An Open Label Phase I Study to Evaluate the Safety and Tolerability of a Modified Vaccinia Ankara (MVA) Based Vaccine Modified to Express Brachyury and T-Cell Costimulatory Molecules (MVA Brachyury-TRICOM)
Phase: PHASE1
Conditions: Lung Cancer, Breast Cancer, Prostate Cancer, Tumors (Others), Ovarian Cancer
Sponsor: National Cancer Institute (NCI)

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
CC Protocol #: 14-C-0142  
 
 
 
 
 
OSP#: 1404-1301 
Version Date: September 14, 2016  
 
 
 
 
IBC#: RD-14-V-01 
Amendment: C 
 
Title: An open-label phase I study to evaluate the safety and tolerability of a modified vaccinia 
Ankara (MVA)-based vaccine modified to express brachyury and T-cell costimulatory molecules 
(MVA-brachyury-TRICOM) 
 
NCI Principal Investigator:  
James L. Gulley, MD, PhD A, B, C, D, E, F 
 
 
 
Genitourinary Malignancies Branch 
 
 
 
National Cancer Institute 
 
 
 
10 Center Drive 
 
 
 
Building 10, Room 12N226 
 
 
 
Bethesda, MD 20892 
 
 
 
Phone: (301) 496-4916 
 
 
 
Fax: (301) 480-5094 
 
 
 
Email: gulleyj@mail.nih.gov 
 
NIH Associate Investigators: 
Ravi A. Madan, M.D., GMB, CCR, NCI A, B, C, D, E, F 
William Dahut, M.D., GMB, CCR, NCI A, B, C, D 
Sheri McMahon, R.N., OCD, CCR, NCI A, B, C, E 
Myrna Rauckhorst, R.N., OCD, CCR, NCI A, B, C, E 
Jennifer Marte, M.D., GMB, CCR, NCI E, F 
 
Non-NIH Associate Investigator: Philip M. Arlen, M.D. [V], GMB, CCR, NCI A, B, E, F 
 
Statistician: 
 
Seth Steinberg, Ph.D. E, F  
 
 
 
 
Biostatistics and Data Management Section 
 
 
 
 
Office of the Director, CCR, NCI 
 
 
 
 
 
9609 Medical Center Drive, Room 2W334 
 
 
 
 
 
Rockville, MD 20850 
 
 
 
 
(240) 276-5563  
 
 
 
 
 
Seth.Steinberg@nih.gov 
 
Referral Contact/  
 
 
Sheri McMahon, R.N., OCD, CCR, NCI 
Study Coordinator:  
 
10 Center Drive, 13N210 
Bethesda, MD 20892 
Phone: 301-496-9812 
Fax: 301-480-1779 
Email: sheri.mcmahon@nih.gov 
 
 

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
Version Date: September 14, 2016 
 
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A. Obtain information by intervening or interacting with living individuals for research 
purposes 
B. Obtaining identifiable private information about living individuals 
C. Obtaining the voluntary informed consent of individuals to be subjects 
D. Makes decisions about subject eligibility 
E. Studying, interpreting, or analyzing identifiable private information or data/specimens for 
research purposes 
F. Studying, interpreting, or analyzing de-identified data or specimens for research purposes 
G. Some/all research activities performed outside NIH 
 
 
 
 
Investigational Agents:  
Drug Name: 
MVA-brachyury-TRICOM 
IND Number: 
15990 
Sponsor: 
Center for Cancer Research 
Manufacturer: 
Bavarian Nordic, Inc. 

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PRÉCIS  
Background: 
• MVA-brachyury-TRICOM is a novel recombinant vector-based therapeutic cancer vaccine 
designed to induce an enhanced immune response against brachyury, which is overexpressed 
in many solid tumor types, such as lung, breast, ovarian, chordoma, prostate, colorectal, and 
pancreatic adenocarcinoma. 
• Modified vaccinia Ankara (MVA) is a replication-deficient, attenuated derivative of vaccinia. 
It is used in the smallpox vaccination and is now being developed as a recombinant viral 
vector to produce vaccines against infectious diseases and cancer.  
• Many MVA vector-based trials conducted in patients with cancer have demonstrated its 
safety and the immunogenicity of its transgenes. 
• Brachyury is a member of the T-box family of transcription factors. It is overexpressed in 
cancer cells compared with normal tissue and has been linked to cancer cell resistance and 
metastatic potential. 
• Brachyury as a vaccine target has been demonstrated to be safe in an ongoing phase I study 
of recombinant yeast-brachyury and to generate brachyury-specific T-cell responses. 
• Poxviral vaccines delivering a triad of three human T-cell costimulatory molecules 
designated TRICOM (B7.1, ICAM-1 and LFA-3) have been extensively studied in both 
preclinical and clinical studies and have demonstrated their ability to induce robust T-cell 
activation and provide evidence of clinical benefit. 
• In vitro, MVA-brachyury-TRICOM is able to effectively expand brachyury-specific CD8+ 
and CD4+ T cells from peripheral blood mononuclear cells. 
• Previous work indicates that MVA-brachyury-TRICOM will induce activation a distinct T-
cell subpopulation from that seen with yeast-brachyury vaccine already in development. 
Objectives: 
• To determine the safety and tolerability of escalating doses of MVA-brachyury-TRICOM 
vaccine.  
Eligibility: 
• Patients must have histologically confirmed malignancy that is metastatic or unresectable 
locally advanced malignant solid tumor. In the case of chordoma, unresectable, locally 
recurrent, or metastatic tumors are acceptable for enrollment, given that this represents 
incurable disease. As much as possible, patients enrolled will have tumor types with known 
increased expression of brachyury (such as lung, breast, ovarian, prostate, colorectal, 
pancreatic, or chordoma). 
• Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1 at study entry  
• Age ≥ 18 years.  
• Prior Therapy: Completed, or disease progression on at least one prior line of disease-
appropriate therapy for metastatic disease, or not a candidate for therapy of proven efficacy 
for their disease. 

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
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Design: 
• This is an open-label, phase I trial with sequential cohorts of patients (3–6 patients per dose 
cohort) with dose escalation of MVA-brachyury-TRICOM vaccine.  
• Three cohorts will receive MVA-brachyury-TRICOM vaccine administered subcutaneously 
as either 1, 2, or 4 injections of study drug (1 injection = 2 x 108 infectious units (IU) at 
monthly (28 days +/– 4 days) intervals for 3 months (treatment).  
• Expansion cohorts of up to 10 patients may be enrolled at the two highest tolerated dose 
levels.  These cohorts will allow certain standard, relatively non-toxic therapies to continue 
while patients receive vaccine. 
• Up to 18 patients may be required to be enrolled in the 3 cohorts, plus an additional 10 at the 
MTD and at the dose level just below it. Thus, up to 38 patients may be theoretically required 
to complete this trial. If 3 patients per month can be accrued, the study is expected to require 
1 year to complete the necessary enrollment. 

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TABLE OF CONTENTS  
PRÉCIS ........................................................................................................................................... 3 
1. 
INTRODUCTION ................................................................. Error! Bookmark not defined. 
1.1 
Study Objectives ............................................................................................................ 10 
1.1.1 
Primary Objectives.................................................................................................. 10 
1.1.2 
Secondary Objectives.............................................................................................. 10 
1.2 
Background and Rationale ............................................................................................. 10 
1.2.1 
Description and Identification of Brachyury .......................................................... 10 
1.2.2 
Brachyury Function in Epithelial-to-Mesenchymal Transition (EMT) .................. 10 
1.2.3 
Analysis of Brachyury Expression in Human Tumors and Normal Tissues .......... 12 
1.2.4 
Brachyury Expression in Lung Carcinomas ........................................................... 14 
1.2.5 
Expression of Brachyury Protein in Breast Tumor Samples .................................. 16 
1.2.6 
Brachyury Expression and Prognosis ..................................................................... 17 
1.2.7 
Brachyury Confers Chemotherapy Resistance ....................................................... 18 
1.2.8 
Immunogenicity of the Brachyury Protein ............................................................. 19 
1.2.9 
Prior Human Experience with Brachyury Vaccine Therapy .................................. 19 
1.2.10 Poxviral Vectors...................................................................................................... 19 
1.2.11 Safety of Poxviral Vectors ...................................................................................... 20 
1.2.12 Modified vaccinia Ankara (MVA) .......................................................................... 21 
1.2.13 Clinical Experience with MVA Vector-Based Trials ............................................. 21 
1.2.14 Safety of Recombinant MVA Vectors .................................................................... 22 
1.2.15 TRICOM Costimulatory Molecules ....................................................................... 23 
1.2.16 MVA-TRICOM in CLL .......................................................................................... 23 
1.2.17 Infection of Human Dendritic Cells with MVA-Brachyury-TRICOM .................. 24 
1.2.18 Induction of Human T Cells by MVA-Brachyury-TRICOM ................................. 25 
1.2.19 MVA-Brachyury-TRICOM-Infected Human DCs Stimulate Brachyury-Specific 
CD4+ T cells ......................................................................................................................... 26 
1.2.20 MVA-Bavarian Nordic (MVA-BN) ....................................................................... 27 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT ....................................................... 27 
2.1 
Eligibility Criteria .......................................................................................................... 27 
2.1.1 
Inclusion Criteria (All Subjects) ............................................................................. 27 

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2.1.2 
Inclusion Criteria (Expansion Phase Only) ............................................................. 28 
2.1.3 
Exclusion Criteria ................................................................................................... 29 
2.1.4 
Recruitment Strategies ............................................................................................ 30 
2.2 
Screening Evaluation...................................................................................................... 30 
2.2.1 
Clinical Evaluation.................................................................................................. 30 
2.2.2 
Laboratory studies ................................................................................................... 30 
2.3 
Baseline Evaluation ........................................................................................................ 31 
2.3.1 
Laboratory studies ................................................................................................... 31 
2.3.2 
Electrocardiogram (ECG) within 28 days prior to protocol enrollment ................. 31 
2.3.3 
Scans and X-rays: ................................................................................................... 31 
2.4 
Registration Procedures.................................................................................................. 31 
3 
STUDY IMPLEMENTATION ............................................................................................ 32 
3.1 
Study Design .................................................................................................................. 32 
3.1.1 
Dose-Limiting Toxicity .......................................................................................... 33 
3.1.2 
DLT Evaluation Period ........................................................................................... 33 
3.1.3 
Schema .................................................................................................................... 34 
3.2 
Drug Administration ...................................................................................................... 35 
3.3 
Study Calendar ............................................................................................................... 35 
3.4 
Criteria for Removal from Protocol Therapy and Off-Study Criteria ............................ 36 
3.4.1 
Criteria for Removal from Protocol Therapy.......................................................... 37 
3.4.2 
Off-Study Criteria ................................................................................................... 37 
3.4.3 
Off-Study Procedure ............................................................................................... 37 
4 
CONCOMITANT MEDICATIONS/MEASURES .............................................................. 37 
5 
BIOSPECIMEN COLLECTION .......................................................................................... 38 
5.1 
Correlative Studies for Research/Pharmacokinetic Studies ........................................... 38 
5.1.1 
Parameters of Immune Activation .......................................................................... 38 
5.1.2 
Brachyury expression in tissue samples ................................................................. 39 
5.2 
Sample Storage, Tracking, and Disposition ................................................................... 39 
5.2.1 
Samples Sent to Clinical Services Program (CSP) ................................................. 39 
5.2.2 
Protocol Completion/Sample Destruction .............................................................. 40 
6 
DATA COLLECTION AND EVALUATION ..................................................................... 41 
6.1 
Data Collection ............................................................................................................... 41 

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6.1.1 
Case Report Forms .................................................................................................. 41 
6.2 
Response Criteria ........................................................................................................... 41 
6.3 
Toxicity Criteria ............................................................................................................. 43 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
 
43 
7.1 
Definitions ...................................................................................................................... 43 
7.1.1 
Adverse Event ......................................................................................................... 43 
7.1.2 
Suspected adverse reaction ..................................................................................... 43 
7.1.3 
Unexpected adverse reaction .................................................................................. 44 
7.1.4 
Serious..................................................................................................................... 44 
7.1.5 
Serious Adverse Event ............................................................................................ 44 
7.1.6 
Disability ................................................................................................................. 44 
7.1.7 
Life-Threatening Adverse Drug Experience ........................................................... 44 
7.1.8 
Protocol Deviation (NIH Definition) ...................................................................... 45 
7.1.9 
Non-compliance (NIH Definition) .......................................................................... 45 
7.1.10 Unanticipated Problem............................................................................................ 45 
7.2 
NCI-IRB Reporting ........................................................................................................ 45 
7.2.1 
NCI-IRB Expedited Reporting of Unanticipated Problems and Deaths ................. 45 
7.2.2 
NCI-IRB Requirements for PI Reporting at Continuing Review ........................... 45 
7.2.3 
NCI-IRB Reporting of IND Safety Reports............................................................ 46 
7.3 
IND Sponsor Reporting Criteria .................................................................................... 46 
7.3.1 
Reporting Pregnancy ............................................................................................... 46 
7.3.2 
Paternal exposure .................................................................................................... 47 
7.4 
FDA Reporting Criteria .................................................................................................. 47 
7.4.1 
IND Safety Reports to the FDA (Refer to 21 CFR 312.32).................................... 47 
7.4.2 
FDA Annual Reports (Refer to 21 CFR 312.33) .................................................... 48 
7.4.3 
Expedited Adverse Event Reporting Criteria to the IND Manufacturer ................. 48 
7.5 
NIH Office of Science Policy (OSP)/Institutional Biosafety Committee (IBC) Reporting 
Criteria ...................................................................................................................................... 48 
7.5.1 
Serious Adverse Event Reports to OSP/IBC .......................................................... 48 
7.5.2 
Annual Reports to OSP/IBC ................................................................................... 49 
7.6 
Data and Safety Monitoring Plan ................................................................................... 50 
7.6.1 
Principal Investigator/Research Team .................................................................... 50 

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7.6.2 
Sponsor Monitoring Plan ........................................................................................ 50 
7.6.3 
Safety Monitoring Committee (SMC) .................................................................... 50 
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STATISTICAL CONSIDERATIONS.................................................................................. 51 
9 
COLLABORATIVE AGREEMENTS ................................................................................. 52 
9.1 
Agreement Type ............................................................................................................. 52 
10 HUMAN SUBJECTS PROTECTIONS ............................................................................... 52 
10.1 
Rationale For Subject Selection ................................................................................. 52 
10.1.1 Inclusion of Women and Minorities ....................................................................... 52 
10.1.2 Exclusions ............................................................................................................... 52 
10.2 
Participation of Children ............................................................................................ 52 
10.3 
Evaluation of Benefits and Risks/Discomforts ........................................................... 53 
10.4 
Consent and Assent Process and Documentation ....................................................... 53 
10.4.1 Telephone (Re-)Consent Procedure ........................................................................ 53 
10.4.2 Informed Consent of Non-English Speaking Subjects ........................................... 54 
11 PHARMACEUTICAL INFORMATION ............................................................................. 54 
11.1 
MVA-Brachyury-TRICOM ........................................................................................ 54 
11.1.1 Source ..................................................................................................................... 54 
11.1.2 Toxicity ................................................................................................................... 54 
11.1.3 Formulation and Preparation................................................................................... 54 
11.1.4 Stability and Storage ............................................................................................... 55 
11.1.5 Administration Procedures...................................................................................... 55 
11.1.6 Incompatibilities ..................................................................................................... 55 
12 REFERENCES ..................................................................................................................... 55 
13 APPENDICES ...................................................................................................................... 60 
13.1 
Appendix A: Performance Status Criteria .................................................................. 60 
13.2 
Appendix B: Immune-Related Response Criteria ...................................................... 61 
13.3 
Appendix C: RECIST 1.1 Criteria .............................................................................. 63 
13.3.1 Confirmation ........................................................................................................... 64 
13.3.2 Duration of Overall Response ................................................................................. 64 
13.3.3 Measurable Disease ................................................................................................ 64 
13.3.4 Malignant Lymph Nodes ........................................................................................ 64 
13.3.5 Non-Measurable Disease ........................................................................................ 64 

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13.3.6 Evaluable Disease ................................................................................................... 65 
13.3.7 Target Lesions ......................................................................................................... 65 
13.3.8 Non-Target Lesions ................................................................................................ 65 
13.4 
Appendix D: Modified Choi Criteria .......................................................................... 66 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1 
INTRODUCTION 
1.1 
STUDY OBJECTIVES 
1.1.1 Primary Objectives 
1.1.1.1 To determine the safety and tolerability of escalating doses of MVA-brachyury-TRICOM 
vaccine.  
1.1.2 Secondary Objectives 
1.1.2.1 To evaluate CD8 and CD4 immunologic response as measured by an increase in 
brachyury-specific T cells. 
1.1.2.2 To evaluate evidence of clinical benefit, such as progression-free survival, RECIST 
criteria, reduction in serum markers, and/or reduction in circulating tumor cells. 
1.1.2.3 Parameters of general immune activation: frequency of immune cell subsets in peripheral 
blood and changes in serum levels of cytokines and antibodies to brachyury. 
1.1.2.4 Correlation of brachyury expression in tissue samples with clinical outcomes.   
 
1.2 
BACKGROUND AND RATIONALE 
 
MVA-brachyury-TRICOM is a novel recombinant vector-based therapeutic cancer vaccine 
developed by the LTIB in collaboration with our CRADA partner Bavarian Nordic, Inc. The 
vaccine is designed to enhance the immune response against brachyury, which is overexpressed 
in many solid tumor types, including lung, breast, ovarian, prostate, chordoma, colorectal, and 
pancreatic adenocarcinoma, as well as some hematologic malignancies.  
1.2.1 Description and Identification of Brachyury  
Using a computer-based differential display analysis tool to conduct global comparison of 
expressed sequence tag (EST) clusters in the Unigene database,1,2 the gene encoding for the 
transcription factor brachyury was identified as highly represented in tumor-derived libraries and 
rarely observed in normal tissue-derived libraries.3 Brachyury is a member of the T-box family 
of transcription factors, characterized by a highly conserved DNA-binding domain designated as 
T-domain.4-7 Brachyury homologs have been reported to be involved in embryonic mesodermal 
development.4,8-11  
1.2.2 Brachyury Function in Epithelial-to-Mesenchymal Transition (EMT)  
Epithelial-to-mesenchymal transition (EMT) is a reversible process during which cells switch 
from a polarized, epithelial phenotype into a highly motile, mesenchymal phenotype.12,13 At the 
biochemical level, the EMT program involves the downregulation of epithelial proteins such as 
E-cadherin and cytokeratins and the induction of mesenchymal proteins, including fibronectin, 
N-cadherin and vimentin.14,15 Numerous observations support the concept that the EMT process 
plays a role in the progression of human carcinomas (Figure 1).16 

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Figure1. Role of brachyury in epithelial-to-mesenchymal transition during tumor progression 
We have demonstrated17 that overexpression of brachyury in human carcinoma cell lines is able 
to drive a switch from an epithelial to a mesenchymal-like phenotype (i.e., EMT). For example, 
stable transfection of a pancreatic cancer cell line, PANC-1, with a plasmid encoding for full-
length human brachyury resulted in cell spreading, decreased epithelial E-cadherin, and 
increased mesenchymal fibronectin expression (Figure 2). Western blot also confirmed the 
overexpression of other mesenchymal markers and downregulation of epithelial markers (Figure 
2), providing evidence that brachyury may facilitate EMT.17 
 
Overexpression of brachyury in epithelial tumor cells also resulted in a concomitant increase in 
tumor cell migration and extracellular matrix invasion (Figure 3A and B). Stable silencing of 
brachyury expression in brachyury-positive human carcinoma cells (lung H460 cells) resulted in 
downregulation of mesenchymal markers and upregulation of epithelial markers, with 
concomitant loss of cell migration and invasion (Figure 3C and D).17 
 
Figure 2. Brachyury alters morphology and 
EMT marker expression in human 
pancreatic cancer epithelial tumor cells. (A) 
Bright field images of PANC-1-pcDNA 
(control) and PANC-1-p/brachyury cells and 
immunofluorescent analysis of EMT markers; 
merged images with DAPI stained nuclei are 
shown. (B) Western blot analysis of brachyury-
induced EMT markers. See (17) for details. 

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Brachyury expression also negatively modulated tumor cell-cycle progression and correlated 
with low expression of cyclin D1, and carcinoma cells silenced for brachyury expression grew at 
a greater rate in vitro (Figure 4A). These brachyury-silenced cells grew at a comparable rate to 
control cells when implanted subcutaneously in athymic mice (Figure 4B); however, they had a 
diminished capacity to metastasize to the lungs from the primary, subcutaneous tumor, or to form 
experimental lung metastases when injected i.v. (Figure 4C).  
Collectively, these results demonstrate that the transcription factor brachyury confers on the 
tumor cells a mesenchymal phenotype as well as migratory and invasive capabilities and 
enhances tumor cell progression.17 
 
 
 
 
 
1.2.3 Analysis of Brachyury Expression in Human Tumors and Normal Tissues 
By using reverse transcription followed by polymerase chain reaction (RT-PCR), 
investigators in the LTIB have identified the overexpression of brachyury in gastrointestinal, 
Figure 3. Brachyury induction of migration and invasion. (A and B) Brachyury induces a 
highly migratory and invasive EMT phenotype in vitro in human PANC-1 pancreatic carcinoma 
cells. (C and D) Silencing brachyury expression in H460 human lung carcinoma cells reduces 
both migratory and invasive capacity. See (17) for details. 
Figure 4. Brachyury inhibition with silencing RNA reduces tumor metastases. Compared to 
control (con.shRNA, circles), human H460 lung carcinoma cells silenced for brachyury 
(Br.shRNA, triangles) (A) proliferate at a faster rate in vitro (p = 0.003); (B) grow at a similar rate 
when injected s.c. in athymic mice; and (C) form less experimental metastases when injected 
intravenously (p = 0.002). See (17) for details. 

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bladder, kidney, ovary, uterus, and testicular carcinomas. Similar studies also found 
overexpression of brachyury mRNA in cell lines of lung, colon and prostate cancers, but not 
in the majority of normal tissues tested, with the exception of expression in the testis and 
low levels of expression in B cells pooled from multiple normal donors. The expression of 
brachyury mRNA in normal B cells was further evaluated in CD19+ B-cell fractions 
isolated from various healthy donors; weak amplification was observed in 4/9 samples 
analyzed by RT-PCR. These results at the RNA level, however, contrasted with data 
obtained by immunohistochemistry analysis in normal spleens and lymph nodes, which 
were negative for the expression of brachyury protein. Moreover, the cytotoxic lysis of 
normal B cells was evaluated by using brachyury-specific T cells as effectors; no lysis was 
observed with any of the normal B cells purified from the blood of 5 different healthy 
donors.3 We have determined that EBV infection of human B cells enhances brachyury 
expression; approximately 1 in 105–106 human B cells have latent EBV infection. 
Immunohistochemistry (IHC) analysis of brachyury expression using an anti-brachyury 
monoclonal Ab confirmed the selective expression in tumors of this transcription factor. No 
expression of brachyury was observed in the majority of normal tissues tested, with the 
exception of testis (3/3 positive) and thyroid (4/6 positive). The expression of brachyury 
protein was also detected in 3 of 4 thyroid tissue lysates evaluated by Western blot. These 
results at the protein level contrasted with the expression of brachyury at the mRNA level, 
which was negative in 3/3 individual thyroid tissues tested by RT-PCR. Altogether, these 
results indicate brachyury expression in 7/13 thyroid tissues analyzed. 
Immunohistochemistry analysis of normal tissues obtained from non-cancer subjects 
demonstrated brachyury expression in 0/5 lung, 0/3 heart, 0/3 brain, 0/3 liver, 0/3 kidney, 
0/3 spleen, 0/3 skeletal muscle, 0/1 adrenal gland, 0/1 skin, 4/6 thyroid, and 3/3 testis 
samples analyzed. 
 
Quantification of brachyury expression has been performed in multiple carcinoma tissues by 
real-time PCR analysis of brachyury mRNA levels. These studies have been performed, for 
example, with lung17,18 and breast carcinomas.19 In the same studies and in Hamilton et al,20 PCR 
analysis of brachyury mRNA expression in multiple normal tissues has also been performed. 
Unlike with carcinomas, the expression of brachyury mRNA was undetectable or very low in the 
majority of normal tissues evaluated. The only normal tissue that showed brachyury mRNA 
levels comparable to those found in some carcinoma tissues was the normal testis. Analysis of 
brachyury protein expression was also conducted by immunohistochemistry.18,19 Expression of 
brachyury protein was demonstrated in lung carcinomas and invasive ductal breast carcinomas, 
both primary and metastatic lesions. Unlike with carcinomas, expression of brachyury protein 
was negative among the normal tissues analyzed, with the exception of testis and some cases of 
normal thyroid. 
The transcription factor brachyury is a driver of EMT. This phenomenon, which is responsible 
for the acquisition of tumor invasiveness and tumor resistance to multiple therapeutics, is a 
manifestation of tumor plasticity and therefore is expected to take place in a transient fashion 
along tumor progression. A few tumor cells in a primary tumor mass might express brachyury at 
a certain time under the influence of environmental factors such as TGF-β, IL-8, and others, and 
then disseminate from the primary tumor into circulation and secondary metastatic sites. The 

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analysis of a primary tumor biopsy or surgical sample might, therefore, be negative for 
brachyury expression due to the transient nature of the phenomenon, while disseminated tumor 
cells might still be positive for expression of the target brachyury. Thus, when analyzing a 
primary tumor by IHC or PCR, it is difficult to determine whether results reflect the state of 
individual tumor cells at one point in time, or whether a subpopulation of cells had previously 
undergone the EMT process via brachyury expression and had already metastasized. 
Additionally, our IHC studies with matched primary/metastatic tumors demonstrated that 
primary tumors with focal brachyury expression corresponded to metastatic sites highly positive 
for the expression of brachyury. These observations constitute the rationale for not including the 
level of expression of brachyury in primary tissue samples as a criterion for the inclusion of 
patients in the study. 
 
1.2.4 Brachyury Expression in Lung Carcinomas 
 
Expression of brachyury was found in approximately 40% of primary lung tumors (Table 1, 
Figure 5A-D). Real-time PCR analysis of brachyury mRNA expression in multiple human lung 
tumor tissues showed that the percentage of tumors positive for brachyury expression increases 
with tumor stage: 30/48 (62.5%) of stage II–IV lung cancer biopsies showed overexpression of 
brachyury vs. 12/32 (37.5%) of stage I lung cancers and 2/16 (12.5%) of histologically normal 
lung biopsies from lung cancer patients (Figure 6).17 
 
 
 
Table 1. Brachyury protein expression analyzed by IHC  
 
Lung tumor tissues 
Brachyury-positive 
Adenocarcinoma 
10/21 (48%) 
Squamous carcinoma 
3/12 (25%) 
Undifferentiated carcinoma 
2/4 (50%) 
Bronchioalveolar carcinoma 
Small cell lung cancer 
1/1 (100%) 
0/1 (0%) 
Total 
16/39 (41%) 
 
 
 

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Figure 6A. Brachyury 
expression in human lung 
tumor tissues. Real-time PCR 
was performed for brachyury on 
lung tumor tissue cDNA from 80 
lung cancer patients of the 
indicated stages of disease. The 
stage II, III, and IV cDNA 
samples are further represented 
by the symbols , ▲, and , 
respectively. As controls, 16 
samples of “normal” lung cDNA 
were analyzed, each obtained 
from a histologically normal 
section of lung from a lung 
cancer patient. See (17) for 
details.  
Figure 5. Tissue sections stained for brachyury expression, corresponding to a lung adenocarcinoma 
(A); a squamous carcinoma (B); and a bronchioalveolar carcinoma (BAC), mucinous type (C). Lung 
tumor cells invading a blood vessel, positive for brachyury expression (D). IHC staining for 
brachyury in a bone metastasis of breast cancer (E). 
Figure 6B. Expression of 
brachyury mRNA in breast tumor 
tissues. Brachyury mRNA 
expression, as determined by real-
time PCR, is shown for breast 
primary tumor tissues grouped by 
histological tumor grade (Nottingham 
grading). 
E

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1.2.5 Expression of Brachyury Protein in Breast Tumor Samples 
The expression of brachyury was further analyzed in breast tissues by IHC with a murine anti-
brachyury monoclonal Ab. Twenty-seven of 30 primary breast tumors (90%) were positive for 
brachyury expression. Sixteen benign breast tissues were negative for brachyury, with the 
exception of 2 cases of fibroadenoma where focal expression of brachyury protein was detected. 
Expression of brachyury was further evaluated in matched primary carcinoma and metastatic 
lymph nodes from 2 patients. As shown in Table 2, brachyury was expressed at higher levels in 
tumor-positive lymph nodes compared to the primary tumor, while the tumor-negative lymph 
node was negative for brachyury expression in both cases. Additional metastatic lesions (pleura, 
bone, and brain) also exhibited high levels of brachyury expression (Table 2, Figure 5E).  
 
Table 2.  Expression of brachyury protein in primary and metastatic breast carcinoma lesions 
by immunohistochemistry utilizing a murine monoclonal anti-brachyury Ab 
 
Pt 
Tissue 
Brachyury 
% Positivity 
Intensity 
6 
Breast primary tumor 
30 
+ 
6 
Met+ lymph node (a) 
90 
+ 
6 
Met+ lymph node (b) 
90 
+ 
6 
Non-met lymph node (c) 
Neg 
Neg 
9 
Breast primary tumor 
Focal 
+ 
9 
Met+ lymph node (a) 
60 
++ 
9 
Met+ lymph node (b) 
60 
++ 
9 
Non-met lymph node (c) 
Neg 
Neg 
31 
Pleura 
90 
+ 
32 
Bone 
90 
++ 
33 
Bone 
90 
+ 
34 
Brain 
70 
++ 
 
 
 
 
Matched breast primary tumor and metastatic lymph nodes from 2 patients and metastatic 
lesions from additional patients were analyzed for brachyury expression by IHC with a 
murine monoclonal anti-brachyury antibody. Two lymph nodes positive for metastasis from 
each patient (a, b) and one lymph node negative for metastasis from the same patient (c) were 
assayed.  

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
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Confidential 
1.2.6 Brachyury Expression and Prognosis 
Breast tumor gene expression data (n = 4010) derived from 23 datasets from the Gene 
Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) was 
compiled. Each sample was assigned into low (first quartile, lowest 25%), intermediate (second 
quartile, intermediate 50%) and high (third quartile, highest 25%) subgroups according to 
brachyury mRNA levels. A Kaplan-Meier estimate of survival was used to evaluate differences 
in prognosis among the 3 subgroups. Among 357 breast cancer patients treated with tamoxifen 
monotherapy as adjuvant therapy for 5 years post-diagnosis, we found that high brachyury 
expression significantly correlated with higher risk of recurrence (p = 0.0283, n = 357, Figure 
7A) and distant metastasis (p = 0.0150, n = 332, Figure 7B). To minimize the effect of clinical 
confounding factors such as tumor size, grade, nodal status, age, HER2, ER and PR status that 
might cause false positive association between gene expression and poor prognosis, a Cox 
Proportional-Hazards Regression (COXPH) survival analysis was conducted (n = 270). When 
mRNA expression signal was used as a continuous variable, increased expression of brachyury 
was significantly associated with higher risk of recurrence (p = 2.77x10-5, n = 270) and distant 
metastasis (p = 0.0001, n = 270). 

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1.2.7 Brachyury Confers Chemotherapy Resistance  
The LTIB has developed human lung carcinoma cell lines that have been either stably knocked 
down for expression of brachyury (sh/brachyury) or transfected to stably overexpress brachyury 
(ph/brachyury). Brachyury downregulation significantly enhances sensitivity to chemotherapy, 
while stable overexpression of brachyury in human lung cancer cells is associated with a 
significant decrease in cell death mediated by various chemotherapy agents in vitro (Figure 8). 
 
Figure 7. Brachyury expression and prognosis. 
A 
B 
Figure 8. Expression of brachyury in 
human carcinoma cells induces 
resistance to chemotherapy. (A) 
Silencing of brachyury (sh/brachyury) 
in H226 human lung carcinoma cells 
resulted in enhanced sensitivity to 
chemotherapy. (B) A549 lung cancer 
cells stably transfected with a 
brachyury vector (ph/brachyury) had 
enhanced resistance to several 
chemotherapeutic agents. 

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1.2.8 Immunogenicity of the Brachyury Protein 
By using an MHC-peptide binding prediction algorithm, a 9-mer HLA-A2 binding epitope of the 
brachyury protein has been identified,3 which was successfully used in vitro to expand human 
brachyury-specific CD8+ T-cell lines from peripheral blood of both normal donors and cancer 
patients. Functional assays demonstrated that these brachyury-specific T-cell lines are able to 
efficiently lyse, in an MHC-restricted manner, human carcinoma cell lines (breast, lung, colon) 
endogenously expressing the brachyury protein.  
 
Utilizing the identified HLA-A2-binding epitope of brachyury, CD8+ brachyury-specific T-cell 
responses were detected in the blood of cancer patients following vaccinations with either CEA- 
or prostate-specific antigen (PSA)-based vaccines, likely due to the phenomenon of antigen 
cascade.21 All together, these results demonstrated that brachyury is an immunogenic protein in 
humans.  
 
1.2.9 Prior Human Experience with Brachyury Vaccine Therapy  
A phase I study using escalating doses of yeast-brachyury vaccine (GI-6301) is ongoing at the 
NCI in 23 subjects with varied malignancies, including chordoma and colorectal cancer. The 
vaccine has been very well tolerated with no observed DLTs.  
  
1.2.10 Poxviral Vectors  
Vaccinia virus has been used for over 200 years as a vaccine for smallpox and has a well-
established safety profile. The virus actively replicates in human cells, resulting in the 
presentation of high levels of antigen to the immune system over a period of 1–2 weeks, 
substantially increasing the potential for immune stimulation. The immune response specific to 
vaccinia then eliminates the virus. As a result of its safety profile and ability to elicit both 
humoral and cell-mediated immunity in humans, the vaccinia virus was chosen as one of the 
vectors to deliver MUC-1, CEA, and TRICOM in previous NCI-sponsored trials.22,23 
 
Immunization with live recombinant poxviral vectors that have been genetically engineered to 
express one or more antigens allows expression of tumor-associated antigens (TAAs) and 
subsequent co-presentation of antigenic peptides with host histocompatibility antigens, a strategy 
that favors the induction of cell-mediated immune responses. Recombinant poxviruses can infect 
antigen-presenting cells, including dendritic cells and macrophages, resulting in efficient 
expression of TAAs simultaneously with costimulatory molecules required to elicit T-cell 
responses. TAAs expressed by recombinant poxviruses are presented to the immune system 
together with highly immunogenic viral proteins, which may act as adjuvants to enhance immune 
responses to the TAAs. Thus, the use of recombinant poxviral vectors to present TAAs to the 
immune system results in the generation of killer T cells that specifically destroy the selected 
tumor with little incremental toxicity. 

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
Version Date: September 14, 2016 
 
Confidential 
1.2.11 Safety of Poxviral Vectors 
A recent report on safety data from NCI clinical trials with poxviral vaccines evaluated all 
vaccine injections from 215 patients in 8 clinical trials involving poxviral vector vaccines. 
Vaccines consisted of recombinant vaccinia and recombinant fowlpox encoded with 3 human 
costimulatory molecules (TRICOM), and prostate specific antigen (PSA), or carcinoembryonic 
antigen, and/or mucin-1. Vaccines were administered at doses between 1.2 x 108 to 2 x 109 pfu, 
subcutaneously, in all patients. 84 patients also received other concurrent treatment modalities, 
such as radiation, celecoxib, ipilimumab, samarium-153, or flutamide on 4 of these trials. All 8 
clinical trials involved granulocyte-macrophage colony-stimulating factor (GM-CSF) 100 mcg, 
or recombinant fowlpox encoding GM-CSF at 1 x 108 pfu as an immune adjuvant. Grade 2 or 
higher adverse events at least possibly attributed to vaccine were reported (Table 3). A total of 
1,348 poxviral injections were given in 215 patients. No contact transmission, inadvertent 
inoculation, or any serious adverse events (AEs) related to vaccinia were observed. Below is the 
summary of proportion of vaccine administrations associated with specific AEs. These data 
demonstrate a favorable safety profile of the poxviral vaccines at a broad range of doses, routes 
of administration, in combination with other treatments, and in various tumor types. Clinical trial 
information: NCT00060528, NCT00096551, NCT00088413, NCT00081848, NCT00113984, 
NCT00450619, NCT00450463. 
 
Table 3. Safety of poxviral vaccines: NCI experience24 
AEs 
Grade 2 
Grade 3 
Grade 4 
% of injections 
associate with an AE 
Injection-site reaction 
454 
0 
0 
25.3 
Injection-site cellulitis 
0 
2 
0 
0.1 
Fever 
22 
2 
0 
1.3 
General symptoms (fatigue, myalgia, 
rash, etc.) 
53 
7 
0 
3.3 
Respiratory AEs (dyspnea, etc.) 
4 
2 
0 
0.3 
Gastrointestinal AEs (nausea, etc.) 
13 
3 
0 
0.9 
Neurologic AEs (headache, etc.)  
9 
2 
0 
0.6 
Hypotension 
3 
0 
0 
0.2 
Myocardial infarction 
0 
0 
1* 
0.1 
Atrial fibrillation 
1 
0 
0 
0.1 

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AEs 
Grade 2 
Grade 3 
Grade 4 
% of injections 
associate with an AE 
Cytopenia 
6 
1 
0 
0.4 
TTP 
0 
0 
1* 
0.1 
Lab abnormalities 
7 
0 
0 
0.4 
Total 
572 
19 
2 
33.1 
* One patient experienced multiple AEs associated with myocardial infarction and thrombotic 
thrombocytopenic purpura (TTP). 
1.2.12 Modified vaccinia Ankara (MVA) 
Despite the desirable features of poxviruses, replication-competent viruses like vaccinia should 
not be administered to severely immunocompromised patients. To address this problem, an 
attenuated vaccinia virus called modified vaccinia Ankara (MVA) was developed for high-risk 
individuals. MVA was generated by over 500 serial passages of a smallpox vaccine from 
Ankara, Turkey, in chicken embryo fibroblasts, resulting in over 15% loss of the vaccinia virus 
genome.25 MVA can infect mammalian cells and express transgenes, but it cannot produce 
infective viral particles.  
 
Bavarian Nordic has further generated a proprietary isolate of MVA designated MVA-BN. 
MVA-BN exhibits a severely restricted host range and fails to replicate in most mammalian cells, 
including primary human cells and most transformed cell lines. 
1.2.13 Clinical Experience with MVA Vector-Based Trials  
Several clinical trials in metastatic renal cell carcinoma have been conducted with the TroVax 
vaccine, which consists of a recombinant MVA expressing the 5T4 TAA. Initial trials 
demonstrated some objective clinical responses, stable disease, and both antibody and T-cell 
responses to 5T4. A phase III randomized, placebo-controlled study employed MVA 5T4 with 
and without cytokines and sunitinib in patients (n = 733) with metastatic renal cell cancer (Table 
5). Treatment arms were well-balanced. There was, however, no significant difference in overall 
survival between the 2 treatment arms. The magnitude of the 5T4-specific antibody response 
post-vaccination was associated with increased patient survival, as seen in previous trials. A 
second vaccine has also been analyzed in patients with metastatic renal cell carcinoma. The 
TG4010 vaccine consists of MVA expressing recombinant MUC-1 and IL-2 transgenes. The 
vaccine was used either alone or in combination with exogenous IFN-α and IL-2. No objective 
responses were noted. Eighteen percent of patients had stable disease for >6 months with vaccine 
alone, and 30% had stable disease for >6 months with vaccine and cytokine. Median overall 
survival was 19.3 months for all patients and 22.4 months for patients receiving vaccine and 
cytokine. 

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
Version Date: September 14, 2016 
 
Confidential 
 
The TroVax vaccine has also been evaluated in 4 small single-arm trials in patients with 
metastatic colorectal cancer. Antibody and T-cell responses, as well as stable disease, were noted 
along with some complete responses and partial responses in the various trials. In a trial 
involving the combination of TroVax with FOLFIRI chemotherapy, 11/17 patients were 
considered evaluable for immunologic evaluation. Of these, 6 had complete or partial responses 
as well as T-cell or antibody responses to vaccine. These immune responses correlated with 
clinical benefit. 
 
The TG4010 vaccine was also evaluated in prostate cancer patients (n = 40) with biochemical 
failure. Thirteen of 40 patients had a more than 2-fold improvement in PSA doubling time, and 
10 patients had their PSA stabilized over 18 months. This vaccine has also been evaluated in 
patients (n = 65) with stage 3/4 non-small cell lung cancer. In this randomized phase II study, 
patients in arm 1 received vaccine in combination with chemotherapy; in arm 2 patients received 
vaccine monotherapy until disease progression, followed by vaccine plus the same 
chemotherapy. The median overall survival in arm 1 was 12.7 months versus 14.9 months for 
arm 2. One-year overall survival rate was 53% for arm 1 and 60% for arm 2. 
 
1.2.14 Safety of Recombinant MVA Vectors  
Safety data from the first clinical trials of recombinant MVA recorded no serious or severe 
adverse events.26 MVA-METRAP (fusion between the string of multiple defined T-cell epitopes 
originally developed for the VLPs (known as ME), and a complete P. falciparum liver stage 
antigen, thrombospondin relative adhesive protein (TRAP)) was administered to 26 healthy 
volunteers aged 18–55 by intradermal injection at a dose of 3 × 107 pfu, in some cases following 
priming with a DNA vaccine also expressing METRAP administered either intramuscularly or 
by gene gun. Most adverse events were mild: flu-like illness, nausea, lethargy, or 
lymphadenopathy. Ten moderate adverse events were reported. In general, the vaccinations were 
well tolerated. Swabs were taken from the surface of the skin following injection and from a 
fluid-filled blister at one injection site and assessed for the presence of MVA-METRAP by PCR, 
which would have detected both live and dead virus, but none was found. 
 
Several clinical trials have since been conducted in patients with varied malignancies, including 
renal, colorectal, NSCLC, prostate, and breast cancer.27,28,29,30,31 MVA vaccines have been well 
tolerated in trials giving multiple doses, with a majority of adverse events reported as mild, 
transient, and following an expected pattern; most resolved within 48 hours of vaccination.  
 
MVA-BN has been used to generate 2 active cancer immunotherapies: MVA-BN-HER2 and 
MVA-BN-PRO. MVA-BN-HER2 expresses a modified version of the HER2 protein and has 
been used to treat women with HER2-positive breast cancer. MVA-BN-PRO expresses both 
PSA and prostatic acid phosphatase and has been used to treat men with non-metastatic prostate 
cancer. 

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
Version Date: September 14, 2016 
 
Confidential 
 
MVA-BN-HER2 was evaluated in 3 phase I studies, BNIT-BR-001, BNIT-BR-002, and BNIT-
BR-003. In total, 45 patients were treated with single or multiple doses of vaccine and the drug 
was well-tolerated. No patients discontinued treatment due to a drug-related adverse event. In the 
BNIT-BR-003 study, one event of cellulitis in one patient was the only SAE reported and was 
considered possibly related. In the BNIT-BR-003 study, immunotherapy-induced HER-2 
transgene-specific and MVA vector-specific T-cell and humoral responses were detected during 
and after treatment, indicating that MVA-BN-HER2 is immunologically active in the adjuvant 
setting of breast cancer.  
 
MVA-BN-PRO was evaluated in prostate cancer patients enrolled in a single phase I clinical 
trial, BNIT-PR-001. The primary objective of this study was to evaluate the safety and 
tolerability of single and multiple injection regimens of MVA-BN-PRO for the treatment of 
androgen-insensitive prostate cancer. There were no dose-limiting toxicities identified and no 
drug-related serious adverse events noted in 24 patients treated. 
 
1.2.15 TRICOM Costimulatory Molecules  
Destruction of immunological targets requires T-cell lymphocyte recognition, via the T-cell 
receptor, of antigenic peptides presented in the context of MHC molecules on APCs. In addition 
to this antigen-specific signal, a second, antigen-independent signal is required for T-cell 
activation.32,33 This second signal is provided by the interaction of specific ligands on the T-cell 
surface with costimulatory molecules expressed on APCs. The most extensively studied pathway 
of costimulation is that involving the interaction of the costimulatory molecule B7.1 expressed 
on APCs with CD28 and CTLA4 on the T cell.34,35 A number of additional costimulatory 
molecules on APCs have been identified, including ICAM-1 and LFA-3, whose ligands are 
LFA-1 and CD2, respectively, on the surface of T cells.36,37, 38  
 
Proper engagement of the T-cell receptor and costimulatory receptors requires the expression of 
both antigen and costimulatory molecules in the same cell. Therefore, coexpression of 
costimulatory molecules using a single recombinant vector presents the potential of cooperation 
among these proteins to enhance T-cell activation. Recombinant vectors coexpressing 3 
costimulatory molecules, LFA-3, ICAM-1, and B7.1, designated TRICOM™, have been shown 
to have synergistic effects on antitumor responses compared to vectors expressing individual 
costimulatory molecules.33 Mice immunized with a recombinant vaccinia virus coexpressing 
carcinoembryonic antigen (CEA) and murine TRICOM exhibited greater immune responses and 
antitumor responses than mice immunized with a recombinant vaccinia virus coexpressing CEA 
and murine B7.1. Enhanced antitumor immunity was also observed in mice that were transgenic 
(tolerant) for CEA.39 MVA-brachyury-TRICOM, therefore, has been designed to simultaneously 
express brachyury together with B7.1, LFA-3, and ICAM-1.  
1.2.16 MVA-TRICOM in CLL  
We previously demonstrated that infection of CLL cells with MVA expressing the costimulatory 
molecules B7.1, ICAM-1, and LFA-3 (TRICOM) increased expression of these costimulatory 

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
Version Date: September 14, 2016 
 
Confidential 
molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. 
We further evaluated an alternative MVA vector platform encoding for human CD40L in 
comparison with MVA-TRICOM for their ability to enhance the immunogenicity of CLL cells, 
in vitro. Our results indicated that MVA-TRICOM-infected and MVA-CD40L-infected CLL 
cells are equally potent at inducing autologous T-cell proliferation.40  
 
1.2.17 Infection of Human Dendritic Cells with MVA-Brachyury-TRICOM 
In order to investigate the ability of MVA-brachyury-TRICOM to infect cells from the blood of 
normal donors, dendritic cells (DCs) were prepared from peripheral blood mononuclear cells 
(PBMCs) by culture for 6 days in the presence of GM-CSF and IL-4, and subsequently incubated 
with wild-type MVA (MVA-WT), MVA-TRICOM (expressing only the 3 costimulatory 
molecules), or MVA-brachyury-TRICOM.  
 
Using MVA-WT alone, no increased expression of CD80, CD54, or CD58 was observed. MVA-
TRICOM resulted in upregulation of each of the 3 costimulatory molecules. Addition of the 
target antigen, brachyury, did not interfere with upregulation of the costimulatory molecules, 
indicating that all 4 transgenes could be inserted into the vector and result in effective infection 
and gene expression (Table 4). 
 
Table 4. In vitro infection of human DCs with MVA-brachyury-TRICOM 
 
 
Brachury expression was present when DCs were infected with MVA-brachyury-TRICOM, but 
was undetectable in uninfected or MVA-WT-infected DCs (Figure 9). Similarly, expression of 
brachyury in DCs infected with MVA-brachyury-TRICOM (MOI = 10) was evaluted by 
immunofluorescence using a monoclonal anti-brachyury Ab (Abcam) (Figure 10). 

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
Version Date: September 14, 2016 
 
Confidential 
 
 
 
 
 
These results indicate that the MVA-brachyury-TRICOM vector is able to infect human DCs, 
resulting in upregulation of brachyury as well as 3 costimulatory molecules (TRICOM).  
1.2.18 Induction of Human T Cells by MVA-Brachyury-TRICOM 
In order to investigate the ability of MVA-brachyury-TRICOM to expand brachyury-specific T 
cells from the blood of normal donors, DCs were prepared from PBMCs and infected with 
MVA-WT, MVA-TRICOM, or MVA-brachyury-TRICOM vectors. Brachyury-specific T cells 
were stimulated with irradiated DCs. Supernatants were collected and evaluated for IFN-gamma 
production by ELISA assay. Shown is the IFN-gamma production in response to MVA-
brachyury-TRICOM vs. MVA-WT-infected DCs (Figure 11).  
Figure 9. Detection of brachyury 
expression by Western blot analysis. 
Expression of brachyury was evaluated 
by Western blot analysis with a 
monoclonal rabbit anti-brachyury Ab 
(clone 54-1). GADPH expression is 
also shown. (MW = molecular weight 
marker). 
Figure 10. Detection of 
brachyury by 
immunofluorescence. 
Expression of brachyury was 
evaluated by 
immunofluorescence with a 
monoclonal anti-brachyury 
Ab (Abcam). (Green 
indicates brachyury 
expression, gray indicates 
DAPI-stained nuclei.)  

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
Version Date: September 14, 2016 
 
Confidential 
 
Figure 11. Brachyury-specific CD8+ T cell stimulation by MVA-brachyury-TRICOM-infected 
DCs compared with MVA-TRICOM-infected DCs. 
1.2.19 MVA-Brachyury-TRICOM-Infected Human DCs Stimulate Brachyury-Specific CD4+ T 
cells  
CD4+ T cells from normal-donor PBMCs were exposed to either MVA-WT or MVA-brachyury-
TRICOM-infected DCs and then exposed to a control (HSA) or brachyury protein. The CD4+ T 
cells exposed to MVA-WT did not proliferate in response to HSA or brachyury, but the CD4+ T 
cells exposed to MVA-brachyury-TRICOM DCs proliferated significantly better when exposed 
to brachyury protein compared with HSA (Figure 12). These results indicate MVA-brachyury-
TRICOM is able to effectively expand CD4+ brachyury-specific T cells from the blood of 
normal donors, as compared to the MVA-WT vector.  
 
 

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Abbreviated Title: Phase I MVA-Brachyury-TRICOM 
Version Date: September 14, 2016 
 
Confidential 
Figure 12. MVA-brachury-TRICOM (and not MVA-WT)-infected human DCs expand CD4+ T 
cells from PBMCs of normal donors that recognize purified brachyury protein. 
 
1.2.20 MVA-Bavarian Nordic (MVA-BN) 
MVA-BN effectively infects mammalian cells. Infection of mammalian cells results in 
transcription of the viral genes, but no MVA-BN is released from the cells due to a genetic block 
in the viral assembly and egress. The infected cells eventually undergo apoptosis (programmed 
cell death). MVA-BN replicates efficiently in CEF cells and probably also in certain other avian 
cell lines. Despite its high attenuation and reduced virulence, in preclinical studies MVA-BN has 
been shown to elicit both humoral and cellular immune responses to vaccinia and genes cloned 
into the MVA genome. MVA-BN is a potent inducer of type I interferon (IFN) in human cells. 
Like other MVA, MVA-BN expresses a soluble interleukin-1 receptor,41 which has been 
implicated as an antivirulence factor for certain poxviruses.42 MVA does not express soluble 
receptors for IFN-γ, IFN-α/β, tumor necrosis factor, or CC chemokines.43 
MVA-BN is a further attenuated MVA strain that has lost its ability to replicate in most 
mammalian cell types, including almost all human cell lines, and is safe in severely 
immunocompromised animals (AGR129 mice). The hallmark of MVA-BN is the fact that it does 
not productively replicate in the human keratinocyte cell line HaCat, the human cervix 
adenocarcinoma cell line HeLa, the human embryo kidney cell line 293 (HEK293), and the 
human bone osteosarcoma cell line 143B. However, like other MVA strains, MVA-BN 
effectively infects mammalian cells. 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT  
2.1 
ELIGIBILITY CRITERIA 
2.1.1 Inclusion Criteria (All Subjects) 
2.1.1.1 Patients must have a metastatic or unresectable locally advanced malignant solid tumor, 
histologically confirmed by the Laboratory of Pathology, NCI. In the case of chordoma, 
unresectable, locally recurrent, or metastatic tumors are acceptable for enrollment, 
given that this represents incurable disease. Efforts will be made, as much as possible, 
to enroll patients with tumor types with known increased expression of brachyury (such 
as lung, breast, ovarian, prostate, colorectal, pancreatic, or chordoma; other tumors may 
be included as data on the level of brachyury in those tumors becomes available). 
2.1.1.2 Patients may have measurable or nonmeasurable but evaluable disease. See Section 6.2 
for the evaluation of measurable disease.  Patients with surgically resected metastatic 
disease at high risk of relapse are also eligible. 
2.1.1.3 Prior therapy: Patients must have completed or had disease progression on at least one 
prior line of disease-appropriate therapy for metastatic disease, or not be candidates for 
therapy of proven efficacy for their disease. 
2.1.1.4 There should be a minimum of 4 weeks from any prior chemotherapy, immunotherapy 
and/or radiation, with the exception of hormonal therapy for prostate and breast 

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Confidential 
cancers, HER2-directed therapy for HER2+ breast cancer (3+ IHC or FISH+), and 
erlotinib in EGFR-mutated lung cancer in the expansion cohort as detailed in section.  
There should be a minimum of 6 weeks from any prior antibody therapies, (such as 
ipilimumab or anti-PD1/PDL1) due to prolonged half-life.  
2.1.1.5 Patients must have recovered (grade 1 or baseline) from any clinically significant 
toxicity associated with prior therapy. Typically, this is 3–4 weeks for patients who 
most recently received cytotoxic therapy, except for the nitrosoureas and mitomycin C, 
for which 6 weeks is needed for recovery.  
2.1.1.6 Age ≥ 18 years. Because no dosing or adverse event data are currently available on the 
use of MVA-brachyury-TRICOM vaccine in patients < 18 years of age, children are 
excluded from this study but will be eligible for future pediatric trials. 
2.1.1.7 ECOG performance status  ≤ 1 (Karnofsky ≥ 70%, see Appendix A, Section 13.1). 
2.1.1.8 Patients must have normal organ and marrow function as defined below:  
o Serum creatinine ≤ 1.5 x upper limit of normal OR creatinine clearance on a 24-h urine 
collection of ≥ 50 mL/min. 
o ALT and AST ≤ 3x the upper limits of normal. 
o Total bilirubin ≤ 1.5 x upper limit of normal OR in patients with Gilbert’s syndrome, a 
total bilirubin ≤ 3.0. 
o Hematological eligibility parameters (within 16 days of starting therapy): 
• Granulocyte count ≥ 1,500/mm3 
• Platelet count ≥ 100,000/mm3 
2.1.1.9 Patients must have baseline pulse oximetry > 90% on room air.  
2.1.1.10 The effects of MVA-brachyury-TRICOM on the developing human fetus are unknown. 
For this reason, women of child-bearing potential and men must agree to use adequate 
contraception (hormonal or barrier method of birth control; abstinence) prior to study 
entry and for the duration of study participation and for a period of 4 months after the 
last vaccination therapy. Should a woman become pregnant or suspect she is pregnant 
while she or her partner is participating in this study, she should inform her treating 
physician immediately. 
2.1.1.11 Patients with prostate cancer must continue to receive GnRH agonist therapy (unless 
orchiectomy has been done). If a patient has refused GnRH therapy, they may be 
enrolled on a dose level for which the safety has already been determined. 
Patients must be able to understand and be willing to sign a written informed consent 
document. 
2.1.2 Inclusion Criteria (Expansion Phase Only)  
The following inclusion criteria apply specifically to patients being considered for the expansion 
phase of the protocol, as described in Section 8. 

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2.1.2.1 Subjects with EGFR-mutated lung cancer may continue erlotinib if they have been on the 
drug for ≥ 3 months with stable disease or a response. Erlotinib may also be continued 
in the case of a progressing tumor after prior response (or > 6 months stable disease).  
2.1.2.2 Patients with ER+ breast cancer being treated with hormonal therapy (selective estrogen 
receptor modulator or aromatase inhibitor) who have rising tumor markers as evidence 
of disease progression or metastatic disease on scans may continue on hormonal therapy 
while being treated with vaccine. 
2.1.2.3 Patients with Her2+ breast cancer receiving Her2-directed therapy (e.g. trastuzumab) may 
continue on that therapy when enrolling into a dose level for which safety has been 
established. 
2.1.2.4 Subjects with mestastatic colorectal cancer may continue “maintenance” therapy with 
capecitabine and/or bevacizumab. 
2.1.3 Exclusion Criteria 
2.1.3.1  Concurrent treatment for cancer, with specific exceptions noted in inclusion criteria.  
2.1.3.2 Chronic hepatitis B or C infection, because potential immune impairment caused by 
these disorders may diminish the effectiveness of this immunologic therapy. 
2.1.3.3 Any significant disease that, in the opinion of the investigator, may impair the patient’s 
tolerance of study treatment.  
2.1.3.4 Significant dementia, altered mental status, or any psychiatric condition that would 
prohibit the understanding or rendering of informed consent. 
2.1.3.5 Active autoimmune diseases requiring treatment or a history of autoimmune disease 
that might be stimulated by vaccine treatment. This requirement is due to the potential 
risks of exacerbating autoimmunity. However, patients with vitiligo or clinically stable 
autoimmune endocrine disease who are on appropriate replacement therapy (if such 
therapy is indicated) are eligible. 
2.1.3.6 Concurrent use of systemic steroids, except for physiologic doses of systemic steroid 
replacement or local (topical, nasal, or inhaled) steroid use. Limited pharmacologic 
doses of systemic steroids (e.g., in patients with exacerbations of reactive airway 
disease or to prevent I.V. contrast allergic reaction or anaphylaxis in patients who have 
known contrast allergies) are allowed. 
2.1.3.7 Patients who are receiving any other investigational agents within 28 days before start 
of study treatment. 
2.1.3.8 Patients with untreated central nervous system metastases or local treatment of brain 
metastases within the last 6 months. Patients with stable brain metastasis for 6 months 
post-intervention are eligible. Subjects with chordoma will be eligible regardless of site 
of disease if other eligibility criteria are met. 
2.1.3.9 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to MVA-brachyury-TRICOM or other agents used in study. 
2.1.3.10 Serious or uncontrolled intercurrent illness including, but not limited to, ongoing or 
active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac 
arrhythmia, or psychiatric illness/social situations that, in the opinion of the 
investigator, would limit compliance with study requirements. 

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2.1.3.11 Pregnant women are excluded from this study due to the unknown effects of the MVA-
brachyury-TRICOM vaccine on the fetus or infant. Because there is an unknown but 
potential risk for adverse events in nursing infants secondary to treatment of the mother 
with MVA-brachyury-TRICOM, breastfeeding should be discontinued if the mother is 
treated with MVA-brachyury-TRICOM. These potential risks may also apply to other 
agents used in this study. 
2.1.3.12 HIV-positive patients are ineligible because of the potential for decreased immune 
response to the vaccine.  
2.1.4 Recruitment Strategies 
This study will be listed on available websites (www.clinicaltrials.gov, 
www.cancer.gov/clinicaltrials, http://clinicalstudies.info.nih.gov) and participants will be 
recruited from the current patient population at NIH. 
2.2 
SCREENING EVALUATION 
Screening evaluation will be conducted within 16 days before starting treatment unless otherwise 
specified. 
2.2.1 Clinical Evaluation 
• History and physical examination including vital signs (blood pressure, pulse, 
respiratory rate, oxygen saturation) 
• Height and weight 
• Performance status determination (see Appendix A, Section 13.1)  
2.2.2 Laboratory studies 
To be performed at any time before protocol enrollment: 
• Confirmation of diagnosis by Laboratory of Pathology at the NIH Clinical Center. 
• Request of a minimum of three unstained slides for expression of brachyury.  
Samples will be preferably from metastatic sites of disease as well as primary, if 
possible. This is optional and not required for enrollment.  
To be performed within 8 weeks prior to protocol enrollment: 
• Screening for HIV 
• Screening for hepatitis B and C. 
 
To be performed within 16 days prior to protocol enrollment: 
• Complete blood count plus differential and platelet count 
• Serum chemistries (Na+, K+, Cl–, CO2, glucose, BUN, creatinine, 
albumin, calcium, magnesium, phosphorus, alkaline phosphatase, ALT, 
AST, total bilirubin, LDH, TSH)  
• Urinalysis 

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• Beta-HCG for women of child-bearing age (repeated within 48 hours prior 
to treatment). A female of child-bearing potential (FCBP) is a sexually 
mature woman who: 
o Has not undergone a hysterectomy or bilateral oophorectomy 
o Has not been naturally postmenopausal for at least 24 consecutive 
months (i.e., has had menses at any time in the preceding 24 
consecutive months). 
2.3 
BASELINE EVALUATION 
2.3.1 Laboratory studies 
To be performed at any time before protocol enrollment: 
• HLA typing 
To be performed within 16 days prior to protocol enrollment: 
• Screening tests noted above will be used as baseline  
 
Additional baseline labs to be performed 
• ANA titer 
• CD4:CD8 ratio, CD3, 4, 8, 19 subsets and NK markers (baseline, about day 29 prior 
to vaccination and around day 85, and then every 85 days thereafter while patients 
remain on study for follow-up) 
2.3.2 Electrocardiogram (ECG) within 28 days prior to protocol enrollment 
2.3.3 Scans and X-rays: 
To be performed within 28 days prior to the protocol enrollment (may include based on site of 
disease): 
• Computerized tomography (CT) of the chest/abdomen/pelvis 
• Brain MRI 
• PET scan 
• Bone scan 
 
2.4 
REGISTRATION PROCEDURES 
Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) 
within 24 hours of signing consent.  A registration Eligibility Checklist from the web site 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and sent via 
encrypted email to:  NCI Central Registration Office (HOIS) 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 

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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.  
 
3 
STUDY IMPLEMENTATION 
3.1 
STUDY DESIGN 
This is an open-label phase I trial with sequential cohorts of patients (3–6 patients per dose 
cohort to establish safety) with dose escalation of MVA-brachyury-TRICOM vaccine (see 
Section 8). Three cohorts will receive MVA-brachyury-TRICOM vaccine administered 
subcutaneously as either 1, 2, or 4 injections of study drug (1 injection = 2 x 108 IU) at monthly 
(28 days– 4 days up to +14 days) intervals for 3 months (treatment).44 A dose will be considered 
missed if not given within 14 days of the original schedule and the patient will resume treatment 
with the next scheduled dose. The time of patient monitoring post injection would be 2 hour 
observation after first dose and 30 minutes after subsequent doses.  
Toxicity in the 28 days following the first vaccination after the 3rd subject (or 6th subject, if 
applicable) in dose levels 1 through 3 will be employed for decision-making (i.e., for dose-
escalation and for maximum tolerable dose estimation). Intrapatient dose escalation will not be 
allowed. In addition, patients may be enrolled as an expansion cohort at the MTD and at the dose 
level just below the MTD after either 0 of 3 or no more than 1 of 6 patients have been treated at 
that dose level and have not experienced a DLT for at least 28 days. Cohorts 2 and 3 will accrue 
up to an additional 10 evaluable patients each (up to 16 patients total in each) for safety and 
correlative analysis.  
Patients will be sequentially enrolled and monitored for safety as described at dose level 2 and 
dose level 3.  If 0 of 3 or no more than 1 of 6 patients treated at each dose level have experienced 
a DLT for at least 28 days, we will enroll patients on the expansion phases.   
 
Patients may be concurrently enrolled in the expansion cohort of dose level 2 while enrolling 
patients on dose level 3 for safety evaluation.  Any patient who is eligible will be prioritized into 
a dose escalation slot.  Once all dose escalation slots are filled, patients will be enrolled 
sequentially onto the expansion phase 2, followed by phase 3.  Expansion slots will only be filled 
by subjects who are not eligible (due to concurrent therapy) for escalation slots until safety is 
established in both dose level 2 and 3. There will not be randomization. 
 
Patients who come off study for any reason other than IND-related toxicity (defined as at least 
possibly related) prior to completion of the first restaging evaluation (about day 85) may be 
replaced. If a patient did not experience DLT and did not complete the DLT evaluation period 
(28 days), he or she will not be evaluable for toxicity and will be replaced in the dose level as 
outlined in Statistical Design (Section 8).  
• If 0/3 patients develop DLT on a dose level, then the dose may be escalated. For purposes 
of obtaining additional safety and efficacy information, cohorts up to 6 patients/cohort may 
be enrolled.  
• If 1/3 patients develops DLT on a dose level, then up to 3 more patients will be enrolled.  
• If no more than 1/6 patients develops DLT, then the dose will be escalated.  

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• If 2 or more patients experience DLT on a dose level, then the MTD has been exceeded 
and dose escalation has been completed. Three patients will be enrolled on the dose level 
below that level, provided that not more than 3 patients were treated on that dose level. 
 (The MTD will be the dose level at which no greater than 1/6 patients has a DLT, and the next 
higher dose level has at least 2 patients with a DLT.) 
 
3.1.1 Dose-Limiting Toxicity 
Dose-limiting toxicity will be defined as any one of the following: Any grade ≥ 3 hematologic 
toxicity or grade ≥ 3 non-hematologic toxicity that is possibly, probably, or definitely related to 
study drug, except transient (≤ 48 hour) grade 3 fatigue, local reactions, flu-like symptoms, 
fever, headache, and laboratory abnormalities that are not associated with organ pathology. Also 
any ≥ grade 2 allergic and ≥ grade 2 autoimmune reaction(s) (except endocrine-related immune 
toxicity) will be defined as a DLT. Any grade 3 autoimmune endocrine-related toxicity that has 
not resolved clinically within 7 days of initiating therapy will also be defined as a DLT. 
Generalized erythroderma or macular or papular rash lasting less than 7 days and not associated 
with desquamation will not be DLTs.  
In addition, study accrual will be halted pending discussions with the IRB and the study sponsor 
if there is an occurrence of a grade 5 toxicity by the NCI-CTCAE (Version 4) attributable to the 
treatment regimen or if the maximum tolerated dose (MTD) is exceeded in dose level 1. 
 
3.1.2 DLT Evaluation Period 
The evaluation period for DLTs will be 28 days following the first injection of vaccine. 
 

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3.1.3 Schema       
 
 
Dose Level 
Dose and Schedule  
1 
N = 3 to 6 
1 site of injection at 2 x 108 IU given every 28 days for 3 doses.  
2 
N = 10-16 
2 sites of injection at 2 x 108 IU given every 28 days for 3 doses.  
3 
N = 10-16  
4 sites of injection at 2 x 108 IU given every 28 days for 3 doses.  
 
Dose escalation will proceed in cohorts of 3–6 patients. The MTD is the dose level at which no 
more than 1 of up to 6 patients have DLT during the first 28 days of treatment, and the dose 
below that at which at least 2 (of ≤ 6) patients have DLT as a result of the drug. If a patient did 
not experience DLT and did not complete the DLT evaluation period (28 days), he or she will 
not be evaluable for toxicity and will be replaced in the dose level as outlined in the Statistical 
Design (Section 8).  
Dose escalation will follow the rules outlined in the table below. 
 

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Number of Patients with 
DLT 
at a Given Dose Level 
Escalation Decision Rule 
0 of 3 
Enter up to 3 patients at the next dose level 
> 2 
Dose escalation will be stopped. This dose level will 
be declared the maximally administered dose 
(highest dose administered). Up to three (3) 
additional patients will be entered at the next lowest 
dose level if only 3 patients were treated previously 
at that dose. 
1 of 3 
Enter up to 3 more patients at this dose level. 
• If 0 of these 3 patients experience DLT, 
proceed to the next dose level. 
• If 1 or more of this group experience DLT, 
then dose escalation is stopped, and this dose 
is declared the maximally administered dose. 
Up to three (3) additional patients will be 
entered at the next lowest dose level if only 3 
patients were treated previously at that dose. 
< 1 of 6 at highest dose 
level below the maximally 
administered dose 
This is the MTD and is generally the recommended 
phase II dose. At least 6 patients must be entered at 
the recommended phase II dose. 
 
 
3.2 
DRUG ADMINISTRATION 
MVA-brachyury-TRICOM is supplied frozen in single-use vials.  The vial must be thawed at 
room temperature for approximately 10-15 minutes prior to preparation, and should not be re-
frozen after thawing. The thawed suspension will appear milky and may contain clumps or 
aggregates.  To ensure homogeneity, the vial should be swirled vigorously, but not shaken, for 
approximately 10 – 20 seconds immediately prior to use. The thawed drug product is to be drawn 
into a syringe with an appropriately sized safety shielded needle suited to patient comfort (e.g. 
22- to 28-gauge). MVA-brachyury-TRICOM is to be administered via subcutaneous injection in 
the upper arm and/or outer thigh per the schedule described in Section 3.1.3. 
3.3 
STUDY CALENDAR 
 
 
Baseline1 
 
Day 1 
 
Day 29 
 
Day 57 
 
Restaging 
Day 85 
Day 169 and every 85 days 
thereafter until progression 
History and 
X 
 
 
 
 
 

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physical1 
Medical 
assessments2 
X 
 
X8 
X8 
X8 
X 
Imaging 
X3 
 
 
 
X 
X 
Urinalysis  
X 
 
X 
X 
X 
X 
Serum HIV 
antibody4 
X 
 
 
 
 
 
Serum hepatitis B 
& C5 
X 
 
 
 
 
 
CBC with 
differential, 
platelet count 
X1 
 
X 
X 
X 
X 
Chemistry6  
X1 
 
X 
X 
X 
X 
EKG  
X1 
 
 
 
X 
X 
Correlative / 
Immunology 
(blood)10 
X 
 
X 
X 
X 
X 
ANA titer 
X 
 
 
 
X 
X 
CD3, 4, 8, 19 
subsets, NK 
markers and 
CD4:CD8 ratio 
X 
 
X 
 
X 
X 
HLA typing 
X 
 
 
 
 
 
Serum beta-
HCG7 
X 
 
 
 
 
 
 
Pulse oximetry  
X 
 
 
 
 
 
MVA-brachyury-
TRICOM 
vaccine9 
 
X 
X 
X 
 
 
Adverse events 
 
 
X 
X 
X 
X 
Concomitant 
medications 
X 
 
X 
X 
X 
X 
1Baseline: H & P and laboratory studies should be completed within 16 days of initiating treatment. Baseline radiographic, EKG, and immunologic 
studies should be obtained within 28 days of initiating treatment.  
2 Medical assessments: interim history (since last visit), vital signs, physical examination and ECOG performance status. To be performed at baseline, within 3 days 
of each dose of vaccine and with each restaging visit (about every 85 days on study). 
3 Radiologic studies consisting of CT chest/abdomen/pelvis (and MRI or bone scan when appropriate for tumor type) will be performed within 28 days 
prior to initiating treatment, on about day 85, and about every 85 days until progression. 
4 Serum HIV antibody should be completed within 8 weeks of initiating treatment. 
5Serum hepatitis B & C antibody should be completed within 8 weeks of initiating treatment. 
6 Chemistry panel: Na+, K+, Cl–, CO2, glucose, BUN, creatinine, albumin, calcium, magnesium, phosphorus, alkaline phosphatase, ALT, AST, total 
bilirubin, TSH, and LDH. 
7In females of child-bearing age, beta-HCG to be done at baseline within 48 hours prior to day 1.  
8 Within 3 days prior to each vaccination. 
9Administered subcutaneously at 1, 2, or 4 sites (days 1, 29, 57 all - 4 days up to +14 days. In the event a dose is delayed, all subsequent doses will be 
scheduled about 28 days from that dose.) 
10 Correlative / Immunology blood: 6 (10mL) green top sodium heparin tubes for PBMC; 2 (8mL) SST tubes for serum samples, and 2 (10mL) green 
top sodium heparin tubes for circulating tumor cell assay. 
3.4 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF-STUDY CRITERIA 
Prior to documenting removal from protocol therapy or off study (if applicable), effort must be 
made to have all subjects complete a safety visit approximately 30 days following the last dose 
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3.4.1 Criteria for Removal from Protocol Therapy 
Subjects will be removed from further vaccine therapy if any one of the following occurs:  
• Disease progression (as defined by irRC in Appendix B, Section 13.2). 
• Intercurrent illness or medical circumstances. If at any time the constraints of this 
protocol are detrimental to the patient’s health, the patient may be removed from protocol 
therapy. In this event, the reasons for withdrawal will be documented. 
• Unacceptable adverse event(s) as described in Section 3.1.1 (Definition of Dose-Limiting 
Toxicity); or there is evidence of any ≥ grade 2 allergic and ≥ grade 2 autoimmune 
reaction(s) attributed to vaccine (except grade 2 endocrine-related immune toxicity or 
grade 3 endocrine-related immune toxicity that resolves to grade ≤ 1 clinically within 7 
days of initiating supportive therapy); or if > grade 2 toxicity attributed to vaccine 
persists for > 28 days, the patient will not receive further vaccine inoculations and will be 
removed from protocol therapy. Patients who come off treatment must be followed on 
study until resolution of toxicity and until disease progression.  
• Subject decides to withdraw from the study therapy. (In this event, the reasons for 
withdrawal will be documented.) 
• Positive pregnancy test 
3.4.2 Off-Study Criteria 
Subjects will be removed from study for any one of the following criteria: 
• Participant requests to be withdrawn from study; in this event, the reasons for withdrawal 
will be documented.  
• A patient who is noncompliant with protocol guidelines may be removed from the study 
at the discretion of the principal investigator. 
• Disease progression, defined by irRC in Appendix B (Section 13.2). 
• Death. 
3.4.3 Off-Study Procedure 
Authorized staff must notify Central Registration Office (CRO) when a subject is taken off-
study. An off-study 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 (HOIS) ncicentralregistration-
l@mail.nih.gov. 
4 
CONCOMITANT MEDICATIONS/MEASURES 
Anti-emetics and antidiarrheal agents may be administered as required, but are not anticipated to 
be needed and should not be used prophylactically.  Selection of the specific antiemetic regimen 
is at the discretion of the treating physician.  Antiemetic regimens should not include 
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Hormonal therapy in breast and prostate cancer, HER2-directed therapy in breast cancer, 
erlotinib in lung cancer, and “maintenance” capecitabine and/or bevacizumab in colorectal 
cancer are allowed only as per the eligibility criteria (Section 2.1). 
Toxicities thought to be autoimmune-related and attributable to vaccine will lead to 
discontinuation of vaccine and symptomatic treatment. This may also include the use of 
immunosuppressive treatments such as glucocorticoids and replacement hormones if indicated. 
Other supportive care with blood components, antibiotics, analgesics, general medical therapy, 
etc., will be delivered as required. Any patients taking antibiotics for any reason must complete 
that course of therapy and be free of evidence of further infection before receiving any dose of 
vaccine. 
Symptomatic anemia should be treated with appropriate red blood cell or erythropoietin support. 
Other investigational therapies will not be allowed while a subject participates in this study. 
5 
BIOSPECIMEN COLLECTION 
5.1 
CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES 
5.1.1 Parameters of Immune Activation  
5.1.1.1 Blood samples to be collected: 
Correlative / Immunology blood: 6 (10mL) green top sodium heparin tubes for PBMC; 2 (8mL) 
SST tubes for serum samples, 2 (10mL) green top sodium heparin tubes for circulating tumor 
cell assay. 
5.1.1.2 Immunologic /correlative testing will include: 
• Specific T-cell responses, evaluated using flow cytometry intracellular staining (ICS) of 
CD4 and CD8 T lymphocytes for cytokines including IFN-γ, tumor necrosis factor, and 
IL-2. Additional markers of specific T-cell activation may be included based on evolving 
results with this assay. 
• CD3, CD4, CD8, and CD19 subsets, NK markers, and CD4:CD8 ratio (baseline, about 
day 29, and about day 85).  
• Analysis for cytokines (IFN-γ, IL-10, IL-12, IL-2, IL-4, TGF-ß, etc.), chemokines, 
antibodies (including yeast and brachyury), TAAs and/or other markers. 
• If sufficient samples are available pre- and post-vaccination, PBMCs will be evaluated as 
follows:  
a. IFN-γ ELISPOT assays for brachyury-specific T lymphocytes using a brachyury-
specific peptide in selected HLA-A2 peptides. 
b. A proliferation assay using brachyury protein in selected patients.  
c. Phenotypic and functional analysis of immune cell subsets (NK cells and Tregs). 
d. For use in a circulating tumor cell analysis in collaboration with Epic Sciences, 
Inc. (10975 N. Torrey Pines Rd, La Jolla, CA 92037, 858-356-6610). 

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5.1.1.3 Sample Processing 
The samples will be processed through:  
Clinical Services Program  
NCI Frederick Cancer Research and Development Center  
PO Box B  
Frederick, MD 21702  
301-846-1000  
On days samples are drawn, Jen Bangh at CSP should be notified (phone: [301] 846-5893; fax 
[301] 846-6222). She will arrange same-day courier delivery of the specimens.  
The research samples will contain labels on the blood tubes that have the patient's initials, date of 
birth, the assigned protocol, and the date the sample was drawn. The transmittal forms 
accompanying the samples also contain the same information.  
Once a patient’s treatment schedule has been determined, it should be faxed to Sandra Doren at 
the Laboratory of Tumor Immunology and Biology/ NIH (Fax: [301] 496-2756; phone: [301] 
496-9573) for planning purposes. 
5.1.2 Brachyury expression in tissue samples 
• Upon enrollment, a minimum of 3-4 unstained slides, preferably from a metastatic site of 
disease, will be requested for analysis of brachyury expression.  Unstained slides from 
the primary will also be requested, if available.  This is not required for enrollment and 
will be analyzed retrospectively.  
• An optional on-study biopsy will also be presented as a means to obtain pathologic 
samples for correlation of clinical outcomes with Brachyury expression.  
 
5.2 
SAMPLE STORAGE, TRACKING, AND DISPOSITION 
Samples will be ordered and tracked through the CRIS Screens.  Should a CRIS screen 
not be available, the NIH form 2803-1 will be completed and will accompany the 
specimen and be filed in the medical record.  Samples will not be sent outside NIH 
without IRB notification and an executed MTA.  
5.2.1 Samples Sent to Clinical Services Program (CSP) 
All data associated with patient samples are protected by a secure database. All samples drawn at 
the NIH Clinical Center will be transported to the NCI Frederick Central Repository by Leidos 
couriers. 
Samples will be tracked and managed by the Central Repository database. All samples will be 
stored in liquid nitrogen. These freezers are located at NCI Frederick Central Repository in 
Frederick, Maryland. 
Fisher BioServices manages the NCI Frederick Central Repositories under subcontract to Leidos 
Biomedical Research, Inc., Frederick. NCI Frederick Central Repositories store, among other 
things, biological specimens in support of NIH clinical studies. All specimens are stored in 

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secure, limited access facilities with sufficient security, back-up and emergency support 
capability and monitoring to ensure long-term integrity of the specimens for research.  
Specimens are stored in accordance with applicable HHS and FDA Protection of Human 
Subjects Regulations in accordance with Fisher BioServices Federal-Wide Assurance. Fisher 
BioServices’ role is limited to clinical research databases and repositories containing patient 
specimens. Fisher BioServices neither conducts nor has any vested interest in research on human 
subjects, but does provide services and supports the efforts of its customers, many of which are 
involved in research on human subjects. The Fisher BioServices 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 Fisher BioServices to accept only de-identified samples and 
sample information. To the best 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 (BSI) System II. This inventory tracking 
system is used to manage the storage and retrieval of specimens and 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 system functions. BSI provides audit tracking for 
processes 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 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. 
5.2.2 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 so long as sample or data analysis continues. Samples from consenting 
subjects will be stored until they are no longer of scientific value or until a subject withdraws 
consent for their continued use, at which time they will be destroyed. Once primary research 
objectives for the protocol are achieved, intramural researchers can request access to remaining 
samples, provided they have an IRB-approved protocol and patient consent.  
The PI will report any loss or destruction of samples to the NCI IRB as soon as he/she is made 
aware of such loss. The PI will report destroyed samples to the IRB if samples become 
unsalvageable because of environmental factors such as a broken freezer or lack of dry ice in a 

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shipping container, or if a patient withdraws consent. Samples will also be reported as lost if they 
are lost in transit between facilities or misplaced by a researcher. Freezer problems, lost samples, 
or other problems associated with samples will also be reported to the IRB, the NCI Clinical 
Director, and the office of the CCR, NCI. 
6 
DATA COLLECTION AND EVALUATION  
6.1 
DATA COLLECTION 
Data will be entered into the NCI C3D data base. 
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. 
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 breech in our plan to protect 
subject confidentiality and trial data has occurred, the IRB will be notified.  
 
6.1.1 Case Report Forms 
The site staff will be required to enter in C3D available patient data from a visit no later than 10 
business days after the visit is completed. The site staff will respond in a timely fashion to all 
queries made to the CRFs by the sponsor team and monitors. 
 
6.2 
RESPONSE CRITERIA 
For the purposes of this study, patients should be re-evaluated for response every 12 weeks. In 
addition to a baseline scan, confirmatory scans should also be obtained between 4 and 8 weeks 
following initial documentation of objective response or concern of progression, per irRC (per 
Appendix B, Section 13.2). 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline 
(version 1.1).45 Changes in the largest diameter (unidimensional measurement) of tumor lesions 
and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria. 
All patients in this study must be assessed for response to treatment, even if there are major 
treatment deviations.  

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Each patient will be assigned one of the following categories: 1) complete response (CR); 2) 
partial response (PR); 3) stable disease (SD); 4) progressive disease (PD); or 5) not evaluable 
(NE) (death prior to restaging from malignant disease, toxicity, or due to other causes; or 
insufficient data). For the purposes of this study, patients will be re-evaluated for response after 
every 3 planned dosesBecause of direct clinical observation of immune-cell influx into tumor 
causing tumor enlargement in some patients prior to sustained response, it has recently been 
suggested that clinical trials involving the use of immunotherapy use alternative guidelines, 
called immune-related response criteria (irRC), to determine radiographic response or 
progression after therapy. These recommendations have been used in recent clinical trials. One 
study of 227 subjects with metastatic melanoma showed that the approximately 10% of patients 
who had PD by modified WHO criteria but either CR, PR, or SD by irRC had overall survival 
similar to those patients who had SD, PR, or CR by both criteria. The irRC were created using 
bidimensional measurements (as previously widely used in the WHO criteria). We have taken 
the concepts of the irRC and combined them with the recently revised RECIST 1.145 to come up 
with the modified irRC used in this protocol (see Appendix B in Section 13.2). Consistent with 
the irRC, the main changes from RECIST 1.1 are (a) a requirement for confirmation of both 
progression and response by imaging at least 4 weeks after initial imaging and (b) not 
automatically calling the appearance of new lesions progressive disease if the total measurable 
tumor burden has not met criteria for progressive disease.  
For irRC, only index and measurable new lesions are taken into account. At baseline tumor 
assessment on this trial, target lesions will be measured along the longest axis and the 
measurements will be summed, called sum of largest diameter (SLD). These lesions must be a 
minimum of 10 mm per lesion, maximum of 5 target lesions, maximum of 2 per organ system. 
At each subsequent tumor assessment, the unidimensional measurement of target lesions and of 
new measureable lesions is added together to provide the total tumor burden. As per the modified 
definitions in Appendix B (Section 13.2), all responses and progression except SD require 
confirmation on a consecutive scan at least 4 weeks from the initial observation.  
Patients who experience rapid disease progression mandating discontinuation of therapy prior to 
completing 12 weeks of therapy (prior to first restaging) will be considered treatment failures. 
When possible, the same imaging studies used to define the extent of tumor at baseline upon 
study entry will be used for restaging. Time to disease progression will be defined as from the 
first date of therapy until the first notation of clinical or radiographic progression. 
In the case of SD, follow-up measurements must have met the SD criteria at least once after 
study entry at a minimum interval (in general, not < 6–8 weeks) defined in the study protocol.  
Because subjects with non-measurable disease (also called evaluable-only disease) are eligible 
for enrollment, those subjects will be evaluated with imaging studies for any evidence of disease 
progression. In these cases, progression will be defined as new lesions found on imaging studies. 
In the spirit of the irRC, any new lesions in these subjects would then require confirmation of the 
new lesion on repeat imaging at least 4 weeks later to ensure that new lesions were not immune-
related phenomena. 
While we will use the irRC to determine disease progression for treatment purposes, we will also 
report radiographic responses by RECIST 1.1 (Appendix C, Section 13.3). For study patients 
with chordoma, modified Choi criteria will be used to evaluate disease progression, stability, or 

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response using MRI and/or CT imaging (Appendix D, Section 13.4) in addition to irRC 
(RECIST 1.1). Both techniques will be used, while irRC will be used to determine progression 
for study purposes. 
 
6.3 
TOXICITY CRITERIA 
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on study. The descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be used to report AEs.All 
appropriate treatment areas should have access to a copy of the CTCAE version 4.0.  A copy of 
the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_40).  
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN 
7.1 
DEFINITIONS 
7.1.1 Adverse Event 
An adverse event is defined as any reaction, side effect, or untoward event that occurs during the 
course of the clinical trial associated with the use of a drug in humans, whether or not the event 
is considered related to the treatment or clinically significant. For this study, AEs will include 
events reported by the patient, as well as clinically significant abnormal findings on physical 
examination or laboratory evaluation. A new illness, symptom, sign or clinically significant 
laboratory abnormality or worsening of a pre-existing condition or abnormality is considered an 
AE.   
All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless 
of severity, will be followed until satisfactory resolution. AEs should be reported up to 30 days 
following the last dose of study drug.   
An abnormal laboratory value will be considered an AE 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. 
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 

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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 
normal life functions 
• 
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. 

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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 REPORTING 
7.2.1 NCI-IRB Expedited Reporting of Unanticipated Problems and Deaths 
The Protocol PI will report to the NCI-IRB: 
• All deaths, except deaths due to progressive disease  
• All protocol deviations 
• All unanticipated problems 
• All non-compliance 
Reports must be received by the NCI-IRB within 7 working 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. 

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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  
An investigator must immediately report 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.  
Study endpoints that are serious adverse events (e.g. all-cause mortality) must be reported in 
accordance with the protocol unless there is evidence suggesting a causal relationship between 
the drug and the event (e.g. death from anaphylaxis).  In that case, the investigator must 
immediately report the death to the sponsor. 
Events will be submitted to Dr. William Dahut, authorized representative for the IND Sponsor 
(CCR) at: 
William Dahut, M.D. 
Bldg. 10, Room 3-2571 MSC 1206 
10 Center Drive 
Bethesda, MD 20892 
Telephone: 301-496-4251 
William.Dahut@nih.gov 
Copy all MedWatch forms to: nciprotocolsupportoffice@mail.nih.gov 
7.3.1 Reporting Pregnancy  
7.3.1.1 Maternal exposure 
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 

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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. 
7.3.2 Paternal exposure 
Male patients should refrain from fathering a child or donating sperm during the study and for 4 
months after the last dose of vaccine. 
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 4 months after the last 
dose should, if possible, be followed up and documented. 
 
7.4 
FDA REPORTING CRITERIA 
7.4.1 IND Safety Reports to the FDA (Refer to 21 CFR 312.32) 
The Sponsor will notify the FDA of any unexpected fatal or life-threatening suspected adverse 
reactions as soon as possible but no later than 7 calendar days of initial receipt of the information 
using the MedWatch Form 3500a.  
The Sponsor is also responsible for reporting any:  
• suspected adverse reaction that is both serious and unexpected 
• any findings from clinical, epidemiological, or pooled analysis of multiple studies or any 
findings from animal or in vitro testing that suggest a significant risk in humans exposed 
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• clinically important increase in the rate of a serious suspected adverse reaction over that 
listed in the protocol or investigator brochure.  
to the FDA and to all investigators no later than 15 calendar days after determining that the 
information qualifies for reporting using the MedWatch Form 3500a. If FDA requests any 
additional data or information, the sponsor must submit it to the FDA as soon as possible, but no 
later than 15 calendars days after receiving the request.  
7.4.2 FDA Annual Reports (Refer to 21 CFR 312.33) 
The study Sponsor will submit a brief report annually of the progress of the trial within 60 days 
of the anniversary date that the IND went into effect as indicated in 21CFR 312.33, and any 
associated FDA correspondences regarding the IND annual report.  
7.4.3 Expedited Adverse Event Reporting Criteria to the IND Manufacturer 
The Sponsor will notify the IND Manufacturer of any unexpected fatal or life-threatening 
suspected adverse reactions as soon as possible but no later than 3 calendar days of initial receipt 
of the information.  The Sponsor will also notify the IND Manufacturer of Serious Adverse 
Events meeting any of the criteria defined by the bulleted list in Section 7.4.1 no later than 
7 calendar days after determining that the information qualifies for reporting. A copy of the draft 
MedWatch Form 3500a may be used to meet these reporting requirements. Notifications will be 
sent to the IND Manufacturer’s Pharmacovigilance department either via email at 
pharmacovigilance@bavarian-nordic.com or via fax at (888) 465-1219. 
A listing of all other Adverse Events will be provided to the IND Manufacturer annually,  
e.g. via provision of a copy of the IND annual report which includes a safety summary and 
Adverse Event Listings. The safety database for this study also will be shared with the IND 
manufacturer at the end of the study. 
 
7.5 
NIH OFFICE OF SCIENCE POLICY (OSP)/INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) 
REPORTING CRITERIA 
7.5.1 Serious Adverse Event Reports to OSP/IBC 
The Principal Investigator will notify OSP via email (HGTprotocols@mail.nih.gov) and IBC of 
any unexpected fatal or life-threatening experience associated with the use of  MVA-brachyury-
TRICOM 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 
MVA-brachyury-TRICOM, but are not fatal or life-threatening, much be reported to NIH 
OSP/IBC as soon as possible, but not later than 15 calendar days after the sponsor’s initial 
receipt of the information.  Adverse events may be reported by using the Adverse Event 
Reporting template available on the NIH OSP website at:  http://osp.od.nih.gov/office-
biotechnology-activities/biomedical-technology-assessment/hgt/gemcris or by using the FDA 
Form 3500a. 

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7.5.2 Annual Reports to OSP/IBC 
The study Principal Investigator will submit to OSP via email (HGTprotocols@mail.nih.gov) 
and IBC a brief report annually of the progress of the trial within 60 days of the anniversary date 
that the IND went into effect unless the IND sponsor has been authorized to submit this report.  
Within 60 days after the one-year anniversary of the date on which the investigational new drug 
(IND) application went into effect, and after each subsequent anniversary until the trial is 
completed, the Principal Investigator (or delegate) shall submit the information described below.  
Alternatively, the FDA annual report can be sent to OSP/IBC in lieu of a separate report.  Please 
include the OSP/IBC protocol number on the annual report, and the updated clinical protocol. 
7.5.2.1 Clinical Trial Information  
A brief summary of the status of each trial in progress and each trial completed during the 
previous year. The summary is required to include the following information for each trial:  
• the title and purpose of the trial  
• clinical site 
• the Principal Investigator 
• clinical protocol identifiers, including the NIH OSP/IBC protocol number, NIH grant 
number(s) (if applicable), and the FDA IND application number;  
• participant population (such as disease indication and general age group, e.g., adult or 
pediatric);  
• 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 
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7.5.2.3 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.  
7.5.2.4 A copy of the updated clinical protocol including a technical abstract.  
 
7.6 
DATA AND SAFETY MONITORING PLAN 
7.6.1 Principal Investigator/Research Team  
The clinical research team will meet on a regular basis (at least every other week) when patients 
are being actively treated on the trial to discuss each patient. Decisions about dose level 
enrollment and dose escalation if applicable 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 a lead 
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 and to the Sponsor.  
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 
This trial will be monitored by personnel employed by Harris Technical Services on contract to 
the NCI, NIH. 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. 
At least 25% of enrolled patients will be randomly selected and monitored at least biannually or 
as needed, based on accrual rate. The patients selected will have 100% source document 
verification done. Additional monitoring activities will include: adherence to protocol specified 
study eligibility, treatment plans, data collection for safety and efficacy, reporting and time 
frames of adverse events to the NCI IRB and FDA, and informed consent requirements. Written 
reports will be generated in response to the monitoring activities and submitted to the Principal 
investigator and Clinical Director or Deputy Clinical Director, CCR, NCI. 
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. 

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8 
STATISTICAL CONSIDERATIONS   
 
This study will be conducted as a standard phase I trial with an expanded cohort at the MTD. 
There are 3 dose levels of vaccine to be evaluated, and initially 3 patients will be enrolled in the 
lowest dose level. If 0/3 have a dose limiting toxicity (DLT) after 28 days from the third patient 
receiving the initial vaccine on study, then subsequent patients will be accrued to the next higher 
dose level. If 1/3 has a DLT, then 3 additional patients will be enrolled at that dose level. If 1/6 
has a DLT, escalation may proceed, while if 2+/6 have a DLT, then the MTD will be considered 
to have been exceeded, and up to 3 additional patients will be enrolled at the next lower dose 
level if 6 patients have not previously been enrolled at that dose. The MTD will be the dose level 
at which no greater than 1/6 patients has a DLT, and the next higher dose level has at least 2 
patients with a DLT. 
 
The MTD will initially consist of 6 patients as indicated, and the study plans to enroll a total of 
10 additional patients each at the MTD and the dose level below MTD (total of 16 at the MTD 
and up to 16 at the dose level just below the MTD) in in an effort to identify significant changes 
in T-cell precursors will be detectable. Potential evaluation techniques include flow-cytometry 
based intracellular cytokine staining after bBrachyury 15-mer stimulation and ELISPOT.  
Evaluations will take place both with respect to absolute change as well as whether a positive 
response is identified.  
 
Because we will be using new evaluation techniques with a first in human study, we will enroll 
10 patients in an expansion cohort, resulting in 16 potential evaluable patients in the two highest 
tolerated dose level cohorts, which will provide 80% power to detect a change in T-cell 
precursors from baseline equal to 1.0 standard deviations of the difference (effect size = 1.0) 
using a 2-tailed 0.05 alpha level paired t-test.  All 10-16 patients in each dose level with 
evaluable T-cell precursor data at this dose level will be included in the analysis, with 10 patients 
providing an illustration of 80% power based on that minimal number. The actual power could 
be greater than 80% if 11-16 patients are evaluable for this determination.  In practice, a 
nonparametric Wilcoxon signed rank test will be used. A positive response using the flow based 
intracellular cytokine staining assay will be defined as the frequency of T lymphocytes positive 
for a cytokine or CD107a post-vaccine being increased greater than two-fold compared to both 
(1) pre-vaccine values and (2) HLA control post-vaccine; the fraction of the patients who have a 
positive response out of those evaluable for this determination will be noted and reported along 
with a corresponding 95% confidence interval. 
 
Although the MTD will have been declared after 6 patients at that dose level have been 
evaluated, monitoring for toxicity in the additional cohort of patients will continue. In the event 
that more than one third of all patients enrolled at the MTD experience a DLT, protocol 
modifications will be undertaken to reduce the likely level of toxicity in future patients prior to 
continuing to enroll additional subjects. 
Clinical response evaluation will take place in the patients enrolled at the MTD and the 
expansion cohort. The results will be considered secondary and will be used to determine the 
parameters for a subsequent phase II trial should the preliminary findings from this trial warrant 
further investigation. 
 

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Up to 18 patients may be required to be enrolled in the 3 cohorts, plus an additional 10 at the 
MTD and the next highest dose level. Thus, up to 38 patients may be theoretically required to 
complete this trial. If 3 patients per month can be accrued, the study is expected to require 1 year 
to complete the necessary enrollment. 
9 
COLLABORATIVE AGREEMENTS  
9.1 
AGREEMENT TYPE 
The MVA-brachyury-TRICOM vaccine is being provided by Bavarian Nordic, Inc. under a 
Collaborative Research and Development Agreement (CRADA) with LTIB, CCR.  
10 HUMAN SUBJECTS PROTECTIONS  
10.1 RATIONALE FOR SUBJECT SELECTION 
Subjects from all racial/ethnic groups and both genders are eligible for this study if they meet the 
eligibility criteria. To date, no information suggests that differences in drug metabolism, immune 
response, 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 ethnic identity are noted, accrual may be expanded or a follow-up 
study may be written to investigate those differences more fully.  
10.1.1 Inclusion of Women and Minorities 
Both men and women and members of all races and ethnic groups are eligible for this trial. 
10.1.2 Exclusions 
Patients with active or chronic infections, including HIV, hepatitis B or C, innate 
immunodeficiency or autoimmune disease are excluded because this agent requires an intact 
immune system. In addition, these patients may be at increased risk of complications and side 
effects due to immunologic changes resulting from this agent.  
The effects of yeast-brachyury vaccine on the developing human fetus are unknown. For this 
reason, women of child-bearing potential and men must agree to use adequate contraception 
(hormonal or barrier method; abstinence) prior to study entry and for the duration of study 
participation.  
10.2 PARTICIPATION OF CHILDREN 
Because no dosing or AE data are currently available on the use of MVA-brachyury-TRICOM 
vaccine in patients < 18 years of age, children are excluded from this study but may be eligible 
for future pediatric trials. 

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10.3 EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS 
All care will be taken to minimize side effects, but they can be unpredictable in nature and 
severity. This study may involve unforeseeable risks to patients. All patients will have blood 
tests, examinations, and CT scans of the chest/abdomen/pelvis or other imaging studies as 
described in the monitoring schedule.  
Because this is a first-in-human study, nothing is known directly about the clinical benefit or risk 
of this vaccine. However, we have previously tested a vaccine targeting the same antigen 
(brachyury) with no significant toxicity. Additionally, the LTIB has a long history with viral-
vector vaccines, including vaccinia, from which MVA is derived. MVA is considered to be a 
safer alternative to vaccinia. The toxicity profile of vaccinia as a vector for therapeutic cancer 
vaccines has been excellent.46 
Given preclinical data, the side effects are expected to be minor and reversible. Efforts to 
minimize risk include subcutaneous administration, close clinical monitoring after dose 
administration, and treatment of any side effects. 
If patients suffer any physical injury as a result of participation in this study, immediate medical 
treatment is available at the Warren Grant Magnuson Clinical Center, 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. 
10.4 CONSENT AND ASSENT PROCESS AND DOCUMENTATION 
The investigational nature and objectives of this trial, the procedures involved, and their 
attendant risks and discomforts, potential benefits, and potential alternative therapies will be 
explained to the patient and a signed informed consent document obtained. Once the patient has 
reviewed the consent, an investigator on this study will discuss the specifics of the consent and 
request the patient sign once all questions are satisfactorily answered. Moreover, any 
experimental invasive procedure will require a separate consent form. All listed associate 
investigators (except those listed on the cover sheet as not being able to make clinical decisions) 
are permitted to obtain informed consent.  
10.4.1 Telephone (Re-)Consent Procedure 
Reconsent 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 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 and a copy of the informed consent document will be kept in the subject’s research 
record. 

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10.4.2 Informed Consent of 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, OSHRP SOP 12, 45 CFR 46.117 (b) (2), 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 (using either the long translated form or the short form). 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 for non-English 
speaking subjects 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 MVA-BRACHYURY-TRICOM  
11.1.1 Source  
MVA-brachyury-TRICOM is an active cancer immunotherapy. This phase I study is being 
conducted under an IND held by the Center for Cancer Research (CCR), National Cancer 
Institute (NCI). Bavarian Nordic, Inc. is the manufacturer of MVA-brachyury-TRICOM and has 
transferred sufficient product to the NCI to complete this phase I study. 
11.1.2 Toxicity  
MVA-BN-derived vectors that encode heterologous (non-vaccinia virus) antigens are being 
developed for the treatment of cancer. In GLP studies, MVA-BN and MVA-BN-derived vectors 
have been administered to 5 different animal species, including primates. In addition, MVA-BN 
and MVA-BN-derived vectors have been administered in clinical trials to over 7800 human 
subjects, including immunodeficient individuals. No marked toxicity and no drug-related serious 
AEs have occurred in any of these studies. 
11.1.3 Formulation and Preparation  
MVA-Brachyury-TRICOM is supplied as a frozen aqueous suspension in 2 mL clear borosilicate 
glass vials. The closure is a sterile bromobutyl rubber stopper, crimped with an aluminum cap 
and covered with a polypropylene closure. Each vial contains one 0.5 mL dose of MVA-
Brachyury-TRICOM, which is 2.0 x 108 Infectious Units (Inf. U.). The thawed product will be a 
slightly turbid to milky suspension which may contain clumps or aggregates.  

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MVA-brachyury-TRICOM is supplied frozen in single-use vials. Each vial must be thawed at 
room temperature for approximately 10–15 minutes prior to preparation, and should not be re-
frozen after thawing. The thawed suspension will appear milky and may contain clumps or 
aggregates. To ensure homogeneity, the vial should be swirled vigorously, but not shaken, for 
approximately 10–20 seconds immediately (within 3 minutes) prior to use. The thawed drug 
product is to be drawn into a syringe with an appropriately sized safety-shielded needle suited to 
patient comfort (e.g., 22- to 28-gauge). 
11.1.4 Stability and Storage  
MVA-brachyury-TRICOM should be stored frozen at -20°C or below and remain frozen until 
use.  
11.1.5 Administration Procedures 
MVA-brachyury-TRICOM is to be administered via subcutaneous injection, using a syringe with 
an appropriately sized safety-shielded needle suited to patient comfort (e.g., 22- to 28-gauge), in 
the upper arm and/or outer thigh per the schedule described in Section 3.1.3.  The injection must 
be delivered within 9 hours of completion of preparation as indicated in Section 11.1.3 to ensure 
stability.  When possible, each subsequent dose should be administered at the same injection site 
of the first dose.  Feasibility will be determined at the discretion of the investigator based on the 
clinical findings at the time of administration. 
11.1.6 Incompatibilities  
There are no known drug interactions associated with the use of MVA-BN and MVA-BN-
derived vectors. As stated in Section 11.1.2, no marked toxicities or serious AEs have been noted 
in clinical trials conducted with MVA-BN and MVA-BN-derived vectors. 
 
 
 
 
 
 
 
 
 
 
 
12 REFERENCES  
 

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Ramlau R, Quoix E, Rolski J, et al: A phase II study of Tg4010 (Mva-Muc1-Il2) 
in association with chemotherapy in patients with stage III/IV Non-small cell lung cancer. J 
Thorac Oncol 3:735-44, 2008 
 
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human MUC1 and IL2 as immunotherapy in patients with breast cancer. J Immunother 23:570-
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74:3815-31, 2000 
 
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McLeod Dea: Phase I dose escalation trial of MVA-BN-PRO in men with 
nonmetastatic castration-resistant prostate cancer. . Journal of Clinical Oncology 31:193, 2013 

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32. 
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signalling in the prevention of human T-cell anergy. Res Immunol 146:140-9, 1995 
 
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Hodge JW, Greiner JW, Tsang KY, et al: Costimulatory molecules as adjuvants 
for immunotherapy. Front Biosci 11:788-803, 2006 
 
34. 
Sharpe AH, Freeman GJ: The B7-CD28 superfamily. Nat Rev Immunol 2:116-26, 
2002 
 
35. 
Riley JL, June CH: The CD28 family: a T-cell rheostat for therapeutic control of 
T-cell activation. Blood 105:13-21, 2005 
 
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ICAM-1 shape unique T cell profiles. Crit Rev Immunol 15:235-53, 1995 
 
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Lebedeva T, Dustin ML, Sykulev Y: ICAM-1 co-stimulates target cells to 
facilitate antigen presentation. Curr Opin Immunol 17:251-8, 2005 
 
38. 
Wang Y, Shibuya K, Yamashita Y, et al: LFA-1 decreases the antigen dose for T 
cell activation in vivo. Int Immunol 20:1119-27, 2008 
 
39. 
Hodge JW, Sabzevari H, Yafal AG, et al: A triad of costimulatory molecules 
synergize to amplify T-cell activation. Cancer Res 59:5800-7, 1999 
 
40. 
Litzinger MT, Foon KA, Tsang KY, et al: Comparative analysis of MVA-CD40L 
and MVA-TRICOM vectors for enhancing the immunogenicity of chronic lymphocytic leukemia 
(CLL) cells. Leuk Res 34:1351-7, 2010 
 
41. 
Blanchard TJ, Alcami A, Andrea P, et al: Modified vaccinia virus Ankara 
undergoes limited replication in human cells and lacks several immunomodulatory proteins: 
implications for use as a human vaccine. J Gen Virol 79 ( Pt 5):1159-67, 1998 
 
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Alcami A, Smith GL: A soluble receptor for interleukin-1 beta encoded by 
vaccinia virus: a novel mechanism of virus modulation of the host response to infection. Cell 
71:153-67, 1992 
 
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Antoine G, Scheiflinger F, Dorner F, et al: The complete genomic sequence of the 
modified vaccinia Ankara strain: comparison with other orthopoxviruses. Virology 244:365-96, 
1998 
 
44. 
McLeod D, Adams G, Gillleman M, et al.: Phase I dose escalation trial of MVA-
BN-PRO in men with nonmetastatic castration-resistant prostate cancer. J Clin Oncol 31 (suppl 
6; abstr 193), 2013 
 
45. 
Eisenhauer EA, Therasse P, Bogaerts J, et al: New response evaluation criteria in 
solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45:228-47, 2009 
 
46. 
Safety profile of recombinant poxviral TRICOM vaccines. 2013 ASCO Annual 
Meeting 31:abstr e16036, 2013 
 
47. 
Wolchok JD, Hoos A, O'Day S, et al: Guidelines for the evaluation of immune 
therapy activity in solid tumors: immune-related response criteria. Clin Cancer Res 15:7412-20, 
2009 

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48. 
Choi H, Charnsangavej C, Faria SC, et al: Correlation of computed tomography 
and positron emission tomography in patients with metastatic gastrointestinal stromal tumor 
treated at a single institution with imatinib mesylate: proposal of new computed tomography 
response criteria. J Clin Oncol 25:1753-9, 2007 
 
49. 
Choi H: Response evaluation of gastrointestinal stromal tumors. Oncologist 13 
Suppl 2:4-7, 2008 
 
50. 
Stacchiotti S, Collini P, Messina A, et al: High-grade soft-tissue sarcomas: tumor 
response assessment--pilot study to assess the correlation between radiologic and pathologic 
response by using RECIST and Choi criteria. Radiology 251:447-56, 2009 
 
51. 
Smith AD, Shah SN, Rini BI, et al: Utilizing pre-therapy clinical schema and 
initial CT changes to predict progression-free survival in patients with metastatic renal cell 
carcinoma on VEGF-targeted therapy: A preliminary analysis. Urol Oncol, 2011 
 
 

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13 APPENDICES 
13.1 APPENDIX A: PERFORMANCE STATUS CRITERIA 
ECOG Performance Status Scale 
 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
0 
Normal activity. Fully active, able 
to carry on all pre-disease 
performance without restriction. 
100 
Normal, no complaints, no 
evidence of disease. 
90 
Able to carry on normal activity; 
minor signs or symptoms of 
disease. 
1 
Symptoms, but ambulatory. 
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work). 
80 
Normal activity with effort; 
some signs or symptoms of 
disease. 
70 
Cares for self, unable to carry on 
normal activity or to do active 
work. 
2 
In bed < 50% of the time. 
Ambulatory and capable of all 
self-care, but unable to carry out 
any work activities. Up and about 
> 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 > 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: IMMUNE-RELATED RESPONSE CRITERIA 
Responses will be evaluated in all cohorts to determine eligibility for subsequent cycles, but only 
responses evaluated in the expanded cohort and in patients enrolled at the MTD will be used for 
statistical analysis. For the purposes of this study, patients will be re-evaluated for response after 
day 85 (after 3 doses of vaccine).  
Because of direct clinical observations of immune-cell influx into tumor causing tumor 
enlargement in some patients prior to sustained response, it has recently been suggested that 
clinical trials involving the use of immunotherapy use alternative guidelines, called immune-
related response criteria (irRC), to determine radiographic response or progression after 
therapy.47 These recommendations have been used in recent clinical trials. One study of 227 
subjects with metastatic melanoma showed that the approximately 10% of patients who had PD 
by modified WHO criteria but either CR, PR, or SD by irRC had overall survival similar to 
patients who had SD, PR, or CR by both criteria. The irRC were created using bidimensional 
measurements (as previously widely used in the WHO criteria). We have taken the concepts of 
the irRC and combined them with the recently revised RECIST 1.145 to come up with the 
modified irRC used in this protocol. Consistent with the irRC, the main changes from RECIST 
1.1 are (a) a requirement for confirmation of both progression and response by imaging at least 4 
weeks after initial imaging and (b) not automatically calling the appearance of new lesions 
progressive disease if the total measurable tumor burden has not met criteria for progressive 
disease.  
For irRC, only index and measurable new lesions are taken into account. At baseline tumor 
assessment on this trial, target lesions will be measured along the longest axis and the 
measurements will be summed, called sum of largest diameter (SLD). These lesions must be a 
minimum of 10 mm per lesion, maximum of 5 target lesions, maximum of 2 per organ system. 
At each subsequent tumor assessment, the unidimensional measurement of target lesions and of 
new measureable lesions is added together to provide the total tumor burden. As per the modified 
definitions below, all responses and progression except SD require confirmation on a 
consecutive scan at least 4 weeks from initial observation.  
Definitions of irRC: 
Response 
irRC 
New measurable lesions  
Incorporated into tumor burden 
New non-measurable 
lesions  
Do not define progression (but preclude irCR) 
Non-index lesions 
Contribute to defining irCR (complete disappearance required) 
          Overall irCR 
100% disappearance of all lesions, whether measurable or not, and no new lesions, in 
2 consecutive observations not less than 4 weeks from the date first documented. 
Also, all measurable lymph nodes must have reduction in short axis to < 10 mm. 
Overall irPR 
≥ 30% decrease in SLD compared with baseline confirmed by a consecutive 
assessment at least 4 weeks after first documentation. 

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Overall irSD 
Not meeting criteria for irCR or irPR, in absence of irPD: 30% decrease in SLD 
compared with baseline cannot be established nor 20% increase compared with nadir. 
Overall irPD 
At least 20% increase in SLD compared with nadir (minimum recorded tumor 
burden) and an increase of at least 5 mm over nadir, confirmed by repeat, consecutive 
observations at least 4 weeks from the date first documented. 
Overall responses derived from changes in index, non-index and new lesions as demonstrated in 
the following table: 
Measurable response 
Non-measurable response 
Overall response 
using irRC 
Index and new, measurable 
lesions (tumor burden)* % 
Non-index lesions 
New, non-measurable 
lesions 
 
Decrease 100 
Absent 
Absent 
irCR& 
Decrease 100 
Stable 
Any 
irPR& 
Decrease 100 
Unequivocal 
progression 
Any 
irPR& 
Decrease ≥ 30 
Absent/stable 
Any 
irPR& 
Decrease ≥ 30 
Unequivocal 
progression 
Any 
irPR& 
Decrease < 30 to increase < 20 
Absent/stable 
Any 
irSD 
Decrease < 30 to increase < 20 
Unequivocal 
progression 
Any 
irSD 
Increase ≥ 20 
Any 
Any 
irPD 
* Decreases assessed relative to baseline  
& Assuming response (irCR) and progression (irPD) are confirmed by a second, consecutive assessment at least 
4 weeks apart. 
 

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13.3 APPENDIX C: RECIST 1.1 CRITERIA 
The best overall response is the best response recorded from the start of treatment until disease 
progression/recurrence (taking as reference for progressive disease the smallest measurements 
recorded since treatment started). The patient's best response assignment will depend on the 
achievement of both measurement and confirmation criteria. 
 
Target lesions 
Non-target 
lesions 
New lesions 
Overall 
response 
Best overall 
response when 
confirmation is 
required* 
CR 
CR 
No 
CR 
> 4 weeks 
confirmation**  
CR 
Non-CR/non-PD 
No 
PR 
> 4 weeks 
confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR/non-
PD/not evaluated 
No 
PR 
SD 
Non-CR/non-
PD/not evaluated 
No 
SD 
Documented at 
least once 4 
weeks from 
baseline** 
PD 
Any 
Yes or no 
PD 
No prior SD, PR, 
or CR 
Any  
PD*** 
Yes or no 
PD 
Any  
Any 
Yes 
PD 
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
** Only for non-randomized trials with response as primary endpoint. 
*** In exceptional circumstances, unequivocal progression in non-target lesions may be accepted 
as disease progression. 
 
a. Patients with a global deterioration of health status requiring discontinuation of treatment 
without objective evidence of disease progression at that time should be reported as 
“symptomatic deterioration.” Every effort should be made to document the objective 
progression even after discontinuation of treatment. 

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b. In some circumstances, it may be difficult to distinguish residual disease from normal 
tissue. When the evaluation of complete response depends on this determination, it is 
recommended that the residual lesions be investigated (fine needle aspirate/biopsy) 
before confirming complete response status. 
13.3.1 Confirmation 
To be assigned a status of PR or CR, changes in tumor measurements must be confirmed by 
repeat assessments that should be performed at least 4 weeks after the criteria for response are 
first met. 
13.3.2 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. 
13.3.3 Measurable Disease 
Measurable lesions are defined as those that can be accurately measured in at least one 
dimension (longest diameter to be recorded) as > 20 mm by chest x-ray, as > 10 mm by CT scan, 
or > 10 mm with calipers by clinical exam. All tumor measurements must be recorded in 
millimeters (or decimal fractions of centimeters). 
Note: Tumor lesions that are situated in a previously irradiated area might or might not be 
considered measurable. 
13.3.4 Malignant Lymph Nodes 
To be considered pathologically enlarged and measurable, a lymph node must be > 15 mm in 
short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater 
than 5 mm). At baseline and follow-up, only the short axis will be measured and followed. 
13.3.5 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 effusions, lymphangitis 
cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or 
MRI), are considered 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. 

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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. 
13.3.6 Evaluable Disease 
Disease that cannot be measured directly by the size of the tumor but can be evaluated by other 
methods. 
13.3.7 Target Lesions 
All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, 
representative of all involved organs, should be identified as target lesions and recorded and 
measured at baseline. Target lesions should be selected on the basis of their size (lesions with the 
longest diameter), be representative of all involved organs, but in addition should be those that 
lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the 
largest lesion does not lend itself to reproducible measurement in which circumstance the next 
largest lesion which can be measured reproducibly should be selected. A sum of the diameters 
(longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be 
calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the 
sum, then only the short axis is added into the sum. The baseline sum diameters will be used as 
reference to further characterize any objective tumor regression in the measurable dimension of 
the disease. 
13.3.8 Non-Target Lesions 
All other lesions (or sites of disease) including any measurable lesions over and above the 5 
target lesions should be identified as non-target lesions and should also be recorded at baseline. 
Measurements of these lesions are not required, but the presence, absence, or in rare cases 
unequivocal progression of each should be noted throughout follow-up. 
 
 

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13.4 APPENDIX D: MODIFIED CHOI CRITERIA 
The Choi Criteria were introduced for radiographic evaluation of gastrointestinal stromal tumors 
treated with imatinib mesylate48,49 and have been evaluated in other soft tissue sarcomas50 and 
renal cell carcinoma.51 After discussion with experts in the field of chordoma as well as the 
Chordoma Foundation, we have agreed that, at this point, there is a consensus to use a modified 
Choi Criteria, with MRI instead of CT, as the best imaging technique to determine radiographic 
response, stability, or progression in patients with chordoma.  
 
The differences between RECIST and Choi Criteria are defined below: