTat dose 
level -2, then the study will be stopped. 
 
In the second part, dose expansion will occur when the MTD has been determined. An additional 
12 subjects will be enrolled in the dose expansion component of the trial, for a total of 18 subjects 
treated at the MTD.  Evaluation of T cell frequencies (CD4, CD8, NK and T Reg cells, etc.) will 
be performed and will result in exploratory comparisons between responders and non-responders. 
If approximately 25-30% of patients experience a clinical response, there should be approximately 
3-4 responders and 8-9 non-responders out of 12 evaluable patients. In these patients, for a given 
measure, considered as an exploratory evaluation, a two-group t-test with a one-sided 0.10 
significance level will have 80% power to detect a difference between the two groups with an 

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effect size of 1.5 (1.5 SDs of the measure of interest, assumed to be approximately the same in 
responders and non-responders), when there are 3 responders and 9 non-responders (total of 12 
evaluable patients). With 4 responders and 8 non-responders, the same comparison would have 
85% power. In practice, an exact Wilcoxon rank sum test will be used instead of a two-group t-
test. 
 
To accrue a total of 18 evaluable patients, it is expected that up to 2 years may be required. Since 
up to three dose levels may be explored, in addition to the 12 evaluable patients at the MTD, the 
accrual ceiling will be set at 30 patients. 
8.3 
POPULATIONS FOR ANALYSIS 
8.3.1 Definitions 
Evaluable for toxicity: All patients will be evaluable for toxicity from the time of their first 
treatment with ETBX vaccines. 
Evaluable for DLT:  The first 6 - 18 subjects enrolled will be evaluable for DLT from the time of 
their first treatment with ETBX-vaccines until 28 days have passed. Subjects who do not complete 
the DLT observation period for reasons other than a DLT will be replaced. 
Evaluable for objective response: Only those patients who have measurable disease present at 
baseline, have received at least one dose of therapy, and have had their disease re-evaluated will 
be considered evaluable for response.  These patients will have their response classified according 
to the definitions stated below. 
Evaluable Non-Target Disease Response:  Patients who have lesions present at baseline that are 
evaluable but do not meet the definitions of measurable disease, have received at least one dose of 
therapy, and have had their disease re-evaluated will be considered evaluable for non-target 
disease.  The response assessment is based on the presence, absence, or unequivocal progression 
of the lesions. 
8.4 
STATISTICAL ANALYSES 
8.4.1 General Approach 
Descriptive analyses of toxicity results and exploratory description of time-to-event and response-
based results, along with comparison of parameters between responders and non-responders using 
a non-parametric test. 
8.4.2 Analysis of the Primary Endpoints 
Not applicable in this Phase 1 Study. The primary objective of evaluating safety is described in 
Section 8.4.4 below. 
8.4.3 Analysis of the Secondary Efficacy Endpoints 
The percentage of subjects that achieve an objective confirmed complete or partial overall tumor 
response using RECIST Version 1.1 will be evaluated by dose cohort and overall. The 95% 
confidence interval of the response rate will be evaluated. Disease control (confirmed response or 
SD lasting for at least 6 months) will be analyzed in a similar manner. 
 

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8.4.3.1 Duration of Response 
The duration of overall response will be evaluated by dose cohort and overall. The duration of 
overall response is measured from the time measurement criteria are met for CR or PR (whichever 
is first recorded) until the first date that recurrent or progressive disease is objectively documented 
(taking as reference for PD the smallest measurements recorded since the treatment started). 
 
8.4.3.2 Progression-Free Survival (PFS) 
PFS will be evaluated by dose cohort and overall using Kaplan-Meier methods. PFS will be defined 
as the time from the date of first treatment to the date of disease progression or death (any cause) 
whichever occurs first. Subjects who do not have disease progression or have not died at the end 
of follow up will be censored at the last known date the subject was progression free. 
 
8.4.3.3 Overall Survival 
OS will be evaluated by dose cohort and overall using Kaplan-Meier methods. OS will be defined 
as the time from the date of first treatment to the date of death (any cause). Subjects who are alive 
at the end of follow up will be censored at the last known date alive. 
 
8.4.3.4 PSA doubling time will be determined using a standard formula. 
8.4.4 Safety Analyses 
DLTs will be evaluated continuously in a cohort. An overall assessment of whether to de-escalate 
to the next dose level will be made at least 1 weeks after the last subject in the previous cohort has 
received their first injection. Safety will be evaluated in 3 or 6 subjects at each dose level in the 
dose de-escalation component of the study.  During the dose de-escalation phase of the study, a 
dose level will be considered safe if < 33% of subjects treated at a dose level experience a DLT 
(i.e., 0 of 3, ≤ 1 of 6).  A DLT is defined in Section 3.1. Safety will continue to be monitored 
among additional subjects treated at the MTD in the dose expansion component of the study. A 
subject will be considered evaluable for safety if treated with at least one injection. DLTs will be 
observed for 4 weeks. 
 
Safety endpoints will be analyzed as summary statistics during treatment and/or as change scores 
from baseline assessments.  AEs will be coded as defined in the Medical Dictionary for Regulatory 
Activities (MedDRA). All AEs will be recorded and tabulated following each treatment (vaccine 
injection). AEs will be recorded by severity, frequency, and relationship to the study intervention 
and will be presented by System Organ Class (SOC) designations and preferred term groupings. 
Information on each AE will include start date, stop date, severity, relationship, expectedness, 
outcome, and duration. Adverse events leading to premature discontinuation from the study 
intervention and serious treatment-emergent AEs will be presented either in a Table or a Listing 
 
Overall safety will be assessed by descriptive analyses using tabulated frequencies of AEs by grade 
using CTCAE Version 5 within dose cohorts and for the overall study population in terms of 
treatment-emergent AEs, SAEs, and clinically significant changes in safety laboratory tests, 
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8.4.5 Baseline Descriptive Statistics 
None will be provided. 
8.4.6 Planned Interim Analyses and Halting Guidelines 
8.4.6.1 Planned Interim Analyses 
Safety data will be examined after 6 patients have been treated to ensure that enrolling the 
remaining patients is allowable. 
8.4.6.2 Halting Guidelines 
The following safety events will trigger a temporary suspension of the study injections: 
x Death possibly related to the study agent. 
x Two Grade 4 toxicity events that are possibly related to the study agent. 
x At any time during the expansion phase > 33% of subjects experience a Grade 3 or 4 
major organ toxicity possibly related to study injections. 
Assessment of these halting rules is a review of cumulative events for all study participants and 
should not be confused with reasons for delaying or terminating the treatment schedule of any 
individual subject. 
Treatment may also be suspended for safety concerns other than those described above if, in the 
judgment of the Investigator , participant safety is threatened. 
8.4.7 Subgroup Analyses 
None will be performed because of the small size of the study. 
8.4.8 Tabulation of Individual Participant Data 
No individual participant data will be tabulated. 
8.4.9 Exploratory Analyses 
The percentage of subjects with a positive immune response will be evaluated by dose cohorts and 
overall. A positive immune response is defined by CMI reactivity in ex vivo stimulation assays, 
with flow cytometric readout (cytokine production or CD107 expression). Antigen-specific 
peptide challenge assays require a readout of > 250 reactive T-cells/million cells above the 
background.(55) 
Immune response will be assessed among the 18 subjects treated at the MTD (6 in dose escalation 
and 12 in dose expansion). The therapy will be considered of further interest if 9 of 18 subjects 
treated at the MTD exhibit an immune response as defined above. The magnitude of response will 
also be described. A subject will be considered evaluable for immune response if they receive at 
least three injections 
T cell frequencies (CD4, CD8, NK, T Reg cells, etc.) will be determined post treatment, and 
compared among the 12 patients in the expansion cohort between responders and non-responders, 
using an exact Wilcoxon rank sum test, with a one-tailed p-value to determine if there is an increase 
in these values being reported, to demonstrate potential trends if identified in a very small number 
of patients. P-values will be presented without adjustment for multiple comparisons, but in the 
context of an exploratory analysis with limited numbers of subjects. 

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9 
COLLABORATIVE AGREEMENTS 
9.1 
COOPERATIVE RESEARCH AND DEVELOPMENT AGREEMENT (CRADA) 
A CRADA is in place between the Laboratory of Tumor Immunology and Biology (LTIB), CCR 
NCI and Etubics Corporation, the manufacturer of ETBX-071 (adenoviral PSA vaccine); ETBX-
061 (adenoviral MUC1 vaccine); and ETBX-051 (adenoviral brachyury vaccine), CRADA# 
02997. 
 
10 HUMAN SUBJECTS PROTECTIONS 
10.1 RATIONALE FOR SUBJECT SELECTION 
10.1.1 Selection Based on Gender, Ethnicity, and Race 
Subjects from all racial/ethnic groups are eligible for this study if they meet the eligibility criteria. 
To date, there is no information that suggests that differences in drug metabolism or disease 
response would be expected in one group compared with another. Efforts will be made to extend 
accrual to a representative population, but in this preliminary study, a balance must be struck 
between patient safety considerations and limitations on the number of individuals exposed to 
potentially toxic and/or ineffective treatments on one hand and the need to explore ethnic aspects 
of clinical research on the other hand. If differences in outcome that correlate with ethnic identity 
are noted, accrual may be expanded or a follow-up study may be written to investigate those 
differences more fully. Women are not eligible for this study as this disease occurs only in men. 
10.1.2 Strategies/Procedures for Recruitment 
Patient accrual for this protocol will be facilitated by Web-based recruitment strategies. This 
protocol will be listed on www.clinicaltrials.gov. 
10.1.3 Justification for Exclusions 
Due to impaired cellular immunity with the concomitant increased risk of serious side effects from 
vaccinations with infectious agents, the Centers for Disease Control and Prevention recommends 
that HIV infected patients be excluded, in addition, patients with chronic hepatitis infection, 
including B and C, because of potential immune impairment. 
10.2 PARTICIPATION OF CHILDREN 
Because no dosing or adverse event data are currently available on the use of the study vaccines 
in patients <18 years of age, children are excluded from this study. 
10.3 PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT 
Adults unable to give consent are excluded from enrolling in the protocol.  However, re-consent 
may be necessary and there is a possibility, though unlikely, that subjects could become 
decisionally impaired. For this reason and because there is a prospect of direct benefit from 
research participation (section 10.4), all subjects ≥ age 18 will be offered the opportunity to fill in 
their wishes for research and care, and assign a substitute decision maker on the “NIH Advance 
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make decisions about their medical care in the event that they become incapacitated or cognitively 
impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent 
Assessment Team (ACAT) for evaluation as needed for the following: an independent assessment 
of whether an individual has the capacity to provide consent; assistance in identifying and 
assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint 
a surrogate. For those subjects that become incapacitated and do not have pre-determined 
substitute decision maker, the procedures described in MAS Policy 87-4 and NIH HRPP SOP 14E 
for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and 
(b) who do not have a legal guardian or durable power of attorney, will be followed. 
10.4 EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS 
Adult subjects, including those that have lost the capacity to consent 
Patients will receive evaluation of their disease at the National Cancer Institute’s Clinical Center.  
This protocol may or may not benefit an individual, but the results may help the investigators learn 
more about the disease and develop new treatments for patients with this disease. 
Potential adverse reactions attributable to the administration of the vaccine utilized in this trial are 
discussed in section 11. All care will be taken to minimize side effects, but they can be 
unpredictable in nature and severity. Patients will be examined and evaluated prior to enrollment. 
All evaluations to monitor the treatment of patients will be recorded in the patient chart. If patients 
suffer any physical injury as a result of the participation in this study, immediate medical treatment 
is available at the Clinical Center, National Cancer Institute, Bethesda, Maryland. 
Although no compensation is available, any injury will be evaluated and treated in keeping with 
the benefits or care to which patients are entitled under applicable regulations. In all publications 
and presentations resulting from this trial, patients’ anonymity will be protected to the maximum 
extent possible. Authorized personnel from the National Cancer Institute (NCI) and Food and Drug 
Administration (FDA) or other regulatory authorities may have access to research files in order to 
verify that patients’ rights have been safeguarded. In addition, patient names will be given to the 
Central Registration to register and verify patients’ eligibility. 
 
10.5 CONSENT PROCESS AND DOCUMENTATION 
The investigational nature and research objectives of this trial, the procedures and treatments 
involved and their attendant risks and discomforts and benefits, and potential alternative therapies 
will be carefully explained to the patient, and a signed informed consent document will be obtained 
by a study investigator prior to entry onto the study. 
The PI or associate investigator will meet with the patient to discuss the protocol treatment and 
alternative options in detail. It will be stated clearly that participation in the research study is 
voluntary and that participants can withdraw from the study without losing benefits they would 
otherwise be entitled to. The patient will be encouraged to ask questions, and additional meetings 
to discuss the treatment options will be arranged as necessary. 
10.5.1 Telephone re-consent procedure 
Re-consent on this study may be obtained via telephone according to the following procedure: the 
informed consent document will be sent to the subject. An explanation of the study will be 

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provided over the telephone after the subject has had the opportunity to read the consent form. The 
subject will sign and date the informed consent. A witness to the subject’s signature will sign and 
date the consent. The original informed consent document will be sent back to the consenting 
investigator who will sign and date the consent form with the date the consent was obtained via 
telephone. A fully executed copy will be returned via mail for the subject’s records. The informed 
consent process will be documented on a progress note by the consenting investigator. 
10.5.2 Consent process for non-English speaking subjects 
If there is an unexpected enrollment of a research participant for whom there is no translated extant 
IRB approved consent document, the principal investigator and/or those authorized to obtain 
informed consent will use the Short Form Oral Consent Process as described in MAS Policy M77-
2, OHSRP SOP 12, 45 CFR 46.117 (b) (2), and 21 CFR 50.27 (b) (2).  The summary that will be 
used is the English version of the extant IRB approved consent document.  Signed copies of both 
the English version of the consent and the translated short form will be given to the subject or their 
legally authorized representative and the signed original will be filed in the medical record. 
Unless the PI is fluent in the prospective subject’s language, an interpreter will be present to 
facilitate the conversation. Preferably someone who is independent of the subject (i.e., not a family 
member) will assist in presenting information and obtaining consent. Whenever possible, 
interpreters will be provided copies of the relevant consent documents well before the consent 
conversation with the subject (24 to 48 hours if possible). 
We request prospective IRB approval of the use of the short form process and will notify the IRB 
at the time of continuing review of the frequency of the use of the Short Form. 
 
11 PHARMACEUTICAL INFORMATION 
11.1 COMBINATION ETBX-071, ETBX-061, AND ETBX-051 VACCINE REGIMEN (IND# 17811) 
11.1.1 Source 
Table 4: Investigational Products 
Table 4a 
Product Name(s): 
ETBX-071 (Ad5 [E1-, E2b-]-PSA Vaccine) 
Dosage Form: 
Suspension for injection 
Unit Dose 
5 x 1011 VP, 1 x 1011 VP, or 5 x 1010 VP. 
Route of Administration 
SC injection 
Physical Description 
Each ETBX vaccine is supplied as a sterile, clear 
solution in a 2-mL single-dose vial. The vaccine is 
provided at a concentration of 5 x 1011 VP per 1 mL and 
contains ARM formulation buffer (20 mM TRIS, 25 
mM NaCl, 2.5% glycerol, pH 8.0). Each vial contains 

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approximately 1.1 mL of the vaccine. The product 
should be stored at ≤ -20°C. 
Manufacturer 
Etubics 
Table 4b 
Product Name(s): 
ETBX-061 (Ad5 [E1-, E2b-]-MUC1 Vaccine) 
Dosage Form: 
Suspension for injection 
Unit Dose 
5 x 1011 VP, 1 x 1011 VP, or 5 x 1010 VP. 
Route of Administration 
SC injection 
Physical Description 
Each ETBX vaccine is supplied as a sterile, clear 
solution in a 2-mL single-dose vial. The vaccine is 
provided at a concentration of 5 x 1011 VP per 1 mL and 
contains ARM formulation buffer (20 mM TRIS, 25 
mM NaCl, 2.5% glycerol, pH 8.0). Each vial contains 
approximately 1.1 mL of the vaccine. The product 
should be stored at ≤ -20°C. 
Manufacturer 
Etubics 
Table 4c 
Product Name(s): 
ETBX-051 (Ad5 [E1-, E2b-]-brachyury Vaccine) 
Dosage Form: 
Suspension for injection 
Unit Dose 
5 x 1011 VP, 1 x 1011 VP, or 5 x 1010 VP. 
Route of Administration 
SC injection 
Physical Description 
Each ETBX vaccine is supplied as a sterile, clear 
solution in a 2-mL single-dose vial. The vaccine is 
provided at a concentration of 5 x 1011 VP per 1 mL and 
contains ARM formulation buffer (20 mM TRIS, 25 
mM NaCl, 2.5% glycerol, pH 8.0). Each vial contains 
approximately 1.1 mL of the vaccine. The product 
should be stored at ≤ -20°C. 
Manufacturer 
Etubics 
 
The ETBX-071, ETBX-061, and ETBX-051 vaccines will be supplied by the manufacturer, 
Etubics Corporation, through a Cooperative Research and Development Agreement (CRADA). 

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The manufacturing department of Etubics will supply the vaccine, which will be distributed to the 
sites by Etubics Corporation. 
11.1.2 Toxicity 
The safety of immunizations (injections) witha combination of three immunotherapeutic vaccines 
(ETBX-071, ETBX-061, and ETBX-051), has not been established and will be determined in this 
phase I clinical trial. 
 
A Phase I/II clinical trial of ETBX-011 (Ad5 [E1-, E2b-]-CEA(6D)) (IND#14325) that expresses 
the tumor-associated antigen carcinoembryonic antigen (CEA) has been performed. A summary 
of the study results on the clinical trial is presented below. 
 
Schedule, Dosing, and Safety of ETBX-011: The primary objective of the Phase I/II dosing trial 
was to assess safety and a secondary objective was to evaluate CEA-specific immune responses to 
CEA and to obtain preliminary data on response rate. The study was performed under an FDA-
approved IND (IND14325) and registered at ClinicalTrials.gov (NCT01147965). Participants 
were recruited from oncology clinics at Duke University Medical Center and Medical Oncology 
Associates, Spokane, WA and provided informed consent that was approved by IRB’s. Patients 
with a histological confirmed diagnosis of metastatic malignancy who were previously treated with 
standard therapy known to have a possible survival benefit were enrolled into the study. For this 
study, the carcinoma must have had over expression of CEA as defined by immunohistochemical 
staining (at least 50% of the tumor with at least moderate intensity of staining) or a carcinoma 
known to be universally CEA positive i.e. colorectal adenocarcinoma). Patients were not treated 
until 4 or more weeks after any prior chemotherapy or radiation therapy.  They could not have a 
history of autoimmune disease, serious intercurrent chronic or acute illness, active hepatitis, 
serologic evidence for HIV infection, or be receiving steroid or immunosuppressive therapy.  All 
patients were >21 years old and had a Karnofsky Performance Score of 70% or higher and a life 
expectancy of at least 3 months. Pregnant women and nursing mothers were excluded. Dose 
Limiting Toxicities (DLTs) were defined as any Grade 3 or 4 immediate hypersensitivity reactions, 
Grade 3 or 4 fever that may possibly be associated with the immunization, Grade ≥2 autoimmune 
events except for vitiligo or fever for less than 2 days and less than 101.5 ºF, Grade ≥2 allergic 
reactions (Grade 2 is defined as generalized urticaria as defined by NCI Common Terminology 
Criteria for Adverse Events (CTCAE version 5), or Grade ≥3 non-hematologic toxicity. The Ad5 
[E1-, E2b-]-CEA(6D) injections were given subcutaneously in the same thigh. The doses were 
administered every 3 weeks for 3 treatments as follows: Cohort 1 (3 patients) 109 VP in 0.5 ml; 
Cohort 2 (3 patients): 1010 VP; Cohort 3 (6 patients) 1011 VP. Following establishment of safety 
in Phase I, 12 additional patients were entered into a Phase II using the 1011 VP/dose. To evaluate 
a higher dose, 6 additional patients (cohort 5) received 5x1011 VP/dose. 
Patient Demographics: One patient with CEA expressing bladder and one patient with lung 
carcinoma was enrolled. Thirty-two patients, median age 57.5 (range 38-77) with metastatic 
colorectal cancer, who had failed a median of three prior chemotherapeutic regimens (range: 2–5), 
had a median performance status of 90% (range 70-100%), and had a median of three sites of 
metastatic disease (range 1-5), were enrolled. The majority was able to receive all three 
immunizations. Five patients who stopped immunizations early did so due to significant disease 

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73 
 
progression. The colorectal adenocarcinoma patient demographics compares favorably with 
previously published studies of patients with chemotherapy-refractory colorectal cancer [44-46]. 
 
Adverse Effects: A total of 94 immunization treatments were administered to all patients. There 
was no dose limiting toxicity and no serious adverse events that resulted in treatment 
discontinuation at any dose level. The most common toxicity was a self-limited, injection site 
reaction. Other reactions that occurred at a low frequency include fever, flu-like symptoms, 
anorexia, chills, nausea, and headache. These symptoms were also self-limiting and did not require 
intervention other than symptomatic measures such as acetaminophen. There were no SAE 
associated with immunizations.  A summary of the adverse events reported on 34 patients treated 
and evaluated for safety are presented below. 
 
Adverse Events: ≥ 5% Frequency Based on Incidence of Total Treatments 
Adverse Events as 
of 07/31/12 
# 
of 
Events 
Unrelated/Unlikely 
Possible 
Probably/ 
Definite 
Incidence 
% 
(based on 94 
total 
treatments) 
 
Injection 
Site 
Reaction 
21 
2 
 
21 
22.3 
Flu-like symptoms 
10 
4 
5 
3 
10.6 
Fever 
9 
6 
1 
4 
9.5 
Fatigue 
8 
6 
2 
 
8.5 
Shortness of Breath 
6 
6 
 
 
6.3 
Pain 
6 
4 
 
 
6.3 
Anorexia 
5 
 
1 
 
5.3 
Chills 
5 
5 
1 
4 
5.3 
Constipation 
5 
5 
 
 
5.3 
Edema 
5 
4 
 
 
5.3 
Nausea 
5 
4 
1 
 
5.3 
 
Grade 3 and Grade 4 Adverse Events in ≥ 2% of Patients* 
Adverse Events 
Number of Grade 3 
(G3) 
 
Number of Grade 4 
(G4) 
Percent (Based on 34 
patients 
evaluated 
for safety) 
Pain (all types) 
2 
 
5.6 
Fatigue 
1 
 
2.9 
Anemia 
1 
 
2.9 
Pleural effusion 
1 
 
2.9 
Alkaline 
Phosphatase 
Increase 
1 
 
2.9 
Abdominal Bloating 
1 
 
2.9 
Bowel obstruction 
1 
 
2.9 
GI disorder 
1 
 
2.9 
Acute renal failure 
1 
 
2.9 

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74 
 
*Represents reported adverse events whether or not related to ETBX-011 vaccine 
 
Of 34 total patients entered into the trial, 28 received all three treatments and the blood hematology, 
chemistry, and ANA values at week 0 (prior to first treatment) were compared with those obtained 
at week 9 (three weeks after the third treatment). There were no biologically significant changes 
in chemistry, hematology, or ANA values. 
11.1.3 Formulation and Preparation 
Each vaccine is a clear colorless liquid filled in a 2-mL amber vial containing 1.0 mL of extractable 
vaccine. There are total of 5x1011 total virus particles (VP) in 1.0 mL of the product. Each vial is 
sealed with a rubber stopper and has a white flip off seal.  End user of the product will need to flip 
the white plastic portion of the cap up/off with their thumb to expose the rubber stopper, and then 
puncture the stopper with an injection needle to withdraw the liquid.  The rubber stopper is secured 
to the vial with an aluminum crimped seal. 
11.1.4 Stability and Storage 
Individual vials (in the desired number) of vaccine will be packaged in a cardboard box and will 
be shipped over dry ice (<-80 °C) by overnight courier with a temperature monitoring device 
included. Upon receipt, one will inspect contents of package for any noticeable damages or defects. 
Unpack the shipment contents and place the cardboard box containing vaccine vials into a freezer 
with a temperature control of d-20 °C. Receiver must stop the temperature monitoring device by 
turning off the power switch (instructions for handling and operation of temperature monitoring 
device will be provided with the package). 
11.1.5 Administration Procedures 
Please see section 3.2.4. Briefly, each ETBX-071, ETBX-061, and ETBX-051 vaccines will be 
administered on Weeks 0, 3, and 6 followed by booster vaccines every 8 weeks for up to a year in 
(only dose expansion cohor)t. All study drug administration treatments should occur within ± 7 
days of the planned visit date. 
Each of the three vaccines should be given by SC injection into a proximal limb, preferably in the 
thigh, after preparation of the site with alcohol. Preferably all three injections on a given day should 
be given to the anterolateral section of the same thigh and preferably the thigh chosen for injections 
should alternate between treatment days. Less preferably it is possible to give three injections to 
different limbs on a given day or to use the same thigh for consecutive treatment days. When the 
3 injections are given on a given day to the same limb (e.g. thigh) the injections must be separated 
by at least 5 cm and it is preferred that ETBX-071 is administerd most proximal, ETBX-061 in the 
middle, and ETBX-051 most distal. 
11.1.6 Incompatibilities 
The vaccines are administered as a separate subcutaneous injection, with no known 
incompatibilities. 
11.1.7 Other Considerations 
The Ad5 [E1-, E2b-] vector is non-replicating and its genome does not integrate into the human 
genome. However, since this is a non-replicating recombinant virus, it is recommended that it be 

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75 
 
handled under Biosafety Level-2 conditions. Any vialed ETBX vaccine material that has been used 
in the study should be autoclaved or incinerated after use according to institutional policy and 
according to local, state and federal regulations. Refer to the Material Safety Data Sheets for 
additional handling instructions. 

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

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
83 
 
13.2 APPENDIX B: INJECTION SITE REACTIONS DIARY CARD 
 
 
Please answer all questions below once per day for the first 7 days after each injection 
 
 
 
Day 0 
(Day of Injection) 
Day +1 
Day +2 
Day +3 
Day +4 
Day +5 
Day +6 
1. Is there 
redness 
at 
the injection 
site? 
Circle:  Yes 
/ No 
 
If 
yes 
measure 
longest 
diameter in 
millimeters 
(mm) 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
 
Yes / No 
 
 
___________mm 
 
2. Is there 
firmness or 
swelling at 
the 
injection 
site? 
Circle:  Yes 
/ No 
 
If 
yes 
measure 
longest 
diameter in 
millimeters 
(mm) 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
Yes / No 
 
 
___________mm 
 
3. Is there 
soreness at 
the 
injection 
site? 
Circle:  Yes 
/ No 
 
If yes tell us 
if 
the 
soreness is 
mild, 
moderate, 
or severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
4. Have you 
experienced 
pain at the 
injection 
site? 
Circle:  Yes 
/ No 
 
If yes tell us 
if the pain is 
mild, 
moderate, 
or severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
To be complete by site: 
Injection #: _________               Date of Injection: _____/_____/_____                 Subject Number: ________________ 
                                                                                     Month/Day/Yr         

Abbreviated Title: Ad5-based vaccines in mCRPC 
NCI Version Date: 09.20.2018 
 
84 
 
 
 
Day 0 
(Day of Injection) 
Day +1 
Day +2 
Day +3 
Day +4 
Day +5 
Day +6 
5. Have you 
taken 
any 
medication 
for injection 
site pain? 
Circle:  Yes 
/ No 
 
Provide 
name 
and 
dose 
of 
medication 
Yes / No 
 
Name: 
___________ 
 
Dose: 
____________ 
 
Yes / No 
 
Name: 
___________ 
 
Dose: 
____________ 
Yes / No 
 
Name: 
___________ 
 
Dose: 
____________ 
Yes / No 
 
Name: 
___________ 
 
Dose: 
____________ 
Yes / No 
 
Name: 
___________ 
 
Dose: 
____________ 
Yes / No 
 
Name: 
___________ 
 
Dose: 
____________ 
Yes / No 
 
Name: 
___________ 
 
Dose: 
____________ 
6. Have you 
experienced 
chills? 
Circle:  Yes 
/ No 
 
If yes tell us 
if the chills 
are 
mild, 
moderate, 
or severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
Yes / No 
 
Mild 
Moderate 
Severe 
7. 
Record 
your 
daily 
temperatur
e 
(Do 
not 
drink 
anything 
within 
5 
minutes 
prior 
to 
taking your 
temperature) 
Circle:  Yes 
/ No 
 
If 
your 
temperatur
e is 104OF 
for 
more 
than 
24 
hours, call 
your study 
doctor 
Record Temp: 
 
 
 
________________
_ 
Record Temp: 
 
 
 
________________
_ 
Record Temp: 
 
 
 
________________
_ 
Record Temp: 
 
 
 
________________
_ 
Record Temp: 
 
 
 
________________
_ 
Record Temp: 
 
 
 
________________
_ 
Record Temp: 
 
 
 
________________
_ 
 
 
 
 
 
 
 
 
Grading injection site pain: 
Mild            Noticeable, does not interfere with activity 
Moderate    Interferes with activity, limiting instrumental activities of daily living 
Severe         Limiting self-care activities of daily living, incapacitating 
Grading Chills: 
Mild             Mild sensation of cold, shivering, chattering of teeth 
Moderate     Moderate tremor of entire body, narcotics indicated 
Severe          Severe or prolonged, not responsive to narcotics