Multi-Targeted Recombinant Ad5 (CEA/MUC1/Brachyury) Based Immunotherapy Vaccine Regimen in People With Advanced Cancer
Phase: PHASE1
Conditions: Neoplasms, Prostate Cancer, Lung Cancer, Breast Cancer, Colon Cancer
Sponsor: National Cancer Institute (NCI)

Abbreviated Title: Ad5-based combination vaccines 
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Abbreviated Title: Ad5-based combination vaccines 
CC Protocol #: 18C0032 A  
 
 
 
 
 
 
Version Date: May 16, 2018 
IBC#: RD-17-VIII-13 
OSP#: 1712-1703 
 
 
NCT #: NCT03384316 
 
 
 
 
 
 
 
 
 
Title: Phase I Trial Using a Multi-Targeted Recombinant Ad5 (CEA/MUC1/Brachyury) 
Based Immunotherapy Vaccine Regimen in Patients with Advanced Cancer  
 
NCI Principal Investigator: 
Julius Strauss, MD 
Laboratory of Tumor Immunology & Biology (LTIB), 
CCR, NCI  
10 Center Drive 
Building 10, Room 13N240A 
Bethesda, MD 20892 
Phone: 301-480-0202  
Email: julius.strauss@nih.gov  
 
 
 
 
 
 
 
 
 
 
 
 
 
Investigational Agents:     
Drug Name: 
ETBX-011; adenoviral 
CEA vaccine 
ETBX-061; adenoviral 
MUC1 vaccine 
ETBX-051; adenoviral 
brachyury vaccine 
IND Number: 
17676 
17676 
17676 
Sponsor: 
Center for Cancer Research 
Center for Cancer Research 
Center for Cancer Research 
Manufacturer: 
Etubics 
Etubics 
Etubics 
 
 
 

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PRÉCIS 
Background: 
• The overall goal of the current project is to expand our immunotherapeutic approach for 
the treatment of advanced cancer employing a multi-targeted approach. 
• Therapeutic cancer vaccines targeting overexpressed proteins offer a potential method to 
activate T cells against tumors.   
• A novel adenovirus based vaccine targeting three (3) human tumor associated antigens 
(TAA), CEA, MUC1, and brachyury, respectively has demonstrated anti-tumor cytolytic 
T cell responses in pre-clinical animal models of cancer. 
 
Objectives: 
• To determine the overall safety and recommended phase 2 dose of a combination of three 
immunotherapeutic vaccines (ETBX-011/ETBX-061/ETBX-051), when administered 
subcutaneously (SC) to subjects with advanced solid tumors 
 
Eligibility: 
• Subjects age 18 and older with cytologically or histologically confirmed locally advanced 
or metastatic solid tumor malignancy who have completed or had disease progression on 
at least one prior line of disease-appropriate therapy or who are not candidates for therapy 
of proven efficacy for their disease. 
• Subjects may have measurable or non-measurable but evaluable disease. Subjects with 
surgically resected metastatic disease at high risk of relapse are also eligible. 
• ECOG performance status < 1 
• Adequate organ and bone marrow function 
• Subjects with active autoimmune diseases requiring systemic treatment and subjects 
requiring systemic steroids (except for physiologic doses for steroid replacement) are not 
allowed 
 
Design: 
• This is a Phase I trial in subjects with advanced cancer. A combination of three therapeutic 
vaccines (ETBX-011, ETBX-51, EBX-61) using the same modified Adenovirus vector 
backbone, separately encoding three well-studied tumor-associated antigens will be 
assessed. The vaccine will be tested at a single dose level, and a dose de-escalation design 
(if required). The dose level of each vaccine tested will be 5x1011 VP. This dose has been 
found in prior phase 1 testing of Ad5 [E1-, E2b-]-CEA(6D) (ETBX-011) to be well 
tolerated (with no dose-limiting toxicities (DLTs) or related Serious adverse events 
(SAEs), and optimal for induction of immune responses. Each of the three vaccines will be 
administered subcutaneously (SC) at separate injection sites (proximal limb, preferably the 
thigh), every 3 weeks for 3 doses, then bi-monthly (every 8 week) boosts for up to a year.  
• Up to six patients will be enrolled at Dose Level 1.  If ≤1 of 6 patients experience a DLT, 
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 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 

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will occur.  If ≥2 of 6 experience DLT at Dose Level -1, then a protocol amendment may 
be written to evaluate a further dose de-escalation. 
• A dose expansion phase of study will be enrolled after the MTD of the combination vaccine 
has been determined. An additional 4 subjects will be enrolled in the dose expansion 
component of the trial, for a total of 10 subjects at the MTD.  
• The ETBX-011, ETBX-51 and ETBX-61 vaccines will be administered SC every 3 weeks 
for 3 doses, and then bi monthly boosts for up to a year. Evaluations including 
immunological assessments will be carried out at baseline, on days of vaccination, and 
after the last vaccination. 
 
 

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TABLE OF CONTENTS 
PRÉCIS 
2 
TABLE OF CONTENTS .................................................................................................................4 
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 RATIONAL ...........................................................................................13 
1.2.1 Overall Objective ........................................................................................................13 
1.2.2 Brachyury Expression in Cancer and Its Use as an Immunotherapy Target ..............13 
1.2.3 CEA Expression in Cancer and Its Use as an Immunotherapy Target .......................14 
1.2.4 MUC1 Expression in Cancer and Its Use as an Immunotherapy Target ....................15 
1.2.5 Adenovirus-Based Vectors .........................................................................................15 
1.2.6 Clinical Experience with an Ad5 [E1-, E2b-]-based Cancer Immunotherapy Vaccine
 
16 
1.2.7 ETBX-081 Preclinical Studies ....................................................................................19 
1.2.8 Clinical Experience .....................................................................................................25 
1.2.9 Rationale .....................................................................................................................25 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT ........................................................25 
2.1 ELIGIBILITY CRITERIA ........................................................................................................25 
2.1.1 Inclusion Criteria ........................................................................................................25 
2.1.2 Exclusion Criteria .......................................................................................................26 
2.1.3 Recruitment Strategies ................................................................................................28 
2.2 SCREENING EVALUATION ...................................................................................................28 
2.3 REGISTRATION PROCEDURES ..............................................................................................28 
2.3.1 Treatment Assignment Procedure ...............................................................................29 
2.4 BASELINE EVALUATION ......................................................................................................29 
3 
STUDY IMPLEMENTATION ..............................................................................................29 

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3.1 STUDY DESIGN ...................................................................................................................29 
3.1.1 Dose Limiting Toxicity ...............................................................................................30 
3.1.2 Dose De-Escalation ....................................................................................................30 
3.2 ADMINISTRATION AND DURATION OF TREATMENT .............................................................33 
3.3 DOSE DELAY OR DISCONTINUATION ...................................................................................33 
3.4 DOSE MODIFICATIONS ........................................................................................................33 
3.5 STUDY CALENDAR ..............................................................................................................34 
3.6 INJECTION SITE REACTIONS ................................................................................................37 
3.7 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA ..............37 
3.7.1 Criteria for removal from protocol therapy ................................................................37 
3.7.2 Off-Study Criteria .......................................................................................................37 
3.7.3 Off Protocol Therapy and Off-Study Procedure .........................................................37 
4 
CONCOMITANT MEDICATIONS/MEASURES ................................................................38 
4.1 CONCURRENT MEDICATIONS/INTERVENTIONS ....................................................................38 
4.1.1 Anticancer Therapy ....................................................................................................38 
4.1.2 Other Medications ......................................................................................................38 
5 
BIOSPECIMEN COLLECTION............................................................................................39 
5.1 CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES ...............................39 
5.1.1 Peripheral Blood Collection .......................................................................................39 
5.1.2 Samples Collected ......................................................................................................39 
5.1.3 Sample Processing ......................................................................................................39 
5.1.4 Sample Tracking, and Disposition ..............................................................................39 
5.1.5 Pharmacodynamic Sample Management and Storage ................................................39 
5.1.6 Sample Analysis .........................................................................................................40 
5.1.7 Protocol Completion/Sample Destruction ..................................................................41 
6 
DATA COLLECTION AND EVALUATION ......................................................................42 
6.1 DATA COLLECTION .............................................................................................................42 
6.2 DATA SHARING PLANS........................................................................................................42 
6.2.1 Human Data Sharing Plan ..........................................................................................42 
6.3 EFFICACY ASSESSMENTS ....................................................................................................43 
6.3.1 Antitumor Response ...................................................................................................43 
6.3.2 Disease Parameters .....................................................................................................43 

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6.3.3 Methods for Evaluation of Measurable Disease .........................................................44 
6.3.4 RECIST Response Criteria .........................................................................................46 
6.3.5 Target Response ..........................................................................................................46 
6.3.6 Duration of Response .................................................................................................48 
6.3.7 Progression-Free Survival (PFS) ................................................................................48 
6.4 TOXICITY CRITERIA ............................................................................................................48 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
 
49 
7.1 DEFINITIONS .......................................................................................................................49 
7.1.1 Adverse Event .............................................................................................................49 
7.1.2 Suspected adverse reaction .........................................................................................49 
7.1.3 Unexpected adverse reaction ......................................................................................49 
7.1.4 Serious ........................................................................................................................49 
7.1.5 Serious Adverse Event ................................................................................................49 
7.1.6 Disability .....................................................................................................................50 
7.1.7 Life-threatening adverse drug experience ..................................................................50 
7.1.8 Protocol Deviation (NIH Definition) ..........................................................................50 
7.1.9 Non-compliance (NIH Definition) .............................................................................50 
7.1.10 Unanticipated Problem ...............................................................................................50 
7.2 NCI-IRB AND CLINICAL DIRECTOR (CD) REPORTING .......................................................50 
7.2.1 NCI-IRB and CD Expedited Reporting of Unanticipated Problems and Deaths .......50 
7.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review ...............................51 
7.2.3 NCI-IRB Reporting of IND Safety Reports ...............................................................51 
7.3 IND SPONSOR REPORTING CRITERIA ..................................................................................51 
7.3.1 Reporting Pregnancy ..................................................................................................51 
7.4 SAFETY REPORTING CRITERIA TO THE PHARMACEUTICAL COLLABORATORS .....................52 
7.4.1 Etubics Corporation ....................................................................................................52 
7.5 INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) REPORTING CRITERIA ...............................53 
7.5.1 Serious Adverse Event Reports to IBC ......................................................................53 
7.5.2 Annual Reports to IBC ...............................................................................................53 
7.6 DATA AND SAFETY MONITORING PLAN ..............................................................................54 
7.6.1 Principal Investigator/Research Team ........................................................................54 
7.6.2 Sponsor Monitoring Plan ............................................................................................54 

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7.6.3 Safety Monitoring Committee (SMC) ........................................................................55 
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STATISTICAL METHODS ...................................................................................................55 
8.1 STATISTICAL HYPOTHESIS ..................................................................................................55 
8.2 SAMPLE SIZE DETERMINATION ...........................................................................................55 
8.3 POPULATION FOR ANALYSES ..............................................................................................56 
8.4 STATISTICAL ANALYSES .....................................................................................................56 
8.4.1 General Approach .......................................................................................................56 
8.4.2 Analysis of Primary Efficacy Endpoints ....................................................................56 
8.4.3 Analyses of Secondary Endpoint ................................................................................56 
8.4.4 Safety Analysis ...........................................................................................................57 
8.4.5 Baseline Descriptive Statistics ....................................................................................58 
8.4.6 Planned Interim Analyses and Halting Guidelines .....................................................58 
8.4.7 Sub-Group Analyses ...................................................................................................58 
8.4.8 Tabulation of Individual Participant Data ..................................................................58 
8.4.9 Exploratory Analyses (Immune Responses) ...............................................................58 
9 
COLLABORATIVE AGREEMENTS ...................................................................................59 
9.1 COOPERATIVE RESEARCH AND DEVELOPMENT AGREEMENT (CRADA) ............................59 
10 HUMAN SUBJECTS PROTECTIONS .................................................................................59 
10.1 RATIONALE FOR SUBJECT SELECTION .................................................................................59 
10.1.1 Selection Based on Gender, Ethnicity, and Race .......................................................59 
10.1.2 Strategies/Procedures for Recruitment .......................................................................59 
10.1.3 Justification for Exclusions .........................................................................................59 
10.2 PARTICIPATION OF CHILDREN .............................................................................................59 
10.3 PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT ..................................................59 
10.4 EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS ........................................................60 
10.5 CONSENT PROCESS AND DOCUMENTATION.........................................................................60 
10.5.1 Telephone re-consent procedure .................................................................................60 
10.5.2 Consent process for non-English speaking subjects ...................................................61 
11 PHARMACEUTICAL INFORMATION ..............................................................................61 
11.1 COMBINATION ETBX-011, ETBX-051, ETBX-061 VACCINE REGIMEN............................61 
11.1.1 Source .........................................................................................................................63 
11.1.2 Toxicity .......................................................................................................................63 

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11.1.3 Formulation and Preparation ......................................................................................65 
11.1.4 Stability and Storage ...................................................................................................65 
11.1.5 ETBX Vaccine Dose Preparation and Administration ...............................................65 
11.1.6 Incompatibilities .........................................................................................................66 
11.1.7 Other Considerations ..................................................................................................66 
12 REFERENCES .......................................................................................................................68 
13 APPENDICES ........................................................................................................................74 
13.1 APPENDIX A: PERFORMANCE STATUS CRITERIA .................................................................74 
13.2 APPENDIX B: INJECTION SITE REACTIONS DIARY CARD .....................................................75 
 
 
 
 

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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation or Specialist Term 
Explanation 
-HCG 
-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 
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 
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 
DLTs 
Dose-limiting toxicities 
E1 
Adenovirus early 1 gene 
E2b 
Adenovirus early 2b gene 

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Abbreviation or Specialist Term 
Explanation 
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-081 
Combination of ETBX-011 + ETBX-051 + ETBX-061 Vaccines 
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 
HTD 
Highest tested dose 
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 
LLD 
Longest lesion diameter 
LLN 
Lower limit of normal 

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Abbreviation or Specialist Term 
Explanation 
MDSC 
Myeloid-derived suppressor cell 
MedDRA 
Medical Dictionary for Regulatory Activities 
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 
NK 
Natural killer (cell) 
ORR 
Objective response rate 
OS 
Overall survival 
PBMC 
Peripheral blood mononuclear cell 
PD 
Progressive disease 
PFS 
Progression-free survival 
PR 
Partial response 
PT 
Prothrombin 
PTT 
Partial thromboplastin time 
aPTT 
Activated partial thromboplastin time 
RECIST 
Response Evaluation Criteria in Solid Tumors 
SAE 
Serious adverse event 
SC 
Subcutaneous 
sCD27 
Soluble CD27 
sCD40L 
Soluble CD40L 
SD 
Stable disease 
SOC 
System Organ Class 
SRC 
Safety Review Committee 
SUSAR 
Suspected unexpected serious adverse reactions 
TAA 
Tumor-associated antigen 
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 
To determine the overall safety and recommended phase 2 dose of a combination of three 
immunotherapeutic vaccines (ETBX-011, ETBX-061, and ETBX-051), when administered 
subcutaneously (SC) to subjects with advanced solid tumors. 
1.1.2 Secondary Objectives  
To make preliminary assessments of: 
• Objective response rate (ORR),  
• Disease control rate (DCR),  
• Duration of response,  
• Progression-free survival (PFS)  
• Overall survival (OS)  
in subjects with advanced solid tumors treated with the combination ETBX-011, ETBX-051, 
ETBX-061 vaccine regimen.  
1.1.3 Exploratory Objectives 
To evaluate the ability of the combination ETBX-011, ETBX-051, ETBX-061 vaccine regimen to 
generate T-cell responses specific for Brachyury, MUC1, and CEA. 
1.1.4 Primary Endpoints 
• Dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) or highest tested 
dose (HTD). 
• Treatment-emergent adverse event (AEs) and serious adverse events (SAEs). 
• Clinically significant changes in safety laboratory tests, physical examinations, 
electrocardiograms (ECGs), and vital signs. 
1.1.5 Secondary Endpoints 
• Objective response rate (ORR; confirmed complete or partial response) according to 
the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1. 
• Disease control rate (DCR; confirmed response or stable disease [SD] lasting for at 
least 6 months). 
• Duration of response. 
• PFS 
• OS 
1.1.6 Exploratory Endpoints 
• Immunogenicity via the induction of antigen-specific T cells of the combination 
ETBX-011, ETBX-051, ETBX-061 vaccine regimen by flow cytometric analysis, and 
analysis in sera of sCD27 and sCD40L. 

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1.2 
BACKGROUND AND RATIONAL 
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, our efforts are focused on 
developing multi-targeted approaches. The overall goal of the current project is to expand our 
immunotherapeutic approach for the treatment of advanced cancer employing a multi-targeted 
approach. Safety and ability to generate CEA-specific T-cell responses in metastatic colorectal 
cancer (mCRC) patients using our Ad5 [E1-, E2b-]-CEA(6D) (ETBX-011) immunotherapeutic as 
a single agent has been achieved in a clinical setting(1, 2). 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(3-7). 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 CEA, MUC1, and/or Brachyury. Preclinical studies were recently published indicating 
that this multi-targeted CEA, MUC1, Brachyury Ad5 combination vaccine induces immune 
responses directed against all three target CEA, MUC1, and Brachyury antigens with minimal to 
no “antigenic competition” in human in vitro studies or in murine vaccination studies(8). Testing 
of a combination multi-targeted CEA, MUC1, and Brachyury targeted adenoviral vector-based 
vaccines in a phase 1 clinical trial is planned to test the safety, ability to generate T-cell responses 
to each tumor antigen, and efficacy of the immunotherapeutic. Subsequent trials will involve the 
use of this vaccine in combination with checkpoint inhibitor monoclonal antibodies (MAbs) and 
other immune modulators.  
1.2.2 Brachyury Expression in Cancer and Its Use 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(9). 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 (10, 11), aberrantly high levels of Brachyury have been observed in the primary 
and/or metastatic sites of non-small cell (NSCLC) and small cell (SCLC) lung cancer (12, 13), 
colon (14), hepatocellular (15), prostate (16) and breast carcinomas (17), including triple 
negative breast cancer (TNBC) (18). High levels of Brachyury are also characteristic of the rare 
tumor type chordoma (19, 20), which is thought to originate from remnants of the embryonic 
notochord where Brachyury is normally found. Recent studies have now characterized the role of 

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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”) (21, 22). Carcinoma cells undergoing this phenotypic transition exhibit 
enhanced motility and invasiveness in vitro, propensity to metastasize in vivo, and features of 
tumor stemness (23), including resistance to a range of therapeutics such as chemotherapy, 
radiation, small molecule therapies and, potentially, immunotherapy (24-27). 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 (28), colon (14), breast (17), triple negative breast (18) and gastrointestinal stromal 
tumor (GIST) (29). Brachyury expression has also been shown to be correlated with advanced 
stage prostate cancer (16).  
 
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 (30, 31). 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 (30, 31). 
 
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(32). 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, 
two recently completed Phase I clinical studies with a recombinant yeast-Brachyury vaccine(33) 
or an MVA-Brachyury-TRICOM vaccine(33, 34) also demonstrated the generation of Brachyury-
specific T cells as well as safety in humans, thus providing further evidence of 
immunogenicity(35). 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. 
1.2.3 CEA Expression in Cancer and Its Use as an Immunotherapy Target  
CEA represents an attractive target for immunotherapy since it is over expressed on all metastatic 
colorectal cancer (mCRC) adenocarcinomas and can be overexpressed in other cancers including 
breast, lung, gastric, pancreatic, bladder, medullary thyroid, head and neck, cervical, hepatic, 
lymphoma, and melanoma. It has also been identified as one of the priority cancer antigens most 
likely to be successful at generating a cancer immunotherapeutic(36). A reason that CEA is a good 
target for T cell-mediated immunity in humans is that it contains known epitopes recognized via 
an MHC-restricted fashion by human cytolytic T lymphocytes (CTL) that bind to MHC loci HLA-

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A2, A3, and A24(8, 37). Of the HLA-A2 restricted epitopes of CEA, CAP1, a nine amino acid 
sequence, has been reported to stimulate CTL in cancer patients immunized with vaccinia-CEA. 
CAP1(6D) is a peptide analog of CAP1. Its sequence includes a heteroclitic (non-anchor position) 
mutation, resulting in an amino acid change from asparagine (Asn) to aspartic acid (Asp), to 
enhance recognition by the T-cell receptor without any change in binding to HLA A2. Compared 
with the non-mutated CAP1 epitope, CAP1(6D) has been shown to enhance sensitization of CTL 
by 100 to 1,000 times(37). The CAP1(6D) epitope has been incorporated into the vector platform 
(Ad5 [E1-, E2b-]-CEA(6D)) and has been safely tested in mCRC patients to induce active cell-
mediated immunity (CMI) against CEA(1, 2).  
1.2.4 MUC1 Expression in Cancer and Its Use as an Immunotherapy Target 
MUC1 (CD227) is a TAA that is overexpressed on a majority of human carcinomas and several 
hematologic malignancies(38-41). MUC1 is normally expressed at the surface of glandular 
epithelial cells(42) and, in carcinomas, it is overexpressed and aberrantly hypoglycosylated(40, 
42, 43). Several clinical trials have been and are being performed to evaluate the use of MUC1 in 
immunotherapeutic vaccines(2, 44-46). Some of these trials have indicated that targeting MUC1 
is safe and may provide survival benefit(2, 45, 47). Multiple enhancer agonist epitopes were 
previously identified, several of which are in the MUC1 C-terminus region(48, 49). This is 
potentially important because studies(47) have demonstrated that 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(48, 49). Therefore, it is believed that MUC1 containing modified 
agonist epitopes has a greater potential as an immunogenic agent for vaccine development. 
1.2.5 Adenovirus-Based Vectors 
Adenoviruses (Ads) have emerged as leading candidate vectors to deliver vaccines designed to 
induce CMI and antibody responses (50-52). Ad vectors infect multiple cell types, including 
dendritic cells which result in priming of a vigorous e response. Ads are a family of DNA viruses 
characterized by an icosahedral, non-enveloped capsid containing a linear double-stranded genome 
(51, 52). 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 (50-53). 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 (51). There 

Abbreviated Title: Ad5-based combination vaccines 
Version Date: 05.16.2018 
 
16 
 
have been over 300 human clinical trials that utilized Ad5 [E1-] vectors, with more than 2000 
subjects given the virus SC, intramuscularly, or intravenously (52). 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 (51, 53). Most humans have antibodies against 
Ad5, with up to two-thirds having lymphoproliferative responses against Ad5 (54, 55). 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. 
1.2.5.2 New Generation Ad5 Vectors (Ad5 [E1-, E2b-]) 
A new and advanced generation of Ad5 vectors has 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-
]) (56-59). 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 (2, 
8, 59-68). 
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(1, 2). 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 
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(1, 2).  
 
 

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17 
 
1.2.6.2 Immune Responses 
A secondary objective of the Ad5 [E1-, E2b-]-CEA(6D) Phase I trial was to evaluate CEA-specific 
immune responses following vaccination. As determined by an ELISA technique(1, 2), no 
antibody activity directed against CEA was observed. CEA-specific cell-mediated immunity 
(CMI) responses were observed 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 3 weeks following the last treatment from patients. CEA-specific ELISpot 
assays were performed on PBMC as previously described(1, 2, 61) to determine the numbers of 
interferon gamma (IFN-) secreting lymphocytes (SFC) after exposure to CEA peptides in vitro. 
This analysis revealed a dose response to increasing levels of vaccine (Figure 1). The highest CMI 
levels occurred in patients that received the highest dose of 5x1011 VP (Cohort 5).  
 
In(1), a population of polyfunctional CD8+ T cells (those that secrete more than 1 cytokine when 
activated) was identified that after immunizations secreted multiple cytokines, a sign of greater 
functionality of T cells induced by the vaccine. In further follow-up analysis(1) 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. 
 
Anti-Ad5 antibody (Ab) and CMI against Ad5 were 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 Ab titer value obtained among all patients was 
1:189 1:71 SEM and the mean pre-treatment Ad5 Ab titer among seropositive patients was 1:308 
 1:108. Analysis of serum samples from patients who received 3 immunizations revealed Ad5 Ab 
titers that were significantly increased (P<0.0001, Mann-Whitney test) by week 9 (mean 1:4767  
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 ± 9.3 SEM IFN-γ 
secreting SFC at week 0 versus 191.1 ± 83.7 IFN- γ SFC at week 9).  
 

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1.2.6.3 Analysis of Clinical Activity 
The Ad5 [E1-, E2b-]-CEA(6D) vaccinated heavily pretreated colorectal cancer patients (total=32) 
were followed for survival and Kaplan-Meier plots and survival proportions performed (PRISM 
software)(1, 2). Events were determined by information from the social security death index 
(SSDI) database, clinical charts and telephone calls (Figure 2).  
 
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 2a) 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 2b) 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 
2c). 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. 
 
Pre
Post
Pre
Post
Pre
Post
Pre
Post
0
100
200
300
400
500
Cohort 1
Cohort 2
Cohort 3/Phase II
Cohort 5
IFN SFC per 106 PBMC
 
Figure 1: CEA-specific T-cell responses (CMI) in treated patients. CMI (IFN- 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 -
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  SEM for each Cohort. 
 

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19 
 
 
 
1.2.7 ETBX-081 Preclinical Studies 
Recombinant Ad5 [E1-, E2b-]–CEA, recombinant Ad5 [E1-, E2b-]–MUC1 and recombinant Ad5 
[E1-, E2b-]–Brachyury were generated and characterized as previously described(8). As seen in 
Figure 3A, Western blot analysis using an anti-Brachyury–specific monoclonal antibody (MAb 
54-1)(11) revealed Brachyury expression when human dendritic cells (DCs) were infected with 
Ad5 [E1-, E2b-]–Brachyury. An Ad5 [E1-, E2b-] vector devoid of any transgene (Ad5 [E1-, E2b-
]–null) was used as a negative control and SW620 human colon carcinoma cells that endogenously 
express Brachyury were used as a positive control. An anti-MUC1–specific MAb was used to 
detect the expression of MUC1 in Ad5 [E1-, E2b-]–MUC1–infected human DCs (Figure 3B). 
SW620 cells, which also express MUC1 endogenously, were used as a positive control. The 
difference in molecular weights seen in the human DCs versus the SW620 human carcinoma cells 
is most likely due to the differential glycosylation of the MUC1 protein. As has been previously 
shown by others(69-72), it would appear that MUC1-C is being expressed in the human DCs 
predominantly as the unglycosylated 17 or 15 kDa form and not the 25-20 glycosylated species. A 
Western blot of Ad5 [E1-, E2b-]–CEA infected human cells is shown in Figure 3. 
 
The generation of Brachyury-, CEA-, and MUC1-specific human CD8+ T cells employing the 
corresponding peptide for each TAA was previously reported(30, 31, 37, 48, 49, 73). As shown in 
Table 1, Ad5 [E1-, E2b-]–null did not activate any of the T cells to produce IFN-γ. Ad5 [E1-, E2b-
]–Brachyury–infected DCs activated Brachyury-specific T cells and not CEA-specific T cells (as 
a negative control). This demonstrates that the Ad5 [E1-, E2b-]–Brachyury–infected DCs could 
process Brachyury in a manner that generates Brachyury–MHC Class I complexes capable of 
specific T-cell activation. Similarly, Ad5 [E1-, E2b-]–CEA–infected DCs specifically activated 
CEA-specific T cells but not MUC1-specific T-cell lines. Both Class I HLA-A2 and -A24 MUC1-
specific T-cell lines have been previously generated(49) and the Ad5 [E1-, E2b-]–MUC1–infected 
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 2: 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 vaccines. Panel c 
represents patients vaccinated 3 times with the 2 highest doses of vaccine. There were 23 events during 
the study. 
  

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Version Date: 05.16.2018 
 
20 
 
DCs were capable of activating both of these T-cell lines but not the CEA-specific T-cell line 
(Table 1). Human DCs were similarly infected with the ETBX-081 vector. As seen in Table 1 A 
and Table 1 B, T cells specific for CEA, MUC1, and Brachyury were each activated to induce 
similar levels of IFN-γ as seen with the use of the individual Ad-5 vectors. 
 
 
 
Studies were also undertaken to determine whether simultaneous infection of human DCs with the 
CEA/MUC1/Brachyury mixture of ETBX-081 could generate T-cell lines specific for all three 
TAAs. As seen in Table 2, when the T cells were activated by incubation with autologous B cells 
pulsed with the corresponding peptide, and not a control peptide, specific T-cell activation was 
observed. For example, the Brachyury-specific T-cell line, generated by infecting human DCs with 
ETBX-081, was stimulated to produce IFN-γ when incubated with autologous DCs pulsed with 
Brachyury peptide, but was not activated with the same autologous DCs pulsed with a CEA 
peptide. Similar results were seen with CEA and MUC1 T-cell lines generated with ETBX-081–
infected DCs. These results indicate the lack of so-called “antigenic competition” in the in vitro 
use of ETBX-081. 
C 
Figure 3: Expression of Brachyury and MUC1 protein in human dendritic cells (DCs) infected with Ad5 
[E1-, E2b-]−Brachyury and Ad5 [E1-, E2b-]−MUC1. SW620 tumor cells were used as positive control. 
Actin was used as a loading control. (A) Expression of Brachyury was robust in DCs infected with Ad5 [E1-
, E2b-]-Brachyury. (B) MUC1 expression was observed in human DCs infected with Ad5 [E1-, E2b-
]−MUC1 vector as compared to DCs infected with Ad5 [E1-, E2b-]–null (no transgene). (C) Expression of 
CEA in A549 cells infected with Ad5 [E1-, E2b-]-CEA.  A549 cells were infected with Ad5 [E1-, E2b-]-
CEA and CEA expression was confirmed by western blot analysis. Recombinant CEA was used as a positive 
control and uninfected A549 cells served as a negative control. The samples are visualized below in the 
following order A. Negative Control, B. Magic Mark XP Western Marker, C. Negative, D. CEA Reference 
Material (30ng), E. Ad5 [E1-, E2b-]-CEA lysate (20uL), F. Ad5 [E1-, E2b-]-CEA lysate (20uL), G. Negative 
A549 cells. 

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21 
 
Table 1 
Table 1 A: Infection of Human Dendritic Cells with Recombinant Adenovirus Vectors 
Encoding CEA, MUC1 or Brachyury Can Activate Antigen-specific T-cell Lines 
Dendritic cells (DCs) infected with 
Antigen-specific T-cell lines 
 
CEA 
MUC1 
(HLA-A2) 
MUC1 
(HLA-A24) 
Brachyury 
Ad5 [E1-, E2b-]–null 
<15.6 
<15.6 
<15.6 
<15.6 
Ad5 [E1-, E2b-]−Brachyury 
<15.6 
-- 
-- 
351.9 
Ad5 [E1-, E2b-]−MUC1 
<15.6 
335.2 
806.4 
-- 
Ad5 [E1-, E2b-]−CEA 
350.0 
<15.6 
<15.6 
-- 
Uninfected DCs 
<15.6 
<15.6 
<15.6 
<15.6 
T cells only 
<15.6 
<15.6 
<15.6 
<15.6 
 
Human DCs (6-day culture in IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) 
2x104 cells/well in 0.5 ml of AIM-V) were infected with indicated adenovirus vectors at 20,000 multiplicity 
of infection (MOI). After 48 hours, DCs were washed and used for stimulation of human antigen-specific 
T cells. Results are expressed in pg/ml of IFN-γ per 1x105 T cells/ml. Numbers in bold indicate a significant 
enhancement of IFN-γ secretion compared to corresponding wells with uninfected DCs. [-- indicates that 
the assay was not performed.] 
 
Table 1 B: Infection of Human Dendritic Cells with ETBX-081 Vectors Encoding 
Transgenes Can Activate Antigen-specific T-cell Lines to Produce IFN-γ 
 
Dendritic cells (DCs) infected with 
Antigen-specific T-cell lines 
 
CEA 
(HLA-A2) 
MUC1 
(HLA-A2) 
MUC1 
(HLA-A24) 
Brachyury 
(HLA-A2) 
ETBX-081  
480 
236 
763 
496 
Ad5 [E1, E2b]–null 
<15.6 
<15.6 
<15.6 
<15.6 
Uninfected DCs 
<15.6 
<15.6 
<15.6 
<15.6 
T cells only 
<15.6 
<15.6 
<15.6 
<15.6 
 
Human DCs (6-day culture in IL-4 and GM-CSF) from an HLA-A2 and –A24 donor were infected with ETBX-
081 vector at 2 x 104 /well (24-well plate) in 0.5 ml of AIM-V. ETBX-081 vectors were used at 20,000 MOI for 
1 hour and then 1.5 ml of AIM-V were added to each well. Infected DCs were incubated for 48 hours and then 
washed and used for stimulation of human antigen-specific T cells. Results are expressed in pg of IFN-γ per 1 x 
105 T cells/ml. Numbers in bold indicate a significant enhancement of IFN-γ secretion compared to 
corresponding wells with uninfected DCs. 
 

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Table 2: Infection of Human Dendritic Cells with ETBX-081 Can Generate Antigen-specific T 
Cells to Brachyury, MUC1 and CEA and Produce IFN-γ When Stimulated with 
Autologous B Cells Pulsed with the Corresponding Peptides 
  
 
Peptides (10 μg/ml) 
Antigen-specific T-cell lines 
CEA 
MUC1 
(A2)  
MUC1 
(A24)  
Brachyury 
T-Brachyury 
<15.6 
-- 
-- 
243 
T-MUC1 (A2) 
<15.6 
174 
-- 
-- 
T-MUC1 (A24) 
<15.6 
-- 
206 
-- 
T-CEA 
211 
<15.6 
-- 
-- 
 
Human dendritic cells (DCs) from a prostate cancer patient (6-day culture in IL-4 and granulocyte-macrophage 
colony-stimulating factor (GM-CSF) 2x104 cells/well in 0.5 ml of AIM-V) were infected with ETBX-081 at 
20,000 MOI. After 48 hours, infected DCs were washed and used to generate specific cytotoxic T lymphocytes 
(CTLs) using autologous peripheral blood mononuclear cells (PBMCs) as effectors. Following 3 cycles of in 
vitro stimulations, autologous peptides-pulsed B cells were used as antigen-presenting cells. Results are 
expressed in pg/ml of IFN-γ. [-- indicates that the assay was not performed.] 
 
 
Table 3: Infection of Human DCs with ETBX-081 Can Generate Brachyury-, MUC1- and 
CEA-specific CTLs That Efficiently Lyse Tumor Cells Expressing All Three 
Antigens 
 
 
 
Antigen-specific T-cell lines 
SW620  
Brachyury+ MUC1+ CEA+ 
(HLA-A2+/A24+) 
ASPC-1  
Brachyury+ MUC1+ CEA+ 
(HLA-A1+/A26+) 
T-Brachyury 
64.4 (3.6) 
8.3 (2.7) 
T-MUC1 (P93L) 
28.5 (1.3) 
2.0 (1.6) 
T-MUC1 (C6A)  
49.3 (3.3) 
5.0 (1.8) 
T-CEA 
42.4 (3.7) 
4.3 (1.9) 
 
Human dendritic cells (DCs) were infected with ETBX-081 at 20,000 MOI. Infected DCs were used to generate 
specific cytotoxic T lymphocytes (CTLs) using autologous peripheral blood monoclonal cells (PBMCs). 
Autologous DCs were used as antigen-presenting cells for three in vitro stimulations (IVS). Autologous peptide-
pulsed B cells pulsed were used to re-stimulate antigen-specific CTLs for two additional IVS. The effector-to-
target ratio used was 30:1; CTLs were used at IVS 5. Results are expressed in % specific lysis (SD). 
 
Studies also investigated whether Brachyury-, MUC1–, and CEA-specific human T cells generated 
using DCs infected with ETBX-081 could lyse human carcinoma cells that endogenously express 
these TAAs. SW620 human colon carcinoma cells express all three TAAs and possess the HLA-

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23 
 
A2 and -A24 Class I alleles. ASPC-1 human pancreatic carcinoma cells were used as a negative 
control since they express the three TAAs but in the context of HLA-A1 and -A26 molecules. The 
results (Table 3) demonstrated that ETBX-081–infected human DCs can generate T cells capable 
of lysing, in an MHC-restricted manner, human tumor cells that endogenously express Brachyury, 
CEA, and MUC1.  
 
Studies were also undertaken to determine whether Ad5 [E1-, E2b-]–Brachyury, Ad5 [E1-, E2b-
]–MUC1, and Ad5 [E1-, E2b-]–CEA could each generate TAA-specific T-cell responses in vivo, 
and whether the ETBX-081 mixture could generate comparable responses. C57Bl/6 mice (n=5 per 
group) were injected subcutaneously (s.c.) three times at 2-week intervals with 1010 viral particles 
(VP) of Ad5 [E1-, E2b-]–CEA, Ad5 [E1-, E2b-]–MUC1, Ad5 [E1-, E2b-]–Brachyury, or ETBX-
081 (1:1:1 mixture of 1010 VP each). An additional group of mice (n=5) received 3x1010 VP of 
Ad5 [E1-, E2b-]–null (an empty vector control). Two weeks after the final vaccination, splenocytes 
from vaccinated mice were stimulated with corresponding Brachyury, CEA, or MUC1 peptide 
pools and analyzed for IFN-γ and IL-2 secreting cells by the enzyme-linked immunospot 
(ELISPOT) assay. Mice vaccinated with singular constructs or with ETBX-081 responded to 
Brachyury, CEA, and MUC1 peptides, respectively, with significant increases in IFN-γ and IL-2 
spot forming cells (SFCs) as compared to control mice (Figure 4A and B). There was no 
significant difference in the average number of IFN-γ SFCs in mice vaccinated with Ad5 [E1-, 
E2b-]–Brachyury or Ad5 [E1-, E2b-]–CEA individually as compared with the ETBX-081 vaccine. 
There was a significant decrease in IFN-γ SFCs in mice treated with the ETBX-081 vaccine as 
compared to Ad5 [E1-, E2b-]–MUC1 alone, although the MUC1–specific immune response 
induced by ETBX-081 remained significantly elevated over control mice (p < 0.0001) (Figure 
4A). IL-2 responses were similar in mice treated with ETBX-081 versus single vaccine constructs; 
moreover, there was a significant increase (p = 0.004) in CEA-specific IL-2 SFCs when mice were 
vaccinated with the ETBX-081 vaccine versus the Ad5 [E1-, E2b-]–CEA vaccine alone (Figure 
4B). Taken together, these data indicate that combining Ad5 [E1-, E2b-]–Brachyury, Ad5 [E1-, 
E2b-]–CEA, and Ad5 [E1-, E2b-]–MUC1 in a ETBX-081 vaccine admixture has the effect of 
generating antigen-specific IFN-γ– and IL-2–producing cells similar to that achieved when using 
each vaccine alone. 
 
Figure 4: Analysis of IFN-γ− and IL-2−expressing splenocytes following vaccination of mice with 
Ad5 [E1-, E2b-]−Brachyury, Ad5 [E1-, E2b-]−CEA, Ad5 [E1-, E2b-]−MUC1, ETBX-081, and Ad5 
[E1-, E2b-]–null. C57Bl/6 mice (n = 5/group) were vaccinated three times at 2-week intervals with 
1010 VP (viral particle) of Ad5 [E1-, E2b-]−Brachyury (white bar), Ad5 [E1-, E2b-]−CEA (grey bar), 
Ad5 [E1-, E2b-]−MUC1 (black bar) or ETBX-081 (1:1:1 mixture of 1010 VP each of Ad5 [E1-, E2b-
]−Brachyury, Ad5 [E1-, E2b-]−CEA, Ad5 [E1-, E2b-]−MUC1) (diagonal hatched bar). Controls 
received 3x1010 VP of Adeno-null (horizontal striped bar). Splenocytes were collected 14 days after 
the final vaccination and assessed for IFN-γ−secreting cells (A) or IL-2-secreting cells (B) by 
ELISPOT assay. For positive controls, splenocytes were exposed to Concanavalin A (Con A) (data not 
shown). Data reported as the number of spot forming cells (SPFs) per 106 splenocytes. The error bars 
depict the SEM. Significant differences (p < 0.05) between columns are reported in p-values, not 
significant = ns. 
 

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Studies were then undertaken to determine whether the ETBX-081 vaccine regimen was as 
effective as the use of a single recombinant adenovirus construct in eliciting an anti-tumor effect. 
It should be noted that unlike human carcinomas of which the majority overexpress the human 
TAAs CEA, MUC1 and Brachyury, no mouse models express all three of these antigens. C57BL/6 
mice (n = 7/group) were implanted s.c. with 1x106 MC38 cells expressing MUC1 (MC38-MUC1) 
in the left flank. Mice were vaccinated weekly with s.c. injections in the opposite flank using 1010 
VP of Ad5 [E1-, E2b-]-MUC1 or ETBX-081, respectively. A control group of mice received 
3x1010 VP of Ad5 [E1-, E2b-]–null (no transgene). Mice vaccinated with Ad5 [E1-, E2b-]–MUC1 
or ETBX-081 had significantly smaller tumors than control mice on days 25 (p < 0.01) and 29 (p 
< 0.05) (Figure 5). There was no significant difference (p > 0.1) in anti-tumor effect for the groups 
of mice vaccinated with Ad5 [E1-, E2b-]–MUC1 vs. ETBX-081 at all time points. 
 
 
 
 
 
 
Figure 5: Comparison of immunotherapy of MUC1-expressing tumors using Ad5 [E1-, E2b-
]−MUC1 vs ETBX-081. C57Bl/6 mice (n=7/group) were inoculated with 106 MC-38-MUC1 cells 
subcutaneously in the left flank. Mice were administered 1010 VP (viral particle) of Ad5 [E1-, E2b-
]−MUC1 or ETBX-081 (1:1:1 mixture of 1010 VP each of Ad5 [E1-, E2b-]−CEA, Ad5 [E1-, E2b-
]−MUC1, and Ad5 [E1-, E2b-]−Brachyury, 3x1010 VP total). A control group of mice received 
3x1010 VP of Ad5 [E1-, E2b-]–null (no transgene). Tumor growth was monitored and volumes 
calculated. (*) indicates days when Ad5 [E1-, E2b-]−MUC1 treated mice had significantly smaller (p 
< 0.05) tumors than control mice and (^) indicates days when ETBX-081–treated mice had 
significantly smaller (p < 0.05) tumors than control mice. There was no significant difference (p > 
0.1) between Ad5 [E1-, E2b-]−MUC1 vs. ETBX-081–treated mice at any time point. Error bars 

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25 
 
1.2.8 Clinical Experience 
The combination of ETBX-011, ETBX-061, and ETBX-051 vaccine has not been tested in 
humans. 
This Phase I study is the  first clinical study of this vaccine combination  in subjects with 
advanced cancer. 
1.2.9 Rationale 
The preclinical data with the combination ETBX-011, ETBX-061, and ETBX-051 vaccine support 
this Phase I study to evaluate the safety, preliminary efficacy, and immunogenicity of the vaccine. 
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 and female). 
2.1.1.2 Ability to understand and provide signed informed consent that fulfills Institutional 
Review Board (IRB)’s guidelines. 
2.1.1.3 Subjects with cytologically or histologically confirmed locally advanced or metastatic 
solid tumor malignancy.  
2.1.1.4 Subjects must have completed or had disease progression on at least one prior line of 
disease-appropriate therapy or not be candidates for therapy of proven efficacy for their 
disease. 
2.1.1.5 Subjects may have measurable or nonmeasurable but evaluable disease as defined in 
section 6.3.1. Subjects with surgically resected locally advanced or metastatic disease at 
high risk of relapse are also eligible. 
2.1.1.6 Eastern Cooperative Oncology Group (ECOG) performance status < 1 (Appendices 
2.1.1.7 Appendix A) 
2.1.1.8 Subjects who have received prior CEA, MUC1, and/or Brachyury-targeted 
immunotherapy (vaccine) are eligible for this trial if this treatment was discontinued at 
least 4 weeks prior to enrollment. 
2.1.1.9 Resolution of clinically significant side effects of prior chemotherapy, radiotherapy, 
immunotherapy or surgical procedures to NCI CTCAE Grade ≤ 1 or grade ≤ 2 for 
neuropathy. 
2.1.1.10 Adequate hematologic function at screening, as follows: 
2.1.1.10.1 Absolute neutrophil count (ANC) ≥1 x 109/L   
2.1.1.10.2 Hemoglobin ≥ 9 g/dL  
2.1.1.10.3 Platelets ≥ 75,000/mcL.  

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2.1.1.11 Adequate renal and hepatic function at screening, as follows: 
2.1.1.11.1 Serum creatinine ≤ 1.5 x upper limit of normal (ULN) OR creatinine clearance 
(CrCl) ≥ 40 mL/min (if using the Cockcroft-Gault formula below): 
i. Female CrCl = [(140 - age in years) x weight in kg x 0.85] / [72 x serum 
 creatinine in mg/dL] 
ii. Male CrCl = [(140 - age in years) x weight in kg x 1.00]/ 1.00] / [72 x serum 
creatinine in mg/dL]  
2.1.1.11.2 Bilirubin ≤ 1.5 x ULN OR in subjects with Gilbert’s syndrome, a total bilirubin ≤ 
3.0 x ULN 
2.1.1.11.3 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 2.5 x 
ULN, unless liver metastases are present, then values must be ≤ 3 x ULN)   
2.1.1.12 The effects of the combination ETBX-011, ETBX-051, ETBX-061 vaccine regimen on 
the developing human fetus are unknown.  For this reason, female subjects of 
childbearing potential defined as any female who has experienced menarche and who 
has not undergone surgical sterilization (hysterectomy or bilateral oophorectomy or 
tubal ligation) or who is not postmenopausal (menopause being defined clinically as 12 
months of amenorrhea in a woman over 45 in the absence of other biological or 
physiological causes) and male patients who are not surgically sterile (vasectomy etc.), 
must agree to use acceptable contraceptive methods for the duration of the study and for 
one month after the last vaccination. Acceptable forms of contraception include oral 
contraceptives, intrauterine device, condom or vaginal diaphragm plus spermicidal 
(gel/foam/cream/vaginal suppository), or total abstinence. 
2.1.1.13 Ability to attend required study visits and return for adequate follow up, as required by 
this protocol. 
2.1.2 Exclusion Criteria 
2.1.2.1 Pregnant and nursing women. Because there is an unknown but potential risk for 
adverse events in nursing infants secondary to treatment of the mother with combination 
ETBX-011, ETBX-051, ETBX-061, breastfeeding should be discontinued if the mother 
is treated with combination ETBX-011, ETBX-051, ETBX-061.  These potential risks 
may also apply to other agents used in this study. 
2.1.2.2 There should be a minimum of 4 weeks from any prior investigational drug, 
chemotherapy, immunotherapy, with the exception of hormonal therapy for prostate and 
breast cancers, HER2-directed therapy for HER2+ breast or stomach cancer (3+ IHC or 
FISH+), drugs targeting  EGFR, ALK or ROS1 in EGFR,ALK, ROS1-mutated lung 
cancer, respectively, or standard maintenance therapies for any solid tumor under the 
condition that subjects are on these therapies for at least two months before start of trial 
treatment. 
2.1.2.3 There should also be a minimum of 4 weeks from any prior radiotherapy except for 
palliative bone directed therapy. 
2.1.2.4 Known active brain or central nervous system metastasis (less than 1 month out from 
definitive radiotherapy or surgery), or seizures requiring anticonvulsant treatment, or 
clinically significant cerebrovascular accident or transient ischemic attack (<3 months). 

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2.1.2.5 Subjects with active autoimmune disease requiring systemic immunosuppressive 
treatment within the past 4 weeks such as but not restricted to inflammatory bowel 
disease, systemic lupus erythematosus, ankylosing spondylitis, scleroderma, or multiple 
sclerosis. A history of autoimmune disease which is not active nor has required recent 
systemic immunosuppressive therapy (< 4 weeks prior to enrollment) is not reason for 
exclusion.  
2.1.2.6 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. 
2.1.2.7 Subjects with clinically significant 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) of ventricular 
arrhythmia. 
2.1.2.8 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. 
2.1.2.9 History of second malignancy within 3 years prior to enrollment except for the 
following: adequately treated non-melanoma skin cancer, cervical carcinoma in situ, 
superficial bladder cancer or other localized malignancy after discussion with the 
medical monitor.  
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 corticosteroid therapy with the exception of 
physiologic doses of corticosteroids (≤ the equivalent of prednisone 10 mg/day) or other 
immunosuppressives such as azathioprine or cyclosporin A are excluded on the basis of 
potential immune suppression. For these subjects these excluded treatments must be 
discontinued at least 2 weeks prior to enrollment for recent short course use (≤ 14 days) 
or discontinued at least 4 weeks prior to enrollment for long term use (> 14 days). In 
addition, the use of corticosteroids as premedication for contrast-enhanced studies is 
allowed prior to enrollment and on study.  
2.1.2.12 Subjects with known allergy or hypersensitivity to any component of the investigational 
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. 

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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 
All screening tests and procedures must be performed within 28 days prior to the first planned 
dosing of the study drug, unless otherwise noted. The following procedures and evaluations will 
be performed and documented in the subject’s source records. 
• History and physical exam including ECOG performance status and vital signs 
• 12-Lead ECG 
• Clinical laboratory tests (within 16 days prior to drug administration) 
− Chemistry: sodium, potassium, chloride, bicarbonate, calcium, glucose, BUN, 
creatinine, ALT, AST, alkaline phosphatase, total protein, albumin, and total and 
direct bilirubin. 
− Hematology: complete blood count (CBC) with differential and platelets  
− Coagulation panel: PT, INR, and PTT. 
− Urinalysis. 
• Serum pregnancy test (β-HCG) for females of childbearing-potential and women 
< 12 months since the onset of menopause (within 16 days prior to enrollment)  
• HBV (HBsAg), HCV (anti-HCV), HIV (anti-HIV) screening (within 3 months prior 
to enrollment) 
• 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. 
• Confirmation of diagnosis.  A report from any CAP or CLIA certified laboratory is 
acceptable.  
2.3 
REGISTRATION PROCEDURES 
Registration will be a two-part process as patients are screened on this protocol. Authorized staff 
must register an eligible candidate with NCI Central Registration Office (CRO) within 24 hours 
of signing consent. To initially register a subject after the participant has signed the consent, 
complete the top portion of the registration Eligibility Checklist from the website 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) indicating that the patient is being 
registered for screening and send via encrypted email to: NCI Central Registration Office  
ncicentralregistration-l@mail.nih.gov. Once eligibility is confirmed after completion of 
screening studies, complete the remainder of the form which is the eligibility checklist, 
indicating that the patient is being registered for treatment and email the completed registration 
checklist to the CRO at 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.  

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29 
 
Subjects that do not meet screening criteria should be removed from the study following the 
procedure in section 3.7.3.  
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 
Combination ETBX-011, ETBX-051, ETBX-061 adenoviral 
vaccine regimen dose de-escalation 
2 
Dose Expansion 
Combination ETBX-011, ETBX-051, ETBX-061 adenoviral 
vaccine regimen dose  
 
Arm Assignment 
Subjects in cohort 1 will be directly assigned to arm 1.  
Subjects in cohort 2 will be directly 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 
the baseline evaluation). The following procedures and evaluations will be performed and 
documented in the subject’s source records. 
• History and physical exam including ECOG performance status and vital signs  
• Serum pregnancy test (β-HCG) for females of childbearing-potential and women 
< 12 months since the onset of menopause. 
• Chemistry: sodium, potassium, chloride, bicarbonate, calcium, glucose, BUN, 
creatinine, ALT, AST, alkaline phosphatase, total protein, albumin, and total and 
direct bilirubin. 
• Hematology: CBC with differential and platelets  
3 
STUDY IMPLEMENTATION 
3.1 
STUDY DESIGN 
This is a Phase I trial in subjects with advanced cancer.  
The combination vaccine regimen is three different adenoviral vaccines (ETBX-011, ETBX -061 
and ETBX-051), administered to patients at the same time.  All utilize the same second generation 
E1(-), E2 (-) vector.  
ETBX-011 is a CEA-targeting vaccine that comprises the Ad5 [E1-, E2b-] vector and a modified 
CEA (CEA(6D)) gene insert. The investigational product ETBX-061 is a MUC1-targeting vaccine 

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30 
 
that comprises the Ad5 [E1-, E2b-] vector and a modified MUC1 (MUC1c) gene insert. The 
investigational product ETBX-051 is a Brachyury-targeting vaccine that comprises the Ad5 [E1-, 
E2b-] vector and a modified Brachyury gene insert. (Table 8).  
  
3.1.1 Dose Limiting Toxicity 
For the purposes of dose escalation a dose limiting toxicity (DLT) is defined as: 
• Any Grade 3 or greater toxicity that is possibly related to the vaccine and as defined 
by Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0 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, skin 
reactions or rash, headache, nausea, emesis that resolves to Grade ≤ 1 or 
asymptomatic grade 3 amylase/lipase elevation. For purposes of dose escalation the 
DLT evaluation period will be for 3 weeks following the first dose of drug.  
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, initiation 
of the dose expansion phase will occur.  If ≥2 of 3 or 6 experience DLT in 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 an amendment may be written to evaluate a further dose de-
escalation. 
In the second part, dose expansion will occur when the MTD has been determined. An additional 
4 subjects will be enrolled in the dose expansion component of the trial, for a total of 10 subjects 
treated at the MTD.  
A schematic overview of the study is shown in Figure 6. 
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 3 
days between enrolling successive subjects. DLTs will be monitored continuously. 
Dose levels are shown in Table 4. No intra-patient dose escalations are permitted. 
 
Table 4: Dose Levels for Each ETBX VP 
Dose Level (DL) 
ETBX VP 
Standard (1) 
5 x 1011 
-1 
1 x 1011 
Standard dose (DL 1) (5 x 1011 VP): 
• If ≤ 1 of the initial 6 subjects experience a DLT, dose expansion will commence. 
• If >1 of the initial 3 subjects, or if ≥ 2 of the 6 total subjects experience a DLT, 
enrollment at DL -1 will commence.  

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31 
 
 
Dose de-escalation DL -1 (1 x 1011 VP): 
• If ≤ 1 of the initial 6 subjects experience a DLT, this dose level will be defined as the 
MTD and dose expansion will commence. 
• If >1 of the initial 3 subjects, or if ≥ 2 of the 6 total subjects experience a DLT, then a 
protocol amendment may be written to evaluate a further dose de-escalation.. 
 
 

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32 
 
Figure 6: Study Design and Treatment Schema 
 

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33 
 
3.2 
ADMINISTRATION AND DURATION OF TREATMENT 
Each of the three ETBX vaccines will be administered on Weeks 0, 3, and 6 followed by booster 
vaccines every 8 weeks for up to a year. 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. For specifics on 
drug administration see section 11.1.5.  
 
Vital sign assessments (temperature, heart rate, blood pressure, respiratory rate) 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. 
3.3 
DOSE DELAY OR DISCONTINUATION 
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. Patients will receive a diary card to record 
injections site reactions (see Appendix B).  
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 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.  
• Dosing of the vaccines injections should be given on schedule every 3 weeks (Week 0, 
3, and 6), and then bi-monthly (every 8 weeks) boosts for up to a year. In the event of 
conflicts, a +/- 1 week window is acceptable.  
• 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. 
 
3.4 
DOSE MODIFICATIONS 
No dose modifications are allowed with these vaccines except for dose de-escalation as 
described in section 3.1.  
 
 

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3.5 
STUDY CALENDAR  
Assessment 
Screening 
Treatment 
(Every 3-Week 
Dosing) 
Bimonthly_Boosters 
(Every 8-Week Dosing) 
End of 
Treatment 
(within 90 
days after 
the last 
vaccine) m  
Follow Upl 
Study Week 
Day 
-28 to -1 n 
Baselin
e/0 
3 
6 
14 
22 
30 
38 
46 
54 
 
 
Clinic Visit 
X 
Xc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Informed Consent 
Xa 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Confirmation of diagnosis (path 
report) 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination, Heightb, 
Weight, ECOG 
Xc 
Xc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Medical Historyd 
X 
Xc 
 
 
 
 
 
 
 
 
 
 
Concomitant Medications 
X 
Xc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Vital Signse 
X 
Xc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
12-Lead ECG 
X 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
Confirm 
Contraceptive 
Measuresf 
X 
 
 
 
 
 
 
 
 
 
 
 
Study Drug Injection/ Injection 
Site Reaction Monitoringg 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 

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Assessment 
Screening 
Treatment 
(Every 3-Week 
Dosing) 
Bimonthly_Boosters 
(Every 8-Week Dosing) 
End of 
Treatment 
(within 90 
days after 
the last 
vaccine) m  
Follow Upl 
Study Week 
Day 
-28 to -1 n 
Baselin
e/0 
3 
6 
14 
22 
30 
38 
46 
54 
 
 
Dispensation of Subject Diary 
Cardh 
 
X 
 
 
 
 
 
 
 
 
 
 
Review of Subject Diary Cardh 
 
 
X 
 
 
 
 
 
 
 
 
 
Tumor Imagingi 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
 
Pregnancy Testj 
Xc,n 
Xc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Urinalysis 
X n 
 
 
 
 
 
 
 
 
 
X 
 
Chemistry Panel 
Xc,n 
Xc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
CBC, Differential, Platelets 
Xc,n 
Xc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Coagulation 
X n 
 
 
 
 
 
 
 
 
 
X 
 
Serum Virology (HIV, HBV, 
HCV)k 
X n 
 
 
 
 
 
 
 
 
 
 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Exploratory Immune Analysis 
 
X 
 
X 
X 
 
X 
 
 
 
X 
 
Telephone Follow Up 
 
 
 
 
 
 
 
 
 
 
 
X 
 
a Informed 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 
b Height will only be assessed at screening. 
c If the assessment is performed within 24 hours prior to the first dosing, a second assessment at baseline/week 0 can be omitted. 

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36 
 
d 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.. 
e 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. 
f Acceptable contraceptive measures are described in Section 2.1.1.12.  
g Injection site reactions will be monitored 
h Subjects will be given a diary card for the self-evaluation and reporting of injection site reactions. 
i 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  
j Serum pregnancy test will be performed on females of childbearing-potential and women < 12 months since the onset of menopause. The pregnancy test will be 
performed at each dosing visit with the result confirmed as negative prior to dosing. 
k 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). 
l 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 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.   
m 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.  
n See section 2.2 for screening criteria which are exceptions to the D-28 to D-1 screening window period.

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3.6 
INJECTION SITE REACTIONS 
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 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 for the seven days after the first set of 
vaccine injections (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. 
3.7 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA 
Regardless of reason for removal from study therapy, patients will be asked to have follow up 
safety visit within 90 days 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.   
3.7.1 Criteria for removal from protocol therapy 
• Completion of protocol therapy. 
• Clinical or radiographic progression of disease Patients with serological progression 
alone will remain on treatment at the discretion of the PI. 
• Unacceptable Toxicity as defined as any serious adverse event that is unexpected 
relative to the known safety profile of the investigational agents in the opinion of the 
investigator or as described in sections 3.1.1 and 3.3 
• Participant requests to be withdrawn from active therapy 
• Investigator discretion 
• Positive pregnancy test 
Also see section 3.3 
3.7.2 Off-Study Criteria 
• PI decides to end study 
• Participant requests to be withdrawn from study. Reasons for withdrawal will be 
documented.  
• Noncompliance with protocol guidelines (patient removed at discretion of Principal 
Investigator)  
• Death 
• Screen failure 
3.7.3 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.  
 

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4 
CONCOMITANT MEDICATIONS/MEASURES 
Subjects must inform the investigators of the current or planned use or all other medications during 
the study (including prescription medications, vitamins, herbal and nutritional supplements, and 
over-the-counter medications).  
For the administration of 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.  
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 
physiologic doses of systemic steroids, inhaled steroids, nasal sprays, and topical creams for small 
body areas is 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 with the exception of treatments 
described in section 3.1 then 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. 

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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 Peripheral Blood Collection 
Subjects will have approximately 70 mL of peripheral blood drawn to evaluate the study drug’s 
effect on the immune response at specific time points during the study and/or after a specified 
injection. Blood draws will be done at baseline and prior to each injection (baseline, week6,14, 30 
and EOT).  
5.1.2 Samples Collected 
Six10-mL green top sodium heparin tubes for PBMC samples and one 8-mL serum-separating 
tube for serum samples will be drawn. Samples will be picked up by courier and delivered to the 
Leidos Biomedical Research facility in Frederick, MD.. 
5.1.3 Sample Processing 
Blood samples will be processed at:  
Leidos Biomedical Research 
Attn: Dr. Ludmila Krymskaya /Theresa Burks  
1050 Boyles Street 
Bldg. 496/Room 121 
Frederick, MD 21702 
5.1.4 Sample Tracking, and Disposition 
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 the National Institutes for Health (NIH) without IRB notification and an 
executed material transfer agreement. 
5.1.5 Pharmacodynamic Sample Management and Storage 
All data associated with the patient samples is protected by using a secure database. All Clinical 
Support Laboratory Staff receive annual training in maintaining records’ confidentiality. All 
samples drawn at the NIH Clinical Center will be transported to the Clinical Support Laboratory 
at the Frederick National Laboratory for Cancer Research by couriers. 
Samples will be tracked and managed by Central Repository database, where there is no link to 
personal identifiable information. All samples will be stored in either a -80ºC freezer or vapor 
phase liquid nitrogen. These freezers are located at NCI Frederick Central Repository in Frederick, 
Maryland. 
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, 

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limited-access facilities with sufficient security, backup, and emergency support capability and 
monitoring to ensure long-term integrity of the specimens for research.  
Specimens are stored in accordance with applicable HHS and FDA Protection of Human 
Subjects Regulations in accordance with the subcontractor’s Federal-wide Assurance.  The 
subcontractor’s role limited to clinical research databases and repositories containing patient 
specimens. The subcontractor does not conduct or have any vested interest in research on human 
subjects, but does provide services and support the efforts of its customers, many of which are 
involved in research on human subjects. The subcontractor’s IRB reviews policies and 
procedures for labeling, data collection and storage, access, and security. The IRB will review 
protection of privacy issues prior to acceptance of any new work and in the event of change 
impacting privacy issues in existing work.  
It is the intent and purpose of the subcontractor to accept only de-identified samples and sample 
information. To the limit of our ability, every effort will be made to ensure that protected 
information is not sent electronically or by hard copy or on vial labels.  
Sample data is stored in the BioSpecimen Inventory System II (BSI). This inventory tracking 
system is used to manage the storage and retrieval of specimens as well as to 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. 
5.1.6 Sample Analysis 
Immune assessments will be performed at the Laboratory of Tumor Immunology and Biology at 
the NCI’s Center for Cancer Research (CCR) and include flow cytometry-based and serum assays. 
Analyses of PBMCs: 
1. 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 CEA, MUC1, and Brachyury. Control peptide 
pools will involve the use of human leukocyte antigen 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 

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will involve the production of IFN-γ, IL-2, tumor necrosis factor, and CD107a. If sufficient 
PBMCs are available, assays may also be performed for the development of T cells to other 
tumor-associated antigens. A detailed description of this assay has been previously 
reported (35). 
2. 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 (74), and for function of 
specific immune cell subsets, including CD4 and CD8 T cells, NK cells, Tregs, and 
MDSCs. 
Analyses of soluble factors: 
Sera will be analyzed pre- and post-therapy for the following soluble factors: sCD27, sCD40 
ligand, and selected patients may be analyzed for cytokines and antibodies to human tumor 
antigens. For selected patients, sera may be analyzed for antibodies to CEA, MUC1, or 
Brachyury and adenovirus.  
 
Additional assays:  
Blood samples may be used for additional research studies, which may include phenotypic and 
functional analysis of immune-cell subsets and analyses for cytokines (IFN-γ, IL-10, IL-12, IL-2, 
IL-4, etc.), chemokines, antibodies, tumor-associated antigens, and/or other markers. In addition, 
assays for antibody or neutralizing antibody titer to adenovirus (type 5) and cell-mediated 
immunity to adenoviral antigens may be assessed.  
Samples will be tracked according to Section 5.1.4.  
5.1.7 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 subject consent or an exemption from the Office of Human Research 
Protections.  
The Investigator will report any loss or destruction of samples to the NCI IRB as soon as he/she is 
made aware of such loss. The Investigator 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 subject withdraws consent. Samples will also be reported as lost 
if they are lost in transit between facilities or misplaced by a researcher. Freezer problems or lost 
samples will also be reported to the IRB, the NCI Clinical Director, and the office of the NCI CCR. 

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6 
DATA COLLECTION AND EVALUATION  
6.1 
DATA COLLECTION 
Data will be entered in C3D. The PI will be responsible for overseeing entry of data into an in-
house password protected electronic system 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. 
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 a minimum of 30 days after removal from study treatment 
or until off-study, whichever comes first. 
An abnormal laboratory value will be recorded in the database as an AE only if the laboratory 
abnormality is characterized by any of the following: 
• 
Results in discontinuation from the study 
• 
Is associated with clinical signs or symptoms  
• 
Requires treatment or any other therapeutic intervention 
• 
Is associated with death or another serious adverse event, including hospitalization.  
• 
Is judged by the Investigator to be of significant clinical impact 
• 
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. 
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.  
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: 
• De-identified data in an NIH-funded or approved public repository.   
• De-identified data in BTRIS (automatic for activities in the Clinical Center) 
• De-identified or identified 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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• An NIH-funded or approved public repository.  Insert name or names:_clinicaltrials.gov_. 
• BTRIS (automatic for activities in the Clinical Center) 
• Approved outside collaborators under appropriate individual agreements. 
• Publication and/or public presentations. 
 
When will the data be shared? 
• Before publication. 
• At the time of publication or shortly thereafter. 
6.3 
EFFICACY ASSESSMENTS 
6.3.1 Antitumor Response 
Tumor assessments will be done at week 6 and then every 8 weeks thereafter and may include the 
following evaluations: physical examination (with photograph and measurement of skin lesions, 
as applicable); cross-sectional imaging using computed tomography (CT) or magnetic resonance 
imaging (MRI) scan of the chest, abdomen, and pelvis (pelvis scan is optional unless known pelvic 
disease is present at baseline); nuclear bone scan for subjects with known/suspected bone lesions; 
and CT or MRI scan of the brain (only as clinically warranted based on symptoms/findings). The 
preferred method of disease assessment is CT with contrast. If CT with contrast is contraindicated, 
CT of the chest without contrast and MRI scan of the abdomen/pelvis with contrast is preferred.  
At baseline, tumor lesions will be selected and categorized as target (measurable disease) or non-
target lesions (non-measurable disease).  
6.3.2 Disease Parameters 
Measurable disease. Measurable lesions are defined as those lesions that can be accurately measured 
in at least one dimension (longest diameter to be recorded) as:  
• By chest x-ray: >20 mm;  
• By CT scan:  
o Scan slice thickness 5 mm or under: as >10 mm 
o Scan slice thickness >5 mm: double the slice thickness  
• 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 the 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 (those with the 
longest diameter), be representative of all involved organs, but in addition should be those that lend 
themselves to reproducible and 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 above 
the 5 target lesions should be identified as non-target lesions and should 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. 
6.3.3 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. The same mode of assessment (e.g., CT) used to 
identify/evaluate lesions at baseline should be used throughout the course of the study unless 
subject safety necessitates a change (e.g., allergic reaction to contrast media). 
Clinical lesions:  Clinical lesions will only be considered measurable when they are superficial 
(e.g., skin nodules and palpable lymph nodes) and 10 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.  

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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).   
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. (75-77) 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. (78) 

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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. 
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. 
6.3.4 RECIST Response Criteria 
Antitumor activity will be evaluated with target and/or non-target lesions according to RECIST 
Version 1.1 (79) as summarized below. 
6.3.5 Target Response 
Percentage change in target lesion size will be evaluated by the following formulae: 
1. When determining complete response or partial response: 
[(Post value – Baseline value)/Baseline value] x 100 
2.   When determining progressive disease: 
[(Post value – Smallest value since treatment started)/(Smallest value since treatment 
started)] x 100 

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Target response will be classified according to the RECIST Version 1.1 Target Lesion Response 
Criteria in Table 5. 
 
Table 5: RECIST Target Response Criteria 
Target Response Criteria 
Definition 
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 diameters of 
target lesions, taking as reference the baseline sum 
diameters. 
Stable Disease (SD) 
Neither sufficient shrinkage to qualify for PR nor 
sufficient increase to qualify for PD, taking as 
reference the smallest sum diameters while on 
study. 
Progressive Disease (PD) 
At least a 20% increase in the sum of diameters of 
target lesions, taking as reference the smallest sum 
diameters while 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 5 mm. (Note: 
the appearance of one or more lesions is also 
considered progression). 
 
Non-target response will be classified according to the RECIST Version 1.1 Non-Target Lesion 
Response Criteria in Table 6. 
Table 6: RECIST Non-Target Response Criteria 
Non-Target Response Criteria 
Definition 
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). 
Non-CR / Non-PD 
Persistence of one or more non-target lesion(s) 
and/or maintenance of tumor marker level above 
the normal limits. 
PD 
Unequivocal progression of existing non-target 
lesions. (Note: the appearance of one or more new 
lesions is also considered progression). 
 

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Overall response will be classified according to the RECIST Version 1.1 Overall Response Criteria 
in Table 7. 
Table 7: RECIST Overall Response Criteria 
 
6.3.6 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.  
6.3.7 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 5.0 will be utilized for AE 
reporting.  All appropriate treatment areas should have access to a copy of the CTCAE version 
5.0.  A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site: 
Target Lesions 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
CR 
No 
CR 
CR 
Non-CR / Non-PD 
No 
PR 
CR 
Not Evaluated 
No 
PR 
PR 
Non-PD 
or 
not 
all 
evaluated 
No 
PR 
SD 
Non-PD 
or 
not 
all 
evaluated 
No 
SD 
Not all evaluated 
Non-PD 
No 
Inevaluable 
PD 
Any 
Yes or No 
PD 
Any 
PD 
Yes or No 
PD 
Any 
Any 
Yes 
PD 

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(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm) 
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. 
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: 
• 
Death, 
• 
A life-threatening adverse drug experience 
• 
Inpatient hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant incapacity or substantial disruption of the ability to conduct 
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• 
A congenital anomaly/birth defect. 
• 
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 experience 
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: 
•  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 documents, and 
(b) the characteristics of the subject population being studied; AND 
• Is related or possibly related to participation in the research; AND 
• 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 
NCI-IRB AND CLINICAL DIRECTOR (CD) REPORTING 
7.2.1 NCI-IRB and CD Expedited Reporting of Unanticipated Problems and Deaths 
The Protocol PI will report in the NIH Problem Form to the NCI-IRB and NCI CD: 
• All deaths, except deaths due to progressive disease  
• All Protocol Deviations 
• All Unanticipated Problems 

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• All non-compliance 
Reports must be received within 7 days of PI awareness via iRIS. 
 
7.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review 
The protocol PI will report to the NCI-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: 
• All Grade 2 unexpected events that are possibly, probably or definitely related to the 
research;  
• All Grade 3 and 4 events that are possibly, probably or definitely related to the research;  
• All Grade 5 events regardless of attribution;  
• All Serious Events regardless of attribution.  
NOTE: Grade 1 events are not required to be reported.  
 
7.2.3 NCI-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 NCI IRB. 
7.3 
IND SPONSOR REPORTING CRITERIA 
During the first 30 days after the subject receives investigational agent/intervention, the  
investigator must report within 1 business day to the sponsor, using the mandatory MedWatch 
form 3500a, 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 reporting per the above guideline: 
• 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.  
• 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. 
7.3.1 Reporting Pregnancy 
7.3.1.1 Maternal Exposure 

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If a patient becomes pregnant during the course of the study, the study treatment should be 
discontinued immediately and the pregnancy reported to the Sponsor. The potential risk of 
exposure of the fetus to the investigational agent(s) or chemotherapy agents (s) should be 
documented in box B5 of the MedWatch form “Describe Event or Problem”. 
Pregnancy itself is not regarded as an SAE. However, as patients who become pregnant on study 
risk intrauterine exposure of the fetus to agents which may be teratogenic, the CCR is requesting 
that pregnancy should be reported in an expedited manner as Grade 3 “Pregnancy, puerperium 
and perinatal conditions - Other (pregnancy)” under the Pregnancy, puerperium and perinatal 
conditions SOC.  
Congenital abnormalities or birth defects and spontaneous miscarriages should be reported and 
handled as SAEs. Elective abortions without complications should not be handled as AEs. The 
outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, 
normal birth, or congenital abnormality) should be followed up and documented. 
If any pregnancy occurs in the course of the study, then the investigator should inform the 
Sponsor within 1 day, i.e., immediately, but no later than 24 hours of when he or she becomes 
aware of it. 
The designated Sponsor representative will work with the investigator to ensure that all relevant 
information is provided to the Sponsor within 1 to 5 calendar days for SAEs and within 30 days 
for all other pregnancies. 
The same timelines apply when outcome information is available. 
7.3.1.2 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 combination ETBX-011, ETBX-051, ETBX-061 . 
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  
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 within 2 business days 
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. 
 

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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 
experience associated with the use of ETBX vaccines as soon as 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 vaccines, 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:  
• 
the title and purpose of the trial  
• 
clinical site  
• 
the Principal Investigator  
• 
clinical protocol identifiers;  
• 
participant population (such as disease indication and general age group, e.g., adult or 
pediatric);  

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• 
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  
• 
the status of the trial, e.g., open to accrual of subjects, closed but data collection ongoing, 
or fully completed,  
• 
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:  
• 
a narrative or tabular summary showing the most frequent and most serious adverse 
experiences by body system  
• 
a summary of all serious adverse events submitted during the past year  
• 
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  
• 
if any deaths have occurred, the number of participants who died during participation in 
the investigation and causes of death  
• 
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.  
 
7.6 
DATA AND SAFETY MONITORING PLAN 
7.6.1 Principal Investigator/Research Team 
The clinical research team will meet on a weekly 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 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 or associate 
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 
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• Informed consent process 
• Eligibility confirmation 
• Drug administration and accountability 
• Adverse events monitoring 
• 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 NCI-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 METHODS 
8.1 
 STATISTICAL HYPOTHESIS 
This is a Phase I trial in subjects with advanced cancer. A combination ETBX-011, ETBX-051, 
ETBX-061 vaccine regimen using the same modified Adenovirus vector backbone, separately 
encoding three well-studied TAAs will be assessed. The vaccine will be tested at standard dose 
level, and a dose de-escalation (if required) design will be employed. The primary endpoint will 
be to determine the overall safety of combined ETBX-011/ETBX-061/ETBX-051 vaccine when 
administered SC to subjects with advanced solid tumors and establish a recommended phase 2 
dose of vaccine. This will be reported when an MTD is determined (expected to be less than 1 year 
from trial start). A secondary endpoint will be to obtain preliminary data on ORR, DCR, duration 
of response, PFS, and OS in treated subjects (preliminary data will be assessed around two years 
following trial start). An exploratory hypothesis (objective) will be to evaluate the immunogenicity 
of the combined ETBX-011/ETBX-061/ETBX-051 vaccine. 
8.2 
SAMPLE SIZE DETERMINATION 
Up to six patients will be enrolled in an initial dose level 1.  If ≤1 of 6 patients experience a DLT, 
then the MTD will be declared, and initiation of the dose expansion phase will occur.  If ≥2 of 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 in this second dose 
level -1, then an amendment may be written to further evaluate dose de-escalation. Once the MTD 
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enrolled at the MTD for a total of 10 subjects to gain additional safety data and exploratory data 
on vaccine immunogenicity. 
We assume 16 patients will have screen failure. Therefore, up to 32 subjects will be enrolled in 
the study. Initial dose evaluation and or dose finding will require 6-12 subjects, followed by 4 
additional subjects treated at a defined dose. 
8.3 
POPULATION FOR ANALYSES 
Evaluable for toxicity:  All patients will be evaluable for toxicity from the time of their first 
treatment with the combination ETBX-011, ETBX-051, ETBX-061 vaccine regimen. 
Evaluable for objective response: Only those patients who have measurable disease present at 
baseline, have received at least one cycle 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.  (Note:  Patients who exhibit objective disease 
progression prior to the end of cycle 1 will also be considered evaluable.) 
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 cycle 
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. The planned analyses for each objective is described in the subsections below. 
8.4.2 Analysis of Primary Efficacy 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 Analyses of Secondary Endpoint  
Analyses of secondary endpoint to obtain preliminary data on objective response rate (ORR), 
disease control rate (DCR), duration of response, progression-free survival (PFS), and overall 
survival (OS) in treated subjects. 
 
Since this is a Phase I trial, only preliminary data will be obtained for evidence of vaccine 
efficacy. Preliminary analyses of vaccine efficacy are described in the Sections 8.4.3.1 to 8.4.3.4 
below. 
8.4.3.1 Objective Tumor Response and Disease Control Rate 
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 level and overall enrollment. If anti-
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Disease control (confirmed response or SD lasting for at least 6 months) will be analyzed in a 
similar manner. 
8.4.3.2 Duration of Response 
The duration of overall response will be evaluated by dose level and overall enrollment. 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 PD is objectively documented 
(taking as reference for PD the smallest measurements recorded since the treatment started). 
 
8.4.3.3 Progression-Free Survival 
PFS will be evaluated by dose level 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. 
8.4.3.4 Overall Survival  
OS will be evaluated by dose level 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. 
8.4.4 Safety Analysis 
DLTs will be evaluated continuously in a dose level. An overall assessment of whether to de-
escalate to the next dose level will be made at least 1 week after the last subject in the previous 
dose level has received their first injection. 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, ≤ 2 of 9, ≤ 3 of 10). 
Safety will be evaluated in 3 or 6 subjects at each dose level in the dose escalation component of 
the study. Safety will continue to be monitored among additional subjects treated at the MTD or 
HTD 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 1 week.  
 
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.  
 
In addition, overall safety will be assessed by descriptive analyses using tabulated frequencies of 
AEs by grade using CTCAE Version 5.0 within dose levels and for the overall study population in 
terms of treatment-emergent AEs, SAEs, and clinically significant changes in safety laboratory 
tests, physical examinations, ECGs, and vital signs. 
 
The safety events listed in section 8.4.6.2 will trigger a temporary suspension of the study 
injections.: 

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8.4.5 Baseline Descriptive Statistics 
Not applicable for this Phase I study. 
8.4.6 Planned Interim Analyses and Halting Guidelines 
8.4.6.1 Planned interim analysis 
Not applicable for this Phase I study. 
8.4.6.2 Halting guidelines 
The following safety events will trigger a temporary suspension of the study injections: 
• Death possibly related to the study agent. 
• Two Grade 4 toxicity events that are possibly related to the study agent. 
• 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 Sub-Group 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 (Immune Responses) 
Exploratory immune analyses will be conducted to evaluate anti-tumor immune responses induced 
by vaccine injections. Immune response will be assessed among all subjects treated in each dose 
level. The magnitude of immune responses will also be described. A subject will be considered 
evaluable for immune response if they receive at least three vaccine injections. The percentage of 
subjects with a positive immune response will be evaluated by dose levels and overall enrollment. 
A positive immune response is defined by CMI reactivity in ex vivo stimulation using a 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 
(35). 
 
If more than one dose level of patients is enrolled and at least three patients are treated in the dose 
level, Statistical analyses of data will be performed. For flow cytometry analyses on PBMC 
samples, Student T tests will be performed on percentages of TNF- and/or IFN- expressing cells 
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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-011 (adenoviral CEA 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 genders and 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 gender and ethnic aspects of clinical research on the other hand. If differences in outcome 
that correlate with gender and ethnic identity are noted, accrual may be expanded or a follow-up 
study may be written to investigate those differences more fully.  
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 
Directive for Health Care and Medical Research Participation” form so that another person can 
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 for evaluation.  For those subjects that become incapacitated and do not have 

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pre-determined substitute decision maker, the procedures described in MAS Policy 87-4 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  
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. 
 
If there is an optional biopsy for research in the protocol, then the patient will consent at the time 
of the procedure.  If the patient refuses the optional biopsy at that time, the refusal will be 
documented in the medical record. 
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 
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 

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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-011, ETBX-051, ETBX-061 VACCINE REGIMEN 
The combination ETBX-011, ETBX-051, ETBX-061 vaccine regimen refers to the combination 
of three investigational adenoviral vaccines.  All utilize the same second generation E1(-), E2 (-) 
vector.  Subjects will each receive the three vaccines at the same time, at the same doses, through 
three separate injections administered in different limbs (proximal limb areas) or separate 
locations. 
ETBX-011 is a CEA-targeting vaccine that comprises the Ad5 [E1-, E2b-] vector and a modified 
CEA (CEA(6D)) gene insert. The investigational product ETBX-061 is a MUC1-targeting vaccine 
that comprises the Ad5 [E1-, E2b-] vector and a modified MUC1 (MUC1c) gene insert. The 
investigational product ETBX-051 is a Brachyury-targeting vaccine that comprises the Ad5 [E1-, 
E2b-] vector and a modified Brachyury gene insert. (Table 8). Refer to the current version of the 
Investigator’s Brochure for detailed investigational product information. 
 
Table 8: Investigational Products 
Table 8a 
Product Name(s): 
ETBX-011 (Ad5 [E1-, E2b-]-CEA(6D) Vaccine) 
 
Dosage Form: 
Suspension for injection 

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Dose 
5 x 1011 VP (standard dose), or 1 x 1011 VP (DL-1). 
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 1.3 mL of the vaccine. The product 
should be stored at  -20+10°C until ready for use. 
Manufacturer 
Etubics 
 
Table 8b 
Product Name(s): 
ETBX-061 (Ad5 [E1-, E2b-]-MUC1 Vaccine) 
 
Dosage Form: 
Suspension for injection 
Dose 
5 x 1011 VP (standard dose), or 1 x 1011 VP (DL-1). 
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 
contains1 mL of extractable vaccine. The product should be 
stored at ≤ -60°C until ready for use. 
Manufacturer 
Etubics 
 
Table 8c 
Product Name(s): 
ETBX-051 (Ad5 [E1-, E2b-]-Brachyury Vaccine) 
Dosage Form: 
Suspension for injection 
Dose 
5 x 1011 VP (standard dose), or 1 x 1011 VP (DL-1). 
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 1 mL of extractable vaccine. The product should be 
stored at ≤ -60°C until ready for use. 
Manufacturer 
Etubics 

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Version Date: 05.16.2018 
 
63 
 
 
11.1.1 Source  
The ETBX vaccines will be supplied by the manufacturer, Etubics Corporation, through a 
Cooperative Research and Development Agreement (CRADA). The manufacturing department of 
Etubics will supply each vaccine in 2-mL single-dose vials. Each single-dose vial contains a sterile, 
clear suspension of the ETBX vaccine at 5 x 1011 VP per 1 mL intended for single dose 
administration and contains ARM formulation buffer (20 mM TRIS, 25 mM NaCl, 2.5% glycerol, 
pH 8.0). 
 
Upon receipt of the investigational product, the Investigator or delegated designee will verify that 
an appropriate shipping temperature was maintained, conduct an inventory, and sign the drug 
receipt form and send a scanned copy to the Sponsor contact as designated on the form. If a 
temperature excursion occurs, the Sponsor should be notified immediately, and the investigational 
product must be quarantined and maintained under the correct storage conditions until further 
instructions are provided by the Sponsor. The temperature excursion form will need to be 
completed and returned to the Sponsor. The original drug receipt form and packing slip must be 
retained in the Investigator’s pharmacy records. 
11.1.2 Toxicity 
The safety of immunizations (injections) with the ETBX-011, ETBX-051, ETBX-061 vaccines 
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 

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64 
 
reactions (Grade 2 is defined as generalized urticaria as defined by NCI Common Terminology 
Criteria for Adverse Events (CTCAE version 5.0), 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 
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 
 
 
 

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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 
*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 supplied as a clear colorless liquid filled in a 2-mL amber vial at a concentration 
of 5x1011 total virus particles (VP) per 1.0 mL. 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  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 
monitored temperature control. ETBX-011 vials should be stored at -20 ± 10℃; ETBX-051 and 
ETBX-061 vials should be stored at ≤ -60℃. 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 ETBX Vaccine Dose Preparation and Administration 
The dose of ETBX vaccine to be injected is 5 x 1011 VP per 1 mL.  A dose level of 1 x 1011 VP per 
mL (dose level -1)is 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–

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66 
 
46°F). The vaccine is stable in the vial for at least 8 hours after removal from the freezer when 
kept refrigerated at 2-8℃ (35-46℉). Once the vaccine has been thawed, it must not be refrozen. 
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 (i.e. within 1 hour) 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. 
11.1.5.1  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 without any further manipulation (refer to the vial label for a 
description of the vialed ETBX vaccine concentration). 
11.1.5.2  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). 
11.1.5.3   Administration 
Each of the three ETBX vaccines will be administered on Weeks 0, 3, and 6 followed by bi-
monthly booster vaccines for up to a year. All study drug administration treatments should occur 
within ± 7 days of the planned visit date.  
ETBX-011 will be administered by subcutaneous injection in the upper arm after preparation of 
the site with alcohol. A 1 to ½ inch, 20- to 25- gauge needle should be used for administration.   
ETBX-051 will be administered by subcutaneous injection in the anterolateral upper thigh after 
preparation of the site with alcohol. A 1 to ½ inch, 20- to 25- gauge needle should be used for 
administration. It is preferred to administer ETBX-051 and ETBX-061 in separate thighs. 
However, if administered in the same thigh, injection sites should be separated by at least 5 cm. 
ETBX-061 will be administered by subcutaneous injection in the anterolateral upper thigh after 
preparation of the site with alcohol. A 1 to ½ inch, 20- to 25- gauge needle should be used for 
administration. It is preferred to administer ETBX-051 and ETBX-061 in separate thighs. 
However, if administered in the same thigh, injections sites should be separated by at least 5 cm.  
11.1.6 Incompatibilities 
The vaccines are administered as separate subcutaneous injections, with no known 
incompatibilities. 
11.1.7 Other Considerations 

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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 
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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induce cell mediated immune responses. Immunol Lett. 2009;122(1):44-51. 
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type 5 vaccine platform induces cellular immunity against HIV-1 Gag, Pol, Nef despite the 
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74 
 
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. 

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13.2 APPENDIX B: INJECTION SITE REACTIONS DIARY CARD 
 
Please answer all questions below once per day for the first 7 days 
 
 
 
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 
5. Have you 
taken any 
medication 
for 
Circle:  Yes 
/ No 
 
Provide 
name and 
Yes / No 
 
Name: 
___________ 
 
Yes / No 
 
Name: 
___________ 
 
Yes / No 
 
Name: 
___________ 
 
Yes / No 
 
Name: 
___________ 
 
Yes / No 
 
Name: 
___________ 
 
Yes / No 
 
Name: 
___________ 
 
Yes / No 
 
Name: 
___________ 
 

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Version Date: 05.16.2018 
 
76 
 
 
 
Day 0 
(Day of Injection) 
Day +1 
Day +2 
Day +3 
Day +4 
Day +5 
Day +6 
injection 
site pain? 
dose of 
medication 
Dose: 
____________ 
 
Dose: 
____________ 
Dose: 
____________ 
Dose: 
____________ 
Dose: 
____________ 
Dose: 
____________ 
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: 
 
 
 
________________
_