Conditions: Breast Cancer Triple Negative Breast Cancer HER2+ Breast Cancer Hormone Receptor Negative Breast Cancer Metastatic Breast Cancer
Keywords: Immuno-oncology Immunotherapy Vaccine Invasive Breast Cancer
Allocation: NON_RANDOMIZED | Intervention model: SEQUENTIAL
Enrollment: 1
Healthy volunteers: no

Brief summary:
Background:

Breast cancer is the second most common cause of United States (U.S.) cancer deaths in women. Immunotherapy drugs use a person's immune system to fight cancer. Researchers want to see if a new combination of immunotherapy drugs can help treat breast cancer that has gone to places in the body outside of the breast (metastasized).

Objective:

To learn if a new combination of immunotherapy drugs can shrink tumors in people with metastatic breast cancer.

Eligibility:

Adults 18 and older who have been diagnosed with metastatic breast cancer, such as Triple Negative Breast Cancer (TNBC) or estrogen receptors (ER)-/progesterone receptors (PR)-/human epidermal growth factor receptor 2 (HER2)+ Breast Cancer (HER2+BC)

Design:

Participants will be screened with:

medical history

physical exam

disease confirmation (or tumor biopsy)

tumor scans (computed tomography, magnetic resonance imaging, and/or bone scan)

blood and urine tests

electrocardiogram (measures the hearts electrical activity)

echocardiogram (creates images of the heart).

Participants will be assigned to 1 of 3 groups. The drugs they get will be based on the group they are in. Drugs are given in cycles. Each cycle = 3 weeks. Participants will be seen in clinic every 3 weeks, prior to the start of a new cycle. At each visit, participants will have an clinical exam, have blood drawn and will be asked about any side effects. They will repeat the screening tests during the study. New scans, like a computed tomography (CT) scan, will be done every 6 weeks to see if the treatment is working.

All participants will get Bavarian Nordic (BN)-Brachyury. It is 2 different vaccines - a prime and a boost. First the priming vaccines, called MVA-BN-Brachyury help to jump start the immune system. Next the boosting vaccines, called fowlpox virus (FPV)-Brachyury help to keep the immune system going. They are injected under the skin during different cycles.

All participants will get M7824 (also known as Bintrafusp alfa), which is an immunotherapy drug. Some participants will get a commonly used drug is HER2+ breast cancer called adotrastuzumab emtansine (also known as T-DM1DM1 or kadcyla). For both, a needle is inserted into a vein to give the drugs slowly.

Some participants will take Entinostat weekly by mouth. It is in tablet form. Participants will keep a pill diary.

Participants will continue on their assigned treatment until their cancer grows, they develop side effects or want to stop treatment. About 28 days after treatment ends, participants will have a follow-up visit or a telephone call. Then they will be contacted every 3 months for 1 year, then every 6 months for 1 year. They may have more tumor scans or continue treatment.

Detailed description:
Background:

* Current greater than or equal to 2nd line treatments for metastatic breast cancer provide modest response rates, and modest improvement in progression-free survival but no treatments are curative.
* Bavarian-Nordic (BN)-Brachyury vaccine is a recombinant poxvirus vaccine against the transcription factor brachyury, which plays an important role in the epithelial-to-mesenchymal transition in breast cancer. A recently completed phase 1 study of BN-Brachyury vaccine showed the vaccine was well tolerated and generated an immune response.
* M7824 (Bintrafusp alfa) is a novel bifunctional fusion protein composed of a monoclonal antibody against human programmed death-ligand 1 (PD-L1) fused to the soluble extracellular domain of human transforming growth factor (TGF)-Beta receptor II (TGF-BetaRII(241-R2), which functions as a TGF-Beta trap.
* Ado-trastuzumab emtansine (T-DM1 or Kadcyla) is an antibody drug conjugate used in second- and third- line treatment of metastatic human epidermal growth factor receptor 2 (HER2+) breast cancer (HER2+BC). T-DM1 activates antibody-dependent cellular cytotoxicity (ADCC), dendritic cell maturation, increases tumor infiltrating lymphocytes (TILs), increased PD-L1 expression, and increased immunomodulatory cytokines.
* Entinostat is a class 1 histone deacetylase inhibitor (HDACi) which suppresses tumor initiating cells, regulatory T-cells and myeloid-derived suppressor cells (MDSCs), as well as enhances cytotoxic T-cell mediated lysis, direct natural killer (NK) lysis, NK cell activation, increases PD-L1 expression and antibody-dependent cellular cytotoxicity (ADCC). In addition, entinostat may also be able to overcome HER2 resistance.
* We propose a Phase 1b trial to evaluate the safety and efficacy of the stepwise combination of the BN-Brachyury vaccine, M7824, T-DM1 and entinostat in metastatic breast cancer.

  * Arm 1 - Triple Negative Breast Cancer (TNBC); M7824 + BN-Brachyury
  * Arm 2 - estrogen receptors (ER)-/progesterone receptors (PR-)/HER2+ Breast Cancer; M7824 + BN-Brachyury + T-DM1
  * Arm 3 - ER-/PR-/HER2+ Breast Cancer; M7824 + BN-Brachyury + T-DM1+ Entinostat

Objectives:

Primary Objectives:

* Arms 1-3: Overall response rate (overall response rate (ORR); partial response (PR)+complete response (CR)
* Arms 1-3: Safety for each of the three combinations of agents explored in the arms

Eligibility:

Selected Inclusion Criteria

* Histologically confirmed metastatic breast cancer with appropriate immunohistochemistry (IHC) testing by a certified lab:

  * For Arm 1: Triple negative breast cancer. Hormone receptor negative is defined by estrogen receptor \< 10% and progesterone receptor \< 10%. HER2 negative breast cancer is defined as HER2 per IHC 0 or 1+ or 2+ with negative fluorescence in situ hybridization (FISH).
  * For Arms 2 and 3: Hormone receptor negative, HER2+ breast cancer as defined by estrogen receptor \< 10% and progesterone receptor \< 10%. HER2 positive as per IHC 3+ or 2+ with positive FISH.
* Prior treatment:

  * For Arm 1: greater than or equal to 1 prior therapy in the metastatic setting. Patients with known PD-L1 positive tumors must have received prior treatment with atezolizumab + nab-paclitaxel. Patients with ER 1-9% must have received treatment with at least two lines of endocrine treatment (selective estrogen receptor modulators (SERM), AI, fulvestrant) with one prior treatment including a cyclin-dependent kinase 4 (CDK4)/6 inhibitor + endocrine therapy for their metastatic cancer and should be considered endocrine therapy resistant.
  * For Arms 2 and 3: greater than or equal to 1 prior treatment in the metastatic setting with a taxane (docetaxel or paclitaxel), herceptin and pertuzumab.
* Females or males greater than or equal to 18 years old
* Eastern Cooperative Oncology Group (ECOG) 0 or 1
* Measurable metastatic disease per Response Evaluation Criteria in Solid Tumors (RECIST) 1.1.
* For Cohort 3, Arms 2 and 3: At least one biopsiable lesion and willingness to undergo up to three research biopsies.
* Adequate hematopoietic, hepatic, renal and cardiac (ejection fraction (EF) greater than or equal to 50%) function.

Selected Exclusion Criteria

* Patients who have received chemotherapy, including trastuzumab and pertuzumab in the previous 3 weeks; other investigational agents within 4 weeks or a programmed cell death protein 1 (PD-1)/PD-L1 antibody within 4 weeks prior to study enrollment; radiotherapy less than or equal to 4 weeks of study entry.
* Symptomatic central nervous system (CNS) metastases and leptomeningeal disease are excluded but treated brain metastases (no radiotherapy within 6 weeks) or asymptomatic brain metastasis are allowed.
* History of invasive malignancy less than or equal to 3 years prior to enrollment.
* History of congestive heart failure (CHF) as defined as New York Heart Association (NYHA) class 3 or 4 or hospitalization for CHF (any NYHA class) within 6 months of trial start.
* Concurrent use of chronic systemic steroids except for physiologic systemic steroids for replacement defined as 10mg of prednisone or an equivalent dose.

Design:

This study contains three separate, single arm phase 1b trials.

* Arm 1 will evaluate M7824 and BN-Brachyury in patients with triple-negative breast cancer (TNBC).
* If this doublet is determined to have acceptable toxicity (0-1 dose limiting toxicities (DLTs) of the first 6 patients), up to 19 patients will be enrolled on Arm 2 in which BN-Brachyury, M7824 and T-DM1 will be evaluated in patients with advanced HR-/HER2+ BC with disease progression after treatment with docetaxel (THP) or intolerance to THP.
* If this triplet is determined to have acceptable toxicity (0-1 DLTs of the first 6 patients), 19 patients will be enrolled on Arm 3 in which BN-Brachyury, entinostat, M7824 and T-DM1 will be evaluated in patients with advanced HR-/HER2+ BC with disease progression after treatment with THP or intolerance to THP.
* Up to 51 evaluable patients will be recruited for this study, with an accrual ceiling set at 65 patients.

Trial Drugs

* BN-Brachyury vaccine every 3 weeks until cycle 9, then every 12 weeks:

  * Recombinant MVA-BN-Brachyury (R2PD): 4 injections of vaccines with 1 given subcutaneous (SC) in each extremity on Day 1 of Cycles 1 and 2. Each injection of MVA-BN-Brachyury consists of 2.0 x 10(8) infectious units (Inf.U).
  * Recombinant fowlpox virus (FPV)-Brachyury: 1 injection given SC in one extremity on Day 1 of Cycles 3 and beyond. Each infection of FPV-Brachyury consists of 1.0 x 10(9) Inf.U.
* adotrastuzumab emtansine (T-DM1) 3.6mg/kg via intravenous (IV) infusion every (q)3 weeks on Day 1 of each cycle.
* M7824 2,400mg via IV infusion q3 weeks on Day 1 of each cycle.
* Entinostat 5mg by mouth weekly recommended phase 2 dose (RP2D) administered by patient on Days 1, 8 and 15 of each cycle.

Arms:
arm 1: Arm 1 - Triple Negative Breast Cancer.

Bifunctional fusion molecule involving programmed death-ligand 1 (PD-L1) with transforming growth factor beta (TGF-b) sequestering agent added to a vaccine for the tumor associated antigen called brachyury. arm 2: Arm 2 - Estrogen receptor (ER)-/progesterone receptor (PR)-/Human Epidermal Growth Factor Receptor 2 (HER2) + Breast Cancer.

Bifunctional fusion molecule involving programmed death-ligand 1 (PD-L1) with transforming growth factor beta (TGF-b) sequestering agent added to a vaccine for the tumor associated antigen called brachyury. These investigational agents will be added to standard of care treatment called Ado-trastuzumab emtansine (T-DM1). arm 3: Estrogen receptor (ER)-/progesterone receptor (PR)-/Human Epidermal Growth Factor Receptor 2 (HER2)+ Breast Cancer.

Bifunctional fusion molecule involving programmed death-ligand 1 (PD-L1) with transforming growth factor beta (TGF-b) sequestering agent added to a vaccine for the tumor associated antigen called brachyury as well as to an oral histone deacetylase (HDAC) inhibitor called entinostat. These investigational agents will be added to standard of care treatment called Ado-trastuzumab emtansine (T-DM1). (T-DM1).

Interventions: intervention 1: Brachyury-TRICOM intervention 2: Entinostat intervention 3: M7824 intervention 4: Ado-trastuzumab emtansine
Intervention descriptions: intervention 1: Every three weeks, until cycle 9, then every 12 weeks: \*Recombinant MVA-Bavarian Nordic (BN)-Brachyury recommended phase 2 dose (R2PD): 4 injections of vaccines with 1 given subcutaneous (SC) in each extremity on Day 1 of Cycles 1 and 2. Each injection of MVA-BN-Brachyury consists of 2.0 x 10\^8 infectious units (Inf.U). \*Recombinant fowlpox virus (FPV)-BN-Brachyury: 1 injection given SC in one extremity on Day 1 of Cycles 3 and beyond. Each injection of FPV-BN-Brachyury consists of 1.0 x 10\^9 Inf.U. intervention 2: 5mg by mouth weekly (RP2D) administered by patient on Days 1, 8 and 15 of each cycle. intervention 3: 1800mg via intravenous (IV) infusion every (q)3 weeks on Day 1 of each cycle. intervention 4: 3.6mg/kg via intravenous (IV) infusion every (q)3 weeks on Day 1 of each cycle.

Protocol document:
Prot_SAP_000.pdf: 
Abbreviated Title: The BrEAsT Trial   
Version Date: 06.01.2021 
1 
 
Abbreviated Title: The BrEAsT Trial   
NIH Protocol #: 20C0056 
IBC #: RD-19-IX-02 
RSC #: 2746 
NCT #: NCT04296942 
Version Date: June 01, 2021 
 
NCI Supplement 
 
Title: A Phase Ib Trial of Sequential Combinations of BN-Brachyury, Entinostat, ado-
trastuzumab emtansine and M7824 in Advanced Stage Breast Cancer (BrEAsT) 
 
NCI Principal Investigator:  Fatima Karzai, MD 
Genitourinary Malignancies Branch (GMB) 
Center for Cancer Research (CCR) 
National Cancer Institute (NCI) 
10 Center Drive 
Building 10, Rm 13N240 
Bethesda, MD 20892 
Phone: 301-480-7174 
E-mail: fatima.karzai@nih.gov  
 
 
 
 
 
Investigational Agents:     
Drug Name: 
BN-Brachyury 
Entinostat 
M7824 
Ado-trastuzumab 
emtansine (also 
known as T-DM1 
or Kadcyla) 
IND Number: 
19165 
19165 
19165 
19165 
Sponsor: 
NCI, CCR 
NCI, CCR 
NCI, CCR 
NCI, CCR 
Manufacturer: 
Bavarian Nordic 
Syndax 
EMD Serono, Inc. 
CC Pharmacy 
 
 
Coordinating Center: Center for Cancer Research 
Responsible Safety Monitoring Committee: NCI Safety Monitoring Committee 


Abbreviated Title: The BrEAsT Trial   
Version Date: 06.01.2021 
2 
 
PRÉCIS  
 
Background: 
• Current >2nd line treatments for metastatic breast cancer provide modest response rates, 
and modest improvement in progression-free survival but no treatments are curative.  
• Bavarian-Nordic (BN)-Brachyury vaccine is a recombinant poxvirus vaccine against the 
transcription factor brachyury, which plays an important role in the epithelial-to-
mesenchymal transition in breast cancer. A recently completed phase 1 study of BN-
Brachyury vaccine showed the vaccine was well tolerated and generated an immune 
response.  
• M7824 is a novel bifunctional fusion protein composed of a monoclonal antibody against 
human PD-L1 fused to the soluble extracellular domain of human TGF-β receptor II 
(TGF-βRII), which functions as a TGF-β “trap.”  
• Ado-trastuzumab emtansine (T-DM1 or Kadcyla) is an antibody drug conjugate used in 
second- and third- line treatment of metastatic HER2+ breast cancer (HER2+BC). T-
DM1 activates ADCC, dendritic cell maturation, increases TILs, increased PD-L1 
expression, and increased immunomodulatory cytokines.  
• Entinostat is a class 1 histone deacetylase inhibitor (HDACi) which suppresses tumor 
initiating cells, regulatory T-cells and myeloid-derived suppressor cells (MDSCs), as well 
as enhances cytotoxic T-cell mediated lysis, direct natural killer (NK) lysis, NK cell 
activation, increases PD-L1 expression and antibody-dependent cellular cytotoxicity 
(ADCC). In addition, entinostat may also be able to overcome HER2 resistance.  
• We propose a Phase 1b trial to evaluate the safety and efficacy of the stepwise 
combination of the BN-Brachyury vaccine, M7824, T-DM1 and entinostat in metastatic 
breast cancer.  
o Arm 1 – Triple Negative Breast Cancer (TNBC); M7824 + BN-Brachyury 
o Arm 2 – ER-/PR-/HER2+ Breast Cancer; M7824 + BN-Brachyury + T-DM1 
o Arm 3 - ER-/PR-/HER2+ Breast Cancer; M7824 + BN-Brachyury + T-DM1+ 
Entinostat 
Objectives: 
Primary Objectives:  
• Arms 1-3: Overall response rate (ORR; PR+CR)  
• Arms 1-3: Safety for each of the three combinations of agents explored in the arms 
Eligibility: 
Selected Inclusion Criteria  
• Histologically confirmed metastatic breast cancer with appropriate IHC testing by a 
certified lab: 
o For Arm 1: Triple negative breast cancer. Hormone receptor negative is defined 
by estrogen receptor < 10% and progesterone receptor < 10%. HER2 negative 
breast cancer is defined as HER2 per IHC 0 or 1+ or 2+ with negative FISH. 
o For Arms 2 and 3: Hormone receptor negative, HER2+ breast cancer as defined 
by estrogen receptor < 10% and progesterone receptor < 10%. HER2 positive as 
per IHC 3+ or 2+ with positive FISH.  


Abbreviated Title: The BrEAsT Trial   
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• Prior treatment:  
o For Arm 1: >1 prior therapy in the metastatic setting. Patients with known PD-L1 
positive tumors must have received prior treatment with atezolizumab + nab-
paclitaxel. Patients with ER 1-9% must have received treatment with at least two 
lines of endocrine treatment (SERM, AI, fulvestrant) with one prior treatment 
including a CDK4/6 inhibitor + endocrine therapy for their metastatic cancer and 
should be considered endocrine therapy resistant.   
o For Arms 2 and 3: > 1 prior treatment in the metastatic setting with a taxane 
(docetaxel or paclitaxel), herceptin and pertuzumab.  
• Females or males > 18 years old  
• ECOG 0 or 1 
• Measurable metastatic disease per RECIST 1.1.  
• For Cohort 3, Arms 2 and 3: At least one biopsiable lesion and willingness to undergo up 
to three research biopsies. 
• Adequate hematopoietic, hepatic, renal and cardiac (EF > 50%) function. 
Selected Exclusion Criteria  
• Patients who have received chemotherapy, including trastuzumab and pertuzumab in the 
previous 3 weeks; other investigational agents within 4 weeks or a PD-1/PD-L1 antibody 
within 4 weeks prior to study enrollment; radiotherapy < 4 weeks of study entry. 
• Symptomatic CNS metastases and leptomeningeal disease are excluded but treated brain 
metastases (no radiotherapy within 6 weeks) or asymptomatic brain metastasis are 
allowed. 
• History of invasive malignancy < 3 years prior to enrollment.  
• History of congestive heart failure (CHF) as defined as NYHA class 3 or 4 or 
hospitalization for CHF (any NYHA class) within 6 months of trial start.   
• Concurrent use of chronic systemic steroids except for physiologic systemic steroids for 
replacement defined as 10mg of prednisone or an equivalent dose.  
Design: 
This multicenter study contains three separate, single arm phase 1b trials.  
• Arm 1 will evaluate M7824 and BN-Brachyury in patients with TNBC.   
• If this doublet is determined to have acceptable toxicity (0-1 DLTs of the first 6 patients), 
up to 19 patients will be enrolled on Arm 2 in which BN-Brachyury, M7824 and T-DM1 
will be evaluated in patients with advanced HR-/HER2+ BC with disease progression 
after treatment with THP or intolerance to THP.  
• If this triplet is determined to have acceptable toxicity (0-1 DLTs of the first 6 patients), 
19 patients will be enrolled on Arm 3 in which BN-Brachyury, entinostat, M7824 and T-
DM1 will be evaluated in patients with advanced HR-/HER2+ BC with disease 
progression after treatment with THP or intolerance to THP.    
• Up to 51 evaluable patients will be recruited for this study, with an accrual ceiling set at 
65 patients. 
Trial Drugs 
• BN-Brachyury vaccine every 3 weeks until cycle 9, then every 12 weeks:  


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4 
 
o Recombinant MVA-BN-Brachyury (R2PD): 4 injections of vaccines with 1 given 
SC in each extremity on Day 1 of Cycles 1 and 2. Each injection of MVA-BN-
Brachyury consists of 2.0 x 108 infectious units (Inf.U).   
o Recombinant FPV-Brachyury: 1 injection given SC in one extremity on Day 1 of 
Cycles 3 and beyond. Each injection of FPV-Brachyury consists of 1.0 x 109 
Inf.U.   
• T-DM1 3.6mg/kg via IV infusion q3 weeks on Day 1 of each cycle.  
• M7824 2,400mg via IV infusion q3 weeks on Day 1 of each cycle. 
• Entinostat 5mg by mouth weekly (RP2D) administered by patient on Days 1, 8, and 15 of 
each cycle.  
 
 
 
 
 
 
 
 
 


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TABLE OF CONTENTS  
PRÉCIS .......................................................................................................................................... 2 
TABLE OF CONTENTS ................................................................................................................ 5 
1 
NUMBER OF PATIENTS TO BE SEEN AT NIH................................................................ 6 
2 
NCI RECRUITMENT AND SCREENING ACTIVITIES .................................................... 6 
2.1 
Recruitment Strategies ..................................................................................................... 6 
2.2 
Screening Evaluation ........................................................................................................ 6 
2.2.1 
Screening activities prior to the obtaining informed consent ................................... 6 
2.2.2 
Screening activities performed after a consent for screening has been signed ......... 6 
3 
NCI Registration Procedures .................................................................................................. 6 
3.1 
Participant Registration and Status Update Procedures ................................................... 6 
3.2 
Screen Failures ................................................................................................................. 6 
4 
NCI TREATMENT ASSIGNMENT PROCEDURES ........................................................... 7 
5 
COST AND COMPENSATION ............................................................................................ 7 
5.1.1 
Costs .......................................................................................................................... 7 
5.1.2 
Compensation ........................................................................................................... 7 
5.1.3 
Reimbursement ......................................................................................................... 7 
6 
CORRELATIVE STUDIES FOR RESEARCH / PHARMACOKINETI STUDIES ............ 7 
6.1 
Biospecimen Collection ................................................................................................... 7 
6.2 
Sample Storage, Tracking and Disposition ...................................................................... 7 
6.2.1 
Sample Management and Storage at Clinical Services Program – Leidos 
Biomedical Research, Inc. (CSP) ............................................................................................ 8 
6.2.2 
Samples Managed by Dr. Figg’s Blood Processing Core (BPC).............................. 9 
7 
NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN .... 9 
7.1 
NCI Clinical Director Reporting ...................................................................................... 9 
7.2 
Institutional Biosafety Committee (IBC) Reporting Criteria ........................................... 9 
7.2.1 
Serious Adverse Event Reports to IBC ..................................................................... 9 
7.2.2 
Annual Reports to IBC............................................................................................ 10 
7.2.3 
Clinical Trial Information ....................................................................................... 10 
7.2.4 
Progress Report and Data Analysis......................................................................... 10 
7.3 
NIH Required Data and Safety Monitoring Plan ........................................................... 11 
8 
COLLABORATIVE AGREEMENTS ................................................................................. 11 
8.1 
Cooperative Research and Development Agreement (CRADA) ................................... 11 
9 
HUMAN SUBJECTS PROTECTIONS ............................................................................... 11 
9.1 
Evaluation of Benefits and Risks/Discomforts .............................................................. 11 
9.2 
Consent Process and Documentation ............................................................................. 11 
10 REGULATORY AND OPERATIONAL CONSIDERATIONS ......................................... 12 
 
 


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1 
NUMBER OF PATIENTS TO BE SEEN AT NIH 
We plan to enroll up to 65 patients on this study.  
We plan to enroll 35 participants at the NIH. 
2 
NCI RECRUITMENT AND SCREENING ACTIVITIES 
2.1 
RECRUITMENT STRATEGIES   
Please refer to Section 2.3 of the Sponsor Protocol for recruitment strategies.  
2.2 
SCREENING EVALUATION 
2.2.1 Screening activities prior to the obtaining informed consent 
Please refer to Section 2.4.1 of the Sponsor Protocol for screening activities prior to the 
obtaining informed consent.   
2.2.2 Screening activities performed after a consent for screening has been signed 
The following screening procedures will be performed only after the subject has signed the study 
consent or the consent for study # 01C0129 (provided the procedures are permitted on that study) 
on which screening evaluations or tests will be done within 14 days prior to enrollment, except 
when specified differently. Assessments performed at outside facilities or on another NIH 
protocol within these timeframes and those below may also be used to determine eligibility once 
a patient has signed the consent. 
2.2.2.1 Pathologic Confirmation  
Pathological confirmation of diagnosis and ER, PR and HER2 expression in the Laboratory of 
Pathology at NIH Clinical Center or Walter Reed National Military Medical Center at Bethesda.  
However, if no pathologic specimen is available, patients may enroll with a pathologists report 
showing a histologic diagnosis of TNBC or HER2-positive breast cancer in a College of 
American Pathologists (CAP) accredited laboratory (or other accrediting entity) and a clinical 
course consistent with the disease.  
2.2.2.2 Screening Visit: Physical Exam, Labs and Imaging   
Please refer to Section 2.4.2.2 of the Sponsor Protocol for the screening visit procedures.   
3 
NCI REGISTRATION PROCEDURES 
3.1 
PARTICIPANT REGISTRATION AND STATUS UPDATE PROCEDURES 
Registration and status updates (e.g., when a participant is taken off protocol therapy and when a 
participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant 
Registration & Status Updates found here. 
3.2 
SCREEN FAILURES 
Screen failures are defined as participants who consent to participate in the clinical trial but are 
not subsequently assigned to the study intervention or entered in the study. A minimal set of 
screen failure information is required to ensure transparent reporting of screen failure 


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participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing 
requirements and to respond to queries from regulatory authorities. Minimal information 
includes demography, screen failure details, eligibility criteria, and any serious adverse event 
(SAE). 
Individuals who do not meet the criteria for participation in this trial (screen failure) because of a 
transient lab abnormality may be rescreened.  
4 
NCI TREATMENT ASSIGNMENT PROCEDURES 
Please refer to Section 2.5 of the Sponsor Protocol for Cohorts, Arms, and Arm Assignment 
procedures.  
5 
COST AND COMPENSATION  
5.1.1 Costs 
NIH does not bill health insurance companies or participants for any research or related clinical 
care that participants receive at the NIH Clinical Center. If some tests and procedures performed 
outside the NIH Clinical Center, participants may have to pay for these costs if they are not 
covered by insurance company. Medicines that are not part of the study treatment will not be 
provided or paid for by the NIH Clinical Center.     
5.1.2 Compensation 
Participants will not receive compensation on this study. 
5.1.3 Reimbursement 
The NCI will cover the costs of some expenses associated with protocol participation. Some of 
these costs may be paid directly by the NIH and some may be reimbursed to the 
participant/guardian as appropriate. The amount and form of these payments are determined by 
the NCI Travel and Lodging Reimbursement Policy.    
6 
CORRELATIVE STUDIES FOR RESEARCH / PHARMACOKINETI STUDIES  
6.1 
BIOSPECIMEN COLLECTION   
Please refer to Section 5.1 of the Sponsor Protocol for the biospecimen collection procedures. 
Biopsy Acquisition  
Samples will be picked up and delivered for processing and storage by the contacts listed below: 
• Primary contact: Ryan Joyce, 240-760-6180, pager 11609 
• Backup contact: Trung Pham, 301-760-6180, pager 11609 
6.2 
SAMPLE STORAGE, TRACKING AND DISPOSITION 
Samples will be ordered in CRIS and tracked through a Clinical Trial Data Management system.  
Should a CRIS screen not be available, the CRIS downtime procedures will be followed.  
Samples will not be sent outside NIH without appropriate approvals and/or agreements, if 
required.  


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6.2.1 Sample Management and Storage at Clinical Services Program – Leidos Biomedical 
Research, Inc. (CSP) 
Clinical Services Program - Leidos Biomedical Research, Inc. 
Attn: Theresa Burks 
1050 Boyles Street 
Bldg. 469/Room 121 
Frederick, MD 21702 
On days samples are drawn, Jen Bangh at CSP (part of NCI Frederick Central Repositories) 
should be notified (phone: [301] 846-5893; fax [301] 846-6222). She will arrange same-day 
courier delivery of the specimens. 
All data associated with the patient samples is protected by using a secure database. All Clinical 
Support Laboratory Staff receive annual training in maintaining records’ confidentiality. All 
samples drawn at the NIH Clinical Center will be transported to the Clinical Support Laboratory 
at the Frederick National Laboratory for Cancer Research by couriers. 
Samples will be tracked and managed by Central Repository database, where there is no link to 
personal identifiable information. All samples will be stored in either a -80ºC freezer or vapor 
phase liquid nitrogen. These freezers are located at NCI Frederick Central Repository in Frederick, 
Maryland. 
NCI Frederick Central Repositories (managed under a subcontract) store, among other things, 
biological specimens in support of NIH clinical studies. All specimens are stored in secure, 
limited-access facilities with sufficient security, backup, and emergency support capability and 
monitoring to ensure long-term integrity of the specimens for research.  
Specimens are stored in accordance with applicable HHS and FDA Protection of Human Subjects 
Regulations in accordance with the subcontractor’s Federal-wide Assurance.  The subcontractor’s 
role limited to clinical research databases and repositories containing patient specimens. The 
subcontractor does not conduct or have any vested interest in research on human subjects, but does 
provide services and support the efforts of its customers, many of which are involved in research 
on human subjects.  
It is the intent and purpose of the subcontractor to accept only coded, linked samples and sample 
information. To the limit of our ability, every effort will be made to ensure that protected 
information is not sent electronically or by hard copy or on vial labels.  
Sample data is stored in the BioSpecimen Inventory System II (BSI). This inventory tracking 
system is used to manage the storage and retrieval of specimens as well as to maintain specimen 
data. BSI is designed for controlled, concurrent access. It provides a real-time, multi-user 
environment for tracking millions of specimens. The system controls how and in what order 
database updates and searches are performed. This control prevents deadlocks and race conditions. 
For security, BSI has user password access, 3 types of user access levels, and 36 user permissions 
(levels of access) that can be set to control access to the system functions. BSI provides audit 
tracking for processes that are done to specimens including shipping, returning to inventory, 
aliquoting, thawing, additives, and other processes. BSI tracks the ancestry of specimens as they 
are aliquoted, as well as discrepancies and discrepancy resolution for specimens received by the 
repository. If a specimen goes out of the inventory, the system maintains data associated with the 


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withdrawal request. Vials are labeled with a unique BSI ID which is printed in both eye-readable 
and bar-coded format. No patient-specific information is encoded in this ID.  
Investigators are granted view, input, and withdrawal authority only for their specimens. They 
may not view specimen data or access specimens for which they have not been authorized. 
Access to specimen storage is confined to repository staff. Visitors to the repositories are 
escorted by repository staff at all times. 
6.2.2 Samples Managed by Dr. Figg’s Blood Processing Core (BPC)  
6.2.2.1 BPC contact information  
Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the 
Friday before is preferred).  
For sample pickup, page 102-11964.  
For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 
240-760-6190 (main clinical pharmacology lab number).  
For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov.  
The samples will be processed, barcoded, and stored in Dr. Figg’s lab until requested by the 
investigator. 
6.2.2.2 Sample Data Collection, Sample Storage, Procedures for Storage of Tissue Specimens 
in the Laboratory of Pathology, Protocol Completion/Sample Destruction 
Please refer to Section 5.2.2.2, 5.2.2.3, 5.2.3, 5.2.4 of the Sponsor Protocol for these procedures. 
7 
NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN 
Please refer to Section 7 of the Sponsor Protocol for reporting requirements and monitoring plan 
procedures.  
7.1 
NCI CLINICAL DIRECTOR REPORTING 
Problems expeditiously reported to the OHSRP/IRB in iRIS will also be reported to the NCI 
Clinical Director. A separate submission is not necessary as reports in iRIS will be available to 
the Clinical Director. 
In addition to those reports, all deaths that occur within 30 days after receiving a research 
intervention should be reported via email to the Clinical Director unless they are due to 
progressive disease.   
To report these deaths, please send an email describing the circumstances of the death to Dr. 
Dahut at NCICCRQA@mail.nih.gov within one business day of learning of the death. 
7.2 
INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) REPORTING CRITERIA 
7.2.1 Serious Adverse Event Reports to IBC 
The Principal Investigator (or delegate) will notify IBC of any unexpected fatal or life-
threatening experience associated with the use of BN-Brachyury 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 BN-Brachyury, but are not fatal or life-


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10 
 
threatening, must be reported to the NIH IBC as soon as possible, but not later than 15 calendar 
days after the investigator’s initial receipt of the information.  Adverse events may be reported 
by using the FDA Form 3500a. 
7.2.2 Annual Reports to IBC 
Within 60 days after the one-year anniversary of the date on which the IBC approved the initial 
protocol, and after each subsequent anniversary until the trial is completed, the Principal 
Investigator (or delegate) shall submit the information described below. Alternatively, the IRB 
continuing review report can be sent to the IBC in lieu of a separate report. Please include the 
IBC protocol number on the report. 
7.2.3 Clinical Trial Information  
A brief summary of the status of the trial in progress or completed during the previous year. The 
summary is required to include the following information:  
• the title and purpose of the trial;  
• clinical site; 
• the Principal Investigator; 
• clinical protocol identifiers;  
• participant population (such as disease indication and general age group, e.g., adult or 
pediatric);  
• 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.  
7.2.4 Progress Report and Data Analysis  
Information obtained during the previous year's clinical and non-clinical investigations, 
including:  
• a narrative or tabular summary showing the most frequent and most serious adverse 
experiences by body system 
• a summary of all serious adverse events submitted during the past year 
• a summary of serious adverse events that were expected or considered to have causes not 
associated with the use of the gene transfer product such as disease progression or 
concurrent medications 
• if any deaths have occurred, the number of participants who died during participation in 
the investigation and causes of death 


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• 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.  
7.3 
NIH REQUIRED DATA AND SAFETY MONITORING PLAN    
Please refer to Section 7.4 of the Sponsor Protocol for the study team and safety monitoring 
committee procedures. 
8 
COLLABORATIVE AGREEMENTS  
8.1 
COOPERATIVE RESEARCH AND DEVELOPMENT AGREEMENT (CRADA) 
Through a collaborative research and development agreement (CRADA), three study drugs have 
been secured for use in this clinical trial.  
The BN-Brachyury vaccines will be provided by Bavarian Nordic through CRADA 05261 with 
LTIB.  
Entinostat will be provided by Syndax through CRADA 03121 with LTIB.  
M7824 will be provided by EMD Serono, Inc. through CRADA 02666 with LTIB. 
9 
HUMAN SUBJECTS PROTECTIONS  
Please refer to Section 12.1 and 12.2 of the Sponsor Protocol for the rationale for subject 
selection and participation of children.  
9.1 
EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS 
Please refer to Section 12.4 of the Sponsor Protocol for the risks and benefits of participation on 
this study.  
9.2 
CONSENT PROCESS AND DOCUMENTATION   
Please refer to Section 12.6 of the Sponsor Protocol for the consent process and documentation.  
Note: For the NIH site,  the initial consent process or re-consent may take place in person or 
remotely (e.g., via telephone or other NIH approved remote platforms used in compliance with 
policy, including HRPP Policy 303) per discretion of the designated study investigator and with 
the agreement of the participant.   
Manual (non-electronic) signature on electronic document: 
When a manual signature on an electronic document is used for the documentation of 
consent at the NIH Clinical Center, this study will use the Adobe platform (which is not 
21 CFR Part 11 compliant) to obtain the required signatures.  
During the consent process, participants and investigators will view individual copies of 
the approved consent document on screens at their respective locations.   
Both the investigator and the subject will sign the document using a finger, stylus or 
mouse.  
 


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Electronic signature on electronic document: 
When permitted by the NIH Clinical Center, an electronic signature may be obtained 
using the iMedConsent platform to obtain the required signatures once it is designated as 
21 CFR Part 11 compliant. 
During the consent process, participants and investigators will view individual copies of 
the approved consent document on screens at their respective locations.   
The identity of the participant will be determined by a prompt which will require the 
provision of information from a form of government-issued identification prior to 
obtaining the signature. If participant does not have such identification available, security 
questions will be used to confirm identity. 
Both the investigator and the subject will sign the document electronically per system 
prompts. 
10 REGULATORY AND OPERATIONAL CONSIDERATIONS 
Please refer to Section 13 of the Sponsor Protocol for study discontinuation and closure, quality 
assurance and quality control, and conflict of interest procedures. 
 
 
 
 
 


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Abbreviated Title: The BrEAsT Trial   
NIH Protocol #: 20C0056 
IBC #: RD-19-IX-02 
RSC #: 2746 
NCT #: NCT04296942 
Version Date:  July 06, 2021 
 
Title: A Phase Ib Trial of Sequential Combinations of BN-Brachyury, Entinostat, ado-
trastuzumab emtansine and M7824 in Advanced Stage Breast Cancer (BrEAsT) 
 
NCI Principal Investigator:  Fatima Karzai, MD 
Genitourinary Malignancies Branch (GMB) 
Center for Cancer Research (CCR) 
National Cancer Institute (NCI) 
10 Center Drive 
Building 10, Rm 13N240 
Bethesda, MD 20892 
Phone: 301-480-7174 
E-mail: fatima.karzai@nih.gov  
 
 
 
 
 
Investigational Agents:     
Drug Name: 
BN-Brachyury 
Entinostat 
M7824 
Ado-trastuzumab 
emtansine (also 
known as T-DM1 
or Kadcyla) 
IND Number: 
19165 
19165 
19165 
19165 
Sponsor: 
NCI, CCR 
NCI, CCR 
NCI, CCR 
NCI, CCR 
Manufacturer: 
Bavarian Nordic 
Syndax 
EMD Serono, Inc. 
Will be purchased 
from commercial 
sources. 
Supplier: 
Bavarian Nordic 
Syndax 
EMD Serono, Inc. 
Local site 
 
 
Coordinating Center: Center for Cancer Research 
Responsible Safety Monitoring Committee: NCI Safety Monitoring Committee 
 


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PRÉCIS  
 
Background: 
• Current >2nd line treatments for metastatic breast cancer provide modest response rates, 
and modest improvement in progression-free survival but no treatments are curative.  
• Bavarian-Nordic (BN)-Brachyury vaccine is a recombinant poxvirus vaccine against the 
transcription factor brachyury, which plays an important role in the epithelial-to-
mesenchymal transition in breast cancer. A recently completed phase 1 study of BN-
Brachyury vaccine showed the vaccine was well tolerated and generated an immune 
response.  
• M7824 is a novel bifunctional fusion protein composed of a monoclonal antibody against 
human PD-L1 fused to the soluble extracellular domain of human TGF-β receptor II 
(TGF-βRII), which functions as a TGF-β “trap.”  
• Ado-trastuzumab emtansine (T-DM1 or Kadcyla) is an antibody drug conjugate used in 
second- and third- line treatment of metastatic HER2+ breast cancer (HER2+BC). T-
DM1 activates ADCC, dendritic cell maturation, increases TILs, increased PD-L1 
expression, and increased immunomodulatory cytokines.  
• Entinostat is a class 1 histone deacetylase inhibitor (HDACi) which suppresses tumor 
initiating cells, regulatory T-cells and myeloid-derived suppressor cells (MDSCs), as well 
as enhances cytotoxic T-cell mediated lysis, direct natural killer (NK) lysis, NK cell 
activation, increases PD-L1 expression and antibody-dependent cellular cytotoxicity 
(ADCC). In addition, entinostat may also be able to overcome HER2 resistance.  
• We propose a Phase 1b trial to evaluate the safety and efficacy of the stepwise 
combination of the BN-Brachyury vaccine, M7824, T-DM1 and entinostat in metastatic 
breast cancer.  
o Arm 1 – Triple Negative Breast Cancer (TNBC); M7824 + BN-Brachyury 
o Arm 2 – ER-/PR-/HER2+ Breast Cancer; M7824 + BN-Brachyury + T-DM1 
o Arm 3 - ER-/PR-/HER2+ Breast Cancer; M7824 + BN-Brachyury + T-DM1+ 
Entinostat 
Objectives: 
Primary Objectives:  
• Arms 1-3: Overall response rate (ORR; PR+CR)  
• Arms 1-3: Safety for each of the three combinations of agents explored in the arms 
Eligibility: 
Selected Inclusion Criteria  
• Histologically confirmed metastatic breast cancer with appropriate IHC testing by a 
certified lab: 
o For Arm 1: Triple negative breast cancer. Hormone receptor negative is defined 
by estrogen receptor < 10% and progesterone receptor < 10%. HER2 negative 
breast cancer is defined as HER2 per IHC 0 or 1+ or 2+ with negative FISH. 
o For Arms 2 and 3: Hormone receptor negative, HER2+ breast cancer as defined 
by estrogen receptor < 10% and progesterone receptor < 10%. HER2 positive as 
per IHC 3+ or 2+ with positive FISH.  


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• Prior treatment:  
o For Arm 1: >1 prior therapy in the metastatic setting. Patients with known PD-L1 
positive tumors must have received prior treatment with atezolizumab + nab-
paclitaxel. Patients with ER 1-9% must have received treatment with at least two 
lines of endocrine treatment (SERM, AI, fulvestrant) with one prior treatment 
including a CDK4/6 inhibitor + endocrine therapy for their metastatic cancer and 
should be considered endocrine therapy resistant.   
o For Arms 2 and 3: > 1 prior treatment in the metastatic setting with a taxane 
(docetaxel or paclitaxel), herceptin and pertuzumab.  
• Females or males > 18 years old  
• ECOG 0 or 1 
• Measurable metastatic disease per RECIST 1.1.  
• For Cohort 3, Arms 2 and 3: At least one biopsiable lesion and willingness to undergo up 
to three research biopsies. 
• Adequate hematopoietic, hepatic, renal and cardiac (EF > 50%) function. 
Selected Exclusion Criteria  
• Patients who have received chemotherapy, including trastuzumab and pertuzumab in the 
previous 3 weeks; other investigational agents within 4 weeks or a PD-1/PD-L1 antibody 
within 4 weeks prior to study enrollment; radiotherapy < 4 weeks of study entry. 
• Symptomatic CNS metastases and leptomeningeal disease are excluded but treated brain 
metastases (no radiotherapy within 6 weeks) or asymptomatic brain metastasis are 
allowed. 
• History of invasive malignancy < 3 years prior to enrollment.  
• History of congestive heart failure (CHF) as defined as NYHA class 3 or 4 or 
hospitalization for CHF (any NYHA class) within 6 months of trial start.   
• Concurrent use of chronic systemic steroids except for physiologic systemic steroids for 
replacement defined as 10mg of prednisone or an equivalent dose.  
Design: 
This multicenter study contains three separate, single arm phase 1b trials.  
• Arm 1 will evaluate M7824 and BN-Brachyury in patients with TNBC.   
• If this doublet is determined to have acceptable toxicity (0-1 DLTs of the first 6 patients), 
up to 19 patients will be enrolled on Arm 2 in which BN-Brachyury, M7824 and T-DM1 
will be evaluated in patients with advanced HR-/HER2+ BC with disease progression 
after treatment with THP or intolerance to THP.  
• If this triplet is determined to have acceptable toxicity (0-1 DLTs of the first 6 patients), 
19 patients will be enrolled on Arm 3 in which BN-Brachyury, entinostat, M7824 and T-
DM1 will be evaluated in patients with advanced HR-/HER2+ BC with disease 
progression after treatment with THP or intolerance to THP.    
• Up to 51 evaluable patients will be recruited for this study, with an overall accrual ceiling 
set at 65 patients. Planned enrollment at the NIH is 35 patients and planned enrollment at 
OSU is 30 patients.  
Trial Drugs 
• BN-Brachyury vaccine every 3 weeks until cycle 9, then every 12 weeks:  


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o Recombinant MVA-BN-Brachyury (R2PD): 4 injections of vaccines with 1 given 
SC in each extremity on Day 1 of Cycles 1 and 2. Each injection of MVA-BN-
Brachyury consists of 2.0 x 108 infectious units (Inf.U).   
o Recombinant FPV-Brachyury: 1 injection given SC in one extremity on Day 1 of 
Cycles 3 and beyond. Each injection of FPV-Brachyury consists of 1.0 x 109 
Inf.U.   
• T-DM1 3.6mg/kg via IV infusion q3 weeks on Day 1 of each cycle.  
• M7824 2,400mg via IV infusion q3 weeks on Day 1 of each cycle. 
• Entinostat 5mg by mouth weekly (RP2D) administered by patient on Days 1, 8, and 15 of 
each cycle.  
 
 
 
 
 
 
 
 
 


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TABLE OF CONTENTS  
PRÉCIS .......................................................................................................................................... 2 
TABLE OF CONTENTS ................................................................................................................ 5 
STATEMENT OF COMPLIANCE .............................................................................................. 10 
1. 
INTRODUCTION ................................................................................................................ 10 
1.1 
Study Objectives ............................................................................................................ 10 
1.1.1 
Primary Objectives .................................................................................................. 10 
1.1.2 
Secondary Objectives .............................................................................................. 10 
1.1.3 
Exploratory Objectives ........................................................................................... 11 
1.2 
Background and Rationale ............................................................................................. 11 
1.2.1 
Current Standards of Care for Advanced Breast Cancer ........................................ 12 
1.2.2 
Immune Modulation and Overcoming HER2 Resistance ....................................... 13 
1.2.3 
Checkpoint Inhibitor Experiences in Breast Cancer ............................................... 15 
1.2.4 
M7824: A bifunctional fusion protein with anti-TGF-β and anti-PD-L1 ............... 15 
1.2.5 
Brachyury Expression in Breast Cancer and Brachyury Vaccine Development .... 18 
1.2.6 
Tumor Infiltrating Lymphocytes and Clinical Outcomes in Breast Cancer ........... 22 
1.2.7 
Combination Therapy Rationale ............................................................................. 22 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT ....................................................... 29 
2.1 
Eligibility Criteria for Arm 1: M7824 + BN-Brachyury in TNBC ................................ 29 
2.1.1 
Inclusion Criteria .................................................................................................... 29 
2.1.2 
Exclusion Criteria ................................................................................................... 31 
2.2 
Eligibility Criteria for Arms 2 and 3: M7824, BN-Brachyury, T-DM1 +/- Entinostat in 
HER2+BC ................................................................................................................................. 32 
2.2.1 
Inclusion Criteria .................................................................................................... 32 
2.2.2 
Exclusion Criteria ................................................................................................... 34 
2.3 
Recruitment Strategies ................................................................................................... 36 
2.4 
Screening Evaluation ...................................................................................................... 36 
2.4.1 
Screening activities prior to the obtaining informed consent ................................. 36 
2.4.2 
Screening activities performed after a consent for screening has been signed ....... 36 
2.5 
Participant Registration and Status Update Procedures ................................................. 38 
2.5.1 
For Participating Site Registration .......................................................................... 38 
2.5.2 
Screen Failure ......................................................................................................... 38 
2.6 
Treatment Assignment Procedures ................................................................................. 39 
2.7 
Baseline Evaluation ........................................................................................................ 39 
3 
STUDY IMPLEMENTATION ............................................................................................ 40 
3.1 
Study Design .................................................................................................................. 40 
3.1.1 
Study Stopping Rule, All Arms .............................................................................. 42 
3.1.2 
Dose Limiting Toxicity ........................................................................................... 43 
3.2 
Drug Administration and Pre-medications ..................................................................... 44 
3.2.1 
MVA-BN-Brachyury and FPV-Brachyury Vaccines ............................................. 44 
3.2.2 
M7824 ..................................................................................................................... 45 
3.2.3 
Ado-trastuzumab emtansine (T-DM1) .................................................................... 46 
3.2.4 
Self-Administered Study Drugs .............................................................................. 47 
3.3 
Study Intervention Compliance ...................................................................................... 47 
3.4 
Dose Modifications ........................................................................................................ 48 
3.4.1 
Dose Modification of Entinostat ............................................................................. 49 


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3.4.2 
Dose Modification of T-DM1 ................................................................................. 50 
3.4.3 
Treatment Discontinuation of BN-Brachyury Vaccines ......................................... 52 
3.4.4 
Treatment discontinuation of M7824 ...................................................................... 52 
3.4.5 
Suggested Evaluation of Suspected Myocarditis .................................................... 53 
3.4.6 
Suggested Evaluation of Suspected Bleeding or Hemorrhage Events and Treatment 
Modification .......................................................................................................................... 53 
3.4.7 
Toxicity Management ............................................................................................. 55 
3.5 
Study Calendar ............................................................................................................... 61 
3.6 
Cost and Compensation .................................................................................................. 66 
3.6.1 
Costs ........................................................................................................................ 66 
3.6.2 
Compensation ......................................................................................................... 66 
3.6.3 
Reimbursement ....................................................................................................... 66 
3.7 
Criteria for Removal from Protocol Therapy and Off Study Criteria ............................ 66 
3.7.1 
Criteria for removal from protocol therapy ............................................................ 66 
3.7.2 
Off-Study Criteria ................................................................................................... 66 
3.7.3 
Lost to Follow-up .................................................................................................... 66 
4 
CONCOMITANT MEDICATIONS / MEASURES ............................................................ 67 
4.1 
Antiemetics ..................................................................................................................... 67 
4.2 
Growth Factor Support ................................................................................................... 67 
4.3 
Restricted Medications ................................................................................................... 67 
4.4 
Contraception ................................................................................................................. 68 
4.5 
Breast Feeding ................................................................................................................ 69 
4.6 
Blood Donation .............................................................................................................. 69 
5 
CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES ......... 69 
5.1 
Biospecimen Collection ................................................................................................. 69 
5.1.1 
Peripheral Blood Collection and Assays ................................................................. 71 
5.1.2 
Tumor Biopsy Collection and Assays..................................................................... 72 
5.1.3 
Genetic Assays ........................................................................................................ 73 
5.2 
Sample Storage, Tracking, and Disposition ................................................................... 73 
5.2.1 
Sample Management and Storage at Clinical Services Program – Leidos 
Biomedical Research, Inc. (CSP) .......................................................................................... 73 
5.2.2 
Samples Managed by Dr. Figg’s Blood Processing Core (BPC)............................ 74 
5.2.3 
Procedures for Storage of Tissue Specimens in the Laboratory of Pathology ....... 75 
5.2.4 
Protocol Completion/Sample Destruction .............................................................. 75 
6 
DATA COLLECTION AND EVALUATION ..................................................................... 75 
6.1 
Data Collection ............................................................................................................... 75 
6.2 
Data Sharing Plans ......................................................................................................... 76 
6.2.1 
Human Data Sharing Plan ....................................................................................... 76 
6.2.2 
Genomic Data Sharing Plan .................................................................................... 77 
6.3 
Response Criteria ........................................................................................................... 77 
6.3.1 
Antitumor Response................................................................................................ 77 
6.3.2 
Disease Parameters ................................................................................................. 77 
6.3.3 
Methods for Evaluation of Measurable Disease ..................................................... 78 
6.3.4 
Response Criteria .................................................................................................... 80 
6.3.5 
Response Review .................................................................................................... 83 
6.4 
Toxicity Criteria ............................................................................................................. 83 


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NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN .. 83 
7.1 
Definitions ...................................................................................................................... 83 
7.2 
OHSRP Office of Compliance and Training / IRB Reporting ....................................... 83 
7.2.1 
Expedited Reporting ............................................................................................... 83 
7.2.2 
IRB Requirements for PI Reporting at Continuing Review ................................... 83 
7.3 
NCI Guidance for Reporting Expedited Adverse Events for Multi-Center Trials ......... 83 
7.4 
NIH Required Data and Safety Monitoring Plan ........................................................... 84 
7.4.1 
Principal Investigator/Research Team .................................................................... 84 
7.4.2 
Safety Monitoring Committee (SMC) .................................................................... 84 
8 
SPONSOR PROTOCOL SAFETY REPORTING ............................................................... 85 
8.1 
Definitions ...................................................................................................................... 85 
8.1.1 
Adverse Event ......................................................................................................... 85 
8.1.2 
Serious Adverse Event (SAE) ................................................................................. 85 
8.1.3 
Life-threatening....................................................................................................... 85 
8.1.4 
Severity ................................................................................................................... 86 
8.1.5 
Relationship to Study Product ................................................................................ 86 
8.1.6 
Adverse Events of Special Interest (AESI) ............................................................. 86 
8.2 
Assessment of Safety Events .......................................................................................... 86 
8.3 
Reporting of Serious Adverse Events ............................................................................ 87 
8.4 
Waiver of Expedited Reporting to CCR ........................................................................ 87 
8.5 
Safety Reporting Criteria to the Pharmaceutical Collaborators ..................................... 87 
8.6 
Reporting Pregnancy ...................................................................................................... 87 
8.6.1 
Maternal Exposure ................................................................................................. 87 
8.6.2 
Paternal Exposure ................................................................................................... 87 
8.7 
Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND .................. 88 
8.8 
Sponsor Protocol Non-Adherence Reporting ................................................................. 88 
9 
CLINICAL MONITORING ................................................................................................. 88 
10 STATISTICAL CONSIDERATIONS.................................................................................. 89 
10.1 
Statistical Hypothesis ................................................................................................. 89 
10.2 
Sample Size Determination ........................................................................................ 90 
10.3 
Populations for Analyses ............................................................................................ 92 
10.3.1 Evaluable for toxicity: ............................................................................................. 92 
10.3.2 Evaluable for objective response: ........................................................................... 92 
10.4 
Statistical Analyses ..................................................................................................... 92 
10.4.1 General Approach ................................................................................................... 92 
10.4.2 Analysis of the Primary Efficacy Endpoint ............................................................ 92 
10.4.3 Analysis of the Secondary Endpoints ..................................................................... 93 
10.4.4 Safety Analyses ....................................................................................................... 93 
10.4.5 Baseline Descriptive Statistics ................................................................................ 93 
10.4.6 Sub-Group Analyses ............................................................................................... 93 
10.4.7 Tabulation of individual Participant Data ............................................................... 93 
10.4.8 Exploratory Analyses .............................................................................................. 93 
11 COLLABORATIVE AGREEMENTS ................................................................................. 94 
11.1 
Multi-Institutional Guidelines .................................................................................... 94 
12 HUMAN SUBJECTS PROTECTIONS ............................................................................... 94 
12.1 
Rationale For Subject Selection ................................................................................. 94 


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12.2 
Participation of Children ............................................................................................ 94 
12.3 
Evaluation of Benefits and Risks/Discomforts ........................................................... 94 
12.4 
Risk/Benefit Assessment ............................................................................................ 95 
12.4.1 Known Potential Risks ............................................................................................ 95 
12.4.2 Known Potential Benefits ....................................................................................... 96 
12.4.3 Assessment of Potential Risks and Benefits ........................................................... 96 
12.5 
Consent Process and Documentation ......................................................................... 96 
13 REGULATORY AND OPERATIONAL CONSIDERATIONS ......................................... 97 
13.1 
Study Discontinuation and Closure ............................................................................ 97 
13.2 
Quality Assurance and Quality Control...................................................................... 97 
13.3 
Conflict of Interest Policy........................................................................................... 98 
13.4 
Confidentiality and Privacy ........................................................................................ 98 
14 PHARMACEUTICAL INFORMATION ............................................................................. 99 
14.1 
BN-Brachyury (IND # 19165) .................................................................................... 99 
14.1.1 Source / Acquisition and Accountability ................................................................ 99 
14.1.2 Toxicity ................................................................................................................... 99 
14.1.3 Formulation and Preparation ................................................................................... 99 
14.1.4 Stability and Storage ............................................................................................. 100 
14.1.5 Administration Procedures.................................................................................... 100 
14.1.6 Incompatibilities ................................................................................................... 100 
14.2 
Entinostat (IND # 19165) ......................................................................................... 100 
14.2.1 Source / Acquisition and Accountability .............................................................. 100 
14.2.2 Metabolism ........................................................................................................... 101 
14.2.3 Toxicity ................................................................................................................. 101 
14.2.4 Formulation and Preparation ................................................................................. 102 
14.2.5 Stability and Storage ............................................................................................. 102 
14.2.6 Administration Procedures.................................................................................... 102 
14.2.7 Incompatibilities ................................................................................................... 102 
14.3 
M7824 (IND # 19165) .............................................................................................. 102 
14.3.1 Source / Acquisition and Accountability .............................................................. 102 
14.3.2 Metabolism ........................................................................................................... 103 
14.3.3 Toxicity ................................................................................................................. 104 
14.3.4 Formulation and Preparation ................................................................................. 105 
14.3.5 Stability and Storage ............................................................................................. 105 
14.3.6 Administration Procedures.................................................................................... 106 
14.3.7 Incompatibilities ................................................................................................... 106 
14.4 
T-DM1 ...................................................................................................................... 106 
14.4.1 Source / Acquisition and Accountability .............................................................. 106 
14.4.2 Administration Procedures.................................................................................... 106 
15 REFERENCES ................................................................................................................... 107 
16 APPENDICIES ................................................................................................................... 116 
16.1 
Appendix A: Performance Status Criteria ................................................................ 116 
16.2 
Appendix B: Oral Medication Diary (Arm 3 only) ................................................... 117 
16.3 
Appendix C: LTIB SOP on Human Core Biopsy Processing for IHC/IF Processing
 
119 


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16.4 
Appendix D: Clinical and Safety Profile of MVA-BN and Recombinant MVA-Based 
Vaccines .................................................................................................................................. 120 
16.4.1 Safety Overview of MVA-BN and Recombinant MVA-based Vaccines ............ 120 
16.4.2 Safety Profile of MVA-BN Smallpox Vaccine in Healthy Population Compared to 
Special Populations ............................................................................................................. 123 
16.4.3 Immunogenicity Overview of MVA-BN .............................................................. 124 
16.5 
Appendix E: CCR Reportable Event Form (REF) ................................................... 125 
 
 


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STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the following:  
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study. In addition, all changes to the consent form will be IRB-approved; an 
IRB determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
1. INTRODUCTION 
1.1 
STUDY OBJECTIVES 
1.1.1 Primary Objectives 
• Arm 1: M7824 + BN-Brachyury in TNBC 
o To determine if the addition of BN-Brachyury and M7824 improves overall 
response rates (ORR) in triple negative breast cancer (TNBC). 
o To determine the clinical safety of BN-Brachyury and M7824 in breast cancer  
• Arm 2: M7824 + BN-Brachyury + T-DM1 in HER2+BC. 
o To determine if the addition of BN-Brachyury and M7824 to T-DM1 improves 
ORR in HER2+ breast cancer (HER2+BC) over T-DM1 alone. 
o To determine the clinical safety of BN-Brachyury, M7824 and T-DM1 in breast 
cancer.  
• Arm 3: M7824 + BN-Brachyury + T-DM1 + Entinostat in HER2+BC 
o To determine if the addition of BN-Brachyury, M7824, and Entinostat to T-DM1 
improves ORR in HER2+BC over T-DM1 alone. 
o To determine the clinical safety of BN-Brachyury, M7824, Entinostat and T-DM1 
in breast cancer. 
1.1.2 Secondary Objectives 
• Arm 1: M7824 + BN-Brachyury in TNBC 
o To determine if adding BN-Brachyury to M7824 improves progression-free 
survival (PFS) in TNBC.  
• Arm 2: M7824 + BN-Brachyury + T-DM1 in HER2+BC 
o To determine if adding BN-Brachyury, M7824 to T-DM1 increases the PFS in 
patients with metastatic HER2+ metastatic breast cancer who progressed on initial 
treatment with THP (or intolerant to THP). 


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o To determine if adding BN-Brachyury, M7824 to T-DM1 increases the absolute 
percentage of stromal TILs (as measured by the Salgado method) in metastatic 
deposits in patients with metastatic HER2+ metastatic breast cancer.  
• Arm 3: M7824 + BN-Brachyury + T-DM1 + Entinostat in HER2+BC 
o To determine if adding BN-Brachyury, entinostat, M7824 to T-DM1 increases 
PFS in patients with metastatic HR-/HER2+ metastatic breast cancer who 
progressed on initial treatment with THP (or intolerant to THP). 
o To determine if adding BN-Brachyury, entinostat, M7824 to T-DM1 increases the 
absolute percentage of stromal TILs (as measured by the Salgado method) in 
metastatic deposits in patients with metastatic HER2+ metastatic breast cancer.  
1.1.3 Exploratory Objectives 
Studies may be performed on selected patients if adequate samples are available: 
• Peripheral blood mononuclear cells (PBMCs):  
o Changes in immune cell subsets. 
o Changes in brachyury-specific, MUC1-specific, and CEA-specific immune cell 
subsets. 
o Histone acetylation as surrogate marker for entinostat pharmacodynamics (Arm 3 
only). 
• Plasma/Serum: 
o Changes in brachyury-specific, MUC1-specific, and CEA-specific antibodies. 
o Changes in soluble factors like sCD27 and sCD40. 
o Changes in cell free DNA. 
o Changes in TGF-beta levels. 
o Changes in pharmacokinetics of M7824. 
• Tissue Biopsy (Cohort 3, Arms 2 and 3 only):  
o Brachyury expression on tumor. 
o T-cell clonality score. 
o Immunohistochemistry/multispectral imaging. 
o Changes in PD-L1 on tumor. 
o Neoepitopes derived from tumor biopsies. 
o HER2 expression on tumor biopsies.  
o Tumor mutational burden. 
1.2 
BACKGROUND AND RATIONALE 
Invasive breast cancer remains the most common malignancy in women in the United States 
(US) with an annual incidence of 123.1 cases per 100,000 women. (1, 2) Treatment of invasive 
breast cancer involves local control with surgery and/or radiation where appropriate, as well as 
systemic control with endocrine therapy, HER2 targeted therapy, and/or chemotherapy. Despite 
advances in early detection and effective treatments, breast cancer is the second most common 
cause of US cancer deaths in women with an annual rate of 21.9 deaths per 100,000 women. (1) 
It is estimated that in 2018, there will be 266,120 new cases of breast cancer with 40,920 deaths.  
(3) Triple negative breast cancer (TNBC) comprises approximately 10-15% of all breast cancers 
and HER2+ breast cancer (HER2+BC) also accounts for approximately 15-20% of breast 
cancers. (4) The majority of breast cancer is hormone receptor positive.  


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1.2.1 Current Standards of Care for Advanced Breast Cancer  
Recurrent/Refractory Advanced Triple Negative Breast Cancer  
TNBC is defined by lack of estrogen (ER) and progesterone (PR) receptor staining on 
immunohistochemistry (IHC) as well as absence of HER2 amplification. (5) Thus, patients with 
TNBC do not benefit from known targeted therapies to endocrine receptors or HER2 
amplification. (6) Standard therapy for metastatic TNBC is single agent chemotherapy and, in 
metastatic patients, ORRs are around 30-35%, with median PFS 4.5-6 months, and median OS 
around 12 months. (7)  Clearly, new approaches to the treatment of TNBC are needed to improve 
outcomes. 
While recent guidelines by the American Society of Clinical Oncology and the American 
College of Pathologists recommend that ER or PR status should be considered positive if 1% or 
more of the tumor cells demonstrate positive nuclear staining with IHC, it is becoming clearer 
that tumors with low ER or PR staining, defined as 1 to 9% on IHC, often behave more like 
TNBC than like hormone receptor positive breast cancers. (8, 9) Only about 6% of breast cancers 
are low ER or PR 1 to 9%. (10) Molecular analysis shows similar ER gene signature scores in 
ER negative tumors and 1 to 9% IHC ER+ tumors. Furthermore, among patients with 1 to 9% 
ER+ tumors, 48% of these tumors had gene expression signatures that were basal-like, with only 
8% of these tumors being identified as luminal B. (8) Clinically these tumors gain likely little 
benefit from endocrine therapy. Due to the molecular profile and clinical course, many clinical 
trials including TNBC now have relaxed the definition of TNBC from <1% on IHC to < 10% ER 
or PR staining on IHC.        
Recurrent/Refractory Advanced HER2+ Breast Cancer  
HER2+BC is defined by IHC staining of 3+ in 10% or more of tumor cells or HER2 gene 
amplification by an in situ hybridization (ISH) method (average HER2 copy number > 6.0 
signals/cell or HER2/chromosome enumeration probe [CEP]17 > 2.0 ratio). (11) Historically, 
HER2+BC is an aggressive tumor and carried a very poor prognosis prior to the advent of HER2 
targeted therapy. (12) With the addition of trastuzumab, and more recently pertuzumab, to a 
taxane backbone, survival has improved but no therapies are curative.  
First line treatment of metastatic HER2+BC with docetaxel, trastuzumab, and pertuzumab (THP) 
carries a median progression free survival (PFS) of 18.7 months and a median overall survival 
(OS) of 56.6 months. (13) Patients remain on trastuzumab +/- pertuzumab maintenance until 
disease progression at which patients switch to another HER2-tagerted therapy like T-DM1, 
lapatinib + capecitabine, or patients can continue with trastuzumab plus another agent (i.e., 
capecitabine, lapatinib or chemotherapy).  
After disease progression on 
initial HER2-targeted 
therapy, subsequent HER2-
targeted therapies carry a 
median PFS of 6 to 9.6 
months and a median OS of 
15-30 months (Table 1). 
(14-19) The EMILIA study 
demonstrated a survival 
Table 1: Clinical benefit of current standard of care metastatic HER2+ 
breast cancer regimens. 


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advantage with second-line ado-trastuzumab emtansine (T-DM1 or Kadcyla) compared to 
lapatinib + capecitabine in patients who were previously treated with a taxane plus trastuzumab. 
(20)  However, second-line studies involving T-DM1 have mainly been performed in patients 
who did not receive pertuzumab in the first line metastatic setting, as these studies were designed 
before a survival advantage was seen with THP in metastatic HER2+BC. Recent retrospective, 
population-based analyses of patients treated with T-DM1 after first line treatment with THP 
shows decreased “real world” efficacy of T-DM1. Patients with HER2+BC who received second 
line T-DM1 after first-line treatment with THP had a response rate of 18% and duration of 
treatment of 4 to 5 months. (21, 22) This is consistent with other reported clinical data and the 
clinical experience of the primary investigator. 
1.2.2 Immune Modulation and Overcoming HER2 Resistance  
HER2-Targeting Agents 
Trastuzumab is a humanized monoclonal antibody directed against domain IV of the HER2 
extracellular domain. (23) It binds to FcyRIII on immune effector cells and is a potent mediator 
of antibody-dependent, cell-mediated cytotoxicity 
(ADCC), increases immunomodulatory cytokines 
and cross-presentation of tumor antigens. (24, 25)   
T-DM1 is an antibody drug conjugate that is 
composed of humanized antibody trastuzumab and 
DM1, a maytansinoid derivative, linked with a non-
reducible thioether linker. Maytansoid are 
antimitotic agents and prevent microtubule 
assembly. (26) Like trastuzumab, T-DM1 binds to 
FcyRIII and activates ADCC. (27) T-DM1 also 
induces dendritic cell and NK cell maturation, 
increases TILs, and increased immunomodulatory 
cytokines like type 1 and type 2 interferons, 
increases cross-presentation of tumor antigens and 
increases PD-L1 on tumor cells. (24, 27, 28) 
Histone Deacetylase Inhibitors  
Histone deacetylase inhibitors (HDACi) are 
important enzymes in epigenetic regulation of gene 
expression. For example, HDAC1 is a key 
determinant in reversal of carcinoma immune 
escape. Multiple studies have demonstrated HDACi 
have anti-cancer activity in multiple tumor types 
through dysregulating cell cycle progression, causing apoptosis and interrupting tumor 
angiogenesis. (29, 30) 


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Entinostat is a class I-selective HDACi that has 
shown activity in multiple subtypes of breast 
cancer. (31) Monotherapy with weekly entinostat is 
well tolerated based on the Phase 1 study in which 
there were no grade 4 toxicities and the only dose-
limiting grade 3 toxicities included 
hypophosphatemia, hyponatremia and 
hypoalbuminemia – all of which were reversible. 
To date, there is an acceptable toxicity profile with > 900 patients treated with entinostat. The 
most common adverse events reported are fatigue, nausea and vomiting. 
A phase II study called ENCORE-301 found that when added to exemestane, weekly entinostat 
(5mg) prolonged median PFS and reduced the risk of disease progression. (32) In this trial, there 
was a similar serious adverse event rate between the two arms (16% exemestane + entinostat vs 
12% exemestane + placebo) with most common AEs reported being neutropenia (13%), fatigue 
(11%), and nausea/vomiting (5%). Entinostat with exemestane is currently being evaluated in a 
Phase III study (NCT02115282). 
Preclinical data has also indicated that entinostat can reverse acquired HER2 resistance (Figure 
1). When combined with trastuzumab, entinostat downregulated HER2 and HER3 and 
inactivated P13K/AKT signaling – the two main mechanisms of trastuzumab resistance. (31, 33) 
There is an on-going trial involving entinostat in combination with trastuzumab +/- lapatinib 
(NCT01434303). Preliminary reports of efficacy demonstrated no benefit but showed an 
appropriate safety profile. (34) When used in combination with trastuzumab and/or lapatinib, 
10mg every 2 weeks resulted in no reported grade 3 or 4 toxicities and no dose reductions (per 
Syndax, 5mg weekly is equivalent to 10 mg every 2 weeks based on PKs).   
In addition, HDACi are known to have 
immunomodulatory effects. HDACi 
upregulate MHC class I and II, resulting in 
enhanced antigen-mediated cancer cell 
killing through a HLA-restricted 
mechanism. (30, 35, 36) They also suppress 
tumor-initiating cells, regulatory T-cells 
and myeloid-derived suppressor cells. 
Entinostat and vorinostat (a pan HDACi) 
both have been shown to increase the 
sensitivity of prostate cancer and breast 
cancer cells to cytotoxic T-cell mediated 
lysis, direct NK lysis and ADCC. (30, 37)  
An immune subset analysis from the 
ENCORE-301 (exemestane +/- entinostat) 
trial demonstrated an increase in HLA-DR-
positive monocytes and a decrease in 
granulocytic and monocytic myeloid-
derived suppressor cells. (38) Furthermore, 
in preclinical studies immunogenic 
modulation was seen against a broad range 
Figure 1: Entinostat (SNDX-275) 
synergizes with trastuzumab and 
overcomes HER2 resistance (A) through 
downregulation of (B) HER2 and HER3 
as well as decreased AKT signaling. 
Image from Huang et al., 2011.  
Figure 2: Carcinoma cells exposed to vorinostat 
are significantly more sensitive to CTL-mediated 
killing. Human prostate (LNCaP) and breast 
(MDA-MB-231) carcinoma cerlls were exposed to 
vorinostat (3 µM, black bars) or to vehicle 
(DMSO, open bars) prior to being used as tarets 
for antigen-specific CTL lysis using CAE-, 
brachyury-, MUC1-, or PSA-specific CD8+ cells 
as effector cells (E:T = 30:1). To verify the 
effector T cells were HLA-restricted, CTLs were 
incubated with HLA-A2 negative AsPC-1 
pancreatic carcinoma cells exposed to vehicle 
(DMSO) or vorinostat. Results are presented as 
mean +/- S.E.M. from 3-6 replicate wells, and are 
representative of 1-4 independent experiments. 
Asterisks denote statistical significance relative to 
controls. Image from Gameiro et al., 2016. 


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of tumor-associated antigens including brachyury, CEA, MUC1 and PSA (Figure 2) and this 
was associated with increased exposure of multiple proteins involved (39) in antigen processing 
and tumor recognition (30), making it an ideal companion for a vaccine.   
Entinostat also increases cell-surface PD-L1 expression in carcinoma in vitro and in vivo. 
Entinostat promotes an inflamed tumor signature and has the potential to convert a non-inflamed 
“cold” tumor to an inflamed “hot” tumor signature through immunogenic modulation. (39)  
HDAC inhibitors synergize with immunotherapy through increased CD8+ T cells and NK cell 
trafficking. Studies have also demonstrated the generation of long-term immunologic memory. 
(40) The Phase 1 study ENCORE-601 evaluated the safety of Entinostat 5mg weekly (starting 
dose in this study) with Pembrolizumab in patients with NSCLC or with melanoma. (41, 42) 
Patients tolerated treatment well with no increase in immune related adverse events (irAEs) over 
what is expected with checkpoints. Furthermore, clinical efficacy is seen in patients with prior 
resistance to checkpoints. 
1.2.3 Checkpoint Inhibitor Experiences in Breast Cancer  
PD-1/L1 antibodies have demonstrated promise in multiple malignancies. However, the results 
of clinical trials evaluating single agent anti-PD-1/L1 antibodies in breast cancer have been 
underwhelming to date with objective response rates (ORR) of 2-10% in unselected breast 
cancers and ORR of 5-44% in patients with PD-L1 positive breast tumors. (43-45) TNBC have 
responded better to anti-PD-1/L1 antibodies than other breast cancer subtypes (TNBC = 15-20% 
in PD-L1+ tumors; TNBC = 8-10% in unselected PD-L1+ tumors; ER+ = 12% in PD-L1+ 
tumors; HER2+ = 3.8% in unselected tumors). Due to lack of efficacy seen in early trials (43), as 
well as initial concerns about combining anti-PD-1/L1 antibodies with HER2-targeted therapies, 
few checkpoint trials have been conducted in HER2+BC. Loi et al. recently demonstrated a good 
safety profile for the combination of pembrolizumab and trastuzumab in the PANACEA study 
for patients with trastuzumab-resistant breast cancer. Furthermore, there was suggestion of 
increased clinical benefit with an ORR of 15% and a durable clinical response (defined as 
complete response, partial response or stable disease > 6 months) of 24% in PD-L1 positive 
tumors. (46) Atezolizumab + T-DM1 compared to T-DM1 + placebo was evaluated in the 
KATE2 study. There was no improvement in the primary trial endpoint, progression free 
survival, between the two arms and overall survival data is still maturing. There were similar 
grade 3 to 5 AEs reported in the two arms (43.1% Atezolizumab + T-DM1 vs 41.2% 
Atezolizumab + placebo); however, there were more patients with adverse events (mostly grade 
1 or 2) reported in the Atezolizumab + T-DM1 arm (32.6% vs 19.1%). (47)  
1.2.4 M7824: A bifunctional fusion protein with anti-TGF-β and anti-PD-L1 
Transforming growth factor β (TGF-β) is a pleiotropic cytokine. In the premalignant state, it has 
tumor-suppressive effects and suppresses tumorigenic inflammation. However, in patients with 
advanced cancers including breast cancer, TGF-β is associated with malignant progression, 
evasion of immune surveillance, invasion, and metastasis. (48-50)  
The role of TGF-β1 signaling differs based on breast cancer subtype. Hormone receptor positive 
breast cancer cell lines are not as sensitive to TGF-β inhibition as hormone receptor negative 
breast cancer cell lines. (51) Higher levels of TGF-β are associated with greater invasive and 
metastatic potential and tamoxifen resistance. (51, 52) Furthermore, a functional synergy 
between TGF-β and HER2 has been described. Higher levels of TGF-β1 and TGF-β3 have been 


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identified as potential contributors to HER2 resistance and blockade of TGF-β:HER2 interaction 
may reverse HER2 resistance leading to improve efficacy of HER2-targeted therapy. (53, 54) 
The direct effects of TGF-β on T cells include decreases in perforin, granzymes, interferon 
gamma, FAS ligand, and natural killer group 2D (NKG2D). It can also decrease NKG2D and 
major histocompatibility complex (MHC) class I polypeptide sequence A (MICA) in NK cells. 
(55) Elevated levels of TGF-β have been found to correlate with poor outcomes in many 
different human cancers. (56-59) Prompted by these observations, antibodies (e.g., 
fresolimumab) and small-molecule inhibitors (e.g., galunisertib) targeting the TGF-β pathway 
have entered clinical development, where they have demonstrated initial signs of efficacy in 
breast cancer, hepatocellular carcinoma, pancreatic cancer, and melanoma. (60, 61) 
PD-L1 expression on tumor cells has also been associated strongly with poor prognosis in a 
variety of human cancers. (62-65) In recent years, a number of agents targeting the PD-1/PD-L1 
pathway have received regulatory approval, demonstrating impressive durations of response for 
multiple tumor types, including melanoma, non-small cell lung cancer, renal cell cancer, and 
head and neck cancer. (66-74) Notably, atezolizumab, durvalumab and avelumab are all anti–
PD-L1 antibodies with proven efficacy and regulatory approval. (75, 76) Unfortunately, not all 
cancer types seem to respond to these agents, and, even among susceptible cancer types, the 
percentage of responding patients is usually < 20%. (77) 
In an effort to increase the rate of response to these therapies, many ongoing trials are evaluating 
anti–PD-1/PD-L1 agents in combination with other immunotherapies. (78) Importantly, 
combined inhibition of PD-L1 and TGF-β is a promising therapeutic strategy because these key 
pathways have independent and complementary immunosuppressive functions; therefore, their 
dual inhibition may result in synergistic antitumor activity.  
M7824 is a novel bifunctional fusion protein composed of a fully human IgG1 monoclonal 
antibody against human PD-L1 fused, via a flexible glycine-serine linker, to the soluble 
extracellular domain of human TGF-β receptor II (TGF-βRII), which functions as a TGF-β 
“trap.” Preclinical studies of M7824 have shown its ability to simultaneously bind PD-L1 and 
TGF-β, as well as appropriately block PD-L1 signaling and TGF-β signaling in vitro. M7824 
was shown to have better antitumor efficacy than anti-PD-L1 or TGF-β trap control (a mutated 
antibody that doesn't bind to PD-L1, linked with the TGF-β trap) monotherapies in both MC38 
and EMT-6 tumor models, performed in both wild-type mice and B cell deficient mice. Because 
M7824 is very immunogenic in mice, due to the fully human antibody and its immune 
stimulatory mechanism of action, host anti-drug antibodies preclude continued dosing. In an 
orthotopic EMT-6 breast cancer model using B cell-deficient Jh mice M7824 showed much 
better antitumor activity than either anti-PD-L1 antibody or TGF-β trap control alone (Figure 
3A). At tumor re-challenge, 13/13 mice previously cured with M7824 treatment were completely 
protected (resistant to tumor; Figure 3B). Furthermore, M7824 therapy conferred a long-term 
protective immunity that extended survival.(79)  


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Figure 3: Data from preclinical studies of EMT-6 Breast Cancer tumors and M7824 rechallenge. 
 
Furthermore, LTIB has shown that M7824 (a) mediates antibody-dependent cellular cytotoxicity 
(ADCC), (b)increases tumor cell gene expression of molecules involved in T-cell trafficking to 
the tumor (e.g., CXCL-11), (c) enhances TRAIL- and antigen-specific CD8+ T-cell lysis of 
tumor cells and (d) reduces TGF--induced signaling in the TME. (80, 81)  
Based on these preclinical data, a Phase I 3+3 dose-escalation study was completed to evaluate 
the pharmacokinetics (PK), safety, tolerability, and biological and clinical activity of M7824 in 
patients with advanced solid tumors (NCT02517398).  
Sixteen heavily pretreated patients received M7824 at 1, 3, 10, or 20 mg/kg once-every-2-weeks. 
M7824 was shown to saturate peripheral PD-L1 at doses ≥ 3 mg/kg and sequester plasma TGF-
β1, -β2, and -β3 throughout the dosing period at ≥1 mg/kg. The only DLT observed was colitis 
with associated anemia (20 mg/kg). No MTD was reached. Grade 3 treatment-related adverse 
events occurred in 3 patients (skin infection secondary to localized bullous pemphigoid (3 
mg/kg), asymptomatic lipase increase (20 mg/kg), and colitis (20 mg/kg) with associated 
anemia). These toxicities are on par with other PD-1/PD-L1 inhibitors. The only added toxicity 
seen over traditional PD-1/PD-L1 inhibitors was the occurrence of keratoacanthomas which have 
been described previously with TGFβ inhibitors in 10-15% of patients. (82, 83)  There were no 
treatment-related grade 4–5 events. (84) 
An expansion cohort in TNBC was recently completed by EMD Serono. This data is not 
published to date, but there are 2 reported responses in the 22 patients on the TNBC expansion 
cohort (ORR = 10%). The ORR of single agent M7824 in TNBC is similar to that of other single 
agent checkpoint inhibitors. 


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1.2.5 Brachyury Expression in Breast Cancer and Brachyury Vaccine Development 
Brachyury is a member of the T-box family of transcription factors. It is overexpressed in cancer 
cells (including breast) compared with normal tissue and has been linked to chemotherapy 
resistance and metastatic 
potential. Brachyury 
induces the phenomenon 
of mesenchymalization in 
human carcinoma cells 
which is associated with 
more invasive metastatic 
tumor behavior and with 
resistance to 
chemotherapy, radiation, 
small molecule-targeted 
therapies and 
immunotherapies 
(Figure 4). (85, 86)  
Analysis of expression of 
indicated transcripts in 
the breast cancer TCGA (The Cancer Genome Atlas) dataset showed that all breast cancer 
subtypes have some degree of brachyury 
expression. (87) Brachyury expression correlates 
most closely with hormone receptor negativity 
(Figure 5). HER2 expression does not appear to 
have a significant impact on brachyury expression. 
Furthermore, brachyury expression is higher in 
tumor-positive lymph nodes than in the primary 
tumor. (85, 87) In addition, metastatic lesions 
(pleura, bone and brain) also exhibited high levels 
of brachyury expression.  
High brachyury expression has been correlated 
with worse clinical outcomes in small sample of 
TNBC patients (n =58). Even after adjusting for 
clinical-pathologic variables like stage and p53 
expression, patients with higher brachyury 
expression experienced significantly shorter 
relapse-free survival compared with patients with 
low brachyury expression (HR 3.92; CI 1.02 to 
15.1; p = .047). (87) 
This association with worse clinical outcome was 
confirmed in another larger study. (85) 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 
Figure 4: Role of the phenomenon of mesenchymalization in tumor 
metastasis and acquisition of therapeutic resistance. Adapted from 
Palena and Hamilton, 2015. 
Figure 5: Percentage of tumors in the 
TCGA database that are positive for 
brachyury mRNA expression in samples 
classified based on their hormone receptor 
classification. Image from Hamilton et al., 
2016.   


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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, high brachyury expression 
significantly correlated with higher risk of recurrence (p = 0.0283, n = 357, Figure 6A) and 
distant metastasis (p = 0.0150, n = 332; Figure 6B). 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. 270x10-5, n=270) and distant 
metastasis (p = 0.0001, n = 270).  
 
Figure 6: Brachyury expression and prognosis. Kaplan-Meier estimated of recurrence-free 
survival (A) and distant metastasis-free (B) according to brachyury mRNA levels among 357 
patients who received adjuvant tamoxifen monotherapy. The low (lowest 25%), intermediate, 
and high (highest 25%) were defined based on the level of brachyury mRNA in a cohort of 4010 
breast cancer samples that included 357 patients analyzed here. All statistical tests were two-
sided. Figure from Palena et al. 2014.  
Brachyury Immunogenicity  
By using a major histocompatibility complex (MHC)-peptide binding prediction algorithm, a 9-
mer HLA-A2 binding epitope of the brachyury protein has been identified, (88) 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.  
In vitro, MVA-BN-Brachyury-infected human dendritic cells activated CD8+ and CD4+ T-cells 
specific against the self-antigen brachyury. (89) A recent dose escalation phase 1 study 
confirmed the development of brachyury-specific CD4+ and/or CD8+ T-cell responses after 
vaccination in over 80% of patients vaccinated with the MVA-BN-Brachyury vaccine. The 
induction of brachyury-specific T-cell responses was rapid with responses developing in 17 of 28 
patients after a single vaccination and in 9 of 28 patients after two vaccinations. (89) 
Brachyury has also been incorporated into a recombinant Saccharomyces cerevisiae vaccine. 
Prior studies demonstrated that this vaccine can be taken up by and induce maturation of human 


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dendritic cells, which in turn activate brachyury-specific CD4+ and CD8+ T-cells. (90) A dose-
escalation phase 1 study demonstrated brachyury-specific immune responses in the majority of 
evaluable patients on trial with 54% developing brachyury-specific CD4+ and/or CD8+ T-cell 
responses after vaccination. (91)  
Furthermore, 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. (92) All together, these results demonstrated that brachyury is 
an immunogenic protein in humans.  
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 Triad of 
Costimulatory Molecules (TRICOM) 
in previous NCI-sponsored trials.  
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 (Figure 7). 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 pathogen associated molecular patterns, 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.  
Overview of Modified Vaccinia Ankara (MVA)  
MVA-BN-Brachyury 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 by 
Figure 7 BN-Brachyury recombinant poxvirus vaccine 
platform. 


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Bavarian Nordic (BN) 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. In total, for MVA-BN and MVA-BN-based 
recombinant vaccines, the exposure sums up to more than 9,600 subjects (more than 7,700 with 
MVA-BN, more than 400 with recombinant vaccines other than MVA-BN Filo, and more than 
1,500 with MVA-BN-Filo), having received more than 15,000 single doses of vaccine.  
In all completed and ongoing clinical trials, vaccinations with MVA-BN have shown to be 
generally safe and well tolerated. No cases of death, assessed as being even possibly related, 
have been reported for a subject in a clinical trial using MVA-BN. Results obtained from 
completed Phase 1 and 2 trials and ongoing trials with several recombinant MVA-BN based 
vaccines in healthy adults and children, HIV infected, and cancer subjects demonstrate a similar 
safety profile as MVA-BN alone. No pattern regarding Serious Adverse Drug Reactions 
(SADRs) could be detected. (See Appendix D: Clinical and Safety Profile of MVA-BN and 
Recombinant MVA-Based Vaccines for complete list of reported adverse effects and serious 
suspected adverse drug reactions possibly related to the vaccine.)  
MVA-BN was tested for safety and immunogenicity among healthy volunteers in 3 Phase 1 and 
2 dose finding trials. (93-95) Across these trials a linear dose relationship was observed between 
the vaccine doses and both vaccinia ELISA and Plaque Reduction Neutralization Test (PRNT) 
titers. Maximum ELISA seroconversion rates and peak titers were reached 2 weeks after the 
second vaccination, with 100% seroconversion after the second dose for all dose groups 
receiving at least 2 x 107 TCID50 per 0.5 mL dose of MVA- BN. Statistical analysis indicated 
lower doses to be inferior to the standard dose tested throughout all dose ranging trials, whereas 
the standard dose achieved ELISA seroconversion rates between 81% and 100% already after the 
first dose. For the PRNT, the same trend was observed with about 77% seroconversion rates 2 
weeks after the second MVA-BN administration in all groups receiving the highest dose.  
The early onset of seroconversion and the higher titers of total and neutralizing antibodies 
combined with an excellent safety profile qualified the dose of at least 5 x 107 TCID50 as the most 
suitable human dose. The final optimal (standard) dose and schedule for the general population 
was decided to be 2 doses of at least 5 x 107 TCID50 MVA-BN administered subcutaneously 
(s.c.) 2-4 weeks apart. 
Overview of the Fowlpox Vaccine  
Fowlpox virus (FPV) is a member of the genus Avipox, which is evolutionarily divergent from 
vaccinia virus. (96) Immune responses to vaccinia are essentially non-cross-reactive with 
fowlpox and do not block infection and immunization with fowlpox vectors. Unlike vaccination 
with vaccinia, fowlpox vectors are replication incompetent with early viral and transgene 
expression but late gene expression is blocked. There is minimal surface antigen made and 
therefore, minimal neutralizing antibody immune responses are induced with fowlpox vectors. 
This allows for multiple boosting efforts with the fowlpox-based vectors. Prior work performed 
within the LTIB has demonstrated the production of neutralizing antibodies with repeated 
vaccinations with vaccinia vectors.  The recently reported PROSTVAC phase 3 trial used a 
similar prime-boost regimen with the recombinant vaccine virus prime (PROSTVAC-V) 
followed by multiple recombinant fowlpox virus boosts (PROSTVAC-F).   


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1.2.6 Tumor Infiltrating Lymphocytes and Clinical Outcomes in Breast Cancer  
Standardized methodology for assessing tumor infiltrating lymphocytes (TILs) in H&E sections 
has been validated by an international working group.(97) TILs are classified as stromal or 
intratumoral and are graded on a continuous scale. TILs are a well-established prognostic and 
predictive marker in breast cancer. Pathological evaluation of pre-treatment breast cancers 
consistently show low numbers of TILs in the tumor, with most breast tumors having 25% or 
less of the cells within the tumor microenvironment (TME) being lymphocytes. TNBC has the 
highest TILs with median 20-25% followed by HER2+BC median 15-20% and hormone 
receptor positive breast cancer (HR+BC) median 5-10%. (98-101) TILs are prognostic in TNBC 
and HER2+BC, with higher levels correlating with a better prognosis. Several large meta-
analyses have consistently found that for each absolute 10% increase in TILs per high power 
field, the risk of relapse decreases by 15-20% and the risk of death decreases by 20-25%. (99, 
100, 102, 103) This association has not been found in hormone receptor positive (HR+BC) 
breast cancer. (99, 100, 104)  
Breast cancer is generally not considered a “hot” or lymphocyte-predominant (LPBC; defined as 
≥ 50 to 60% lymphocyte infiltration) tumor with only 10-20% of breast cancers being 
lymphocyte predominant. (98) LPBCs have significantly improved pathologic complete response 
(pCR; 40% LPBC vs 5% in breast cancers with fewer TILs) with neoadjuvant chemotherapy. 
(98, 105) Recently, the CLEOPATRA trial evaluated baseline TILs in patients with advanced 
HER2+BC treated with THP and confirmed that higher TIL values are associated with better 
overall survival in metastatic HER2+BC. (106)  
1.2.7 Combination Therapy Rationale  
As described above, checkpoint inhibitor monotherapy does not seem to work well in breast 
cancer despite multiple efforts in the clinical setting. In tumors like breast cancer, it is 
hypothesized that intrinsic resistance to immunotherapy may be due to too few neoantigens 
being present, defective antigen presentation, or suppressive signals (like TGF-β) in the TME 
that exclude T cells and NK cells in the TME. (107-110) Together, these defects create an 
immunosuppressive TME that does not allow for an effective tumor immune response.   
The three key components of a successful anti-tumor immune response are: 
1) presence of effector cells in the form of antigen specific T lymphocytes and/or natural 
killer cells 
2) the ability of those cells to traffic to the tumor  
3) effective cytotoxicity of those cells within the TME 
The use of a multimodality approach may help to shift the TME back to an immunopermissive 
TME and allow engagement of the immune system to eradicate the tumor. In this study, the 
reasoning for trial drug selection is as follows: 
A) Brachyury Vaccine:  
Given the known expression of the TAA brachyury on breast tumors, (87) the BN-
brachyury vaccine was chosen to help generate brachyury-specific T cells, as well as T 
cells against the cascade antigens CEA and MUC1.  
Clinical data produced by LTIB with various brachyury vaccines have consistently 
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• Phase 1 trial with MVA-BN-Brachyury (used in this trial): 5 of 5 breast cancer 
patients generated a brachyury-specific T cell response. (111) 
• Phase 1 trial with yeast-Brachyury: 2 of 5 breast cancer patients generated a 
brachyury-specific T cell response. (91) 
• Prolonged responder to PANVAC: metastatic ER+/PR+/HER2+ breast cancer 
patient generated a robust brachyury-specific T cell response to PANVAC 
(MUC1, CEA pox virus vaccine) at the time of her complete response, where 
brachyury was a cascade antigen generated from the vaccine (data not yet 
published). This complete response is on-going at 9 years.  
 
Yeast Brachyury Vaccine Trial 
Heery
 l
et a
 
2015. Cancer Immunol Res.(91) 
Brachyury-TRICOM Vaccine Trial 
Heery et al. 2017. Clin Cancer Res.(111) 
Study Size  
34 patients , heavily pretreated 
38 patients , heavily pretreated 
Study Design 
Phase 1, 3+3 dose escalation  
Phase 1, 3+3 dose escalation  
Primary Outcome 
Safety and Brachyury-specific T-cell 
responses 
Safety and Brachyury-specific T-cell 
responses 
Safety  
(G = grade) 
No DLTs. No G3 reactions.  
G1/2 injection site reactions common 
No DLTs. G3 diarrhea (n=1) 
G1/2 injection site reactions and G1/2 flu-
like symptoms common 
Immune Responses 
17 of 31 evaluable patients (54%) developed 
Brachyury-specific CD4 and/or CD8 T-cell 
responses; 78% had responses at highest dose 
level 
28 of 34 evaluable patients (82%) 
developed Brachyury-specific CD4 and/or 
CD8 T-cell responses; 87% had responses 
at highest dose level 
Immune Responses in 
Breast Cancer 
Patients 
2 of 5 breast cancer patients (40%) developed 
Brachyury-specific CD4 and/or CD8 T-cell 
responses 
5 of 5 breast cancer patients (100%) 
developed Brachyury-specific CD4 and/or 
CD8 T-cell responses 
 
In addition to the increased immune responses seen with the poxviral-based vaccine 
compared to yeast-based vaccines, unpublished data from the LTIB suggest that 
vaccinating with a poxviral vector containing a tumor associated antigen, like brachyury, 
leads to upregulation of PD-L1 in T-cell poor murine tumor models. We suspect this is 
likely due to trafficking of activated T-cells to the tumor and subsequent release of IFN-. 
This may explain why therapeutic vaccines alone rarely lead to objective responses and 
provides reasoning for combining PD-1/L1 inhibitors with poxviral-based vaccines.  
Clinically, we have seen the benefits of adding poxviral-based vaccines to PD-L1 
inhibitors in immunogenically cold tumors, like breast cancer. In the BN-CV301 phase 1 
study (BN-CV301 is a poxviral vaccine that contains the TAA MUC1 and CEA), two of 
12 patients enrolled had prolonged stable disease when treated with anti-PD-L1 antibody 
following administration of BN-CV301. Both patients had KRAS mutated, colorectal 
cancer, progressed on the single agent vaccine, were transitioned to a anti-PD-L1 
antibody and then had a decrease in tumor markers by >35%. Both patients had 
prolonged stable disease and remained on the checkpoint inhibitor for 41 weeks and on-
going at 85 weeks (data unpublished). Their time on anti-PD-L1 with stable disease far 
exceeded expectations given that prior trials have found a median progression-free 


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survival of 10 weeks (2.2 months) in patients with MSS, mCRC who receive an anti-PD-
L1 antibody. (112)     
The BN-Brachyury vaccine will help to generate more brachyury-specific T-cell 
responses in these breast cancer patients and increase the presence of effector cells in the 
tumor/TME. Due to the stronger immune responses seen with poxviral-based vaccines, 
the preclinical data that demonstrated upregulation of PD-L1 in the tumor, and the 
clinical experiences of benefit seen with checkpoint inhibitor after poxviral-based 
vaccine, BN-Brachyury was chosen as the vaccine backbone for this trial.  
B) M7824  
Preclinical data of M7824 in the EMT-6 breast cancer model in B-cell deficient mice was 
extremely promising with extended survival in a dose-dependent manner and 10 of 10 
cures (See Figure 3). Furthermore, when M7824 was combined with an Ad-Twist 
vaccine (murine tumor associated antigen TWIST1) in a syngeneic model, there was 
improvement in survival (Figure 8) compared to monotherapy and increased CD8+ T 
cell and NK cell activation.(79)     
 
   (A)  
 
 
 
      
 


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  (B) 
Figure 8: Improved anti-tumor activity with M7824 combination (A) M7824 plus 
vaccination with Ad-TWIST significantly improves anti-tumor efficacy verse M7824 
monotherapy in a syngeneic mouse model (B) M7824 plus Ad-TWIST has better anti-
tumor efficacy than Avelumab plus Ad-TWIST or Ad-TWIST alone in a syngeneic mouse 
model.   
 
Clinical activity of single agent M7824 in breast cancer appears to be equivalent to other 
checkpoint inhibitors (10% response rate in TNBC patients; data unpublished, personal 
communication with EMD Serono). Preclinical data shows that M7824 may be better than 
anti-PD-L1(Figure 8A) and when combined with Ad-TWIST vaccine, M7824 was better 
than avelumab (Figure 8B). LTIB has extensively evaluated M7824 preclinically and has 
demonstrated that compared to other anti-PD-L1 antibodies, M7824 has unique immune 
effects. As previously mentioned, M7824 mediates ADCC, increases gene expression of 
molecules involved T-cell trafficking, and enhances antigen-specific CD8+ T-cell lysis of 
tumor cells. (80, 81) Furthermore, TGF- sequestration in the TME not only leads to 
improved CD8+ T-cell lysis  and NK cell trafficking, the blockade of TGF-β:HER2 
interaction may also reverse HER2 resistance leading to improve efficacy of HER2-
targeted therapy. (53, 54) 
M7824 was chosen as the PD-L1 checkpoint inhibitor backbone of this regimen due to its 
synergy with the BN-Brachyury vaccine as well as the added benefits of TGF-β 
sequestration in the TME (increased CD8+ trafficking, increased NK cell infiltration, 
reversal of HER2 resistance, reversal of mesenchymalization, etc). In the GMB/LTIB 
branch at the NCI, one ongoing trial currently employs this combination – The QuEST 
trial (NCT03493945; IND 17851; PI J. Gulley). To date, 15 patients have received one or 
more doses of BN-Brachyury + M7824 (10 patients BN-Brachyury + M7824; 5 patients 


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BN-Brachyury + M7824 + ALT803). There has been one grade 3 pancreatitis (elevation 
in pancreatic enzymes in the absence of clinical symptoms but with imaging concerning 
for pancreatic inflammation; grade 3 due to use of steroids) which was attributed to 
M7824 and is known immune related adverse event of anti-PD-L1 antibodies. There have 
been no other grade 3 or higher adverse events or immune related adverse events 
attributed to the combination of M7824 and BN-Brachyury +/- ALT803. The BrEAsT 
trial uses a higher dose of M7824 than that of the QuEST trial (2400mg flat dose every 3 
weeks vs 1200mg flat dose every 2 weeks) which is why the safety evaluation is being 
conducted in Arm 1. However, the 2400mg dose every 2-week regimen as well as a 
similar regimen of 30mg/kg dose every 2 weeks have been evaluated in several patients. 
In the phase 1 trial of M7824, which was conducted at the NCI in this branch, there was 
no evidence of more SAEs or irAEs with higher doses of M7824 (i.e., 20mg/kg or 
30mg/kg dose levels).(113) There is also an ongoing, unpublished dose expansion cohort 
of the phase 1 trial with the 2400mg flat dose being given every 2 weeks and to date, 
there has not an increase in severe adverse events or irAEs over what is expected with the 
1200mg every 2-week dose (unpublished data). 
C) Entinostat 
As demonstrated in Figure 2, LTIB has demonstrated that treatment with entinostat 
makes breast cancer cells more sensitive to T-cell mediated lysis in vitro. This 
immunogenic modulation was observed against a broad range of TAA including 
brachyury and was associated with increase expression of antigen processing and tumor 
immune recognition. HDAC inhibitors restore protein expression of MHC class I and 
antigen processing and presentation molecules. (30) Entinostat exposure also increases 
the sensitivity of breast cancer cells (MDA-MB-231) to NK-mediated attack through 
direct lysis (increased NK activation and function) and through anti-PD-L1 mediated 
ADCC. (114) 
Furthermore, the LTIB has also demonstrated that entinostat increases PD-L1 expression 
in mice implanted with PC-3 (prostate) carcinoma cells (Figure 9A). (114) Others have 
shown upregulation of PD-L1 on tumors with entinostat treatment. This has been 
attributed to the epigenetic modulation that is seen with HDAC inhibitors. Preliminary 
results of the ENCORE-601 trial involving entinostat with pembrolizumab in checkpoint 
refractory NSCLC demonstrated clinical efficacy in multiple patients regardless of prior 
checkpoint treatment or PD-L1 status. (41) The addition of entinostat to checkpoint 
refractory tumors or to cold tumors is promising. There are currently multiple on-going 
clinical trials involving HDAC inhibitors with checkpoint inhibitors (NCT02915523; 
NCT02909452; NCT02708680; NCT02437136; NCT02453620; NCT02697630; 
NCT03250273; NCT01928576).    
Preclinical data performed by Christmas et al, demonstrates synergy with the 
combination of entinostat, an anti-PD-1 antibody and a HER2-targeted in HER2/neu 
transgenic breast cancer models. This combination  significantly reduced tumor size and 
improved overall survival in mice (Figure 9B, C; see 5th line from top). (115)  In 
addition, the triplet significantly decreased suppression of granulocytic MDSCs in the 
TME, increased CD8+ effector T cells and reduced Tregs compared to single agent 
HER2-targeted therapy.  


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Figure 9 
 
As above, preclinically entinostat improves T-cell mediated cytotoxic killing of breast 
cancer cells through increased MHC expression, improved NK activation and function, 
improved NK-mediated lysis and improved antigen processing and trafficking, making it 
an ideal companion for a therapeutic cancer vaccine like BN-Brachyury. Furthermore, 
epigenetic modulation leading to increased PD-L1 expression makes it an ideal 
companion for a checkpoint inhibitor.  
D) T-DM1 
As discussed above, T-DM1 is a standard of care regimen for metastatic HER2+BC 
patients. While many of its immune effects are similar to trastuzumab (ADCC, increased 
immunomodulatory cytokines, and increased antigen presentation), T-DM1 also has the 
added benefit of increased PD-L1 expression on tumor cells. Furthermore, a maytansine-
containing drug antibody conjugate (similar to T-DM1) induces immunogenic cell death 
in tumor cells.(116) The on-going KATE2 trial (NCT02924883) is evaluating T-DM1 
with Atezolizumab. Preliminary evidence showed no improvement in PFS but survival 
data is still maturing.  Other on-going trials with checkpoints and T-DM1 include a phase 
1b trial combining pembrolizumab and T-DM1 (NCT03032107) and the phase 2 trial, 
PembroMab, which is evaluating pembrolizumab and T-DM1 in patients with metastatic 
HER2+BC (NCT02318901). Efficacy data from these trials have not been reported to 
date.  


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T-DM1 was chosen as the HER2+BC due to the added benefits of increased PD-L1 and 
immunogenic cell death. This will help to increase presence of effector cells in the form 
of antigen specific T lymphocytes and/or natural killer cells, increase immune cell 
trafficking and improve cytotoxicity of these cells within the TME. 
Summary Statement 
We hypothesize that combining these four agents will lead to a robust immune response against 
HR-/HER2+BC with improved response rates. The immune effects of the standard of care 
therapy T-DM1 may be enhanced through combination with entinostat, M7824 and the BN-
Brachyury vaccine. In a tumor that generally does not respond to checkpoint monotherapy, this 
combination of agents will address the three key components of a successful anti-tumor immune 
response. Furthermore, the use of novel combination approaches is in keeping with the Cancer 
Moonshot Taskforce’s mandate which called for the use of innovative strategies to rapidly 
translate new agents from bench to bedside. Rational combination of immune therapies is a 
plausible strategy to achieve this aim and is especially warranted in treating patients who have 
exhausted most, if not all, therapeutic options. Enhancing immunity via several different 
mechanisms is a promising means to produce objective responses in a substantially increased 
portion of patients. Preclinical data in an orthotopic HER2+ TuBO model demonstrates a 
significant tumor reduction in mice who received the four agents (red box in Figure 10) 
proposed in this trial (entinostat, M7824 ,vaccine and T-DM1; Figure 10). For anti-tumor 
studies, TuBo tumor cells (4105, s.c.) were orthotopically implanted into the mammary fat pad 
of female Balb/c mice on day 0. Mice were randomized based on tumor size and treatment 
initiated when tumors reached 200mm3 (n=10/group) based on tumor size and treatment initiated 
when tumors reached 200mm3 (day 18), as per schematic (Figure 10). Tumors were measured 
twice weekly using calipers, with tumor volume being determined as length x length x width/2. 
Animals were monitored by the veterinarian staff for signs of toxicity, including weight loss. No 
toxicity was reported in this study. Animals were sacrificed on day 32 and immune populations 
in the tumor were examined. 
 


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Figure 10: Combination Immunotherapy Reduces Tumor Volume in Orthotopic HER2+ Breast 
Cancer Mouse Model 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT  
2.1 
ELIGIBILITY CRITERIA FOR ARM 1: M7824 + BN-BRACHYURY IN TNBC 
2.1.1 Inclusion Criteria 
2.1.1.1 Patients must have histologically or cytologically confirmed metastatic TNBC, defined 
as ER < 10%, PR < 10% per immunohistochemistry (IHC) and HER 2 negative. HER2 
negative or unamplified breast cancer is defined as IHC 0 or 1+ or IHC 2+ with FISH 
average HER2 copy number < 4.0 signals per cell or HER2/CEP17 < 2.0 with average 
HER2 copy number < 4.0 signals per cell. (117) HER2 testing must have been 
performed in a laboratory accredited by the College of American Pathology (CAP) or 
another accrediting entity. 
2.1.1.2 Patients must have measurable disease, per RECIST 1.1. See Section 6.3 for the 
evaluation of measurable disease. 
2.1.1.3 Patients must have received at least one prior treatment for metastatic disease and 
progressed on treatment or been intolerant to treatment. Patients with known PD-L1 
positive tumors must have received prior treatment with atezolizumab + nab-paclitaxel. 
Patients with ER 1-9% must have received treatment with at least two lines of endocrine 
treatment (SERM, AI, fulvestrant) with one line including a CDK4/6 inhibitor + 
endocrine therapy for their metastatic cancer and should be considered endocrine 
therapy resistant.   
2.1.1.4 Female or male >18 years. 
2.1.1.5 ECOG performance status 0 or 1 (see Appendix A). 
2.1.1.6 Patients must have adequate bone marrow function as defined below:  
− absolute neutrophil count 
>1,500/mcL (> 1.5X 106/L) 
− platelets  
 
 
 
 
 
>100,000/mcL 
− hemoglobin  
 
 
 
 
> 9 mg/dL (transfusion to obtain 
hemoglobin > 9 mg/dL within 24 hours prior 
to dosing is allowed) 


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2.1.1.7 Patients must have adequate renal function, defined as: 
• serum creatinine < 1.5 X upper limit of normal (ULN) OR  
• measured or calculated creatinine clearance ≥ 60 mL/min for participant with 
creatinine levels > 1.5 X institutional ULN (GFR can also be used in place of 
creatinine or CrCl). 
2.1.1.8 Patients must have adequate hepatic function, defined as AST and ALT levels ≤ 3 X 
ULN and total bilirubin < 1.5 X ULN, unless known diagnosis of Gilbert’s syndrome, 
where bilirubin ≤ 5 mg/dL will be permitted. Gilbert’s syndrome will be defined as 
elevated unconjugated bilirubin, with conjugated (direct) bilirubin within the normal 
range and less than 20% of the total. Total bilirubin will be permitted up to 5 mg/dL, if 
patients have historical readings consistent with the definition of Gilbert’s syndrome 
prior to entering study. Adequate hepatic function for patients with known liver 
metastases is defined as AST and ALT levels ≤ 5 X ULN.  
2.1.1.9 The effects of BN-Brachyury, entinostat, M7824 and T-DM1 on the developing human 
fetus are unknown.  However, the two components of T-DM1 are known to have 
negative fetal effects including oligohydramnios and oligohydramnios sequence 
(pulmonary hypoplasia, skeletal malformations and neonatal death). For this reason: 
• Women of child-bearing potential must agree to use adequate contraception (see 
Table 17) at study entry, for the duration of study participation and for 7 months 
after the last dose of study medication. 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.  
• Men should refrain from fathering a child or donating sperm during the study and 
for 4 months after the last dose of study medications.  
2.1.1.10 Patients with well-controlled HIV infection are eligible for trial as long as: 
• On an effective anti-retroviral therapy (ART) ˃ 4 weeks and with evidence of viral 
suppression as defined as HIV viral load ˂ 400 copies/mL within the last 3 months; 
• CD4 > 200 cells/L within the last 3 months; and  
• No reported opportunistic infections within 6 months prior to enrollment, except for 
the following which will be allowed:  
i. Esophageal candidiasis treated within last 6 months or currently improving with 
antifungal treatment. 
ii. Oral and/or genital HSV treated within last 6 months or currently improving 
with antiviral treatment. 
iii. Mycobacterium avium infection in last 6 months or that has been treated for at 
least 1 month.  
2.1.1.11 Patients with evidence of chronic hepatitis B virus (HBV) infection are eligible for trial 
as long as the HBV viral load is undetectable on suppressive therapy, if indicated.  
2.1.1.12 Patients with history of hepatitis C virus (HCV) infection must have been treated and 
cured. For patients with HCV infection who are currently on treatment, they are eligible 


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if they have an undetectable or unquantifiable HCV RNA 12 weeks or longer after 
definitive treatment completion.  
2.1.1.13 Patients must be able to understand and willing to sign a written informed consent 
document.  
2.1.2 Exclusion Criteria 
2.1.2.1 Patients who have received chemotherapy in the prior 3 weeks (6 weeks for 
nitrosoureas or mitomycin); other investigational agents or a PD-1/L1 agent within 4 
weeks prior to study enrollment.  
2.1.2.2 Patients who have received radiotherapy within 4 weeks prior to study entry. 
2.1.2.3 Patients with active brain metastases or leptomeningeal metastases should be excluded 
from this clinical trial because of their poor prognosis and because they often develop 
progressive neurologic dysfunction that would confound the evaluation of neurologic 
and other adverse events. However, patients with treated brain metastases are eligible if 
there is no magnetic resonance imaging (MRI) evidence of progression for 6 weeks 
after treatment is complete and within 28 days prior to the first dose of trial drug. 
Patients requiring immunosuppressive doses of systemic corticosteroids (> 10mg/day 
prednisone equivalent) for palliation are excluded.   
2.1.2.4 Patients with a history of another invasive malignancy < 3 years prior to enrollment 
(patients with non-melanoma skin cancers, carcinoma in situ of the breast or cervix are 
eligible). 
2.1.2.5 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to agents used in study. For example, reaction to prior vaccination with 
vaccinia virus or prior hypersensitivity reaction to fully humanized monoclonal 
antibodies (Grade > 3 NCI-CTCAEv5). 
2.1.2.6 Uncontrolled intercurrent illness including, but not limited to, ongoing active infection 
that requires systemic treatment with ongoing antibiotics (eligible if can stop antibiotics 
on day of enrollment), unstable angina pectoris, cardiac arrhythmia, or psychiatric 
illness/social situation that in the opinion of the primary investigator would prohibit the 
patient from complying with study requirements.  
2.1.2.7 Patients with bone metastases who have initiated denosumab or a bisphosphonate 
therapy within 28 days prior to or after Cycle 1 Day 1. Continuation of prior therapy is 
allowed.  
2.1.2.8 Patients with inherited bleeding disorders, a history of bleeding diathesis such as vWF 
deficiency or recent (within 3 months prior to enrollment) clinically significant bleeding 
events that, in the judgment of the investigator, would interfere with patient’s ability to 
carry out the treatment program. 
2.1.2.9 Participants unwilling to accept blood products as medically indicated.  
2.1.2.10 Patients should have no evidence of being immunocompromised as listed below:  
- 
Active, known or suspected autoimmune disease. Patients are permitted to enroll if 
they have vitiligo, type I diabetes mellitus, residual hypothyroidism due to an 
autoimmune condition only requiring hormone replacement, psoriasis not requiring 
systemic treatment or conditions not expected to recur in the absence of an external 
trigger in the opinion of the primary investigator.  


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- 
Altered immune function that in the judgement of the PI that may affect a patient’s 
ability to adequately engage the immune system and respond to the immunotherapy 
agents being administered, including but not limited to: inflammatory bowel 
disease; active infectious enteritis; eosinophilic enteritis; lupus erythematous; 
ankylosing spondylitis; scleroderma; multiple sclerosis. These criteria do not 
include all disease with an immune-related component but are not autoimmune in 
nature or have a primary alteration in the general immune function that may 
interfere with the vaccine mechanism of action, for example celiac disease.  
- 
Immunosuppressive therapy post-organ transplant. 
2.1.2.11 Concurrent use of chronic use of systemic steroids, except for physiologic doses of 
systemic steroids for replacement, defined as 10mg of prednisone per day or equivalent, 
or local (topical, nasal, ophthalmic or inhaled) steroid use or prior concomitant use with 
chemotherapy. Systemic steroids must have been discontinued >2 weeks prior to trial 
start. Prior use of corticosteroids in short-term schemes (duration shorter than 3 days) 
for indications such as prophylaxis of reactions to intravenous contrast for imaging 
studies or chemotherapy-related AEs are not considered part of this exclusion. Prior use 
of corticosteroids for brain metastasis ending at least 14 days prior to enrollment is not 
considered part of this exclusion criteria. 
2.1.2.12 Pregnant and breastfeeding women are excluded from this study because of the 
potential for teratogenic or abortifacient effects with all of the agents involved in this 
trial.  
2.1.2.13 Clinically significant cardiomyopathy, coronary disease, chronic heart failure (CHF; 
New York Heart Association class III or IV or hospitalization for CHF), or 
cerebrovascular accident within 6 months prior to enrollment.  
2.1.2.14 Patients with a history of myocarditis are excluded due to the potential of myocarditis 
with anti-PD-L1 antibodies.  
2.1.2.15 Patients with pulse oximetry < 92% on room air will be excluded due to the potential of 
pneumonitis with anti-PD-L1 antibodies.  
2.1.2.16 Any other condition, which would, in the opinion of the Principal Investigator or 
Medical Monitor, indicated the subject is a poor candidate for the clinical trial or would 
jeopardize the subject or the integrity of the data obtained. 
2.2 
ELIGIBILITY CRITERIA FOR ARMS 2 AND 3: M7824, BN-BRACHYURY, T-DM1 +/- 
ENTINOSTAT IN HER2+BC 
2.2.1 Inclusion Criteria 
2.2.1.1 Patients must have histologically or cytologically confirmed metastatic HER2+ breast 
cancer. HER2 positive or amplified breast cancer is defined as IHC 3+ or FISH average 
HER2 copy number ≥ 6 signals per cell or HER2/CEP17 ≥ 2.0. (117) HER2 testing 


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must have been performed in a laboratory accredited by the College of American 
Pathology (CAP) or another accrediting entity.  
2.2.1.2 Patients must have hormone receptor negative, HER2+ breast cancer. Hormone receptor 
negative is defined as estrogen receptor < 10% by IHC and progesterone receptor < 
10% by IHC. 
2.2.1.3 Patients must have measurable disease, per RECIST 1.1. See Section 6.3 for the 
evaluation of measurable disease. 
2.2.1.4 Patients in Cohort 3 must have at least one lesion deemed safe to biopsy and be willing 
to undergo up to three biopsies while on trial. 
 
2.2.1.5 Patients must have received front-line treatment for metastatic disease with a taxane, 
trastuzumab and pertuzumab (THP; docetaxel or paclitaxel allowed) and progressed on 
treatment or were intolerant to treatment. Patients must have received at least one prior 
therapy in the metastatic setting. Prior T-DM1 therapy is allowed. 
2.2.1.6 Female or male >18 years.  
2.2.1.7 ECOG performance status 0 or 1 (see Appendix A). 
2.2.1.8 Patients must have adequate bone marrow function as defined below:  
− absolute neutrophil count 
 
>1,500/mcL (> 1.5X 106/L) 
− platelets  
 
 
 
 
 
>100,000/mcL 
− hemoglobin  
 
 
 
 
> 9 mg/dL (transfusion to obtain 
hemoglobin > 9 mg/dL within 24 hours prior 
to dosing is allowed) 
2.2.1.9 Patients must have adequate renal function, defined as: 
• serum creatinine < 1.5 X upper limit of normal (ULN) OR  
• measured or calculated creatinine clearance ≥ 60 mL/min for participant with 
creatinine levels > 1.5 X institutional ULN (GFR can also be used in place of 
creatinine or CrCl). 
2.2.1.10 Patients must have adequate hepatic function, defined as AST and ALT levels ≤ 3 X 
ULN and total bilirubin < 1.5 X ULN, unless known diagnosis of Gilbert’s syndrome, 
where bilirubin ≤ 5 mg/dL will be permitted. Gilbert’s syndrome will be defined as 
elevated unconjugated bilirubin, with conjugated (direct) bilirubin within the normal 
range and less than 20% of the total. Total bilirubin will be permitted up to 5 mg/dL, if 
patients have historical readings consistent with the definition of Gilbert’s syndrome 
prior to entering study. Adequate hepatic function for patients with known liver 
metastases is defined as AST and ALT levels ≤ 5 X ULN.  
2.2.1.11 Patients must have adequate cardiac function as defined by an ejection fraction > 50%. 
2.2.1.12 The effects of BN-Brachyury, entinostat, M7824 and T-DM1 on the developing human 
fetus are unknown. However, the two components of T-DM1 are known to have 
negative fetal effects including oligohydramnios and oligohydramnios sequence 
(pulmonary hypoplasia, skeletal malformations and neonatal death). For this reason:  
• Women of child-bearing potential must agree to use adequate contraception (see 
Table 17) at study entry, for the duration of study participation and for 7 months 
after the last dose of study medication. Should a woman become pregnant or 


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suspect she is pregnant while she or her partner is participating in this study, she 
should inform her treating physician immediately. 
• Men should refrain from fathering a child or donating sperm during the study and 
for 4 months after the last dose of study medications.  
2.2.1.13 Patients with well-controlled HIV infection are eligible for trial as long as: 
• On an effective anti-retroviral therapy (ART) ˃ 4 weeks and with evidence of viral 
suppression as defined as HIV viral load ˂ 400 copies/mL within the last 3 months; 
• CD4 > 200 cells/L within the last 3 months; and  
• No reported opportunistic infections within 6 months prior to enrollment, except for 
the following which will be allowed:  
i. Esophageal candidiasis treated within last 6 months or currently improving with 
antifungal treatment. 
ii. Oral and/or genital HSV treated within last 6 months or currently improving 
with antiviral treatment. 
iii. Mycobacterium avium infection in last 6 months or that has been treated for at 
least 1 month.  
2.2.1.14 Patients with evidence of chronic hepatitis B virus (HBV) infection are eligible for trial 
as long as the HBV viral load is undetectable on suppressive therapy, if indicated.  
2.2.1.15 Patients with history of hepatitis C virus (HCV) infection must have been treated and 
cured. For patients with HCV infection who are currently on treatment, they are eligible 
if they have an undetectable or unquantifiable HCV RNA 12 weeks or longer after 
definitive treatment completion. 
2.2.1.16 Patients must be able to understand and willing to sign a written informed consent 
document.  
2.2.2 Exclusion Criteria 
2.2.2.1 Patients who have received chemotherapy, including herceptin and/or pertuzumab in the 
prior 3 weeks (6 weeks for nitrosoureas or mitomycin); other investigational agents or a 
PD-1/L1 agent within 4 weeks prior to study enrollment.  
2.2.2.2 Patients who have received radiotherapy within 4 weeks prior to study entry. 
2.2.2.3 Patients with active brain metastases or leptomeningeal metastases should be excluded 
from this clinical trial because of their poor prognosis and because they often develop 
progressive neurologic dysfunction that would confound the evaluation of neurologic 
and other adverse events. However, patients with treated brain metastases are eligible if 
there is no magnetic resonance imaging (MRI) evidence of progression for 6 weeks 
after treatment is complete (no radiotherapy within 6 weeks) and within 28 days prior to 
the first dose of trial drug or asymptomatic brain metastasis. Patients requiring 


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immunosuppressive doses of systemic corticosteroids (> 10mg/day prednisone 
equivalent) for palliation are excluded.  
2.2.2.4 Patients with a history of another invasive malignancy < 3 years prior to enrollment 
(patients with non-melanoma skin cancers, carcinoma in situ of the breast or cervix are 
eligible). 
2.2.2.5 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to agents used in study. For example, reaction to prior vaccination with 
vaccinia virus or known hypersensitivity reaction to fully humanized monoclonal 
antibodies (Grade > 3 NCI-CTCAEv5). 
2.2.2.6 Uncontrolled intercurrent illness including, but not limited to, ongoing active infection 
that requires systemic treatment with ongoing antibiotics (eligible if can stop antibiotics 
on day of enrollment), unstable angina pectoris, cardiac arrhythmia, or psychiatric 
illness/social situation that in the opinion of the primary investigator would prohibit the 
patient from complying with study requirements.  
2.2.2.7 Known history of a gastrointestinal illness that in the investigator’s opinion would 
prevent the absorption of entinostat, which is an oral agent. (Arm 3 ONLY) 
2.2.2.8 Patients with bone metastases who have initiated denosumab or a bisphosphonate 
therapy within 28 days prior to or after Cycle 1 Day 1. Continuation of prior therapy is 
allowed.  
2.2.2.9 Patients with inherited bleeding disorders, a history of bleeding diathesis such as vWF 
deficiency or recent (within 3 months prior to enrollment) clinically significant bleeding 
events that, in the judgment of the investigator, would interfere with patient’s ability to 
carry out the treatment program. 
2.2.2.10 Participants unwilling to accept blood products as medically indicated.  
2.2.2.11 Patients should have no evidence of being immunocompromised as listed below:  
- 
Active, known or suspected autoimmune disease. Patients are permitted to enroll if 
they have vitiligo, type I diabetes mellitus, residual hypothyroidism due to an 
autoimmune condition only requiring hormone replacement, psoriasis not requiring 
systemic treatment or conditions not expected to recur in the absence of an external 
trigger in the opinion of the primary investigator.  
- 
Altered immune function, that in the judgement of the PI that may affect a patient’s 
ability to adequately engage the immune system and respond to the immunotherapy 
agents being administered, including but not limited to: inflammatory bowel 
disease; active infectious enteritis; eosinophilic enteritis; lupus erythematous; 
ankylosing spondylitis; scleroderma; multiple sclerosis. These criteria do not 
include all disease with an immune-related component, but are not autoimmune in 
nature or have a primary alteration in the general immune function that may 
interfere with the vaccine mechanism of action, for example celiac disease.  
- 
Immunosuppressive therapy post-organ transplant. 
2.2.2.12 Concurrent use of chronic use of systemic steroids, except for physiologic doses of 
systemic steroids for replacement, defined as 10mg of prednisone per day or equivalent, 
or local (topical, nasal, ophthalmic or inhaled) steroid use or prior concomitant use with 
chemotherapy. Systemic steroids must have been discontinued >2 weeks prior to trial 
start. Prior use of corticosteroids in short-term schemes (duration shorter than 3 days) 


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for indications such as prophylaxis of reactions to intravenous contrast for imaging 
studies or chemotherapy-related AEs are not considered part of this exclusion. Prior use 
of corticosteroids for brain metastasis ending at least 14 days prior to enrollment is not 
considered part of this exclusion criteria. 
2.2.2.13 Pregnant and breastfeeding women are excluded from this study because of the 
potential for teratogenic or abortifacient effects with all of the agents involved in this 
trial.   
2.2.2.14 Clinically significant cardiomyopathy, coronary disease, chronic heart failure (CHF; 
New York Heart Association class III or IV or hospitalization for CHF), or 
cerebrovascular accident within 6 months prior to enrollment. 
2.2.2.15 Patients with a history of myocarditis are excluded due to the potential of myocarditis 
with anti-PD-L1 antibodies.  
2.2.2.16 Patients with pulse oximetry < 92% on room air will be excluded due to the potential of 
pneumonitis with anti-PD-L1 antibodies.  
2.2.2.17 Any other condition, which would, in the opinion of the Principal Investigator or 
Medical Monitor, indicated the subject is a poor candidate for the clinical trial or would 
jeopardize the subject or the integrity of the data obtained. 
2.3 
RECRUITMENT STRATEGIES   
This study will be listed on www.clinicaltrials.gov, on NIH social media platforms, and on OSU 
websites and social media platforms. We will also engage in local outreach to local hospitals and 
breast cancer advocacy groups (such as SHARE, Koman, and breast cancer research foundation) 
with IRB-approved recruitment materials.   
2.4 
SCREENING EVALUATION 
2.4.1 Screening activities prior to the obtaining informed consent 
Minimal risk activities that may be performed before the subject has signed a consent include the 
following:  
• Email, written, in person or telephone communications with prospective subjects.  
• Review of existing medical records to include H&P, laboratory studies, etc.   
• Review of existing MRI, x-ray, or CT images. 
• Review of existing photographs or videos. 
• Review of existing pathology specimens/reports from a specimen obtained for diagnostic 
purposes. 
2.4.2 Screening activities performed after a consent for screening has been signed 
The following screening procedures will be performed only after the subject has signed the study 
consent within 14 days prior to enrollment, except when specified differently. Assessments 
performed at outside facilities or at the local site, but off-protocol within these timeframes and 
those below may also be used to determine eligibility once a patient has signed the consent. 
2.4.2.1 Pathologic Confirmation  
Pathological confirmation of diagnosis and ER, PR and HER2 expression will be obtained at the 
local-site or participating facilities.  


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However, if no pathologic specimen is available, patients may enroll with a pathologists report 
showing a histologic diagnosis of TNBC or HER2-positive breast cancer in a College of 
American Pathologists (CAP) accredited laboratory (or other accrediting entity) and a clinical 
course consistent with the disease.  
2.4.2.2 Screening Visit: Physical Exam, Labs and Imaging   
History and Physical Exam: 
A thorough medical history and physical examination with height, weight and vital signs 
including pulse oximetry will be conducted at screening. Results of the physical examination 
including any abnormalities will be documented and can be used for the baseline examination if 
conducted within 3 days prior to initiating therapy. Abnormal findings will be reassessed at 
subsequent visits. On subsequent visits, any newly diagnosed or worsening conditions, signs and 
symptoms, whether related or unrelated to the trial, will be reported as adverse events. 
Performance Status:  
The ECOG performance status (Appendix A) will be assessed at screening and documented in 
the clinical record. 
Laboratory Assessment at Screening: 
• CBC with differential;  
• PT/INR/PTT;  
• Biochemical profile for Screening: 14 comprehensive metabolic panel (sodium, 
potassium, chloride, carbon dioxide, BUN, creatinine or measured creatinine clearance, 
glucose, AST (SGOT), ALT (SGPT), total bilirubin, serum creatinine, reflex Free T4 and 
TSH;  
• CD4 if clinically indicated (within 3 months prior to enrollment); 
• HBV, HCV, HIV testing including viral load if clinically indicated (within 3 months 
prior to enrollment).  
Pregnancy Testing: 
For female subjects of childbearing potential, urine or serum beta-HCG will be performed on 
initial screening.  
Patients who are postmenopausal (age-related amenorrhea for 12 or more consecutive months, or 
documented FSH > 40 mIU/mL / Estradiol < 20mIU/mL), or who had undergone hysterectomy 
or bilateral oophorectomy are exempt from pregnancy testing. Patients who experienced 
chemotherapy-induced menopause within the past 2 years and are considered to be of 
childbearing potential must undergo pregnancy screening.  
Imaging for Disease Evaluation at Screening: May be completed within 28 days 
• CT of chest/abdomen /pelvis (MRI may be substituted if clinically indicated). An attempt 
to be consistent with imaging modality for evaluation throughout trial must be made.  
• Brain MRI (preferred) in patients with known CNS disease or concern for CNS 
metastasis as described in Sections 2.1.2.3, 2.2.2.3 or head CT with contrast, if clinically 
indicated.   
Additional Screening 


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• A 2D Echocardiogram will be performed within 28 days prior to prior to enrollment 
for all patients in Arms 2 and 3 due to the use of T-DM1.  
• An EKG will be performed within 28 days prior to enrollment for all patients.  
2.5 
PARTICIPANT REGISTRATION AND STATUS UPDATE PROCEDURES 
Registration and status updates (e.g., when a participant is taken off protocol therapy and when a 
participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant 
Registration & Status Updates found here. 
2.5.1 For Participating Site Registration 
Registration will be a two-part process as participants are screened on this protocol. A protocol 
registration form will be supplied by the CCR study coordinator and updates will be provided as 
needed. To initially register a subject, after the participant has signed consent, complete the top 
portion of the form and send to CCR study coordinator. Once eligibility is confirmed, after 
completion of screening studies, complete the remainder of the form which is the eligibility 
checklist, indicating that the participant is being registered for treatment and send to CCR study 
coordinator. In addition, source documents supporting the eligibility criteria must be sent to the 
CCR study coordinator. The CCR study coordinator will notify you either by e-mail or fax that 
the protocol registration form has been received which will include the unique patient/subject ID 
number. Questions about eligibility should be directed to the CCR study coordinator or PI. 
Questions related to registration should be directed to the CCR study coordinator.  
When a participant has a status change (e.g., subject screened on the study, does not meet 
eligibility criteria and is removed from the study, participant is taken off protocol therapy or off 
study, etc.), the Participant Status Update Form will be supplied by the CCR study coordinator. 
Send the completed form to the CCR study coordinator. 
 
2.5.2 Screen Failure 
Screen failures are defined as participants who consent to participate in the clinical trial but are 
not subsequently assigned to the study intervention or entered in the study. A minimal set of 
screen failure information is required to ensure transparent reporting of screen failure 
participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing 
requirements and to respond to queries from regulatory authorities. Minimal information 
includes demography, screen failure details, eligibility criteria, and any serious adverse event 
(SAE). 
 
Individuals who do not meet the criteria for participation in this trial (screen failure) because of a 
transient lab abnormality may be rescreened.  


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2.6 
TREATMENT ASSIGNMENT PROCEDURES 
Cohorts 
Number Name 
Description 
1 
First Cohort 
Patients with metastatic triple negative breast cancer.  
2 
Second Cohort 
Direct assignment of patients with metastatic hormone 
receptor negative, HER2 positive breast cancer  
3 
Third Cohort 
Alternating direct assignment of patients with metastatic 
hormone receptor negative, HER2 positive breast 
cancer. 
 
Arms 
Number Name 
Description 
1 
M7824  
+ BN-Brachyury 
Bifunctional fusion molecule involving PD-L1 with 
TGF-b sequestering agent added to a vaccine for the 
tumor associated antigen called brachyury.  
2 
M7824  
+ BN-Brachyury  
+ T-DM1 
Bifunctional fusion molecule involving PD-L1 with 
TGF-b sequestering agent added to a vaccine for the 
tumor associated antigen called brachyury. These 
investigational agents will be added to standard of care 
treatment called T-DM1. 
3 
M7824  
+ BN-Brachyury  
+ T-DM1 
+ Entinostat 
Bifunctional fusion molecule involving PD-L1 with 
TGF-b sequestering agent added to a vaccine for the 
tumor associated antigen called brachyury as well as to 
an oral HDAC inhibitor called entinostat. These 
investigational agents will be added to standard of care 
treatment called T-DM1. 
 
Arm Assignment 
• Patients in Cohort 1 will be directly assigned to Arm 1. 
• Following the completion of the DLT period for Arm 1, patients in Cohort 2, will be directly 
assigned to Arm 2. Six patients will be assessed for safety. Patients in Arm 1 will continue to 
enroll on this arm until 8 – 13 patients are accrued.  
• Following completion of the DLT period for Arm 2, 6 additional patients in Cohort 2 will be 
directly assigned to Arm 3 to be assessed for safety.  
• Following completion of the DLT period for Arm 3, patients in Cohort 3 will be directly 
assigned on an alternating basis between Arms 2 and 3.  
2.7 
BASELINE EVALUATION  
Baseline evaluation is the C1D1 evaluation. Data collected during screening may count for the 
baseline evaluation if conducted within the below specified time periods and do not need to be 
repeated prior to first treatment:  
• History and physical examination with vital signs (including pulse oximetry), ECOG 
evaluation (Appendix A: Performance Status Criteria), skin assessment, history of prior 
therapy, review of concurrent medications, and review of baseline symptoms. Must be 
completed within 3 days of C1D1.   


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• Laboratory Assessment. Must be completed within 7 days of C1D1 except where noted 
below.  
o CBC with differential 
o Sodium, potassium, chloride, CO2, creatinine or measured creatinine clearance, 
glucose, BUN 
o Mineral panel (magnesium, calcium, phosphorus and albumin) 
o Hepatic panel (alkaline phosphatase, AST/ALT/total bilirubin) with total protein 
o PT/INR/PTT – Must be completed < 72 hours prior to biopsy if being performed 
o Urine or serum β-HCG test in women of childbearing potential (or endocrine 
evaluation with LH, FSH and estradiol) – Must be completed on C1D1.  
o Troponin, CK, CK-MB 
o Tumor Markers: – CEA, CA15-3, CA 27.29  
o Urinalysis 
• Assessment of disease by imaging (may include CT chest, abdomen and pelvis or MRI). 
Must be completed within 28 days prior to C1D1.   
• EKG. Must be completed within 7 days prior to C1D1. 
• Research Blood. Refer to tables in Section 5.1 for additional information.  
• Tumor Biopsy. Required in Cohort 3 only, optional in others. Refer to tables in Section 
5.1 for additional information. Must be collected within 7 days prior to C1D1. 
3 
STUDY IMPLEMENTATION 
3.1 
STUDY DESIGN 
This is an open label, multi-center, phase 1b trial with three arms to evaluate BN-Brachyury, 
entinostat, M7824 +/- T-DM1 in advanced breast cancer. Arm 1 will involve BN-Brachyury and 
M7824 in TNBC. Arms 2 and 3 will involve BN-Brachyury, M7824, T-DM1 +/- entinostat in 
ER-/PR-/HER2+ breast cancer that have previously progressed on or was intolerant to a taxane 
plus trastuzumab and pertuzumab (THP; paclitaxel can have been used in place of docetaxel). Up 
to 51 patients will be treated on this study with an accrual ceiling set at 65 patients to allow for a 
small number of inevaluable patients and up to 10 screening failures. Each cycle is 21 days in 
duration. 
Arm 1: TNBC 
Since M7824 and BN-Brachyury have not previously been used together in breast cancer, an 
eight – thirteen patient phase 1b (Arm 1) study will be conducted to evaluate safety of the 
combination as well as to evaluate efficacy. Patients in Arm 1 must have measurable disease but 
are not required to have biopsiable disease. Since no HER2-targeted agent will be used, this arm 
is limited to TNBC patients only.  
On C1D1, patients will receive M7824 2,400mg IV every 3 weeks (a higher dose of M7824 than 
used in the ongoing QuEST trial referenced above). Patient will receive the first MVA-BN-
Brachyury dose on C1D1 and return to clinic for the 2nd MVA-BN-Brachyury dose on C2D1. On 
C3D1, patients will then transition to the FPV-Brachyury vaccine every 21 days until Cycle 9. 
After that, FPV-Brachyury will be administered every 12 weeks. No biopsies are required. 
Patients will undergo staging with imaging every 9 weeks (i.e., every 3 cycles).  


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After the first 6 patients are enrolled and evaluated for safety, this arm will continue to accrue 
patients until 8 patients have received at least 2 cycles of treatment. If there are 2 or more 
objective responses, this arm will be expanded to a total of 13 patients. If there is an additional 1 
response (total 3 responses in 13 patients), this arm may be further expanded.   
If there are 0 to 1 DLTs in the first 6 patients on Arm 1, then Arms 2 and 3 of the trial will open. 
Patients in Cohort 2 will be assigned to Arm 2. Following the completion of the DLT period for 
Arm 2, additional patients will be assigned to Arm 3. The first six patients in both arms will be 
evaluated for DLTs. Following the completion of the DTL period for Arm 3, patients in Cohort 3 
will be directly assigned in an alternating fashion to Arms 2 and 3.  
Arm 2: HER2+ 
Fourteen patients will be enrolled in Arm 2. All patients in Cohort 2, Arm 2 must have 
measurable disease based on RECIST criteria. All patients in Cohort 3, Arm 2 must have 
measurable disease based on RECIST criteria as well metastatic deposits that can be safely 
biopsied for the study secondary objective of TIL evaluation. Patients in Arm 2 may have an 
optional tumor biopsy at baseline and an optional biopsy after 6 weeks on treatment. The study 
will require a baseline biopsy and a biopsy after 6 weeks on treatment for patients in Cohort 3. 
There will also be an optional biopsy at the time of progression.   
On C1D1, patients will receive T-DM1 3.6mg/kg IV every 3 weeks per standard of care. Patients 
will also receive M7824 2,400mg IV every 3 weeks (see Section 3.2).   
Patients will receive the first MVA-BN-Brachyury on C1D1 and return to clinic for the 2nd 
MVA-BN-Brachyury dose on C2D1.  
On C3D1, patients will undergo the 2nd biopsy (Optional in Cohort 2, Required in Cohort 3). 
Patients will then transition to the FPV-Brachyury vaccine q 21 days until cycle 9. After that, 
FPV-Brachyury will be administered every 12 weeks. Patients will undergo staging every 9 
weeks (i.e., every 3 cycles).  
Arm 2 will continue to accrue patients until 14 patients have received at least 2 cycles of 
treatment to evaluate for efficacy of the combination. If there are 4 or more objective responses, 
this arm will be expanded to a total of 19 patients. If there are an additional 2 responses (total of 
at least 6 responses of 19 patients), this arm may be further expanded.   
Arm 3: HER2+ 
Fourteen patients will be enrolled in Arm 3. All patients in Cohort 2, Arm 3 must have 
measurable disease based on RECIST criteria. All patients in Cohort 3, Arm 3 must have 
measurable disease based on RECIST criteria as well metastatic deposits that can be safely 
biopsied for the study secondary objective of TIL evaluation. Patients in Cohort 2 can undergo 
an optional biopsy at baseline and an optional biopsy after 6 weeks on treatment. The study will 
require a baseline biopsy and a biopsy after 6 weeks on treatment for patients in Cohort 3. There 
is also be an optional biopsy at the time of progression.  
On C1D1, patients will receive T-DM1 3.6mg/kg IV every 3 weeks per standard of care. Patients 
will also receive M7824 2,400mg IV q 3 weeks (see Section 3.2).   
Patients will receive the first MVA-BN-Brachyury on C1D1 and return to clinic for the 2nd 
MVA-BN-Brachyury dose on C2D1. Patients will start entinostat 5mg weekly on C1D1 and 
continue with self-administration on a weekly basis.  


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On C3D1, patients will undergo the 2nd biopsy (Optional in Cohort 2, Required in Cohort 3). 
Patients will then transition to the FPV- Brachyury vaccine every 21 days until cycle 9. After 
that, FPV-Brachyury will be administered every 12 weeks. Patients will undergo staging every 9 
weeks (i.e., every 3 cycles).  
Arm 3 will continue to accrue patients until 14 patients have received at least 2 cycles of 
treatment to evaluate for efficacy of the combination. If there are 4 or more objective responses, 
this arm will be expanded to a total of 19 patients. If there are an additional 2 responses (total of 
at least 6 responses of 19 patients), this arm may be further expanded. 
Patients will be dispensed enough entinostat by the local site pharmacy to complete 1 cycle (i.e., 
three entinostat 5mg will be dispensed at the start of each cycle; if dose reduced then nine 
entinostat 1mg tablets will be dispensed at the start of each cycle). See Section 3.2.4.1 for the 
specific administration procedures of entinostat. 
NOTE: Participating sites will be made aware of participant arm/cohort assignment at the time of 
study registration. 
3.1.1 Study Stopping Rule, All Arms 
Accrual will be halted if there is an occurrence of a grade 5 toxicity (patient death) within 30 
days of receiving any study treatment that is not due to disease progression. Prior to resumption 
of the study, an expedited safety report will be sent to and reviewed by the FDA. Any SAE also 
must be evaluated by the clinical investigator.  
In each arm, for toxicity that is not a DLT and does not require discontinuation of the IND agent, 
these agents may be held at the discretion of the investigator and may be reinitiated after toxicity 
has improved at the same or lower dose level (if applicable). In the even that one drug is 
discontinued due to toxicity, patients may continue treatment with the other drug(s) at the 
discretion of the investigator. 
  


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Figure 11: Trial Schema 
 
3.1.2 Dose Limiting Toxicity 
For the pre-specified safety monitoring plans below, a dose-limiting toxicity (DLT) is defined as 
an adverse event or abnormal laboratory value assessed as suspected to be trial treatment related 
(possible, probable or definite) and unrelated to disease or disease progression that occurs within 
the 30 days of the first treatment. Toxicities and lab values will be categorized and graded 
according to CTCAE v5.0. 
1. Any grade ≥3 non-hematologic, non-hepatic adverse event with the following 
exceptions: 
a. Nausea and/or vomiting that persists for 48 hours despite supportive care 
b. Electrolyte imbalances that persist for 48 hours despite supportive care 
2. Grade ≥ 3 fatigue lasting ≥  7 days 
3. Grade ≥ 4 neutropenia (ANC < 500uL) lasting > 7 days 
4. Grade ≥ 4 thrombocytopenia  
5. Grade ≥ 3 bleeding events 
6. Grade ≥ 3 febrile neutropenia 
7. Grade ≥ 4 elevation in AST or ALT 
8. Grade ≥ 4 elevation in bilirubin 
Monitoring of dose-limiting toxicities  
Up until the time the first 3 patients complete at least one cycle of treatment on each arm, accrual 
will proceed slowly (Arm 1: no more than 1 patient per 6 days; Arms 2 and 3: no more than 1 


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patient per 28 days) to more closely monitor safety, and after 3 patients receive at least 1 cycle of 
drugs, a safety review will be conducted before additional patients are enrolled. If there is one or 
more DLT in the first 3 patients on a given arm, accrual of the next 3 patients on the associated 
arm will proceed slowly with no more than 1 patient per 21 days (1 cycle) to continue closely 
monitor safety. On the basis of the monitoring criteria, if 2 of the first 2 patients or if 3 patients 
experience DLT within the first 6 patients, then the trial will halt accrual and the treatment 
regimen may be modified. The review will include a per-patient listing of all reported AEs to 
date, including actions required for dosing, to more fully review the nature, frequency, severity 
and timing of the events. This information combined with fewer DLTs may also result in 
modification of the treatment regimen. These reviews and decisions will be made by the 
Principal Investigator/ clinical research team as outlined in Section 7.4.1. 
Throughout enrollment of all arms, DLTs within the 30 days of treatment will be summarized 
with pre-specified criteria based on sequential boundaries to pause enrollment to more fully 
review safety if excessive numbers of DLTs are observed. The criteria are such that the 
probability of crossing the boundary is at most 0.05 if the true DLT rate is equal to 10%, and the 
probabilities of pausing are 0.67, 0.90 and 0.98 if the true DLT rate is equal to 30%, 40% or 
50%. 
The accrual will be paused if excessive numbers of DLTs are seen, that is, if the number of DLTs 
is equal to or exceeds bn out of n patients in the population evaluable for toxicity. Table 2 gives 
the criteria for pausing enrollment to more fully evaluate safety. 
Table 2. Criteria for pausing enrollment because of DLTs to more fully review safety, for sample 
size up to 19 patients.  
Number of patients 
n 
1-2 
3-6 
7-12 
13-19 
Number of patients for whom 
DLT is reported 
bn 
2 
3 
4 
5 
 
3.2 
DRUG ADMINISTRATION AND PRE-MEDICATIONS 
The BN-Brachyury vaccines will be given on the day of treatment with M7824 and T-DM1 
(Arms 2 & 3 only). The preferred order of drug administration is vaccine, then M7824 
administration, then T-DM1 if applicable; When possible, administer the vaccine within 15 
minutes prior to the M7824 administration; however, the exact timing of vaccine administration 
is flexible. Patients in Arm 3 will be provided with a take-home supply of entinostat and will 
self-administer entinostat around bedtime on the day of the infusions. Patients will also self-
administer entinostat on subsequent designated days of the cycle (Days 8 and 15; may be taken 
up to 24 hours late before considered a missed dose).  
A window of -1 or +3 days for every 3 week cycle is allowed in the event of scheduling issues 
(i.e., holiday, bad weather, family responsibilities, security alerts or other scheduling issues). 
This can also extend to complications of disease not attributable to disease progression or 
protocol therapy. These delays will not be considered protocol deviation. Any dose that cannot 
be accommodated within this window will be skipped and the dose not made up.  
3.2.1 MVA-BN-Brachyury and FPV-Brachyury Vaccines  
MVA-BN-Brachyury is administered via subcutaneous injection. The preferred injections sites 
include the left upper arm, the right upper arm, the left outer thigh, and the right outer thigh. One 


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dose consists of 4 separate injections: one injection in each of the listed injection sites. Each 
0.5mL injection of MVA-BN-Brachyury consists of a nominal virus titer of 2.0 x 108 infectious 
units (Inf.U).  
FPV-Brachyury is administered via subcutaneous injection as a single injection per dose. 
Options for administration sites include the upper arm or outer thigh. When possible, each 
subsequent dose should be administered at the same injection site as the first dose. Each injection 
of FPV-Brachyury consists of a nominal virus titer 1.0 x 109 Inf.U. 
Ideally, the BN-Brachyury vaccines will be administered prior to the start of the M7824 infusion.  
3.2.2 M7824 
Subjects will be scheduled to receive M7824 at a flat dose of 2,400 mg IV once every 3 weeks. 
M7824 can be administered through a central line (preexisting) or a peripheral IV. There is no 
need to prime the line and purge the air prior to administration in this protocol.  A filter is 
required for administration of M7824. The preference is for M7824 to be given via peripheral 
line. However, if the research team documents that the main material of the port is titanium, 
M7824 can be administered via mediport. It is the responsibility of the research team to confirm 
and document this in CRIS. Once documented, the ordering physician can change the route of 
administration to mediport.  
Please refer to the pharmacy manual for a list of acceptable materials known to be compatible 
with M7824. 
As T-DM1 is dosed every 3 weeks, a q3 week dosing regimen for M7824 was selected based on 
PK modeling and Phase 1 trial safety data. Subjects will receive an IV infusion of M7824 over 1 
hour (-10 minutes / +20 minutes, that is, 50 to 80 minutes). As a routine precaution, subjects 
enrolled in this trial must be observed for 60 minutes post end of infusion, in an area with 
resuscitation equipment and emergency agents. During cycle 1, vital signs will be monitored  
every15 minutes during infusion and until completion of observation period. Starting with cycle 
2, vital signs will be monitored every 30 minutes during the infusion and until the completion of 
the observation period.  
Current experience revealed that infusion related reactions (IRRs) to M7824 seldom occur and 
are generally mild to moderate in severity. Therefore, administration of a premedication is 
generally not required. 
If an Investigator deems it necessary to administer a premedication, an antihistamine (for 
example, 25-50 mg diphenhydramine) and acetaminophen 500-650 mg intravenously or 
equivalent oral dose is recommended approximately 30 to 60 minutes prior to each dose of 
M7824. If Grade ≥ 2 infusion reactions are seen during the first two infusions, premedication 
should not be stopped. Steroids as premedication are not permitted. 
Management of symptoms should follow the guidelines shown in Table 10. 
3.2.2.1 Immediate Hypersensitivity Reaction 
Hypersensitivity reactions may require immediate intensive care. M7824 should be administered 
in a setting that allows for immediate access to an intensive care unit or equivalent environment 
and administration of therapy for anaphylaxis, such as the ability to implement immediate 
resuscitation measures. Steroids (dexamethasone 10 mg), epinephrine (1: 1,000 dilution), allergy 


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medications (IV antihistamines), bronchodilators, or equivalents, and oxygen should be available 
for immediate access.  
A complete guideline for the emergency treatment of anaphylactic reactions according to the 
Working Group of the Resuscitation Council United Kingdom and can be found at 
https://www.resus.org.uk/pages/reaction.pdf 
3.2.2.2 Flu-Like Symptoms 
For prophylaxis of flu like symptoms, a nonsteroidal anti-inflammatory drug (NSAID), e.g., 
ibuprofen 400 mg or comparable NSAID dose, may be administered 2 hours before and 8 hours 
after the start of each IV infusion. 
Patients can receive M7824 for up to 12 months should they remain on trial.     
3.2.3 Ado-trastuzumab emtansine (T-DM1)  
Patients in Arms 2 and 3 will receive T-DM1 3.6 mg/kg by IV infusion every 3 weeks. The first 
infusion will be administered over 90 minutes and subsequent infusions will be administered 
over 30 minutes, if the first infusion was well tolerated. If the first infusion is not well tolerated, 
then the next infusion will be administered over 90 minutes (as per the T-DM1 package insert). 
Infusion times may be adjusted for patients experiencing any infusion-related reaction. There 
does not need to be an observation time between the completion of M7824 and the start of T-
DM1, however, vital signs should be recorded following the completion of M7824 and 
immediately prior to the start of T-DM1. As routine and as described in the FDA package insert 
for T-DM1, subjects who receive T-DM1 should be observed following completion of T-DM1 
infusion.  
Per the FDA package insert for T-DM1,  
• First infusion: Patients should be observed during the infusion and for at least 90 minutes 
following the initial dose for fever, chills, or other infusion related reactions.  
• Subsequent infusions: Patients should be observed during the infusion and for at least 30 
minutes after infusion if the first infusion was well tolerated or for 90 minutes if not well 
tolerated. 
During cycle 1, vital signs will be monitored every15 minutes during the infusion and until 
completion of observation period. Starting with cycle 2, vital signs will be monitored every 30 
minutes during the infusion and until the completion of the observation period.  
Conventional trastuzumab and ado-trastuzumab emtansine products are NOT interchangeable. T-
DM1 should be prepared according to the FDA-approved prescribing information using an 
infusion bag containing 250mL of 0.9% sodium chloride injection (do NOT use 5% dextrose). 
Infusions must be administered through a 0.2 or 0.22 micron nonprotein adsorptive 
polyethersulfone filter. Diluted T-DM1 may be stored in the refrigerator (2 to 8℃) for up to 24 
hours prior to use.  
Pre-medications 
In order to mitigate potential infusion-related reactions, premedication with an antihistamine and 
with paracetamol (acetaminophen) (for example, 25-50 mg diphenhydramine and 500-650 mg 
paracetamol [acetaminophen] IV or oral equivalent) approximately 15 to 60 minutes prior to 
dosing of M7824 is optional and at the discretion of the Investigator. If Grade ≥ 2 infusion 


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reactions are seen during the first two infusions, premedication should not be stopped. 
Premedication can be given to the patient at the same time as the BN-Brachyury vaccine. 
Since T-DM1 is considered a drug with low emetic risk by both ASCO and NCCN, the use of a 
prophylactic single dose of a 5-HT3 antagonist or dopamine blocker (i.e., prochlorperazine) is 
encouraged. The antiemetic regimen may be modified based on patient specific factors or 
standard institutional practices.   
3.2.4 Self-Administered Study Drugs  
The entinostat used in this study is a self-administered investigational agent. Such agents are 
dispensed from the pharmacy to a participant, participant’s representative or to a Patient Care 
Unit for self-administration and a record of the dispensed investigational agent is generated and 
kept by the dispensing pharmacy.  
3.2.4.1 Entinostat (Arm 3 only) 
At the start of each cycle, patients will be dispensed enough entinostat by the local site pharmacy 
to complete 1 cycle (i.e., three entinostat 5mg will be dispensed at the start of each cycle; if dose 
reduced then nine entinostat 1mg tablets will be dispensed at the start of each cycle). Entinostat 
is to be administered on an empty stomach, at least 2 hours after a meal and 1 hour before the 
next meal on Days 1, 8 and 15 of each cycle. If a patient forgets to take a dose on the assigned 
day, the dose can be taken up to 1 day (24 hours) later. If a dose is more than 1 day late, that dose 
will be skipped. Do NOT split, crush or chew entinostat tablets. In case of vomiting after the 
entinostat tablet is swallowed, the dose should not be taken again unless the expelled tablet is 
visualized in the vomitus and is entirely intact. Patients should discuss with the study team 
before attempting to re-take the dose. 
For Cycle 1 Day 8, patients will be asked to take the entinostat tablet in the morning of the day 
of the clinical visit in order to assess for pharmcokinetic data 4-6 hours after taking the 
medication. (118) There is also an optional research lab visit on Cycle 1 Day 15 where patients 
will be instructed to do the same. 
Patients will be asked to keep a medication diary and bring it with them on each study visit (See 
Appendix B). Patients will also bring any remaining pills to each study visit. The research nurse 
will review and validate the completeness and accuracy of the participant’s diary with the 
participant.  
If a participant goes off study while at home, the research nurse will document and ensure the 
return of the unused oral investigational agents from the participant. Unused investigational 
agent will be destroyed per dispensing pharmacy procedure. 
3.3 
STUDY INTERVENTION COMPLIANCE 
Participants in Arm 3 will be provided with a medication diary (Appendix B: Oral Medication 
Diary (Arm 3 only)) and asked to complete this at the time of self-administration of entinostat. 
Research nurses will collect this medication diary at the start of each cycle and will verify that 
entinostat was taken within the appropriate time period specified by the protocol. Medication 
diaries will be used to calculate study intervention compliance.   


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3.4 
DOSE MODIFICATIONS  
• In the case of toxicity, appropriate medical treatment should be used (including anti-emetics, 
anti-diarrheals, etc.).  
• Once a patient has a dose reduction for toxicity, the dose will not be increased.  
• Any of the trial drugs may be stopped for toxicity per the discretion of the primary 
investigator if is it felt that the toxicity can be directly linked to a specific drug or drugs. 
However, if 2 drugs or more must be stopped, the patients will be taken off treatment.  
• Participants continuing to experience toxicity at the off-treatment visit will be contacted for 
additional assessments until the toxicity has resolved or is deemed irreversible. Patients must 
remain on the study to have additional assessments completed.  
• For AEs that are unrelated to the study drugs, study drug may be held for up to 28 days at the 
discretion of the PI. Any dose that would have occurred within this window will be skipped 
and the dose not made up. 
• If any treatment related AE observed was grade > 2, treatment should be delayed by 1 week 
(except for hyperbilirubinemia where drug will be held for grade 2 – see Table 7; except for 
electrolyte abnormalities that can be easily replenished or other lab abnormalities that have 
no clinical consequence) for up to 4 consecutive weeks and possibly patients should be 
reduced by 1 dose level (See Table 3, Table 4, Table 5, Table 6, Table 7).  
• If, after 4 weeks of delay, all treatment related AEs have still not resolved (to grade ≤ 1), then 
any further treatment should be stopped.  
• If any treatment-related AE observed is grade 4, then all treatments should be stopped. The 
one exception is grade 4 thrombocytopenia in which the drug will be held and then reduce 
one dose level. 
 
Table 3A: Recommended Dose Reduction Schedule for Adverse Events for Arm 2 
Dose Level 
Dose Reduction Schedule 
T-DM1 Dose 
1 
Starting dose 
3.6 mg/kg IV q3 weeks 
-1 
First dose reduction 
3.0 mg/kg IV q3 weeks 
-2 
Second dose reduction 
2.4mg/kg IV q3 weeks 
-3 
Third dose reduction 
Discontinue treatment  
 
* There will be no dose reductions of the BN-Brachyury vaccines. 
** M7824 may be reduced at any time after the DLT period in the study from 2,400mg to 
1,800mg at discretion of the PI for any side effects possibly attributed to the TGF-beta 
component of the drug (i.e., mucosal bleeding, excessive keratoacanthomas). 
 
Table 3B: Recommended Dose Reduction Schedule for Adverse Events for Arm 3 
Dose Level 
Dose Reduction Schedule 
T-DM1 Dose 
Entinostat 
1 
Starting dose 
3.6mg/kg IV q3 weeks 
5mg po q7 days 
-1 
First dose reduction 
3.6mg/kg IV q3 weeks 
3mg po q7 days 
-2 
Second dose reduction 
3.0mg/kg IV q3 weeks 
3mg po q7 days 
-3 
Third dose reduction 
2.4mg/kg IV q3 weeks 
2mg po q7 days 
-4 
Requirement for further 
dose reduction 
Discontinue treatment 
 


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* There will be no dose reductions of the BN-Brachyury vaccines. 
** M7824 may be reduced at any time after the DLT period in the study from 2,400mg to 
1,800mg at the discretion of the PI for any side effects possibly attributed to the TGF-beta 
component of the drug (i.e., mucosal bleeding, excessive keratoacanthomas). 
3.4.1 Dose Modification of Entinostat  
Table 4: Non-hematologic Toxicity 
Toxicity 
Dose Modifications 
Grade 4 
Administer symptomatic remedies/ start prophylaxis. 
Hold dose until recovery to Grade 1 or baseline under the following directions: 
1. If recovered within 4 weeks of onset (i.e., ≤3 missed doses), resume 
study drug as follows: 
• If receiving 5 mg, restart study drug at 3 mg. 
• If receiving 3 mg, restart study drug at same dose or reduce to 2 mg  
(see Table 3B). 
• If receiving 2 mg, discontinue study treatment. 
2. If not recovered within 4 weeks, permanently discontinue study drug. 
Grade 3 
Administer symptomatic remedies / start prophylaxis. Hold dose until 
recovery to Grade 1 or baseline under the following directions: 
1. If recovered within 1 week, resume study drug at prior dose. If not 
recovered within 1 week, continue to hold dose. 
2. If recovered within 2-4 weeks, resume study drug as follows: 
• 
If receiving 5 mg, restart study drug at 3 mg. 
• 
If receiving 3 mg, restart study drug at same dose or reduce to 2 mg 
(see Table 3B). 
• 
If receiving 2 mg, permanently discontinue study drug. 
3. If not recovered within 4 weeks, permanently discontinue study drug. 
Recurrence of 
the 
same  ≥ Grade 3 
toxicity despite 
dose reduction 
  
If the same ≥ Grade 3 event recurs: 
1. Administer symptomatic remedies/ start prophylaxis. Hold1 dose until 
recovery to Grade 1 or baseline. 
2. If recovered within 2 weeks, resume study drug as follows: 
• If receiving 5 mg, restart study drug at 3 mg. 
• If receiving 3 mg, restart study drug at same dose or reduce to 2 mg  
(see Table 3B). 
• If receiving 2 mg, permanently discontinue study drug. 
3. If the same ≥ Grade 3 event recurs (i.e., third occurrence) despite 
entinostat dose reduction to 2 mg, as described above, discontinue study 
drug. 
≤Grade 2 
Administer symptomatic remedies / start prophylaxis. 
Dosing of study drug may be interrupted at the Investigator’s discretion. 
• If dose is held for 4 consecutive weeks, permanently discontinue 
study drug. 
• If toxicity resolves, resume entinostat at the original dose. 


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PIf greater than 50% of doses are missed during any 6 week period, discontinue from study drug treatment. 
  
Table 5: Hematologic Toxicity 
Toxicity 
Dose Modifications 
≥ Grade 3 
neutropenia,   
≥ Grade 3 
uncomplicated 
thrombocytopenia, 
or 
Grade 2 complicated 
thrombocytopenia 
  
Administer symptomatic remedies / start prophylaxis. 
Hold dose1 until recovery to Grade 1 or study baseline under the following 
direction: 
1. If not recovered by next scheduled dose, skip the dose. If recovered 
by next scheduled dose, resume study drug at prior dose. 
2. If receiving 2 mg dose, and not recovered by either of the next 2 
scheduled doses, permanently discontinue study treatment. 
Otherwise, skip each dose. If recovered for either of these doses, 
resume study drug as follows:   
• If receiving 5 mg, restart study drug at 3 mg. 
• If receiving 3 mg, restart study drug at 2 mg (see Table 3B). 
3. If not recovered within 4 weeks, permanently discontinue study drug. 
Recurrence of the 
same hematologic 
toxicity 
 If the same hematologic toxicity recurs: 
1. Administer symptomatic remedies/ start prophylaxis. Hold1 dose until 
recovery to Grade 1 or baseline. 
2. If recovered within 2 weeks, resume study drug as follows: 
• If receiving 5 mg, restart study drug at 3 mg 
• If receiving 3 mg, restart study drug at 2 mg. (See Table 3B) 
• If receiving 2 mg, permanently discontinue study drug 
3. If the same ≥ Grade 3 event recurs (i.e., third occurrence) despite 
entinostat dose reduction to 2 mg, as described above, permanently 
discontinue study drug. 
  
PIf greater than 50% of doses are missed during any 6 week period, discontinue from study drug treatment. 
3.4.2 Dose Modification of T-DM1  
Dose modifications for T-DM1 according to published FDA recommendations. Please see 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/125427lbl.pdf for full details.  
Once the dose of T-DM1 is reduced, the dose should not be re-escalated.   
If a planned dose is delayed or missed, it should be administered at the next planned cycle. The 
infusion may be administered at the dose and rate the patient tolerated in the most recent 
infusion.  
The infusion rate of T-DM1 should be slowed or interrupted if the patient develops an infusion-
related reaction. Permanently discontinue T-DM1 for life-threatening infusion-related reactions.  
Management of increased serum transaminases, hyperbilirubinemia, left ventricular dysfunction, 
thrombocytopenia, pulmonary toxicity or peripheral neuropathy may require temporary 


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interruption, dose reduction or treatment discontinuation of T-DM1 as per guidelines provided in 
6, Table 7, Table 8, Table 9. 
Hepatotoxicity 
Table 6: Dose Modification Guidelines for Increased Serum Transaminase (AST/ALT) 
Grade 2 
(> 2.5 to < 5x ULN) 
Grade 3 
(> 5 to < 20x ULN) 
Grade 4 
(> 20 x ULN) 
Treat at same dose level. 
Do not administer T-DM1 until 
AST/ALT recovers to Grade < 2 
and then reduce one level. 
Permanently discontinue T-
DM1. 
ALT = alanine transaminase; AST = aspartate transaminase; ULN = upper limit of normal.  
Table 7: Dose Modification for Hyperbilirubinemia 
Grade 2 
(> 1.5 to < 3x ULN) 
Grade 3 
(> 3 to < 10x ULN) 
Grade 4 
(> 10 x ULN) 
Do not administer T-DM1 until 
total bilirubin recovers to Grade 
<1 and then treat at same dose 
level.  
Do not administer T-DM1 until 
total bilirubin recovers to Grade 
< 1 and then reduce one level. 
Permanently discontinue T-
DM1. 
Permanently discontinue T-DM1 treatment in patients with serum transaminases > 3x ULN and 
concomitant total bilirubin > 2x ULN.  
Permanently discontinue T-DM1 in patients diagnosed with nodular regenerative hyperplasia 
(NRH). 
Left Ventricular Dysfunction  
Table 8: Dose Modification for Left Ventricular Dysfunction  
Symptomatic 
CHF 
LVEF < 40% 
LVEF 40% to 
≤45% AND 
decrease is       > 
10% points from 
baseline 
LVEF 40% to 
≤45% AND 
decrease is       < 
10% points from 
baseline 
LVEF > 45% 
Discontinue T-
DM1. 
Do not administer    
T-DM1. 
Repeat LVEF 
assessment within 
3 weeks. If LVEF 
< 40% is 
confirmed, then 
discontinue T-
DM1. 
Do not administer    
T-DM1. 
Repeat LVEF 
assessment within 
3 weeks. If the 
LVEF has 
recovered to 
within 10% points 
from baseline, 
discontinue T-
DM1. 
Continue with T-
DM1.  
 
Repeat LVEF 
assessment within 
3 weeks. 
Continue with T-
DM1. 
CHF = congestive heart failure; LVEF = left ventricular ejection fraction 


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Thrombocytopenia 
A reduction in dose is recommended in the case of Grade 4 thrombocytopenia (platelets < 
25,000/mm3).  
Table 9: Dose Modification Guidelines for Thrombocytopenia 
Grade 3 
PLT 25,000/mm3 to 50,000/mm3 
Grade 4 
PLT < 25,000/mm3 
Do not administer T-DM1 until PLT recovers 
to Grade < 1 (> 75,000/mm3) and then treat at 
same dose level. 
Do not administer T-DM1 until PLT recovers 
to Grade <1(> 75,000/mm3), and then reduce 
one dose level. 
PLT = platelets  
 
Pulmonary Toxicity  
T-DM1 should be permanently discontinued in patients with interstitial lung disease (ILD) or 
pneumonitis.  
Peripheral Neuropathy 
T-DM1 should be temporarily discontinued in patients experiencing Grade 3 or 4 peripheral 
neuropathy until resolution to < Grade 2. 
3.4.3 Treatment Discontinuation of BN-Brachyury Vaccines  
No dose modification. If unable to tolerate due to side effects, the vaccine will be removed from 
the treatment regimen.  
3.4.4 Treatment discontinuation of M7824  
• Any Grade 4 adverse drug reactions (ADRs), as defined by CTCAE v5 and assessed as 
related to M7824 by the Investigator, except for laboratory values that are determined to 
not be clinically significant or single laboratory valued that resolve to Grade ≤ 1 or baseline 
grade within 7 days with adequate medical management. 
• Any Grade 3 ADRs possibly, probably or definitely related to M7824 except for any of the 
following: 
o Transient (≤ 48 hours) Grade 3 flu-like symptoms or fever, which is controlled with 
medical management.   
o Transient (≤ 48 hours) Grade 3 fatigue, local reactions, headache, nausea, emesis 
which is controlled with medical management. 
o Tumor flare phenomenon defined as local pain, irritation, or rash localized at sites 
of known or suspected tumor. 
o Any Grade ≥ 3 drug-related amylase or lipase abnormality that is not associated 
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o Grade 3 Hemoglobin decrease (< 8.0 g/dL) that is clinically manageable with blood 
transfusions or erythroid growth factor use does not require treatment 
discontinuation.  
o Keratoacanthoma and squamous cell carcinoma of the skin.  
o Any endocrinopathy that can be medically managed with hormone replacement. 
o Any grade 3 adverse drug reaction which in the opinion of the investigator is not 
clinically relevant or can be medically managed with minimal risk to the patient 
(e.g., placement of a pleural catheter for recurrent inflammatory pleural effusions). 
3.4.5 Suggested Evaluation of Suspected Myocarditis  
If a patient has symptoms concerning for a diagnosis of myocarditis or myopericarditis, 
cardiology will be consulted immediately. In addition, the following clinical evaluation may be 
performed depending on clinical symptoms and clinical suspicion of the investigator: 
• Troponin-I and CK, CK-MB x 1 
• EKG in triplicate 
• 2E Echo to evaluate ejection fraction and TDI-derived longitudinal strain 
• Cardiac MRI if applicable 
3.4.6 Suggested Evaluation of Suspected Bleeding or Hemorrhage Events and Treatment 
Modification 
For anemia or hemorrhage events assessed as treatment-related, items queried may include but are 
not limited to detailed relevant past medical and treatment history, bruising tendency, history of 
blood transfusions and/or dependency, and a request for an updated patient history including 
details such as concomitant medications, all laboratory data, updated dosing information and 
recent tumor evaluation scans.  
In this protocol, anemia may be due to M7824 (documented in 29% of patients) and/or T-DM1 
(documented in approximately 10 to 15% of patients). M7824 treatment-related anemia is an AESI 
(see Investigators’ Brochure). Notably, there are many reasons for hemorrhage and/or anemia in 
patients with advanced cancer, and Hb level of at least 9 g/dl is required for this study. A thorough 
investigation of new anemia cases of unspecified etiology is recommended.  
General Guidance for anemia management and evaluation: 
• Participants must enter the study with Hgb values at least 9 g/dL and baseline anemia evaluation 
is conducted per recommendations below.  
• Consider hematology consult for severe and or refractory anemias.  
• All relevant hematologic testing for treatment related anemias should be done prior to blood 
transfusion, if clinically feasible. 
• Transfusion should be performed at the discretion of the investigator, based on clinical 
assessment and considered when participant experiences significant anemia.  
• Guidance for evaluation of baseline anemia or suspected treatment-related anemias is provided 
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o Hb and CBC with differential (e.g., MCV, RDW, ANC, hematocrit, reticulocytes 
counts) 
o Peripheral blood smear for cell morphological assessment 
o Complete metabolic panel including liver panel-LFTs, bilirubin, LDH, renal function, 
and serum folate, B12 values and other chemistries 
o Coagulation factors (PT, PTT, INR) 
o Urinalysis including culture 
o Iron panel (TIBC, ferritin, Fe) 
 
• Discuss further management with Principal Investigator for clinically significant treatment 
related anemias.  
Multiple protocols using M7824, several mucosal bleeding events ranging from low grade 
gingival bleeding and epistaxis to more serious hemoptysis, GI bleeding and hematuria have 
been observed. Some of these events can be attributed to bleeding events related to cancer 
directly and others bleeding events can be attributed to an inflammatory process (e.g. colitis) 
which is a known toxicity of anti-PD-L1 agents including M7824. However, there remains the 
possibility that M7824 may increase the overall risk of bleeding in ways that may not be directly 
related to direct tumor bleeding or inflammatory bleeding events described with checkpoint 
inhibitors like M7824. However, there is no evidence of a negative effect on coagulation or 
platelet number or function. It is hypothesized that this possible increased mucosal bleeding risk 
may be due to the known mucoprotective effects of TGF. Accordingly, patients will be closely 
monitored for mucosal bleeding (e.g., gum bleeding, nose bleeds, coughing up blood, blood in 
their urine, or blood in the stool).  
• If Grade 3 mucosal bleeding occurs in the absence of thrombocytopenia (> 75,000), liver 
enzyme abnormalities (< Grade 3 AST or ALT elevation +/- abnormal bilirubin) or 
coagulation abnormalities (INR. < 2.5), M7824 will be held with BN-Brachyury, T-DM1 
+/- entinostat being allowed to continue per PI discretion. See Tables 5, 6, 7 and 9 for 
specific dosing instructions for agents. M7824 can be resumed once mucosal bleeding is 
< Grade 1 and the dose may be reduced to 1800mg q3 weeks per PI discretion.  
• If Grade 3 non-mucosal bleeding or hemorrhage occurs in the absence of 
thrombocytopenia (>75,000), liver enzyme abnormalities (< Grade 3 AST or ALT 
elevation +/- abnormal bilirubin) or coagulation abnormalities (INR < 2.5), T-DM1 will 
be held with BN-Brachyury, M7824 +/- entinostat being allowed to continue per PI 
discretion. See Tables 5, 6, 7 and 9 for specific dosing instructions for agents.  
• If  Grade 3 bleeding or hemorrhage occurs in the presence of thrombocytopenia 
(<75,000), liver enzyme abnormalities (> Grade 3 AST or ALT elevation +/- abnormal 
bilirubin) or coagulation abnormalities (INR > 2.5) T-DM1, M7824 and entinostat will be 
held until a formal workup is completed with BN-Brachyury being allowed to continue 
per PI discretion.   
• If a drug is held for hemorrhage or bleeding and the hemorrhage or bleeding is attributed 
to a trial drug, a dose reduction should be considered if the drug is to be reintroduced 
once AEs resolve (< Grade 1 within 2 cycles).  
o If bleeding or hemorrhage is attributed to a specific drug, then that drug should be 
reduced by 1 dose level if is it resumed.  


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o If bleeding or hemorrhage cannot be attributed to a specific drug, then the PI 
should consider reducing all suspected drugs by 1 dose level if the patient is to 
remain on treatment.   
o If mucosal bleeding occurs, the PI should consider reducing M7824 to 1800mg q3 
weeks.  
 
3.4.7 Toxicity Management   
Table 10: Treatment Modification Guidance for Symptoms of Infusion-Related Reactions 
including Immediate Hypersensitivity  
Guidelines below are merely suggestions. If an immune-mediated reaction occurs, the subject 
should be treated according to the best available medical practice.  
Infusion-Related Reactions (IRR) are an important risk for M7824. 
NCI-CTCAE Grade 
Treatment Modification for M7824 
Grade 1 – mild 
Mild transient reaction; in general, 
infusion interruption not indicated; 
intervention not indicated. 
• Increase monitoring of vital signs as medically 
indicated as participants are deemed medically 
stable by the attending Investigator. 
• Hold infusion if deemed necessary by the 
investigator. 
Grade 2 – moderate 
Therapy or infusion interruption 
indicated but responds promptly to 
symptomatic treatment (for example, 
antihistamines, nonsteroidal anti 
inflammatory drugs, narcotics, IV 
fluids); prophylactic medications 
indicated for  24 h. 
• Stop the infusion of the study intervention.  
• Increase monitoring of vital signs as medically 
indicated as participants are deemed medically 
stable by the attending Investigator. 
• If symptoms resolve quickly, resume infusion at 
50% of original rate with close monitoring of any 
worsening signs and symptoms, otherwise dosing 
held until resolution of symptoms with mandated 
premedication for the next scheduled visit. 
• If not improving, consider administration of 
glucocorticoids and stop the infusion for that day. 
• If the participant has a second IRR Grade ≥ 2 on 
the slower infusion rate despite premedication, the 
infusion should be stopped, and the investigator 
may consider withdrawal of this participant from 
the study. 


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NCI-CTCAE Grade 
Treatment Modification for M7824 
Grade 3 or Grade 4 – severe or 
life-threatening 
Grade 3: Prolonged (for example, 
not rapidly responsive to 
symptomatic medication and/or brief 
interruption of infusion); recurrence 
of symptoms following initial 
improvement; hospitalization 
indicated for clinical sequelae. 
Grade 4: Life-threatening 
consequences; urgent intervention 
indicated. 
• Stop the infusion of study intervention immediately 
and disconnect infusion tubing from the participant 
with additional appropriate medical measures and 
closely monitor until deemed medically stable by 
the attending Investigator. Hospitalization and/or 
close monitoring is recommended. 
• Administration of glucocorticoids may be required. 
• For Grade 3 or 4 IRRs, permanent discontinuation 
of study intervention is mandated. 
Once the infusion is interrupted or rate reduced to 50% of previous infusion rate, it must 
remain decreased for all subsequent infusions.  
For all types and grades of infusion reactions, details about drug physical constitution, 
method of preparation, and infusion must be recorded. 
Participants should be instructed to report any delayed reaction immediately. 
Once the infusion is interrupted or rate reduced to 50% of previous infusion rate, it must 
remain decreased for all subsequent infusions.  
For all types and grades of infusion reactions, details about drug physical constitution, 
method of preparation, and infusion must be recorded. 
Participants should be instructed to report any delayed reaction immediately. 
 
 
  
 
 
Table 11 Immune-related adverse events (irAEs) 
Immune-related AEs are specific to immunotherapies and vary by organ system.  
The following immune-related AEs are important identified risks for M7824:  
• 
Immune-related pneumonitis 
• 
Immune-related hepatitis 
• 
Immune-related colitis 
• 
Immune-related nephritis and renal dysfunction 
• 
Immune-related endocrinopathies  
• 
(thyroid disorders, adrenal insufficiency, type 1 diabetes mellitus, pituitary disorders) 
• 
Immune related rash 
• 
Other immune-related events (myositis, myocarditis, encephalitis) 
The following immune-related AEs are important potential risks for M7824: 


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• Guillain-Barré syndrome 
• Uveitis 
• Pancreatitis 
• Myasthenia gravis/myasthenic syndrome 
Recommended guidance and management for specific irAEs are provided in the current 
NCCN (guideline available at http://www.nccn.org). 
Requirements in addition to NCCN guidelines: 
• Permanent treatment discontinuation is required in case of immune-related Grade 4 
rash/inflammatory dermatitis, nephritis, autoimmune hemolytic anemia, hemolytic 
uremic syndrome, aplastic anemia, immune thrombocytopenia, acquired thrombotic 
thrombocytopenic purpura inflammatory arthritis, myositis and polymyalgia-like 
syndrome. 
• For Grade 4 immune-related lymphopenia, permanent treatment discontinuation will be 
required, if lymphopenia is considered immune-related in nature, no clear alternative 
explanation exists for the event, and it does not resolve within 14 days. Permanent 
treatment discontinuation is not required when the AE is manifested by a single 
laboratory value out of normal range without any clinical correlates. In this case, 
treatment should be held until the etiology is determined. If the event is not considered 
immune-related and resolves to Grade ≤ 1, restarting treatment may be considered. 
• For Grade 1 immune-related pneumonitis: continue treatment. If clinically indicated, 
monitor participants weekly or more frequently as needed with history, physical 
examination and pulse oximetry. If symptoms appear and/or changes in the physical 
exam are noted, treat as Grade 2. 
• For myositis: in case of management with rituximab, treatment should be discontinued. 
• For Grade 3 or 4 endocrinopathies: withhold until clinically stable or permanently 
discontinue depending on severity. 
• For hepatitis with no tumor involvement of the liver: withhold if total bilirubin 
increases to more than 1.5 and up to 3 times ULN, permanently discontinue if more 
than 3 times ULN. 
• Hepatitis with tumor involvement of the liver: permanently discontinue if total 
bilirubin increases to more than 3 times ULN. 
 
 
Table 12 Management of M7824 mediated Skin Reactions 
Skin reactions are considered important identified risk for M7824. 
• Hyperkeratosis 
• Keratoacanthoma 
• Cutaneous squamous cell carcinoma (cSCC) 
• Basal cell carcinoma 


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• Actinic keratosis 
Management 
• Discontinuation or termination not required in most cases. Continuation of treatment 
should be evaluated by the Investigator. 
• Emollients may be used 
• Develop diagnostic and treatment plan in collaboration with Investigator and 
dermatologist 
• Treatment follow-up will depend on number and localization of lesions. 
o Single lesion: full excision may be recommended 
o Multiple lesion or location not suitable for full excision: Mohrs surgery, 
cryotherapy or other standard treatment options depending on pathology. 
Retinoids may be used after discussion with Investigator. 
• Close clinical follow-up for re-evaluation, resolution and potential recurrence should be 
implemented 
• In general, treatment of skin lesions should be based on local guidelines/standard of 
care. 
Additional consideration: Keratoacanthoma lesions may resolve spontaneously without 
surgical intervention within weeks after discontinuing M7824. 
Consult with Medical Monitor as needed for management of skin lesions. 
 
Table 13 Management of Treatment-Related Anemia 
Anemia is considered an important identified risk for M7824. 
• All relevant hematological testing for treatment-related anemias should be done prior to 
a blood transfusion, if clinically feasible 
Basic Anemia Evaluation 
• CBC with emphasis on red cell indices 
• If indicated and at clinical discretion, the following should be considered: 
o Iron studies 
o Serum Folate and Vit B12 values 
o Coagulation factors 
o Fecal occult blood 
o Urinalysis 
o Hormone panel: TSH, Erythropoietin 
o Peripheral blood smear 
Further Recommendation Based on Suspected Etiology (in Addition to Basic Anemia 
Testing) 
• Suspected Hemolysis 
o bilirubin, LDH, Coombs test, haptoglobin 
• Suspected bleeding:  


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o Consider imaging/interventional radiology consultation as indicated 
o Consider imaging and/or endoscopy as clinically indicated 
• Suspected aplastic anemia: 
o Hematology consultation 
o Consider bone marrow aspiration/morphologic evaluation 
Additional consideration:  
In general, blood transfusions and erythroid growth factors are permitted as clinically 
indicated. 
 
Table 14 Management of Bleeding Adverse Events 
Bleeding Adverse Events 
• Bleeding adverse events are considered important identified risk for M7824. 
• In general, mild and moderate mucosal bleedings resolve without discontinuation of 
treatment. 
• These events may include, but are not limited to the following: 
o Epistaxis 
o Hemoptysis  
o Gingival bleeding 
o Hematuria 
Non-tumor Bleeding 
Grading 
Management 
Grade 2 
• If resolves to Grade ≤ 1 by the day 
before the next infusion, study 
intervention may be continued 
• If not resolved to Grade ≤ 1 by the day 
before the next infusion, but is 
manageable and /or not clinically 
relevant, assessment if clinically 
reasonable to administer the following 
infusion will be per PI discretion. 
Grade 3 
 
• Permanently discontinue treatment 
unless an alternative explanation can be 
identified (such as concomitant use of 
antithrombotic agents, traumatic events, 
etc.)  
• In case of alternative explanations, hold 
study treatment until the event recovers 
to Grade ≤ 1 
Grade 4 
• Treatment must be permanently 
discontinued if no alternative 
explanation is identified. 
Tumor Bleeding 


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Grade ≥ 2 
• Study treatment must be held till the 
event recovers to Grade ≤ 1 
• Permanently discontinue treatment if the 
Investigator considers the participant to 
be at risk for additional severe bleeding. 
 
Table 15 Impaired Wound Healing 
• Impaired wound healing is considered important potential risk for M7824. 
• Elective surgery on study will be allowed per PI discretion. 
• It is recommended to hold study intervention for approximately 4 weeks post major 
surgery for observation. 
Post-operative wound healing should be closely monitored. 
 
 


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3.5 
STUDY CALENDAR 
1 cycle = 21 days
 
Procedure 
Screening 
Cycle 1 
 
 
Cycle 2 
Cycle 3 
Cycle 5 
Cycle 7 
Cycle 4 
Cycle 6 
Cycle 8 
Cycle 9 
And beyond 
End of 
treatment 
visit13 
Safety visit13 
Long term 
follow up14 
Day 1  
Day 8 
Day 15 
Day 1 
 (-1/+3days) 
Day 1  
(-1/+3days) 
Day 1  
(-1/+3days) 
Day 1  
(-1/+3days) 
(≤ 30 days 
from last 
dose) 
(28 days after 
removal from 
study therapy) 
 
Informed consent 
X 
X1 
 
 
 
 
 
 
 
 
 
History and PE2 
X 
X 
(< 3 days) 
X 
 
X 
X 
X 
X 
X 
X 
 
Vital signs15 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
 
Performance Score 
X 
 
 
 
 
 
 
 
 
 
 
Skin assessment16 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
 
Clinical Labs  
X 
X4 (< 7 days) 
X4 
 
X4 
X4 
X4 
X4 
X4 
X 
 
Pathology 
confirmation5 
X 
 
 
 
 
 
 
 
 
 
 
Tumor block 
 
X6 
 
 
 
 
 
 
 
 
 
Tumor biopsy7 
 
 
X 
(< 7 days) 
 
 
 
X 
(Cycle 3 
only) 
 
 
X 
(optional) 
 
 
 
 
Research Blood8  
 
 
See Table below 
Urine or serum 
pregnancy testing in 
women of 
childbearing 
potential9 (2.4.2.2) 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
 
Advance Directives 
Form 
X 
(optional) 
 
 
 
 
 
 
 
 
 
 
Radiological 
Assessments10 
X  
X  
(< 28days) 
 
 
 
X 
(q9 wks) 
 
X 
(q9 wks) 
 
 
X 
Adverse Events 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medications 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
 
Electrocardiogram11 
X 
X  
(< 7 days) 
X 
 
 
 
 
 
 
 
 


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Procedure 
Screening 
Cycle 1 
 
 
Cycle 2 
Cycle 3 
Cycle 5 
Cycle 7 
Cycle 4 
Cycle 6 
Cycle 8 
Cycle 9 
And beyond 
End of 
treatment 
visit13 
Safety visit13 
Long term 
follow up14 
Day 1  
Day 8 
Day 15 
Day 1 
 (-1/+3days) 
Day 1  
(-1/+3days) 
Day 1  
(-1/+3days) 
Day 1  
(-1/+3days) 
(≤ 30 days 
from last 
dose) 
(28 days after 
removal from 
study therapy) 
 
2D Echo every ~84 
days 
Arms 2 and 3 only 
X  
(< 28 
days) 
 
 
 
 
 X every 84 days or 4 cycles  → → →  
 
 
 
MVA-BN-
Brachyury12 
 
X 
 
 
X 
 
 
 
 
 
 
FPV-Brachyury12 
 
 
 
 
 
X 
X 
X 
Every 12 
wks 
 
 
 
Entinostat weekly12 
Arm 3 only 
 
X 
X 
X 
Take on Days 1, 8, and 15 of each cycle.  
Can be taken up to 1 day late. 
 
 
 
T-DM112 
Arms 2 and 3 only 
 
X 
 
 
X 
X 
X 
X 
 
 
 
M782412 
 
X 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
1 Registration must be completed within 24 hours of signing consent. 
2 History including prior treatments and review of baseline adverse events and concurrent medications. Physical examination includes 
breast and lymph node exam. Height (at screening only) and weight are also included. Full history and physical to be performed at 
initial clinical visit. On subsequent visits, abbreviated exam, review of symptoms and review of medications.  
3 CBC with differential, PT/INR/PTT, comprehensive metabolic panel (see 2.4.2.2),  and anti-HIV, anti-HCV, and Hepatitis B Surface 
Ag (within 3 months prior to enrollment). If applicable, CD4 T cell count within 3 months. 
4 Labs must be obtained < 7 days of initiating treatment: CBC with differential, PT/INR/PTT (will be repeated if ≥ 72 hours prior to 
biopsy for Arms 2 and 3), sodium, potassium, chloride, carbon dioxide, BUN, creatinine or measured creatinine clearance, glucose, 
AST, ALT, bilirubin, calcium, total protein, albumin, alkaline phosphatase, phosphorus, magnesium, urinalysis. Troponin-I and CK, 
CK-MB will be evaluated at, C1D1, C1D8, C2D1 and C3D1. Tumor Markers (CEA, CA15-3, CA27.29). TSH and reflexed Free T4 
(if TSH abnormal) will be performed at screening and at restaging visits (approximately every 9 weeks) unless there is a clinical 
concern that warrants evaluation on a more frequent basis.  


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5 Pathological confirmation of diagnosis of either TNBC (Arm 1) or ER-/PR-/HER2+  (Arms 2 and 3). Patients may enroll with a 
pathologist’s report showing a histologic diagnosis of TNBC or HER+BC in a College of American Pathologists (CAP) accredited 
laboratory and a clinical course consistent with the disease. If report not available, disease will be confirmed at the local site.  
6 Primary tumor paraffin block will be requested at C1D1 to perform comparative genomic analysis as part of correlative studies 
planned for this clinical trial. 
7 Planned biopsy after consent but within 7 days prior to first dose of drug on Cohort 3 as well as a planned biopsy on C3D1. There is 
also an optional biopsy at the time of progression on all arms. Patients in Cohorts 1 and 2 may also have optional biopsies at baseline 
and at C3D1.  
8 Where feasible, research blood for all study assessments will be collected as per Table 16. The exact collection time for PK samples 
will be recorded. 
9 For female subjects of childbearing potential, urine or serum beta-HCG will be performed on initial screening. A urine or serum 
beta-HCG will be performed before each administration of the trial drugs during the treatment phase (must be performed on C1D1), at 
the end-of-treatment visit, and at the post-treatment follow-up visit.  
10 Baseline imaging at screening (or within 28 days prior to enrollment): CT of chest/abdomen /pelvis (MRI may be used when CT 
scan is not an option to follow the disease clinically). Brain MRI may be ordered in patients with known CNS disease or if there is 
concern for CNS metastasis. Gadolinium will be used with MRI. Head CT with contrast may be done instead of MRI. Other imaging 
on study: CT of chest/abdomen/pelvis to be repeated every 9 weeks +/- 3 days after start of treatment. Bone scan and additional 
imaging assessments may also be performed as clinically indicated at screening or during the study. 
11 Baseline12-lead EKG at screening (within 28 days prior to enrollment), at baseline, and repeat EKG on Cycle 1 Day 8 with repeats 
only for abnormal baseline EKG, Cycle 1 Day 8 EKG or if patient is symptomatic.  
12 Day 1 Dosing Schedule for each Cycle: BN-Brachyury vaccine is preferred to be given prior to the M7824 infusion, which will be 
followed by T-DM1 administration. Patients will be provided with a take-home supply of entinostat and will self-administer entinostat 
after the completion of the T-DM1 infusion on day 1 of each cycle, preferably prior to bed. On C1D8, patients will take entinostat at 
the time of their clinical appointment. If patients return for the optional C1D15 labs, patients will self-administer entinostat upon 
arrival at the clinical center. Patients will self-administer entinostat on Days 1, 8, and 15 of each subsequent cycle.  
Allowance for scheduling changes: brief interruption and delay in the 21-day cycle may occasionally be required due to travel delays, 
airport closure, inclement weather, family responsibilities, security alerts, and government holidays, etc. This can also extend to 
complications of disease not attributable to disease progression or protocol therapy. These delays will not be considered protocol 
deviation. 
13 End of treatment visit: Where feasible or logistically possible, on the day of or within 30 days of the decision to discontinue treatment 
prematurely before completion of one year of treatment. Does not need to be completed if drug is withheld after one year of treatment. 


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14 Long term follow up: Every 3 months for a year followed by every 6 months for an additional year. During this time: patients who 
have progressed on treatment will be followed by phone or email for survival, adverse events and further tumor therapy. Patients who 
have not progressed on treatment will be scanned until progression. 
15 Vital signs will be monitored every 15 minutes (Cycle 1) and every 30 minutes (Cycle 2 and beyond) during M7824 infusion and 
until completion of the observation period in all Arms. Vital signs should also be recorded immediately prior to the start of T-DM1 in 
Arms 2 and 3. (See 3.2.2 and 3.2.3 for more detail.) 
16 Skin assessment must be performed at baseline, every 6 weeks, and at the end of treatment or at the 28 (±7 days) day safety follow-
up (if not performed in the previous 6 weeks), if feasible.  
 


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Table 16 Research Blood Draws 
 
Week (Day) Time 
M7824 
Entinostat 
Immune and Other Analyses 
  
  
PK ADA 
TGFβ1 levels 
Histone 
Deacetylation 
Immune 
phenotyping, 
Antigen 
specific 
responses, 
immune 
subsets 
T cell 
clonality Soluble 
Factors CfDNA 
Accounted for in the 60-80ml for 
PBMCs 
Cycle 1 Day 1 predose 
X 
X 
X 
X 
X 
X 
X 
X 
  
EOI 
X  
 
 
 
 
 
 
Cycle 1 Day 8   
 
 
X 
X 
X 
X 
X 
 
Cycle 1 
Day15 
Optional   
 
X 
X 
X 
X 
X 
 
Cycle 2 Day 1 predose 
X  
X1 
X1 
X1 
X1 
X1 
 
Cycle 3 Day 1 predose 
X 
X 
X 
 
X 
X 
X 
X 
 
EOI 
X 
 
 
 
 
 
 
 
Cycle 6 Day 1 predose 
X 
X 
 
 
 
 
 
 
End of 
Treatment 
  
X 
X 
 
X 
X 
X 
X 
X 
 
1Cycle 2 Day 1 labs to be drawn only if patient did not do Cycle 1 Day 15 optional labs. Do not 
need both timepoints 
 


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3.6 
COST AND COMPENSATION 
3.6.1 Costs 
Subjects costs will be based on local guidelines as described in the site-specific consent.     
3.6.2 Compensation 
Participants will not receive compensation on this study. 
3.6.3 Reimbursement 
Reimbursement will be provided per local guidelines as indicated in the site consent document.    
3.7 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA 
Regardless of reason for removal from study therapy, patients will be asked to have a 28 day 
follow-up safety visit. Patients who refuse to return for this visit will be asked to review any 
safety concerns by phone within this time period.  
3.7.1 Criteria for removal from protocol therapy 
• Clinical or radiographic progression of disease except when the investigator feels the 
subject is still benefiting from treatment. (It is generally preferable for patients to 
remain on treatment past initial radiographic progression in case there is pseudo -
progression, except when the investigator feels that the clinical picture warrants 
changing therapy at initial progression.); 
• Participant requests to be withdrawn from active therapy; 
• Unacceptable toxicity as defined in Sections 3.1.2 and 3.3; 
• Start of another systemic anticancer treatment or participation in another 
investigational therapeutic trial; 
• Use of restricted medication (Section 4.3); 
• Investigator discretion; 
• Positive pregnancy test;  
• Permanent loss of capacity to give consent. 
3.7.2 Off-Study Criteria 
• Screen failure; 
• PI decision to end the study; 
• Participant requests to be withdrawn from study; 
• Completion of follow up period; 
• Participant lost to follow-up; 
• Investigator discretion; 
• Disease progression; 
• Permanent loss of capacity to give consent; 
• Death. 
3.7.3 Lost to Follow-up 
A participant will be considered lost to follow-up if he or she fails to return for 4 clinic visits 
scheduled visits and is unable to be contacted by the study site staff.  


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The following actions must be taken if a participant fails to return to the clinic for a required 
study visit: 
• The site will attempt to contact the participant and reschedule the missed visit within the 
next 4 weeks and counsel the participant on the importance of maintaining the assigned 
visit schedule and ascertain if the participant wishes to and/or should continue in the 
study. 
• Before a participant is deemed lost to follow-up, the investigator or designee will make 
every effort to regain contact with the participant (where possible, 3 telephone calls and, 
if necessary, a certified letter to the participant’s last known mailing address or local 
equivalent methods). These contact attempts should be documented in the participant’s 
medical record or study file.  
• Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
4 
CONCOMITANT MEDICATIONS / MEASURES 
Any medications (other than those excluded by the clinical trial protocol) that are considered 
necessary to protect subject welfare or alleviate symptoms and will not interfere with the trial 
medications may be given at the Investigator’s discretion. 
4.1 
ANTIEMETICS  
Due to low rates of nausea with M7824, BN-Brachyury vaccine and entinostat, no antiemetic 
prophylaxis will be given. However, if a patient does experience nausea/vomiting, antiemetics 
may be used.  
Since T-DM1 is considered a drug with low emetic risk by both ASCO and NCCN, the use of a 
single dose of a 5-HT3 antagonist or dopamine blocker (i.e., prochlorperazine) is encouraged. 
Due to the effect of CYP3A4 inhibitors on T-DM1, avoid aprepitant, fosaprepitant, and 
netupitant, as well as other CYP3A4 inhibitors. The use of steroids for antiemetic effect is 
allowed per the discretion of the primary investigator. The antiemetic regimen may be modified 
based on patient specific factors or standard institutional practices. 
4.2 
GROWTH FACTOR SUPPORT  
Significant neutropenia is uncommon with single agent T-DM1, M7824 or entinostat. However, 
growth factor support is allowed for grade 3 neutropenia or at the discretion of the primary 
investigator after the completion of the DLT period for each trial arm. If neutropenia or a 
neutropenic fever requiring growth factor support occurs during the DLT period, this will be 
counted as a DLT.  
4.3 
RESTRICTED MEDICATIONS  
The following medications should NOT be administered during the trial: 
• Other immunotherapies or immunosuppressive drugs for example, chemotherapy or 
systemic corticosteroids except for prophylaxis or treatment of allergic reactions, 
endocrine replacement therapy at low dose prednisone [≤ 10 mg daily] or equivalent, for 
the treatment of irAEs, or for short courses (≤ 14 days) as appropriate medical therapy for 


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unrelated medical conditions (e.g., asthma). Steroids with no or minimal systemic effect 
(topical, inhalation) are allowed. 
• Prophylactic use of corticosteroids for infusion related reactions. Corticosteroid 
administration prior to CT scans in patients with intravenous contrast allergy is allowed. 
• Any live vaccine therapies for the prevention of infectious disease. Administration of 
inactivated vaccines is allowed (for example, inactivated influenza vaccines or locally 
approved COVID vaccines). 
• Systemic anticancer treatment other than the investigational agents in this trial. 
• Herbal supplements. 
No formal drug-drug interaction studies with the BN-Brachyury-vaccines, M7824 or entinostat 
have been performed. Use caution when administering entinostat concurrently with medications 
that have a narrow therapeutic index. 
No formal drug-drug interaction studies with T-DM1 have been conducted. In vitro studies 
indicate that DM1, the cytotoxic component of T-DM1, is metabolized mainly by CYP3A4 and 
to a lesser extent by CYP3A5. Concomitant use of strong CYP3A4 inhibitors (e.g., ketoconazole, 
itraconazole, clarithromycin, atazanavir, indinavir, nefazodone, nelfinavir, ritonavir, saquinavir, 
telithromycin, and voriconazole) with T-DM1 should be avoided due to the potential for an 
increase in DM1 exposure and toxicity. Consider an alternate medication with no or minimal 
potential to inhibit CYP3A4. If concomitant use of strong CYP3A4 inhibitors is unavoidable, 
consider delaying T-DM1 treatment until the strong CYP3A4 inhibitors have cleared from the 
circulation (approximately 5 elimination half-lives of the inhibitors) when possible. If a strong 
CYP3A4 inhibitor is co-administered and T-DM1 treatment cannot be delayed, patients should 
be closely monitored for adverse reactions (https://www.accessdata.fda.gov). 
4.4 
CONTRACEPTION 
The effects of BN-Brachyury, entinostat, M7824 and T-DM1 on the developing human fetus are 
unknown. However, both components of T-DM1 are known to cause fetal defects including 
oligohydramnios and oligohydramnios syndromes. For this reason, women of childbearing 
potential must agree to use two methods of adequate contraception (see Table 17) at study entry, 
for the duration of study participation and for at least 7 months for women and 4 months for men 
after the final dose of any study-related medications (T-DM1 has a prolonged half-life). 
Cessation of birth control after this point should be discussed with a responsible physician. 
Periodic abstinence, the rhythm method and the withdrawal method are not acceptable methods 
of birth control. Women with hormone receptor positive breast cancer should avoid hormonal 
contraceptive agents.  
 


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Table 17: Effective methods of contraception (two methods must be used for men and women of 
child-bearing potential) 
Barrier Methods 
Intrauterine Device 
Methods 
Hormonal Methods* 
Male condom plus spermicide 
Copper T 
Implants 
Cap plus spermicide 
Progesterone Ta 
Hormone shot or injection 
Diaphragm plus spermicide 
Levonorgestrel-releasing 
intrauterine system           
(e.g., Mirena) 
Combined pill 
 
 
Minipill 
 
 
Patch 
aThis is also considered a hormonal method of birth control  
*Women with hormone receptor positive breast cancer should avoid hormonal agents; however, 
since all participants in this study will have hormone receptor negative (or low hormone receptor 
positivity) breast cancer, these agents can be used.  
4.5 
BREAST FEEDING 
It is not known whether BN-Brachyury, entinostat, M7824 or T-DM1 is excreted in breast milk. 
It is recommended that women do not breastfeed during treatment on trial and for at least 7 
months after the last dose due to the prolonged half-life of T-DM1 and M7824. 
4.6 
BLOOD DONATION  
Patients should not donate blood while participating in this study or for 7 months following the 
last treatment on trial due to the prolonged half-life of T-DM1 and M7824.  
5 
CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES  
5.1 
BIOSPECIMEN COLLECTION  
Test/assay 
Volume 
(approx.) 
per 
timepoint 
Type of tube 
Collection 
point  
Location of specimen 
analysis* 
(processing/storage 
location) 
M7824 
Pharmacokinetics 
 
4 mL blood 
for serum 
 
 
Serum 
Separator tubes 
(SST) 
 
 
 
 
 
 
 
 
 
 
 
 
 
EMD Serono 
(BPC) 
ADA by ELISA 
 4mL blood 
for serum 
Standard and 123 
immune cell subsets by 
FACS 
 
 
 
 
 
 
 
 
 


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Test/assay 
Volume 
(approx.) 
per 
timepoint 
Type of tube 
Collection 
point  
Location of specimen 
analysis* 
(processing/storage 
location) 
Functional Analysis of 
immune cell subsets by 
FACS 
 
 
 
60-80 mL 
blood for 
PBMCs 
 
 
 
 
 
 
Sodium heparin 
(green top) 
tubes  
 
 
 
 
 
See Section 
3.5 
 
 
LTIB 
(CSP) 
 
Antigen Specific 
Immune Response by 
cytokine staining assay 
Histone acetylation 
T cell clonality by 
immunoSeq platform  
 
NCI Frederick Genomic 
Core Facility, Nanostring 
(CSP) 
RNA expression by 
Nanostring 
 
Soluble Factors by 
ELISA  
 
8 mL blood 
for serum 
Serum 
Separator tubes 
(SST) 
LTIB 
(CSP) 
TGFβ1-3 levels by 
ELISA or bead based 
multiplex assays 
6mL blood 
for plasma 
EDTA 
(lavender top) 
tubes 
 
LTIB, Dr. Liang Cao’s Lab 
(CSP) 
Circulating free tumor 
DNA (cfDNA) by PCR 
system 
10 mL 
blood for 
plasma 
EDTA 
(lavender top) 
tubes 
Dr. Liang Cao’s Lab 
(CSP) 
Immune Markers by 
IHC 
Tumor 
samples 
N/A 
NCI – GMB TIME Lab 
(LP) 
RNA expression by 
Nanostring 
Tumor 
samples 
N/A 
NCI Frederick Genomic 
Core Facility, Nanostring 
(LP) 
T cell clonality by 
immunoSeq platform 
Tumor 
samples 
N/A 
LTIB and NCI Frederick 
Genomic Core Facility 
(LP) 
*Research samples will be sent to the Clinical Services Program – Leidos Biomedical Research, 
Inc. (CSP) (Section 5.2.1), the Blood Processing Core (BPC) (Section 5.2.2), or Laboratory of 
Pathology (LP) (Section 5.2.3) for barcoding, initial processing and storage. From these 
facilities, coded, linked samples will be sent to the designated labs for analysis in batch 
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5.1.1 Peripheral Blood Collection and Assays  
The following tests may be performed on selected patients’ peripheral blood samples if there are 
adequate samples. If collected samples are not sufficient, the correlative studies will be 
performed in order of priority as detailed below. These correlative studies will be done in an 
exploratory fashion. The amount of blood that may be drawn from adult patients and volunteers 
(i.e., those persons 18 years of age or older) for research purposes shall not exceed 10.5 mL/kg 
or 550 mL, whichever is smaller, over any eight week period.  
Pharmacokinetics  
To evaluate M7824 PK, 4ml of serum will be collected as per Section 3.5. Serum samples will be 
analyzed by a validated immunoassay to quantitate M7824 concentration. Samples may be 
further tested in qualified or validated PK characterization assays. Coded, linked samples will be 
shipped to EMD Serono for analysis. 
ADA 
To evaluate ADA (anti-drug antibody), 4ml of blood will be collected as per Section 5.1.1. 
Serum samples will be analyzed by a validated electrochemiluminescence immunoassay (ECLA) 
to detect the presence of anti-M7824. Samples that screen positive will be subsequently tested in 
a confirmatory assay. Those confirmed positive will be titered for a quasi-quantitative result. 
Samples may be further tested in qualified or validated immunogenicity characterization 
methods. The investigation will be done by EMD Serono using ELISA. Coded, linked samples 
will be shipped to EMD Serono for analysis. 
Immune Phenotyping 
Exploratory immunologic studies will be conducted to evaluate the study drug’s effect on the 
immune response before and after treatment, to gain insight into potential biomarkers, and help 
improve the administered therapy. Blood will be collected as per Section 5.1.1. The following 
immune assays may be performed at the Laboratory of Tumor Immunology and Biology (LTIB) 
at the NCI’s Center for Cancer Research (CCR) in select patients where adequate samples are 
available: 
1. Pre- and post-therapy PBMCs, separated by Ficoll-Hypaque density gradient separation, 
will be analyzed for antigen-specific immune responses using an intracellular cytokine 
staining assay. PBMCs will be stimulated in vitro with overlapping 15-mer peptide pools 
encoding the tumor-associated antigen Brachyury; if sufficient PBMCs are available, the 
cascade antigens CEA, and MUC1 will be included. Control peptide pools will involve the 
use of human leukocyte antigen peptide as a negative control and CEFT peptide mix as a 
positive control. CEFT is a mixture of peptides of CMV, Epstein-Barr virus, influenza, and 
tetanus toxin. Post-stimulation analyses of CD4 and CD8 T cells will involve the 
production of IFN-γ, IL-2, tumor necrosis factor, and CD107a. If sufficient PBMCs are 
available, assays may also be performed for the development of T cells to other tumor-
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2. If sufficient PBMCs are available, PBMCs from selected subjects may be analyzed for 
changes in standard immune cell types (CD4 and CD8 T cells, NK cells, Tregs, myeloid-
derived suppressor cells [MDSCs], and dendritic cells) as well as 123 immune cell subsets, 
as described elsewhere (120) and for function of specific immune cell subsets, including 
CD4 and CD8 T cells, NK cells, Tregs, and MDSCs. 
Analyses of soluble factors: 
1. Sera may be analyzed pre- and post-therapy for the following soluble factors: sCD27, 
sCD40 ligand, and selected patients may be analyzed for cytokines and antibodies to 
human tumor antigens such as Brachyury, CEA, or MUC1.  
Additional assays:  
Blood samples may be used for additional research studies, which may include phenotypic and 
functional analysis of immune-cell subsets, TCR clonality, and analyses for cytokines (IFN-γ, IL-
10, IL-12, IL-2, IL-4, etc.), chemokines, antibodies, tumor-associated antigens, and/or other 
markers.  
Plasma analysis for TGFβ1 levels and circulating free DNA: 
Plasma may be analyzed pre- and post-therapy in Dr. Liang Cao’s lab for TGFβ1-3 levels using 
ELISA or bead based multiplex assays and circulating free tumor DNA using PCR-based 
techniques. 
Exploratory pharmacodynamics for entinostat (Arm 3 only): 
1. Histone acetylation has been evaluated as a surrogate for entinostat pharmacodynamics. 
Protein lysine acetylation will be measured by multiparameter flow cytometry in PBMCs 
(CD19 B-cells, CD3T-cells, and CD14 monocytes) to explore the association with PFS. 
(121) This will be collected at baseline, Cycle 1 Day 8 (within 4-6 hours of taking 
entinostat) and Cycle 1 Day 15 (optional; within 4-6 hours of taking entinostat). This will 
be performed on PBMCs collected for the immune analyses. No additional blood collection 
is needed.  
5.1.2 Tumor Biopsy Collection and Assays 
Biopsy Acquisition  
Biopsies will be performed as per the Study Calendar 3.5. Attempts will be made to obtain up to 
four cores (18G or 20G preferred) if safe and feasible. These tumor core biopsies will be 
obtained percutaneously by interventional radiology as long as considered minimal surgical risk. 
Two 3-millimeter punch biopsies of skin will be acceptable in lieu of 18-gauge core biopsies for 
patients with skin involvement. Inability to get tissue with a reasonable attempt will not preclude 
treatment and the patient will remain eligible for all other translational components. 
• Cores 1 and 2: Formalin-Fixed Paraffin-Embedded (FFPE)  
• Cores 3 and 4: Fresh frozen  
Tissue samples will be sent to Laboratory of Pathology for disease evaluation first, remaining 
samples will be processed and stored for research (Section 5.2.3). If fresh-frozen cores are 
obtained, they will be processed and stored in CSP (Section 5.2.1). See site specific instructions 
for collection shipping procedures.  


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Immune Markers by Immunohistochemistry 
Sections of formalin-fixed, paraffin-embedded (FFPE) tissues from before and after therapy may 
be stained using antibodies for multiple immune cell and tumor cell markers (e.g. CD4, CD8, 
FOXP3, PD-1, PD-L1) and analyzed using the Vectra Polaris platform/multispectral imaging in 
selected patients where tumor samples are adequate.  
HER2/Brachyury Expression 
HER2 testing may be performed and if so, will be performed according to the recommendations 
made by the College of American Pathologists (CAP). (122) FFPE samples may be evaluated for 
brachyury expression by using a rabbit monoclonal anti-brachyury antibody MAb 54-1 at a 
1:500-1:2000 dilution, as previously described. (123, 124) Nuclear, cytoplasmic and total 
brachyury staining will be independently scored, with brachyury being observed either in the 
nucleus, the cytosol or both compartments of the tumor cells. The relative staining intensity will 
be scored as weak (+) for pale intensity, moderate (++) for intermediate intensity, and strong 
(+++) for intense, dark immunoprecipitate. Immunoreactivity index will be calculated by 
multiplying the percentage of positive cells by the staining intensity. 
5.1.3 Genetic Assays 
T-cell Clonality 
DNA may be extracted from cryopreserved PBMC or tumor tissue. TCR Vb CDR3 sequencing 
will be performed at the CCR Genomics Core using the Immunoseq kit from Adaptive 
Biotechnologies at the survey (tumor) or deep (PBMC) resolution. In this assay, a multiplex PCR 
system amplifies and quantifies the rearranged CDR3b sequences from sample DNA.  
RNA Expression 
RNA may be extracted from cryopreserved PBMC or tumor tissue. Sequencing of select gene 
panels may be performed at the CCR Genomics Core in collaboration with Nanostring. 
5.2 
SAMPLE STORAGE, TRACKING, AND DISPOSITION 
See site specific instructions for collection and shipping procedures for research blood samples.  
5.2.1 Sample Management and Storage at Clinical Services Program – Leidos Biomedical 
Research, Inc. (CSP) 
All data associated with the patient samples is protected by using a secure database. All Clinical 
Support Laboratory Staff receive annual training in maintaining records’ confidentiality. All 
samples drawn at the NIH Clinical Center will be transported to the Clinical Support Laboratory 
at the Frederick National Laboratory for Cancer Research by couriers. 
Samples will be tracked and managed by Central Repository database, where there is no link to 
personal identifiable information. All samples will be stored in either a -80ºC freezer or vapor 
phase liquid nitrogen. These freezers are located at NCI Frederick Central Repository in Frederick, 
Maryland. 
NCI Frederick Central Repositories (managed under a subcontract) store, among other things, 
biological specimens in support of NIH clinical studies. All specimens are stored in secure, 
limited-access facilities with sufficient security, backup, and emergency support capability and 
monitoring to ensure long-term integrity of the specimens for research.  


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Specimens are stored in accordance with applicable HHS and FDA Protection of Human Subjects 
Regulations in accordance with the subcontractor’s Federal-wide Assurance.  The subcontractor’s 
role limited to clinical research databases and repositories containing patient specimens. The 
subcontractor does not conduct or have any vested interest in research on human subjects, but does 
provide services and support the efforts of its customers, many of which are involved in research 
on human subjects.  
It is the intent and purpose of the subcontractor to accept only coded, linked samples and sample 
information. To the limit of our ability, every effort will be made to ensure that protected 
information is not sent electronically or by hard copy or on vial labels.  
Sample data is stored in the BioSpecimen Inventory System II (BSI). This inventory tracking 
system is used to manage the storage and retrieval of specimens as well as to maintain specimen 
data. BSI is designed for controlled, concurrent access. It provides a real-time, multi-user 
environment for tracking millions of specimens. The system controls how and in what order 
database updates and searches are performed. This control prevents deadlocks and race conditions. 
For security, BSI has user password access, 3 types of user access levels, and 36 user permissions 
(levels of access) that can be set to control access to the system functions. BSI provides audit 
tracking for processes that are done to specimens including shipping, returning to inventory, 
aliquoting, thawing, additives, and other processes. BSI tracks the ancestry of specimens as they 
are aliquoted, as well as discrepancies and discrepancy resolution for specimens received by the 
repository. If a specimen goes out of the inventory, the system maintains data associated with the 
withdrawal request. Vials are labeled with a unique BSI ID which is printed in both eye-readable 
and bar-coded format. No patient-specific information is encoded in this ID.  
Investigators are granted view, input, and withdrawal authority only for their specimens. They 
may not view specimen data or access specimens for which they have not been authorized. 
Access to specimen storage is confined to repository staff. Visitors to the repositories are 
escorted by repository staff at all times. 
5.2.2 Samples Managed by Dr. Figg’s Blood Processing Core (BPC)  
 
5.2.2.2 Sample Data Collection  
All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and 
stored in the Labmatrix utilized by the BPC. This is a secure program, with access to Labmatrix 
limited to defined Figg lab personnel, who are issued individual user accounts. Installation of 
Labmatrix is limited to computers specified by Dr. Figg. These computers all have a password 
restricted login screen.  
Labmatrix creates a unique barcode ID for every sample and sample box, which cannot be traced 
back to patients without Labmatrix access. The data recorded for each sample includes the 
patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, 
as well as box and freezer location. Patient demographics associated with the clinical center 
patient number are provided in the system. For each sample, there are notes associated with the 
processing method (delay in sample processing, storage conditions on the ward, etc.).  
5.2.2.3 Sample Storage  


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Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80 C 
according to stability requirements. These freezers are located onsite in the BPC and offsite at 
NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are 
required to be accompanied by laboratory staff at all times. Access to stored clinical samples is 
restricted.  
Samples will be stored until requested by a researcher named on the protocol. All requests are 
monitored and tracked in Labmatrix. All researchers are required to sign a form stating that the 
samples are only to be used for research purposes associated with this trial (as per the IRB 
approved protocol) and that any unused samples must be returned to the BPC. It is the 
responsibility of the NCI Principal Investigator to ensure that the samples requested are being 
used in a manner consistent with IRB approval.  
5.2.3 Procedures for Storage of Tissue Specimens in the Laboratory of Pathology 
Tissues designated for clinical diagnostics are transported to the Laboratory of Pathology (LP) 
where they are examined grossly and relevant portions are fixed, embedded in paraffin and 
sectioned and stained for diagnostic interpretation. Following completion of the diagnostic 
workup, the slides and tissue blocks are stored indefinitely in the LP’s clinical archives. All 
specimens are catalogued and retrieved utilizing the clinical laboratory information systems, in 
accordance with CAP/JCAHO regulations. The use of any stored specimens for research 
purposes is only allowed when the appropriate IRB approval has been obtained. 
Specimens/recuts can then be requested with the permission of the appropriate Principal 
Investigator using the Tissue Resource Committee request for Human Biological Materials for 
Research form. 
5.2.4 Protocol Completion/Sample Destruction 
All specimens obtained in the protocol are used as defined in the protocol. Any specimens that 
are 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 if a subject 
withdraws consent for their continued use, at which time they will be destroyed. If the patient 
withdraws consent the participant’s data will be excluded from future distributions, but data that 
have already been distributed for approved research use will not be able to be retrieved. 
 
The site PI will record any loss or unanticipated destruction of samples as a deviation. Reports 
will be made per the requirements of Section 7.2. 
6 
DATA COLLECTION AND EVALUATION  
6.1 
DATA COLLECTION 
The CCR PI and all site PIs will be responsible for data collection. Site PIs will be trained on use 
of the CCR remote data capture system C3D. The CCR PI and the site PIs will be responsible for 
overseeing entry of data into an in-house password protected electronic system (C3D) and 
ensuring data accuracy, consistency and timeliness. The principal investigator, associate 
investigators/research nurses and/or a contracted data manager will assist with the data 
management efforts. Primary and final analyzed data will have identifiers so that research data 
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All adverse events, including clinically significant abnormal findings on laboratory evaluations, 
regardless of severity, will be followed until return to baseline or stabilization of event.  
Document AEs from the first study intervention, Study Day 1, through 28 days after the study 
intervention was last administered. Beyond 28 days after the last intervention, only adverse 
events which are serious and related to the study intervention need to be recorded. 
An abnormal laboratory value will be recorded in the database as an AE only if the laboratory 
abnormality is characterized by any of the following: 
• 
Results in discontinuation from the study. 
• 
Is associated with clinical signs or symptoms.  
• 
Requires treatment or any other therapeutic intervention. 
• 
Is associated with death or another serious adverse event, including hospitalization.  
• 
Is judged by the Investigator to be of significant clinical impact. 
• 
If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome. 
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH 
Intramural Records Retention Schedule as applicable.  
Loss or destruction of data: Should we become aware that a major breach in our plan to protect 
subject confidentiality and trial data has occurred, this will be reported expeditiously per Section 
7.2.1.  
6.2 
DATA SHARING PLANS 
6.2.1 Human Data Sharing Plan 
What data will be shared? 
I will share human data generated in this research for future research as follows: 
• Coded, linked data in an NIH-funded or approved public repository.  
• Coded, linked data in BTRIS (automatic for activities in the Clinical Center) 
• Identified or coded, linked data with approved outside collaborators under appropriate 
agreements. 
How and where will the data be shared?  
Data will be shared through: 
• An NIH-funded or approved public repository.  Clinicaltrials.gov  
• BTRIS (automatic for activities in the Clinical Center). 
• Approved outside collaborators under appropriate individual agreements. 
• Publication and/or public presentations. 
When will the data be shared?  
• Before publication. 


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• At the time of publication or shortly thereafter. 
6.2.2 Genomic Data Sharing Plan 
Unlinked genomic data will be deposited in public genomic databases such as dbGaP in 
compliance with the NIH Genomic Data Sharing Policy. 
6.3 
RESPONSE CRITERIA 
6.3.1 Antitumor Response 
For the purposes of this study, patients should be re-evaluated for response every 9 weeks +/- 3 
days. In addition to a baseline scan, confirmatory scans can also be obtained 3 to 6 weeks 
following initial documentation of objective response per the discretion of the primary 
investigator. 
At baseline, tumor lesions will be selected and categorized as target or non-target lesions. Target 
lesions include those lesions that can be accurately measured in at least 1 dimension as ≥ 20 mm 
with conventional techniques or ≥ 10 mm with CT scan. Malignant lymph nodes with a short axis 
diameter ≥ 15 mm can be considered target lesions. Up to a maximum of 2 target lesions per organ 
and 5 target lesions in total will be identified at baseline. These lesions should be representative of 
all involved organs and selected based on their size (those with the longest diameter) and their 
suitability for accurate repeated measurements. A sum of the longest lesion diameter (LLD) for all 
target lesions will be calculated and reported as the baseline sum LLD. For malignant lymph nodes 
identified as target lesions, the short axis diameter will be used in the sum of LLD calculation. All 
other lesions (or sites of disease) should be identified as non-target lesions (including bone lesions). 
All post-baseline response assessments should follow the same lesions identified at baseline. The 
same mode of assessment (e.g., CT) used to identify/evaluate lesions at baseline should be used 
throughout the course of the study unless subject safety necessitates a change (e.g., allergic reaction 
to contrast media). 
For the primary endpoint antitumor activity will be evaluated with target and/or non-target 
lesions according to the revised Response Evaluation Criteria in Solid Tumors (RECIST) 
guideline (Version 1.1). (125) 
6.3.2 Disease Parameters 
Measurable disease:  Measurable lesions are defined as those that can be accurately measured in 
at least one dimension (longest diameter to be recorded) as: 
• By chest x-ray: >20 mm; 
• By CT scan: 
o Scan slice thickness 5mm or under: as >10 mm; 
o Scan slice thickness ˃5mm: double the slice thickness 
• With calipers on clinical exam: >10 mm.   
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). 
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a lymph 
node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness 
recommended to be no greater than 5 mm).  At baseline and in follow-up, only the short axis will 
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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 as non-measurable. 
Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts should not 
be considered as malignant lesions (neither measurable nor non-measurable) since they are, by 
definition, simple cysts. 
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, 
if they meet the definition of measurability described above. However, if non-cystic lesions are 
present in the same patient, these are preferred for selection as target lesions. 
Target lesions.  All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in 
total, representative of all involved organs, should be identified as target lesions and recorded 
and measured at baseline.  Target lesions should be selected on the basis of their size (lesions 
with the longest diameter), be representative of all involved organs, but in addition should be 
those that lend themselves to reproducible repeated measurements.  It may be the case that, on 
occasion, the largest lesion does not lend itself to reproducible measurement in which 
circumstance the next largest lesion which can be measured reproducibly should be selected.  A 
sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target 
lesions will be calculated and reported as the baseline sum diameters.  If lymph nodes are to be 
included in the sum, then only the short axis is added into the sum.  The baseline sum diameters 
will be used as reference to further characterize any objective tumor regression in the measurable 
dimension of the disease. 
Non-target lesions.  All other lesions (or sites of disease) including any measurable lesions over 
and above the 5 target lesions should be identified as non-target lesions and should also be 
recorded at baseline.  Measurements of these lesions are not required, but the presence, absence, 
or in rare cases unequivocal progression of each should be noted throughout follow-up.  
6.3.3 Methods for Evaluation of Measurable Disease 
All measurements should be taken and recorded in metric notation using a ruler or calipers.  All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 4 weeks before the beginning of the treatment. 
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam. 
Clinical lesions:  Clinical lesions will only be considered measurable when they are superficial 
(e.g., skin nodules and palpable lymph nodes) and 10 mm diameter as assessed using calipers 
(e.g., skin nodules).  In the case of skin lesions, documentation by color photography, including a 
ruler to estimate the size of the lesion, is recommended.  


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Chest x-ray:  Lesions on chest x-ray are acceptable as measurable lesions when they are clearly 
defined and surrounded by aerated lung.  However, CT is preferable.  
Conventional CT and MRI:  This guideline has defined measurability of lesions on CT scan 
based on the assumption that CT slice thickness is 5 mm or less.  If CT scans have slice thickness 
greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness.  
MRI is also acceptable in certain situations (e.g. for body scans).   
Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which greatly 
impact image quality, lesion conspicuity, and measurement.  Furthermore, the availability of 
MRI is variable globally.  As with CT, if an MRI is performed, the technical specifications of the 
scanning sequences used should be optimized for the evaluation of the type and site of disease.  
Furthermore, as with CT, the modality used at follow-up should be the same as was used at 
baseline and the lesions should be measured/assessed on the same pulse sequence.  It is beyond 
the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all 
scanners, body parts, and diseases.  Ideally, the same type of scanner should be used and the 
image acquisition protocol should be followed as closely as possible to prior scans.  Body scans 
should be performed with breath-hold scanning techniques, if possible. 
PET-CT:  At present, the low dose or attenuation correction CT portion of a combined PET-CT 
is not always of optimal diagnostic CT quality for use with RECIST measurements.  However, if 
the site can document that the CT performed as part of a PET-CT is of identical diagnostic 
quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be 
used for RECIST measurements and can be used interchangeably with conventional CT in 
accurately measuring cancer lesions over time.  Note, however, that the PET portion of the CT 
introduces additional data which may bias an investigator if it is not routinely or serially 
performed.   
Ultrasound:  Ultrasound is not useful in assessment of lesion size and should not be used as a 
method of measurement.   
Tumor markers:  Tumor markers alone cannot be used to assess response.   
Cytology, Histology:  These techniques can be used to differentiate between partial responses 
(PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as 
germ cell tumors, where known residual benign tumors can remain). 
The cytological confirmation of the neoplastic origin of any effusion that appears or worsens 
during treatment when the measurable tumor has met criteria for response or stable disease is 
mandatory to differentiate between response or stable disease (an effusion may be a side effect of 
the treatment) and progressive disease. 
FDG-PET:  While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis of 
FDG-PET imaging can be identified according to the following algorithm:  


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


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Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor 
marker level above the normal limits. 
Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal 
progression of existing non-target lesions.  Unequivocal progression should not normally trump 
target lesion status. It must be representative of overall disease status change, not a single lesion 
increase.     
Although a clear progression of “non-target” lesions only is exceptional, the opinion of the 
treating physician should prevail in such circumstances, and the progression status should be 
confirmed at a later time by the review panel (or Principal Investigator). 
6.3.4.3 Evaluation of Best Overall Response 
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started).  The patient's best response assignment will 
depend on the achievement of both measurement and confirmation criteria. 
For Patients with Measurable Disease (i.e., Target Disease) 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
> 3 to 6 wks. Confirmation** 
CR 
Non-
CR/Non-PD 
No 
PR 
>3 to 6 wks. Confirmation** 
CR 
Not 
evaluated 
No 
PR 
PR 
Non-
CR/Non-
PD/not 
evaluated 
No 
PR 
SD 
Non-
CR/Non-
PD/not 
evaluated 
No 
SD 
Documented at least once >4 
wks. from baseline** 
PD 
Any 
Yes or 
No 
PD 
no prior SD, PR or CR 
Any 
PD*** 
Yes or 
No 
PD 
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. 


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Note: Patients with a global deterioration of health status requiring 
discontinuation of treatment without objective evidence of disease 
progression at that time should be reported as “symptomatic 
deterioration.”  Every effort should be made to document the objective 
progression even after discontinuation of treatment. 
As an exploratory endpoint antitumor activity will also be evaluated according to iRECIST 
(REF). Using iRECIST criteria the following will be incorporated into the assessment: 
• An increase in the sum of target lesions of more than 20%, unequivocal increase in the 
non-target lesions or new lesions result in iUPD (unconfirmed progression disease); 
iUPD can be assigned multiple ties as long as iCPD (confirmed progressive disease) is 
not confirmed at the next assessment.  
•  Progression is confirmed in the target lesion category if the next imaging assessment 
after iUPD (4-8 weeks later) confirms a further increase in sum of measures of target 
disease from iUPD, with an increase of at least 5mm. Progression is confirmed in the 
non-target lesion category if subsequent imaging, done every 4-8 weeks after iUPD 
shows a further unequivocal increase in non-target lesions. Progression is confirmed in 
the new lesions category if at next assessment additional new lesions appear or an 
increase in the size of previously seen new lesions is seen (>5 mm for sum of new target 
lesion).  
• However, the criteria for iCPD (after iUPD) are not considered to have been met if 
complete response, partial response or stable disease criteria (compared with baseline and 
as defined by RECIST 1.1) are met at the next assessment after iUPD. The status is reset 
(unlike RECIST 1.1, in which any progression precludes later complete response, partial 
response, or stable disease). iCR, iPR, or iSD should then be assigned; and if no change is 
detected, then the timepoint response is iUPD. 
 
For Patients with Non-Measurable Disease (i.e., Non-Target Disease) 
 
6.3.4.4 Duration of Response 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first date that 
recurrent or progressive disease is objectively documented (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started). 
The duration of overall CR is measured from the time measurement criteria are first met for CR 
until the first date that progressive disease is objectively documented. 
 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
*  ‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease 
since SD is increasingly used as an endpoint for assessment of efficacy in some 
trials so to assign this category when no lesions can be measured is not advised 


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Duration of stable disease:  Stable disease is measured from the start of the treatment until the 
criteria for progression are met, taking as reference the smallest measurements recorded since the 
treatment started, including the baseline measurements.  
6.3.4.5 Progression-Free Survival 
Progression Free Survival (PFS) is defined as the duration of time from start of treatment to time 
of progression or death, whichever occurs first. In the absence of progression or death, PFS is 
censored at the date of last disease evaluation 
6.3.5 Response Review 
All imaging studies will be reviewed by an independent radiologist and will be assess according 
to RECIST 1.1. 
6.4 
TOXICITY CRITERIA 
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on the study.  The descriptions and grading scales found in the revised NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE 
reporting.  All appropriate treatment areas should have access to a copy of the CTCAE version 
5.0.  A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc).  
7 
NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN 
7.1 
DEFINITIONS 
Please refer to definitions provided in Policy 801: Reporting Research Events found at: 
https://irbo.nih.gov/confluence/display/IRBO/Policies+and+SOPs. 
7.2 
OHSRP OFFICE OF COMPLIANCE AND TRAINING / IRB REPORTING 
7.2.1 Expedited Reporting 
Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 
802 Non-Compliance Human Subjects Research found at: 
https://irbo.nih.gov/confluence/display/IRBO/Policies+and+SOPs. 
Note: Only IND Safety Reports that meet the definition of an unanticipated problem will need to 
be reported per these policies. 
7.2.2 IRB Requirements for PI Reporting at Continuing Review 
Please refer to the reporting requirements in Policy 801: Reporting Research Events found at: 
https://irbo.nih.gov/confluence/display/IRBO/Policies+and+SOPs.   
7.3 
NCI GUIDANCE FOR REPORTING EXPEDITED ADVERSE EVENTS FOR MULTI-CENTER 
TRIALS 
Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 
802: Non-compliance in Human Subjects Research, found here. Until such time as direct 
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report to the coordinating center PI any deaths possibly related to the research within 24 hours of 
PI awareness of the event. The Site PI must also report any other events required by Policy 801 
to the coordinating center PI within 7 days of PI awareness. 
The participating site Reportable Event Form is attached as Appendix E: CCR Reportable Event 
Form (REF). 
Once direct electronic reporting mechanisms are available, these will be utilized. Please also 
notify the coordinating center PI and study coordinator of your submission at the time you make 
it. 
For IND studies, the site PI will also directly submit reports to the CCR as IND sponsor per 
Section 8.3, 8.6 and 8.8. 
7.4 
NIH REQUIRED DATA AND SAFETY MONITORING PLAN    
7.4.1 Principal Investigator/Research Team  
The clinical research team will meet on a regular basis (approximately weekly) 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. Events meeting requirements for expedited reporting as described in 
Section 7.2.1 will be submitted within the appropriate timeline.  
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.  
7.4.2 Safety Monitoring Committee (SMC) 
This protocol will be periodically reviewed by an intramural Safety Monitoring Committee 
comprised of physicians, biostatisticians and a lay member selected based on experience, area of 
expertise, reputation for objectivity, absence of conflicts of interest and knowledge of or 
experience with clinical trial research. Initial review will occur as soon as possible after the 
annual NIH Intramural 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 based on the risks presented in the study. For initial and subsequent 
reviews, protocols will not be reviewed if there is no accrual within the review period.  
The SMC will operate under the rules of an approved charter that will be written and reviewed at 
the organization meeting of the SMC. Each review will focus on unexpected protocol safety 
issues that are identified during the conduct of the clinical trial.  
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 
SPONSOR PROTOCOL SAFETY REPORTING 
8.1 
DEFINITIONS 
8.1.1 Adverse Event  
Any untoward medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally associated with the 
use of a medicinal (investigational) product, whether or not related to the medicinal 
(investigational) product (ICH E6 (R2)). 
8.1.2 Serious Adverse Event (SAE) 
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 event (see Section 8.1.3); 
• 
Inpatient hospitalization or prolongation of existing hospitalization; 
o A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery 
arranged prior to the start of the study), a planned hospitalization for pre-existing 
condition, or a procedure required by the protocol, without a serious deterioration in 
health, is not considered a serious adverse event. 
o A hospitalization/admission that is solely driven by non-medical reasons (e.g., 
hospitalization for patient or subject convenience) is not considered a serious adverse 
event.  
o Emergency room visits or stays in observation units that do not result in admission to 
the hospital would not be considered a serious adverse event. The reason for seeking 
medical care should be evaluated for meeting one of the other serious criteria. 
• 
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. 
8.1.3 Life-threatening  
An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of 
either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of 
death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a 
more severe form, might have caused death (21CFR312.32). 


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8.1.4 Severity 
The severity of each Adverse Event will be assessed utilizing the CTCAE version 5.0. 
8.1.5 Relationship to Study Product 
All AEs will have their relationship to study product assessed using the terms:  related or not 
related.   
• Related – There is a reasonable possibility that the study product caused the adverse 
event. Reasonable possibility means that there is evidence to suggest a causal relationship 
between the study product and the adverse event. 
• Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event. 
8.1.6 Adverse Events of Special Interest (AESI) 
Adverse events of special interest (AESIs) are serious or nonserious AEs that are of clinical 
interest and should be closely followed. 
AESIs include following: 
• Infusion-related reactions including immediate hypersensitivity. 
• Immune-related adverse events. 
• TGFβ inhibition/M7824 mediated skin reactions. 
• Anemia. 
• Bleeding AEs. 
8.2 
ASSESSMENT OF SAFETY EVENTS 
AE information collected will include event description, date of onset, assessment of severity 
and relationship to study product and alternate etiology (if not related to study product), date of 
resolution of the event, seriousness and outcome. The assessment of severity and relationship to 
the study product will be done only by those with the training and authority to make a diagnosis 
and listed on the Form FDA 1572 as the site principal investigator or sub-investigator. AEs 
occurring during the collection and reporting period will be documented appropriately regardless 
of relationship. AEs will be followed through resolution. 
SAEs will be: 
• 
Assessed for severity and relationship to study product and alternate etiology (if not 
related to study product) by a licensed study physician listed on the Form FDA 1572 as 
the site principal investigator or sub-investigator. 
• 
Recorded on the appropriate SAE report form, the medical record and captured in the 
clinical database. 
• 
Followed through resolution by a licensed study physician listed on the Form FDA 1572 
as the site principal investigator or sub-investigator. 
For timeframe of recording adverse events, please refer to Section 6.1. All serious adverse events 
recorded from the time of first investigational product administration must be reported to the 
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8.3 
REPORTING OF SERIOUS ADVERSE EVENTS 
Any AE that meets a protocol-defined serious criteria or meets the definition of Adverse Event 
of Special Interest that require expedited reporting must be submitted immediately (within 24 
hours of awareness) to OSRO Safety using the CCR SAE report form. Any exceptions to the 
expedited reporting requirements are found in Section 8.4 
All SAE reporting must include the elements described in Section 8.1.6. 
SAE reports will be submitted to the Center for Cancer Research (CCR) at: 
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. CCR SAE report form and 
instructions can be found at: 
https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions  
Following the assessment of the SAE by OSRO, other supporting documentation of the event 
may be requested by the OSRO Safety and should be provided as soon as possible. 
8.4 
WAIVER OF EXPEDITED REPORTING TO CCR 
As death due to disease progression is part of the study objectives, and captured as an endpoint in 
this study, they will not be reported in expedited manner to the sponsor. However, if there is 
evidence suggesting a causal relationship between the study drug and the event, report the event 
in an expedited manner according to Section 8.3. 
8.5 
SAFETY REPORTING CRITERIA TO THE PHARMACEUTICAL COLLABORATORS 
Reporting will be per the collaborative agreement.  
8.6 
REPORTING PREGNANCY  
All required pregnancy reports/follow-up  to OSRO will be submitted to: 
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. Forms and instructions 
can be found here: 
https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions 
8.6.1 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 no later than 24 hours of 
when the Investigator becomes aware of it. The Investigator should notify the Sponsor no later 
than 24 hours of when the outcome of the Pregnancy become known. Pregnancy itself is not 
regarded as an SAE. However, congenital abnormalities or birth defects and spontaneous 
miscarriages that meet serious criteria (Section 8.1.2) should be reported as SAEs.  
The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic 
pregnancy, normal birth, or congenital abnormality) should be followed up and documented. 
8.6.2 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 study drugs. 
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 


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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. 
8.7 
REGULATORY REPORTING FOR STUDIES CONDUCTED UNDER CCR-SPONSORED IND 
Following notification from the investigator, CCR, the IND sponsor, will report any suspected 
adverse reaction that is both serious and unexpected in expedited manner to the FDA in 
accordance to 21 CFR 31.2.32. CCR will report an AE as a suspected adverse reaction only if 
there is evidence to suggest a causal relationship between the study product and the adverse 
event. CCR will notify FDA and all participating investigators (i.e., all investigators to whom the 
sponsor is providing drug under its INDs or under any investigator’s IND) in an IND safety 
report of potential serious risks from clinical trials or any other source, as soon as possible, in 
accordance to 21 CFR Part 312.32.  
All serious events will be reported to the FDA at least annually in a summary format. 
8.8 
SPONSOR PROTOCOL NON-ADHERENCE REPORTING 
Protocol non-adherence is defined as any noncompliance with the clinical trial protocol, GCP, or 
protocol-specific procedural requirements on the part of the participant, the Investigator, or the 
study site staff inclusive of site personnel performing procedures or providing services in support 
of the clinical trial. 
It is the responsibility of the study Staff to document any protocol non-adherence identified by 
the Staff or the site Monitor on the OSRO Site Protocol Non-Adherence Log.  The protocol-
specific, cumulative non-adherence log should be maintained in the site essential documents file 
and submitted to OSRO via OSROMonitoring@mail.NIH.gov on the first business day of each 
month over the duration of the study. In addition, any non-adherence to the protocol should be 
documented in the participant’s source records and reported to the local IRB per their guidelines. 
OSRO required protocol non-adherence reporting is consistent with E6(R2) GCP: Integrated 
Addendum to ICH E6(R1): 4.5 Compliance with Protocol; 5.18.3 (a), and 5.20 Noncompliance; 
and ICH E3 16.2.2 Protocol deviations. 
9 
CLINICAL MONITORING 
Clinical site monitoring is conducted to ensure that the rights of the participants are protected, 
that the study is implemented per the approved protocol, Good Clinical Practice and standard 
operating procedures, and that the quality and integrity of study data and data collection methods 
are maintained.  Monitoring for this study will be performed by NCI CCR Office of Sponsor and 
Regulatory Oversight (OSRO) Monitoring based on OSRO standards, FDA Guidance E6(R2) 
Good Clinical Practice: Integrated Addendum to ICH E6(R1) March 2018, and applicable 
regulatory requirements. 
Details of clinical site monitoring will be documented in a Clinical Monitoring Plan (CMP) 
developed by OSRO. CMPs will be protocol-specific, risk-based and tailored to address human 
subject protections and integrity of the study data. The intensity and frequency of monitoring 
will be based on several factors, including study type, phase, risk, complexity, expected 
enrollment rate, and any unique attributes of the study and the site. OSRO Monitoring visits and 
related activities will be conducted throughout the life cycle of each protocol, with the first 
activity being before study start to conduct a Site Assessment Visit (SAV) (as warranted), 


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followed by a Site Initiation Visit (SIV), Interim Monitoring Visit(s) (IMVs), and a study Close-
Out Visit (COV). 
Some monitoring activities may be performed remotely, while others will take place at the study 
site(s).  Monitoring visit reports will describe visit activities, observations, findings of protocol 
non-adherence and associated action items or follow-up required for resolution of findings.  
Monitoring reports will be distributed to the study PI, NCI CCR QA, coordinating center (if 
applicable) and the OSRO regulatory file. 
If protocol non-adherence is identified by the Monitor (i.e., any noncompliance with the clinical 
trial protocol, GCP, or protocol-specific procedural requirements on the part of the participant, 
the Investigator, or the site Staff) the Monitor will note the observation, review with site Staff 
and if unresolved, request that the Staff document the non-adherence on the protocol-specific 
OSRO Site Protocol Non-Adherence Log (see Section 8.8). 
10 STATISTICAL CONSIDERATIONS     
10.1 STATISTICAL HYPOTHESIS 
We hypothesize that sequentially evaluated combinations of BN-Brachyury, M7824, T-DM1 and 
entinostat will enhance the clinical and immunologic response in patients with advanced breast 
cancer, specifically for HER2+BC patients who previously progressed after treatment (or were 
intolerant to) with a taxane, trastuzumab and pertuzumab. The trial will enroll patients initially to 
BN-Brachyury + M7824, and then add TDM-1 and finally entinostat. 
• Primary Endpoints:  
o Objective response rate (ORR; PR+CR), as defined in Section 6.3.3, for each of 
the three treatment arms enrolled sequentially as described below.  
o Safety for the combinations explored in the arms. 
• Secondary Endpoints: 
o Progression free survival, as designed in Section 6.3.4.5. 
o Change in absolute percentage of stromal TILs quantified according to the 
Salgado method in serial tumor biopsies collected at baseline and on C3D1 (Arms 
2 and 3 only). 
• Exploratory Endpoints (studies may be performed in selected patients if adequate 
samples are available): 
o Peripheral blood mononuclear cells (PBMCs):  
➢ Changes in immune cell subsets.  
➢ Changes in brachyury-specific, MUC1-specific, and CEA-specific T cells 
measured by intracellular cytokine staining (IFN, IL2, TNF, CD107a) . 
➢ Histone acetylation as surrogate for entinostat pharmacodynamics (Arm 3 
only). 
o Plasma/Serum: 
➢ Changes in brachyury-specific , MUC1-specific, and CEA-specific  
antibodies. 
➢ Changes in soluble factors like sCD27 and sCD40. 
➢ Changes in cell free DNA. 
➢ Changes in TGF-beta levels.  


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➢ Changes in pharmacokinetics of M7824. 
o Tissue Biopsy (Cohort 3, Arms 2 and 3):  
➢ Brachyury expression on tumor. 
➢ T-cell clonality score. 
➢ Immunohistochemistry/multispectral imaging. 
➢ Changes in PD-L1 on tumor. 
➢ Neoepitopes derived from tumor biopsies.  
➢ HER2 expression changes. 
➢ Tumor mutational burden. 
10.2 SAMPLE SIZE DETERMINATION 
The primary objective of this trial , on which the sample size is based, is to determine the overall 
clinical response rate of BN-Brachyury, entinostat, M7824 and T-DM1 (PR+CR) evaluated in 
arms with increasing numbers of agents as follows: 
Arm 1:  
In similar subjects with TNBC (unselected for PD-L1 expression) who received a checkpoint 
inhibitor, the clinical response rate was approximately 8-10%. (43-45) The goal would first be to 
determine if using BN-Brachyury plus M7824 could be shown in a small pilot arm to improve 
upon this by a modest amount. Accrual of the first 3 patients on trial in this arm will be slow (no 
more than 1 patient per 6 days) in order to allow for monitoring of toxicity. If there is one or more 
DLT in the first 3 patients on this arm, accrual of the next 3 patients on the associated arm will 
proceed slowly with no more than 1 patient per 21 days (1 cycle) to continue to closely monitor 
safety. If there are 0-1 patients with DLTs among the 6 patients enrolled on this arm, to do so, 
accrual to this arm will be continue by using a Simon optimal two-stage phase II trial design (126) 
to rule out an unacceptably low PR+CR rate of 10% (p0=0.10) in favor of an improved response 
rate of 35% (p1=0.35). With 1-sided alpha=0.10 (probability of accepting a poor treatment=0.10) 
and beta=0.20 (probability of rejecting a good treatment=0.20), the first stage will enroll 8 
evaluable subjects, and if 0 to 1 of the 8 have a clinical response, then no further subjects will be 
accrued. If 2 or more of the first 8 subjects have a response, then accrual would continue until a 
total of 13 evaluable subjects have been enrolled. As it may take 3 to 6 months to determine if a 
subject has experienced a response, a temporary pause in the accrual may be necessary to ensure 
that enrollment to the second stage is warranted. If there are 2 subjects with a response out of 13 
subjects, this would be an uninterestingly low response rate. If there were 3 or more of 13 (23.1%) 
who experienced a response, this would be sufficiently interesting to warrant further study in later 
trials. Under the null hypothesis (10% response rate), the probability of early termination is 81.3%.  
Arms 2 and 3:  
Following determination of safety in arm 1 (0-1 with DLT among first 6 patients enrolled), patients 
who are HER-2 positive will undergo an initial safety evaluation and then will be assigned in an 
alternating fashion (1:1) to the second and third arms: BN-Brachyury, M7824 and T-DM1, or BN-
Brachyury, M7824, TDM-1, and entinostat. Provided that there are 0-1 patients with DLTs among 
the 6 patients enrolled on Arm 2, this arm will be temporarily closed and patients will be enrolled 
on Arm 3 for a safety evaluation. Provided that there are 0-1 patients with DLTs among the 6 
patients enrolled on Arm 3, patients with  HER2 positive breast cancer will be directly allocated1:1 
to Arms 2 and 3. In Arms 2 and 3, accrual of the first 3 patients in each arm will proceed slowly 


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with no more than 1 patient enrolled every 28 days in order to allow for safety monitoring. If there 
is one or more DLT in the first 3 patients on any arm, accrual of the next 3 patients on the associated 
arm will proceed slowly with no more than 1 patient per 21 days (1 cycle) to closely monitor 
safety. If there are 2 or more patients with a DLT among the first 6 treated on either arm, the trial 
will halt accrual pending an amendment to detail how the study will proceed from that point 
forward.  
The primary objective is to determine if either arm could improve upon the 18% response rate by 
a modest amount. Each arm will be conducted using a Simon minimax two-stage phase II trial 
design (126) to rule out an unacceptably low PR+CR rate of 18% (p0=0.18) in favor of an 
improved response rate of 40% (p1=0.40). With 1-sided alpha=0.10 (probability of accepting a 
poor treatment=0.10) and beta=0.20 (probability of rejecting a good treatment=0.20), the first stage 
will enroll 14 evaluable subjects in each arm, and if 0 to 3 of the 14 have a clinical response, then 
no further subjects will be accrued. If 4 or more of the first 14 subjects have a response in an arm, 
then accrual would continue until a total of 19 evaluable subjects have been enrolled in that arm. 
As it may take 3 to 6 months to determine if a subject has experienced a response, a temporary 
pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. 
If there are 4 to 5 subjects with a response out of 19 subjects in an arm, this would be an 
uninterestingly low response rate. If there were 6 or more of 19 (31.6%) who experienced a 
response, this would be sufficiently interesting to warrant further study that combination in later 
trials. If accrual ends to one arm because of insufficient activity, the other arm will remain open to 
enroll patients directly. Under the null hypothesis (18% response rate), the probability of early 
termination is 76.5%.  
There will be no adjustment for the multiplicity of the three arms. 
To allow for a small number of inevaluable patients, up to 42 patients may be enrolled in Cohort 
2 (21 per Arm, if necessary) to explore this primary objective. 
With arms requiring up to 13+19+19=51 evaluable patients and allowing for up to 4 total 
inevaluable patients and up to 10 screening failures, the accrual ceiling will be set at 65 patients. 
Safety will also be assessed as a primary objective. All patients will be assessed for safety as 
described in Sections 10.3.1 and 10.4.4.  
Secondary Objectives:  
Progression Free Survival (PFS) will be evaluated as a secondary objective. This will be 
estimated using a Kaplan-Meier curve and the median PFS will be reported along with a 95% 
confidence interval, separately by arm, based on the patients who are enrolled in the evaluation 
of clinical response.  
Change in TILs will also be evaluated as a secondary objective. Data from 678 patients with 
evaluable TILs enrolled on the CLEOPATRA trial in HR-/HER2+ breast cancer indicated a 
mean baseline stromal TIL value of 21% (SD=22.4%).  In the present study with HR-/HER2+ 
patients, it is anticipated that the baseline TIL value will be assumed to be approximately 25%, 
with the same 22% standard deviation. A very conservative estimate of the SD associated with a 
change between two time points is the baseline SD x square root (2), or 22 x 1.41 =31%, based 
on an assumption of no correlation between the values at baseline and at the time of biopsy.  An 
assumption of the correlation=0.5 would result in the SD of the change being baseline SD x 1.0, 
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The goal will be to estimate if the absolute change in TIL percentage from baseline is 
meaningfully large, as well as noting the magnitude and reporting that with appropriate 
descriptive statistics and graphical presentation. With 15 evaluable patients for TILs in the two 
larger arms, there will be 82% power to detect a difference between a mean baseline TIL value 
of 25% (0.25) and mean post-biopsy TIL value of 50% (0.50), assuming a standard deviation of 
the change of 31% (effect size= 25/31=0.806), using a paired t-test with a two-sided 0.05 
significance level. More conservatively if only 13 or 11 patients have paired samples assessed 
(from any of the three arms), then there will be 82% and 84% power to detect a mean difference 
of 27% (effect size=0.871) or 31% (effect size=1.0), respectively. In practice, the changes in the 
TIL% may not be normally distributed, so the study will plan to test the effect using a Wilcoxon 
signed rank. In practice, the changes in the TIL% may not be normally distributed, so the study 
will plan to test the effect using a Wilcoxon signed rank test instead of a paired t-test. 
Progression Free Survival (PFS) will be evaluated as a secondary objective. This will be 
estimated using a Kaplan-Meier curve and the median PFS will be reported along with a 95% 
confidence interval, separately by arm. based on the patients who are enrolled in the evaluation 
of clinical response. 
10.3 POPULATIONS FOR ANALYSES 
10.3.1 Evaluable for toxicity: 
All patients who are treated with any amount of the combination of agents will be evaluable for 
toxicity from the time of their first treatment; thus, all patients will be evaluable for toxicity from 
the time of their first treatment with the arm-specific combinations of BN-Brachyury, entinostat, 
M7824 and T-DM1. Any patient who receives some but not all treatments in each given arm will 
be summarized separately. 
10.3.2 Evaluable for objective response:  
Only those patients who have measurable disease present at baseline, have received at least two 
cycles of therapy, and have had their disease re-evaluated will be considered evaluable for 
response.  These patients will have their response classified according to the definitions stated 
below. Patients who have measurable disease at baseline, have received at least 1 cycle of 
therapy and have clinical/objective disease progression prior to the first restaging will be 
considered evaluable.  
10.4 STATISTICAL ANALYSES 
10.4.1 General Approach 
Data will be summarized descriptively and graphically, with reporting the outcome measures 
indicated along with two-tailed 95% confidence intervals (CIs), as well as two-tailed 80% CIs 
(corresponding to the design with one-sided α=0.10) for median PFS.  
10.4.2 Analysis of the Primary Efficacy Endpoint 
The ORR will be estimated in each arm using two-sided 80% and 95% CI about the observed 
percentages of response (PR+CR).  


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10.4.3 Analysis of the Secondary Endpoints 
Stromal TILs will be summarized descriptively and graphically by time point and as absolute and 
relative change from baseline. The absolute change in TILs values from baseline following two 
cycles of treatment will be compared via Wilcoxon signed rank test.  
The distribution of PFS will be estimated using the Kaplan-Meier (KM) method, among patients 
evaluable for efficacy, and appropriate confidence intervals (80% as well as 95% two-sided 
confidence intervals) at the median and selected time points will be provided to help interpret 
results relative to the expected results.  This will be done by arm and may be evaluated by 
combining patients in arms 2 and 3 if they are sufficiently similar (p>0.30 by global two-tailed 
log-rank test).  
The fraction of patients who experience a DLT during the single dose level safety phases of the 
trial will be reported, along with a summary, confidence interval, and description of any 
toxicities noted of grade 3 or higher. 
10.4.4 Safety Analyses 
Adverse events (AE) recorded throughout treatment will initially be summarized as the 
maximum grade, for each AE type. The number and frequency of patients experiencing each AE 
type and grade will tabulated, among patients evaluable for toxicity. The number (%) of patients 
experiencing at least one grade 3 to 5 AE will be reported with 95% CI.  This will be done 
separately for the initial safety cohort and for the patients in the primary efficacy cohort. Patients 
who receive at least 1 dose of all drugs in their cohort will be evaluable for safety analyses.  
10.4.5 Baseline Descriptive Statistics 
Continuous variables will be summarized as mean±SD, median and interquartile range, and 
range of values. Categorical variables will be summarized as number (%) of patients. 
10.4.6 Sub-Group Analyses 
Analyses will be performed separately by arm. 
10.4.7 Tabulation of individual Participant Data 
Given the sample size, graphical summaries of individual data will often be used, for example 
scatter plot TILs according to time point with the paired values connected by lines. 
10.4.8 Exploratory Analyses 
Blood sample collection (as specified in Section 5.1.1), as well as biopsy specimens at baseline 
and cycle 3 will yield a set of immune subsets. Values will be summarized descriptively and 
graphically over time. Given the sample size we cannot investigate association of baseline or 
change from baseline to cycle 1 or cycle 2 in relation to PFS using Cox modeling; therefore, 
these investigations would focus on KM plots according to dichotomized values of biomarker 
values, without statistical hypothesis testing. In order to avoid guarantee-time bias, the 
assessments of changes to cycle 1 or cycle 2 may need to take a landmark analysis approach 
because patients will need to be alive and progression free at cycle 1 or cycle 2 in order to have a 
blood sample and be part of the analysis. The changes in the biomarkers will also be summarized 
descriptively and graphically for responders and non-responders. 


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11 COLLABORATIVE AGREEMENTS  
11.1 MULTI-INSTITUTIONAL GUIDELINES 
Until an electronic submission system is available to participating sites, documents requiring 
submission to the reviewing IRB per reliance agreement, including local consent documents 
generated from an approved model consent, should be provided to the coordinating center for 
submission to the IRB. Thereafter, consents may be submitted directly to the IRB using iRIS. 
12 HUMAN SUBJECTS PROTECTIONS  
12.1 RATIONALE FOR SUBJECT SELECTION 
Both men and women are eligible for this trial if they meet eligibility criteria. Subjects from all 
racial and ethnic groups are eligible for this trial if they meet the eligibility criteria. Efforts will 
be made to extend the accrual to a representative population. If differences in outcome that 
correlate to racial or ethnic identity are noted, accrual may be expanded or additional studies may 
be performed to investigate those differences more fully. 
As there is a risk of severe bleeding with this study drug, participants must be willing to receive 
blood transfusions if medically necessary for their own safety. Participants must be able to 
receive blood transfusions in order to minimize the risks of receiving M7824. Another reason for 
this is that including these patients could compromise the scientific validity of the study. For 
example, death from blood loss could make it difficult to assess other aspects of the 
investigational immunotherapy’s safety—a primary scientific goal in this early-phase 
immunotherapy trial, which are carried out in small numbers of participants. 
12.2 PARTICIPATION OF CHILDREN 
The age group for enrollment on this trial is 18 or more years of age. Because no dosing or 
adverse event data are currently available on the use of BN-Brachyury, entinostat, M7824 or T-
DM1 in participants < 18 years of age, children are excluded from this study. 
12.3 EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS 
BN-Brachyury, entinostat, and M7824 are investigational agents for use in metastatic breast 
cancer. Furthermore, BN-Brachyury, entinostat, M7824, and T-DM1 have never been used in 
combination. The protocol provides for detailed and careful monitoring of all patients to assess 
for toxicity. Toxicity data will be collected and reviewed to ensure that there were no severe 
toxicities that would preclude further patient enrollment. Patients will be treated with therapeutic 
intent and response to the therapy will be closely monitored.  
Risks include the possible occurrence of any of a range of side effects which are listed in the 
Consent Document and in Section 12.4.1. Frequent monitoring for adverse effects will help to 
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12.4 RISK/BENEFIT ASSESSMENT 
12.4.1 Known Potential Risks 
Some of the procedures performed on this study are not known to be associated with risk. These 
include urine tests and EKGs. Below are a list of procedures and study interventions that are 
associated with risk. 
12.4.1.1 Risk of MRI 
If there is concern for CNS metastasis or history of CNS metastasis, the PI may request a brain 
MRI.  
People are at risk for injury from the MRI magnet if they have some kinds of metal in their body. 
People with fear of confined spaces may become anxious during an MRI. Those with back 
problems may have back pain or discomfort from lying in the scanner.  The noise from the 
scanner is loud enough to damage hearing, especially in people who already have hearing loss.  
There are no known long-term risks of MRI scans. 
12.4.1.2 Risk of Gadolinium Enhanced MRI 
The gadolinium infusion may cause mild symptoms such as coldness in the arm during the 
injection, a metallic taste, headache, and nausea. There are risks of an IV catheter include 
bleeding, infection, or inflammation of the skin and vein with pain and swelling.   
Procedure-related risks from MRI and gadolinium enhanced MRI will be explained fully during 
informed consent. 
12.4.1.3 Risks of Exposure to Ionizing Radiation 
This research study involves the possibility of 7 CT CAP scans, 4 CT guided biopsies, and 1 head 
CT collected for research purposes per year.  
The amount of radiation exposure received from these procedures is equal to approximately 11 
rem. The CT scans and CT guided biopsies in this study will expose the research participant to 
36.7 years’ worth of background radiation. This level of exposure results in an increased risk of 
cancer.  
12.4.1.4 Risk of Biopsy 
CT-guided biopsies will be performed as clinically indicated. Up to 3 CT-guided biopsies may be 
performed on this study for research purposes per year. 
Patients in all cohorts may be required to undergo a biopsy at screening in order to confirm their 
diagnosis 
Patients in Cohort 3: 2 required biopsies while on treatment, 1 optional biopsy at progression. 
Patients in Cohort 1 and 2: 3 optional biopsies (2 while on treatment and 1 at progression).  
All care will be taken to minimize risks that may be incurred by tumor sampling. However, there 
are procedure-related risks (such as bleeding, infection and visceral injury) that will be explained 
fully during informed consent. 
12.4.1.5 Additional Risks of CT Scans 
In addition to the radiation risks addressed above, CT scans that employ contrast may cause 
allergic reactions, injection site reactions abdominal discomfort and fainting. 


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12.4.1.6 Research Blood Collection Risks 
Risks of blood draws include pain and bruising in the area where the needle is placed, 
lightheadedness, and rarely, fainting. When large amounts of blood are collected, low red blood 
cell count (anemia) can develop.  
12.4.1.7 Other Risks 
Risks include the possible occurrence of any of a range of side effects which are listed in the 
Consent Document or this protocol document. Frequent monitoring for adverse effects will help 
to minimize the risks associated with administration of the study agents. 
12.4.2 Known Potential Benefits 
The potential benefits from this therapy are stabilization or shrinkage of the tumor and a 
reduction in chances of developing new lesions with decrease of symptoms caused by 
progressive disease.  
12.4.3 Assessment of Potential Risks and Benefits 
Metastatic or refractory/recurrent breast cancers are in need of improved therapy options. No 
current therapies are curative. Preclinical studies suggest that the use of a combination of 
multiple immunotherapy agents may have improved anti-tumor efficacy.  
A number of clinically appropriate strategies to minimize risks to patients have been built into 
the protocol through the means of inclusion/exclusion criteria, monitoring strategies, and 
management guidelines. Overall, the potential benefits of the combination of a therapeutic 
vaccine against brachyury (BN-brachyury), a bifunctional fusion protein targeting PD-L1 and 
TGF beta (M7824), an immune and epigenetic modifier (entinostat) and a standard of care 
antibody-drug conjugate (T-DM1) in subjects with advanced breast cancer for patients retaining 
the ability to consent and those who lose capacity to consent during the course of the trial 
outweigh the risks associated with this combination immunotherapy proposed in this study. 
The potential benefit to a patient that goes onto this study is a reduction in the bulk of their tumor 
which may or may not have favorable impact on symptoms and/or survival. 
Potential adverse reactions attributable to the administration of the study drug utilized in this trial 
are discussed in Section 14. All care will be taken to minimize side effects, but they can be 
unpredictable in nature and severity.  
12.5 CONSENT PROCESS AND DOCUMENTATION 
The informed consent document will be provided as a physical or electronic document to the 
participant for review prior to consenting. A designated study investigator will carefully explain 
the procedures and tests involved in this study, and the associated risks, discomforts and benefits. 
In order to minimize potential coercion, as much time as is needed to review the document will 
be given, including an opportunity to discuss it with friends, family members and/or other 
advisors, and to ask questions of any designated study investigator. A signed informed consent 
document will be obtained prior to entry onto the study. 
The initial consent process as well as re-consent, when required, may take place in person or 
remotely (e.g., via telephone or other permissible secure remote platforms per policy, including 
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the participant.  Whether in person or remote, the privacy of the subject will be maintained. 
Consenting investigators (and participant, when in person) will be located in a private area (e.g., 
clinic consult room). When consent is conducted remotely, the participant will be informed of 
the private nature of the discussion and will be encouraged to relocate to a more private setting if 
needed. 
Note: When required, witness signature will be obtained similarly as described for the 
investigator and participant as described below. 
Consent will be documented with required signatures on the physical document (which includes 
the printout of an electronic document sent to participant) or on the electronic document.  
Signatures on electronic documents are described below.  Note: FDA only regulates electronic 
signatures (i.e., an electronic timestamp is generated at the time of signature) in FDA regulated 
research.    
Please see site specific supplement for electronic signature requirements for participating site. 
For the optional biopsies in the protocol, the patient will be asked to sign a separate consent at 
the time of the procedure. If the patient refuses the optional biopsy at that time, the refusal will 
be documented in the medical record and in the research record. 
13 REGULATORY AND OPERATIONAL CONSIDERATIONS 
13.1 STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause. Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to study participants, 
investigator, funding agency, the Investigational New Drug (IND) sponsor and regulatory 
authorities. If the study is prematurely terminated or suspended, the Principal Investigator (PI) 
will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and 
will provide the reason(s) for the termination or suspension. Study participants will be contacted, 
as applicable, and be informed of changes to study visit schedule. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to participants 
• Demonstration of efficacy that would warrant stopping    
• Insufficient compliance to protocol requirements 
• Data that are not sufficiently complete and/or evaluable 
• Determination that the primary endpoint has been met 
• Determination of futility 
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA). 
13.2 QUALITY ASSURANCE AND QUALITY CONTROL 
Each clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion.  An individualized quality management 
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Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution. 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the 
clinical trial is conducted and data are generated and biological specimens are collected, 
documented (recorded), and reported in compliance with the protocol, International Conference 
on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements 
(e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)).  
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local 
and regulatory authorities. 
13.3 CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed 
and managed. Furthermore, persons who have a perceived conflict of interest will be required to 
have such conflicts managed in a way that is appropriate to their participation in the design and 
conduct of this trial. The study leadership in conjunction with the National Cancer Institute and 
other participating sites has established policies and procedures for all study group members to 
disclose all conflicts of interest and will establish a mechanism for the management of all 
reported dualities of interest. 
13.4 CONFIDENTIALITY AND PRIVACY 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants. 
Therefore, the study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor.  
All research activities will be conducted in as private a setting as possible. 
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and 
records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study. The 
clinical study site will permit access to such records. 
The study participant’s contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a 
secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or 
sponsor requirements. 
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored at the NCI CCR. This will not include the 
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data will be identified by a unique study identification number. The study data entry and study 
management systems used by the clinical site(s) and by NCI CCR research staff will be secured 
and password protected. At the end of the study, all study databases will be archived at the NIH. 
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by the National Institutes of Health (NIH). This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research participation in any 
civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or 
local level. By protecting researchers and institutions from being compelled to disclose 
information that would identify research participants, Certificates of Confidentiality help achieve 
the research objectives and promote participation in studies by helping assure confidentiality and 
privacy to participants. 
14 PHARMACEUTICAL INFORMATION   
14.1 BN-BRACHYURY (IND # 19165) 
14.1.1 Source / Acquisition and Accountability 
BN-Brachyury is an active cancer immunotherapy. This phase 1b 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 BN-Brachyury and will supply product for this 
phase 1b study.  
MVA-brachyury and FPV-brachyury will be provided to each clinical trial site by the 
manufacturer. The investigator or designee (e.g. pharmacist) will maintain an ongoing inventory 
of the investigational product supply according to standard site procedures. The investigational 
product will be dispensed at the direction of an investigator for administration to a study 
participant enrolled on the clinical trial. Disposal of expired or unused product will be returned to 
the manufacturer or disposed of according to standard site procedures based on agreement 
between the site and the manufacturer or IND holder.   
14.1.2 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. 
14.1.3 Formulation and Preparation  
MVA-BN-Brachyury is supplied as a frozen aqueous suspension in 2 mL clear borosilicate glass, 
single use vials. The closure is a sterile bromobutyl rubber stopper, crimped with an aluminum 
cap and covered with a polypropylene closure. Each 0.5mL injection will contain a nominal virus 
titer of 2.0 x 108 Inf.U. 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 product will 
be a slightly turbid to milky suspension which may contain clumps or aggregates. To ensure 
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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).  
FPV-Brachyury is a liquid-frozen, highly attenuated, live recombinant virus. FPV-Brachyury is a 
clear to milky suspension. The product needs to be free from visible extraneous particles 
considering that very small particles or clumps/aggregates are product related. A turbid 
appearance including the tendency towards sedimentation is based on the product-inherent 
characteristics. Additional details can be found in the Pharmacy Manual.  
Packages and vials are labeled according to the respective product specification. One FPV-
Brachyury vial has sufficient volume to deliver 0.5 mL and contains a nominal virus titer of 2 x 
109 Inf.U per mL. 
Detailed trial-specific instructions on the preparation and administration of MVA-BN-Brachyury 
and FPV-Brachyury are provided in the clinical trial protocol and/or a separate Pharmacy 
Manual supplied to each clinical trial site. 
14.1.4 Stability and Storage  
MVA-BN-Brachyury and FPV-Brachyury are stable at -80°C ± 10°C and -20°C ± 5°C, 
respectively. MVA-BN-Brachyury and FPV-Brachyury should be stored frozen and remain 
frozen until use. After the completion of preparation, the injection must be administered within 9 
hours. Avoid exposure to direct light and product may not be refrozen 
14.1.5 Administration Procedures 
Administration of BN-Brachyury is described in Section 3.2.1. 
14.1.6 Incompatibilities   
There are no known drug interactions associated with the use of MVA-BN and MVA-BN-
derived vectors. 
14.2 ENTINOSTAT (IND # 19165) 
14.2.1 Source / Acquisition and Accountability 
Entinostat is a class 1-selective histone deacetylase inhibitor. This phase 1b study is being 
conducted under an IND held by the CCR, NCI. Syndax is the manufacturer of entinostat and 
will supply product for this phase 1b study. 
Entinostat will be provided to each clinical trial site by the manufacturer. The investigator or 
designee (e.g. pharmacist) will maintain an ongoing inventory of the investigational product 
supply according to standard site procedures. The investigational product will be dispensed at the 
direction of an investigator for administration to a study participant enrolled on the clinical trial. 
Disposal of expired or unused product will be returned to the manufacturer or disposed of 
according to standard site procedures based on agreement between the site and the manufacturer 
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14.2.2 Metabolism 
Th exact mechanism of metabolism is unknown at this time. However, initially a renal and 
biliary clearance of entinostat was suspected. Subsequent studies have shown that hepatic 
metabolism seems to be a minor component of drug elimination in humans. (118, 127) 
The pharmacokinetics of entinostat were linear over the dose range of 2 to 12 mg/m2 given 
weekly. Considerable inter-patient variation in absorption was observed. The elimination half-
life of entinostat ranged from 54 to 161 hours. The median elimination half-life of entinostat 
under fasted conditions is 140 hours. Data from in vitro experiments showed that, while 
entinostat inhibited cytochrome P-450 (CYP) enzymes 2B6 and 3A4, the degrees of the 
inhibition make it unlikely that any in vivo systemic interactions would occur. Intestinal CYP 
3A4 may be inhibited by entinostat. However, entinostat did not inhibit any tested P 
glucuronosyltransferase (UGT) enzymes. In addition, entinostat was found to induce CYP 1A2, 
CYP 2C6, and CYP 2B8 as well as UGT 1A4. Finally, entinostat is a substrate for P-
glycoprotein (P-gp) and breast cancer resistance protein (BCRP) transporters.  
In pharmacodynamic studies, entinostat-dependent protein lysine hyperacetylation was observed 
at all doses tested to date, with preliminary data suggesting that hyperacetylation may be 
associated with improved clinical outcome when entinostat is administered in combination with 
exemestane in breast cancer. PK and PD data will be evaluated on cycle 1 Day 8 and cycle 1 Day 
15 (optional). 
14.2.3 Toxicity  
The most common AEs (>20%) for entinostat based on 217 patients who received monotherapy 
were fatigue (48%), nausea (47%), thrombocytopenia (45%), anemia (40%), hypoalbuminemia 
(37%), hypophosphatemia (32%), neutropenia (32%), vomiting (28%), anorexia (25%), 
headache (24%), diarrhea (21%), hyponatremia (21%). Grade 3 and 4 events (>10%) included 
thrombocytopenia (22%), anemia (19%), neutropenia (16%), and hypophosphatemia (15%). 
Serious adverse events (>5%) included fatigue (9%), dyspnea (7%), anorexia (6%), neutropenic 
infection (6%) and dehydration (6%). 
The safety profile of entinostat when given in combination was similar to that seen when given 
as monotherapy. Overall, 97% of the 740 patients who received entinostat in combination with 
another agent experienced at least 1 TEAE. In general, the AEs reported most frequently among 
patients receiving entinostat in combination, regardless of indication, were fatigue, nausea, 
anemia, thrombocytopenia, leukopenia, diarrhea, vomiting, and neutropenia.  
As would be expected, the AE profiles of entinostat when given in combination varied somewhat 
based on the agent with which it is given and the corresponding patient population. Entinostat in 
combination with 5-azacitidine was generally associated with an increased number of AEs 
relative to its use in combination with an aromatase inhibitor (AI), erlotinib, or other agents. 
Consistent with the overall AE profile of entinostat, nausea with or without vomiting, fatigue, 
and anemia were the most prevalent AEs regardless of the patient population or the agent given 
in combination.  
Overall, among the 535 patients with solid tumors receiving entinostat in combination, the AEs 
reported most frequently were as follows: fatigue (66%); nausea (58%); anemia (44%); vomiting 
and diarrhea (each 34%); thrombocytopenia and anorexia (each 30%); leukopenia (25%); 
dyspnea (22%); and injection site reaction (20%).  


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Overall, among the 205 patients with hematologic malignancies receiving entinostat in 
combination, the SAEs reported most frequently were: fatigue (70%); thrombocytopenia (67%); 
anemia (65%); neutropenia (62%); leukopenia (60%); nausea (58%); anorexia (41%); diarrhea 
(29%); hypoalbuminaemia (36%); injection site reaction (35%); febrile neutropenia (34%); 
vomiting (33%); constipation (32%); hyperglycemia; hypocalcaemia and hyponatraemia (each 
31%); edema peripheral and dyspnea (each 30%); and headache and hypophosphataemia (each 
20%). 
14.2.4 Formulation and Preparation  
Entinostat is supplied in two strengths of film-coated tablets containing 1 mg (pink to light red) 
or 5 mg (yellow) of entinostat, produced using the polymorph B form of the drug. The film 
coating is derived from an aqueous suspension consisting of hypromellose, talc, titanium 
dioxide, and ferric oxide pigments (red and yellow) as coloring. Each tablet also contains 
mannitol, sodium starch glycolate, hydroxypropylcellulose, potassium bicarbonate, and 
magnesium stearate as inert fillers. 
14.2.5 Stability and Storage  
Entinostat tablets are to be stored at up to 25°C (77°F); excursions are permitted from 15°C to 
30°C (59°F to 86°F). Entinostat tablets are to be stored in a secure, locked storage area to which 
access is limited. 
14.2.6 Administration Procedures 
Administration of Entinostat is described in Section 3.2.4.1. 
14.2.7 Incompatibilities   
At this time, there are no known drug interactions or incompatibilities with entinostat.  
It is thought that entinostat does inhibit CYP450 enzymes but at higher concentrations than 
clinically achievable. (127)  However, no formal drug-drug interactions have been completed. It 
is recommended to use caution when administering entinostat concurrently with medication that 
have a narrow therapeutic index. 
14.3 M7824 (IND # 19165) 
14.3.1 Source / Acquisition and Accountability 
M7824 is a novel bifunctional fusion protein composed of a monoclonal antibody against human 
PD-L1 fused to the soluble extracellular domain of human TGF-β receptor II (TGF-βRII), which 
functions as a TGF-β “trap.” This phase 1b study is being conducted under an IND held by the 
CCR, NCI. EMD Serono is the manufacturer of M7824 and will supply product for this phase 1b 
study. 
M7824 will be provided to each clinical trial site by the manufacturer. The investigator or 
designee (e.g., pharmacist) will maintain an ongoing inventory of the investigational product 
supply according to standard site procedures. The investigational product will be dispensed at the 
direction of an investigator for administration to a study participant enrolled on the clinical trial. 
Disposal of expired or unused product will be returned to the manufacturer or disposed of 
according to standard site procedures based on agreement between the site and the manufacturer 
or IND holder. 


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14.3.2 Metabolism  
This preliminary PK analysis was based on data from 12 subjects (3 subjects each at 1, 3, 10, and 
20 mg/kg) from EMR200647-001 as of the data cutoff date of 04 May 2016. Additional PK data 
from EMR200647-001 and MS200647-0008 will be reported at a later time. Samples for 
measurement of M7824 serum concentrations in Study EMR200647-001 were 
collected and mean M7824 serum concentrations were plotted against time after dosing by dose 
group. The PK profiles at the 3, 10, 20 mg/kg dose groups indicate a one-phase 
elimination with no obvious distribution phase after 1 hour iv infusion of M7824. Similar PK 
profile characteristics were observed at the lowest dose groups (1 mg/kg) with a slightly faster 
elimination rate. 
Non-compartmental analyses were performed for each dose group with PK parameters 
summarized. Based on data from 12 patients in the dose escalation part of EMR200647-001, 
M7824 demonstrated approximately dose-proportional PK from 3 to 20 mg/kg. The mean 
apparent terminal half-life (t1/2) after the 1st dose was about 6 to 7 days for dose levels above 3 
mg/kg. In the 1 mg/kg dose group, a mean terminal t1/2 of about 5 days was observed. When 
compared to the exposure at NOAEL in the 13-weeks repeat dose toxicity study in cynomolgus 
monkeys (140 mg/kg biweekly), the exposure at the highest clinical dose tested to date in 
humans (20 mg/kg biweekly) is 6.6-fold lower in Cmax and 4.3-fold lower in AUC, which 
indicate a safety margin of at least 4-fold. 
As T-DM1 is dosed every 3 weeks, a dosing interval of every 3 weeks was preferred to the 
standard every 2 week dosing for M7824. The dose-level was informed by extensive population 
PK modeling performed by the EMD Serono pharmacokinetics team after reviewing data from 
the ongoing M7824 -001 and -0008 trials. 
For M7824 monotherapy, 1200 mg  every 2 weeks was selected as a recommended phase 2 dose.  
(128) In combination studies in which concomitant therapies are administered q3w, the same 
dosing interval for M7824 is preferred for convenience and compliance. The steady-state Ctrough 
(Ctrough,ss) at 1200 mg q2w is considered the target efficacious Ctrough,ss  for q3w dosing of M7824 
in combination studies with q3w dosing. Based on population PK modeling, median Ctrough,ss 
achieved with 2400 mg q3w dosing is similar to that projected with 1200 mg q2w dosing. In 
addition, for >90% of the participants to be dosed with 2400 mg q3w, Ctrough,ss is  estimated to be 
above the lower bound of 95% CI for Ctrough,ss projected with 1200 mg q2w. Since Ctrough,ss  is 
sim2400 mg q3w is expected to achieve target Ctrough,ss and full PD-L1 target occupancy 
and  TGFβ trapping in blood in > 90% of the participants.  
Safety data to support 2400 mg q3w dosing: The highest dose for M7824 tested in EMR200647-
001 was 30 mg/kg once every 2 weeks and the maximum tolerated dose was not reached. Three 
(3) participants in EMR200647-001 dose escalation cohorts received the actual dose of at least 
2400 mg q2w: 2 in 30 mg/kg cohort and 1 in 20 mg/kg.  All 3 participants who received ≥ 
2400 mg q2w had no DLTs. For the 2400 mg q3w dosing, both the maximum and average 
concentrations at steady state are not expected to exceed those at 2400 mg q2w. The overall 
safety profile of M7824 in Phase I was considered well tolerated, can be adequately managed 
and consistent across various tumor types. Refer to Investigator’s Brochure for more details. 


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14.3.3 Toxicity  
In a phase 1, open-label 3+3 dose-escalation study of M7824 in 16 patients, 3 patients 
experienced grade 3 drug-related adverse events including skin infection secondary to grade 2 
bullous pemphigoid, lipase increased, and colitis with associated anemia. There were no grade 4 
– 5 treatment related adverse events. Please see table below for details: 
Treatment-related adverse events 
 
3 mg/kg (n = 3) 
10 mg/kg (n = 3) 
20 mg/kg (n = 7) 
Total (n = 16) 
 
Any 
Grade 
Grade 
3 
Any 
Grade 
Grade 3 
Any 
Grade 
Grade 3 
Any 
Grade 
Grade 3 
Patients with any 
event** 
2 
(66.7) 
1 
(33.3) 
1 (33.3) 
0 (0.0) 
4 (57.1) 
2 (28.6) 
7 (43.8) 
3 (18.8) 
Anemia 
 
 
 
 
1 (14.3) 
1 (14.3) 
1 (6.3) 
1 (6.3) 
Bullous pemphigoid  
1 
(33.3) 
 
 
 
 
 
1 (6.3) 
 
Colitis 
 
 
 
 
1 (14.3) 
1 (14.3) 
1 (6.3) 
1 (6.3) 
Dermatitis 
acneiform 
 
 
1 (33.3) 
 
 
 
1 (6.3) 
 
Dyspnea 
exertional*** 
 
 
 
 
1 (14.3) 
 
1 (6.3) 
 
Hyperthyroidism 
 
 
 
 
1 (14.3) 
 
1 (6.3) 
 
Hypophosphatemia 
 
 
 
 
1 (14.3) 
 
1 (6.3) 
 
Hypothyroidism 
 
 
1 (33.3) 
 
1 (14.3) 
 
2 (12.5) 
 
Infusion-related 
reaction 
 
 
 
 
1 (14.3) 
 
1 (6.3) 
 
Keratoacanthoma 
 
 
 
 
1 (14.3) 
 
1 (6.3) 
 
Lipase increase 
 
 
 
 
1 (14.3) 
1 (14.3) 
1 (6.3) 
1 (6.3) 
Nausea 
1 
(33.3) 
 
 
 
 
 
1 (6.3) 
 
Pruritus 
1 
(33.3) 
 
 
 
 
 
1 (6.3) 
 
Rash maculo-
papular 
1 
(33.3) 
 
1 (33.3) 
 
 
 
2 (12.5) 
 
Skin infection 
1 
(33.3) 
1 
(33.3) 
 
 
 
 
1 (6.3) 
1 (6.3) 
Vomiting 
1 
(33.3) 
 
 
 
 
 
1 (6.3) 
 
**There were no treatment-related AEs in the 3 patients treated with 1 mg/kg M7824. 
***The differential for this dyspnea was pneumonitis vs. lymphangitic spread of disease (disease 
progression). 
 
The emergent immune-related AE (irAEs) associated with M7824 in more than 670 treated 
patients with different tumor types in Phase I studies is consistent with PD-(L)1 agents. The 
other important identified risks for M7824 are dermatological AEs related to TGFβ inhibition 
and include keratoacanthomas and cutaneous squamous cell carcinoma. These AEs were similar 
to those observed in Ferguson-Smith Syndrome, in which TGFβ is genetic mutated, as well as 
phase I studies with fresolimumab (a pan TGFβ inhibitor). (129, 130) In the EMR200647-001/ 
MS200647-0008 studies, these skin AEs were observed in approximately 7% of participants and 
were adequately managed via excision and some spontaneously regressed without requiring 


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treatment discontinuation. In the context of encouraging clinical activity in various advanced 
cancer types, these skin lesions were overall manageable and no new safety signals emerged in 
the EMR200647-001/MS200647-0008 studies compared with established therapies targeting PD-
(L)1 or TGFβ blockade. These lesions have not been a criterion for treatment discontinuation, 
but thus far have all spontaneously regressed following treatment discontinuation. 
After discussion among NCI investigators on multiple protocols using M7824, multiple bleeding 
events ranging from low grade gingival bleeding and epistaxis to more serious hemoptysis, GI 
bleeding and hematuria have been observed. Some of these events can be attributed to bleeding 
events related to cancer directly and others bleeding events can be attributed to colitis or cystitis 
which is a known toxicity of anti-PD-L1 agents including M7824. However, there remains the 
possibility that M7824 may increase the overall risk of bleeding in ways that may not be directly 
related to direct tumor bleeding or inflammatory bleeding events described with checkpoint 
inhibitors like M7824. It is hypothesized that this possible increased bleeding risk may be due to 
TGF beta inhibition which has an effect on angiogenesis; bleeding has also been observed in 
patients receiving M7824 and may be drug-related (e.g., gum bleeding, nose bleeds, coughing up 
blood, blood in their urine, or blood in the stool). Accordingly, patients will be closely monitored 
for mucosal bleeding (e.g., gum bleeding, nose bleeds, coughing up blood, blood in their urine, 
or blood in the stool). 
A preliminary analysis of QT data from about 130 triplicate ECGs in 19 subjects from 
EMR200647-001 dose escalation cohorts up to a dose of 20 mg/kg showed that M7824 has no 
effect on QTcF intervals or heart rate, with QT-concentration slope close to 0 (0.002 ms per 
μg/mL) with 90% CI including zero (-0.01 to 0.01). The predicted upper bound of the 2-sided 
90% CI of delta QTc at the average Cmax after the first 20 mg/kg dose (493 μg/mL) was 1.35 
msec. while the predicted upper bound of 90% CI of delta QTcF change from baseline at Cmax 
value was less than 5 ms. Additionally, nonclinical safety study revealed no identified 
proarrhythmic risk. 
14.3.4 Formulation and Preparation  
M7824 drug product is provided as a sterile liquid formulation and packaged at a 10mg/ml 
concentration in USP/Ph Eur type I 50R vials that are filled with drug product solution to allow 
an extractable volume of 60ml (600mg/60ml). The vials are closed with rubber stoppers in serum 
format complying with USP and Ph Eur with an aluminum crimp seal closure. Each single-use 
vial contains 600mg of M7824, formulated as 10mg/mL of active, 6% (w/v) Trehalose, 40mM 
NaCl, 5mM Methionine, 0.05% (w/v) Tween 20, 10 mM L Histidine at pH 5.5.  
The liquid formulation is diluted directly with 0.9% sodium chloride solution for injection. The 
estimated volumes of delivery are anticipate to be no more than 250mL. The verified 
concentration range in the infusion solution is 0.16mg/mL to 9.6 mg/mL.  
14.3.5 Stability and Storage  
M7824 drug product must be stored at 2°C to 8°C until use. The storage condition is based on 
data from ongoing long term stability studies with M7824. M7824 drug product stored at room 
(23°C to 27°C) or higher temperatures for extended periods of time might be subject to 
degradation. 
The chemical and physical in-use stability for the infusion solution of M7824 in 0.9% saline 
solution has been demonstrated for a total of 72 hours at room temperature. However, from a 


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microbiological point of view, the diluted solution should be used immediately and is not 
intended to be stored unless dilution has taken place in controlled and validated aseptic 
conditions. If not used immediately, in-use storage times and conditions prior to 
administration are the responsibility of the user. Do not freeze or shake the diluted solution. 
No other drugs should be added to the infusion containers containing M7824. 
14.3.6 Administration Procedures 
Administration of M7824 is described in Section 3.2.2.  
14.3.7 Incompatibilities   
No formal drug interaction studies have been conducted with M7824 in humans. 
14.4 T-DM1  
Please see package insert for T-DM1 (also known as Kadcyla and Ado-Trastuzumab Emtansine) 
available at the FDA website: www.accessdata.fda.gov   
14.4.1 Source / Acquisition and Accountability 
Ado-trastuzumab emtansine (also known as T-DM1 or Kadcyla) will be purchased by the site 
pharmacy from commercial sources. 
T-DM1 will be purchased from commercial sources by the site pharmacy and stored according to 
product labeling and standard site procedures. T-DM1 will be dispensed at the discretion of an 
investigator for administration to a study participant enrolled on the clinical trial. Disposal of 
expired product will be according to standard site procedures. 
14.4.2 Administration Procedures 
Administration of T-DM1 is described in Section 3.2.3. 
 
 
 
 
 
 


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15 REFERENCES  
1. 
Ryerson AB, Eheman CR, Altekruse SF, Ward JW, Jemal A, Sherman RL, et al. Annual 
Report to the Nation on the Status of Cancer, 1975-2012, featuring the increasing incidence of 
liver cancer. Cancer. 2016;122(9):1312-37. 
2. 
Kummar S, Gutierrez M, Gardner ER, Donovan E, Hwang K, Chung EJ, et al. Phase I 
trial of MS-275, a histone deacetylase inhibitor, administered weekly in refractory solid tumors 
and lymphoid malignancies. Clin Cancer Res. 2007;13(18 Pt 1):5411-7. 
3. 
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7-
30. 
4. 
Howlader N, Altekruse SF, Li CI, Chen VW, Clarke CA, Ries LA, et al. US incidence of 
breast cancer subtypes defined by joint hormone receptor and HER2 status. J Natl Cancer Inst. 
2014;106(5). 
5. 
Brenton JD, Carey LA, Ahmed AA, Caldas C. Molecular classification and molecular 
forecasting of breast cancer: Ready for clinical application? Journal of Clinical Oncology. 
2005;23(29):7350-60. 
6. 
Bauer KR, Brown M, Cress RD, Parise CA, Caggiano V. Descriptive analysis of estrogen 
receptor (ER)negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast 
cancer, the so-called triple-negative phenotype - A population-based study from the California 
Cancer Registry. Cancer. 2007;109(9):1721-8. 
7. 
Tutt A, Ellis P, Kilburn L, Gilett C, Pinder S, Abraham J, et al. The TNT trial: A 
randomized phase III trial of carboplatin (C) compared with docetaxel (D) for patients with 
metastatic or recurrent locally advanced triple negative or BRCA1/2 breast cancer 
(CRUK/07/012). Cancer Research. 2015;75(9):2. 
8. 
Iwamoto T, Booser D, Valero V, Murray JL, Koenig K, Esteva FJ, et al. Estrogen 
receptor (ER) mRNA and ER-related gene expression in breast cancers that are 1% to 10% ER-
positive by immunohistochemistry. J Clin Oncol. 2012;30(7):729-34. 
9. 
Landmann A, Farrugia DJ, Zhu L, Diego EJ, Johnson RR, Soran A, et al. Low Estrogen 
Receptor (ER)-Positive Breast Cancer and Neoadjuvant Systemic Chemotherapy: Is Response 
Similar to Typical ER-Positive or ER-Negative Disease? Am J Clin Pathol. 2018;150(1):34-42. 
10. 
Allred DC, Carlson RW, Berry DA, Burstein HJ, Edge SB, Goldstein LJ, et al. NCCN 
Task Force Report: Estrogen Receptor and Progesterone Receptor Testing in Breast Cancer by 
Immunohistochemistry. J Natl Compr Canc Netw. 2009;7 Suppl 6:S1-S21; quiz S2-3. 
11. 
Singh K, Tantravahi U, Lomme MM, Pasquariello T, Steinhoff M, Sung CJ. Updated 
2013 College of American Pathologists/American Society of Clinical Oncology (CAP/ASCO) 
guideline recommendations for human epidermal growth factor receptor 2 (HER2) fluorescent in 
situ hybridization (FISH) testing increase HER2 positive and HER2 equivocal breast cancer 
cases; retrospective study of HER2 FISH results of 836 invasive breast cancers. Breast Cancer 
Res Treat. 2016;157(3):405-11. 
12. 
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast 
cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. 
Science. 1987;235(4785):177-82. 
13. 
Swain SM, Baselga J, Kim SB, Ro J, Semiglazov V, Campone M, et al. Pertuzumab, 
trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N Engl J Med. 
2015;372(8):724-34. 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
108 
 
14. 
Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, et al. Lapatinib 
plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med. 2006;355(26):2733-
43. 
15. 
Cetin B, Benekli M, Dane F, Boruban C, Gumus M, Oksuzoglu B, et al. Lapatinib plus 
Capecitabine for HER2-Positive Advanced-Stage Breast Cancer in Elderly Women: Review of 
the Anatolian Society of Medical Oncology (ASMO) Experience. Breast Care (Basel). 
2013;8(1):67-70. 
16. 
Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, et al. Trastuzumab 
emtansine for HER2-positive advanced breast cancer. N Engl J Med. 2012;367(19):1783-91. 
17. 
Bartsch R, Wenzel C, Altorjai G, Pluschnig U, Rudas M, Mader RM, et al. Capecitabine 
and trastuzumab in heavily pretreated metastatic breast cancer. Journal of clinical oncology : 
official journal of the American Society of Clinical Oncology. 2007;25(25):3853-8. 
18. 
von Minckwitz G, du Bois A, Schmidt M, Maass N, Cufer T, de Jongh FE, et al. 
Trastuzumab beyond progression in human epidermal growth factor receptor 2-positive 
advanced breast cancer: a german breast group 26/breast international group 03-05 study. Journal 
of clinical oncology : official journal of the American Society of Clinical Oncology. 
2009;27(12):1999-2006. 
19. 
Di Lauro V, Torrisi E, Bidoli E, Quitadamo D, Cecco S, Veronesi A. Trastuzumab and 
Gemcitabine in Pretreated HER2 Overexpressing Metastatic Breast Cancer Patients: 
Retrospective Analysis of Our Series. J Oncol. 2012;2012:198412. 
20. 
Diéras V, Miles D, Verma S, Pegram M, Welslau M, Baselga J, et al. Trastuzumab 
emtansine versus capecitabine plus lapatinib in patients with previously treated HER2-positive 
advanced breast cancer (EMILIA): a descriptive analysis of final overall survival results from a 
randomised, open-label, phase 3 trial. Lancet Oncol. 2017;18(6):732-42. 
21. 
Katz D, Feldhamer I, Greenberg-Dotan S, Hammerman A. Duration of second-line T-
DM1 therapy: Is it associated with duration of first-line anti-Her2 therapy in metastatic HER2 + 
breast cancer? Journal of Clinical Oncology. 2017;35(15_suppl):e12512-e. 
22. 
Dzimitrowicz H, Berger M, Vargo C, Hood A, Abdelghany O, Raghavendra AS, et al. T-
DM1 Activity in Metastatic Human Epidermal Growth Factor Receptor 2-Positive Breast 
Cancers That Received Prior Therapy With Trastuzumab and Pertuzumab. J Clin Oncol. 
2016;34(29):3511-7. 
23. 
Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB, Denney DW, et al. Structure of 
the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature. 
2003;421(6924):756-60. 
24. 
Bianchini G, Gianni L. The immune system and response to HER2-targeted treatment in 
breast cancer. Lancet Oncol. 2014;15(2):e58-68. 
25. 
Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo 
cytotoxicity against tumor targets. Nat Med. 2000;6(4):443-6. 
26. 
Krop I, Winer EP. Trastuzumab emtansine: a novel antibody-drug conjugate for HER2-
positive breast cancer. Clin Cancer Res. 2014;20(1):15-20. 
27. 
Junttila TT, Li G, Parsons K, Phillips GL, Sliwkowski MX. Trastuzumab-DM1 (T-DM1) 
retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib 
insensitive breast cancer. Breast Cancer Res Treat. 2011;128(2):347-56. 
28. 
Müller P, Kreuzaler M, Khan T, Thommen DS, Martin K, Glatz K, et al. Trastuzumab 
emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade. 
Sci Transl Med. 2015;7(315):315ra188. 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
109 
 
29. 
Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase 
inhibitors. Nat Rev Drug Discov. 2006;5(9):769-84. 
30. 
Gameiro SR, Malamas AS, Tsang KY, Ferrone S, Hodge JW. Inhibitors of histone 
deacetylase 1 reverse the immune evasion phenotype to enhance T-cell mediated lysis of prostate 
and breast carcinoma cells. Oncotarget. 2016;7(7):7390-402. 
31. 
Huang X, Gao L, Wang S, Lee CK, Ordentlich P, Liu B. HDAC inhibitor SNDX-275 
induces apoptosis in erbB2-overexpressing breast cancer cells via down-regulation of erbB3 
expression. Cancer Res. 2009;69(21):8403-11. 
32. 
Yardley DA, Ismail-Khan RR, Melichar B, Lichinitser M, Munster PN, Klein PM, et al. 
Randomized phase II, double-blind, placebo-controlled study of exemestane with or without 
entinostat in postmenopausal women with locally recurrent or metastatic estrogen receptor-
positive breast cancer progressing on treatment with a nonsteroidal aromatase inhibitor. J Clin 
Oncol. 2013;31(17):2128-35. 
33. 
Huang X, Wang S, Lee CK, Yang X, Liu B. HDAC inhibitor SNDX-275 enhances 
efficacy of trastuzumab in erbB2-overexpressing breast cancer cells and exhibits potential to 
overcome trastuzumab resistance. Cancer Lett. 2011;307(1):72-9. 
34. 
Lim   B, Murthy   R, Jackson   S, Willey   J, Lee   J, Alvarez   R, et al. Open-label phase 
Ib study of entinostat (E), and lapatinib (L) alone, and in combination with trastuzumab (T) in 
patients (pts) with HER2+ metastatic (mHER2+) breast cancer after progression on 
trastuzumab.: J Clin Onc; 2011. p. 609. 
35. 
Trapani D, Esposito A, Criscitiello C, Mazzarella L, Locatelli M, Minchella I, et al. 
Entinostat for the treatment of breast cancer. Expert Opin Investig Drugs. 2017;26(8):965-71. 
36. 
Orillion A, Hashimoto A, Damayanti N, Shen L, Adelaiye-Ogala R, Arisa S, et al. 
Entinostat Neutralizes Myeloid-Derived Suppressor Cells and Enhances the Antitumor Effect of 
PD-1 Inhibition in Murine Models of Lung and Renal Cell Carcinoma. Clin Cancer Res. 
2017;23(17):5187-201. 
37. 
Gameiro SR, Hodge JW, Jochems C. Potential of HDAC Inhibitors for Combination 
Therapy with Immunotherapy Platforms. 2016. 
38. 
Tomita Y, Lee MJ, Lee S, Tomita S, Chumsri S, Cruickshank S, et al. The interplay of 
epigenetic therapy and immunity in locally recurrent or metastatic estrogen receptor-positive 
breast cancer: Correlative analysis of ENCORE 301, a randomized, placebo-controlled phase II 
trial of exemestane with or without entinostat. Oncoimmunology. 2016;5(11):e1219008. 
39. 
Johnson M, Adjei A, Ramalingam S, Janne P, Dominguez G, Gabrilovich D, et al. 
Preliminary results of ENCORE 601, a phase 1b/2, open-label study of entinostat (ENT) in 
combination with pembrolizumab (PEMBRO) in patients with non-small cell lung cancer 
(NSCLC). JCO; 2016. 
40. 
Christiansen AJ, West A, Banks KM, Haynes NM, Teng MW, Smyth MJ, et al. 
Eradication of solid tumors using histone deacetylase inhibitors combined with immune-
stimulating antibodies. Proc Natl Acad Sci U S A. 2011;108(10):4141-6. 
41. 
Hellman M, Janne P, M O, Hafez N, Raez L, Gabrilovich D, et al., editors. 
Efficacy/safety of entinostat and pembrolizymab in NSCLC patients previously treated with anti-
PD-(L)1 therapy. IASLC; 2018; Toronto, Canada. 
42. 
Agarwala S, Moschos S, Johnson M, Opyrchal M, Gabrilovich D, Danaher P, et al., 
editors. Efficacy and safety of entinostat and pembrolizumab in patients with melanoma 
progressing on or after PD-1/L1 blocking activity. ASCO; 2018; Chicago, IL. 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
110 
 
43. 
Dirix LY, Takacs I, Jerusalem G, Nikolinakos P, Arkenau HT, Forero-Torres A, et al. 
Avelumab, an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: 
a phase 1b JAVELIN Solid Tumor study. Breast Cancer Res Treat. 2017. 
44. 
Nanda R, Chow LQ, Dees EC, Berger R, Gupta S, Geva R, et al. Pembrolizumab in 
Patients With Advanced Triple-Negative Breast Cancer: Phase Ib KEYNOTE-012 Study. J Clin 
Oncol. 2016;34(21):2460-7. 
45. 
Rugo HS, Delord JP, Im SA, Ott PA, Piha-Paul SA, Bedard PL, et al. Safety and 
Antitumor Activity of Pembrolizumab in Patients with Estrogen Receptor-Positive/Human 
Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer. Clin Cancer Res. 
2018;24(12):2804-11. 
46. 
Loi S, Giobbie-Hurder A, Gombos A, Bachelot T, Hui R, Cuigliano G, et al., editors. 
Phase Ib/II Study Evaluating Safety and Efficacy of Pembrolizumab and Trastuzumab in Patients 
with Trastuzumab-Resistant HER2-positive Advanced Breast Cancer: Results from the 
PANACEA Study (IBCSG 45-13/BIG 4-13/KEYNOTE-014). San Antonio Breast Cancer 
Symposium; 2017; San Antonio, TX. 
47. 
Emens L, Esteva F, Beresford M, Saura C, De Laurentiis M, Kim S-B, et al. Results from 
KATE2, a randomized phase 2 study of atezolizumab (atezo)+trastuzumab emtansine (T-DM1) 
vs placebo (pbo)+T-DM1 in previously treated HER2+ advanced breast cancer (BC) [abstract]. 
In: Proceedings of the 2018 San Antonio Breast Cancer Symposium: Cancer Res; 2018. p. 
Abstract nr PD3-01. 
48. 
Jakowlew SB. Transforming growth factor-beta in cancer and metastasis. Cancer 
Metastasis Rev. 2006;25(3):435-57. 
49. 
Derynck R, Akhurst RJ, Balmain A. TGF-beta signaling in tumor suppression and cancer 
progression. Nat Genet. 2001;29(2):117-29. 
50. 
Zarzynska JM. Two faces of TGF-beta1 in breast cancer. Mediators Inflamm. 
2014;2014:141747. 
51. 
Tan AR, Alexe G, Reiss M. Transforming growth factor-beta signaling: emerging stem 
cell target in metastatic breast cancer? Breast Cancer Res Treat. 2009;115(3):453-95. 
52. 
Tian M, Schiemann WP. TGF-β Stimulation of EMT Programs Elicits Non-genomic ER-
α Activity and Anti-estrogen Resistance in Breast Cancer Cells. J Cancer Metastasis Treat. 
2017;3:150-60. 
53. 
Wang SE. The Functional Crosstalk between HER2 Tyrosine Kinase and TGF-β 
Signaling in Breast Cancer Malignancy. J Signal Transduct. 2011;2011:804236. 
54. 
Chow A, Arteaga CL, Wang SE. When tumor suppressor TGFβ meets the HER2 
(ERBB2) oncogene. J Mammary Gland Biol Neoplasia. 2011;16(2):81-8. 
55. 
Massagué J. TGFbeta in Cancer. Cell. 2008;134(2):215-30. 
56. 
Teicher BA. Transforming growth factor-beta and the immune response to malignant 
disease. Clin Cancer Res. 2007;13(21):6247-51. 
57. 
Reis ST, Pontes-Junior J, Antunes AA, Sousa-Canavez JM, Abe DK, Cruz JA, et al. Tgf-
beta1 expression as a biomarker of poor prognosis in prostate cancer. Clinics (Sao Paulo, Brazil). 
2011;66(7):1143-7. 
58. 
Bruna A, Darken RS, Rojo F, Ocana A, Penuelas S, Arias A, et al. High TGFbeta-Smad 
activity confers poor prognosis in glioma patients and promotes cell proliferation depending on 
the methylation of the PDGF-B gene. Cancer Cell. 2007;11(2):147-60. 
59. 
Gao N, Zhai Q, Li Y, Huang K, Bian D, Wang X, et al. Clinical Implications of TbetaRII 
Expression in Breast Cancer. PLoS One. 2015;10(11):e0141412. 


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111 
 
60. 
Colak S, Ten Dijke P. Targeting TGF-beta Signaling in Cancer. Trends Cancer. 
2017;3(1):56-71. 
61. 
Formenti SC, Lee P, Adams S, Goldberg JD, Li X, Xie M, et al. Focal Irradiation And 
Systemic Transforming Growth Factor β Blockade in Metastatic Breast Cancer. Clin Cancer Res. 
2018. 
62. 
Phan K, Tsai YC, Niranjan N, Bouchard D, Carrel TP, Dapunt OE, et al. Sutureless aortic 
valve replacement: a systematic review and meta-analysis. Ann Cardiothorac Surg. 
2015;4(2):100-11. 
63. 
Pyo JS, Kang G, Kim JY. Prognostic role of PD-L1 in malignant solid tumors: a meta-
analysis. Int J Biol Markers. 2017;32(1):e68-e74. 
64. 
Hu YG, Shen YF, Li Y. Effect of Pin1 inhibitor juglone on proliferation, migration and 
angiogenic ability of breast cancer cell line MCF7Adr. J Huazhong Univ Sci Technolog Med 
Sci. 2015;35(4):531-4. 
65. 
Wu P, Wu D, Li L, Chai Y, Huang J. PD-L1 and Survival in Solid Tumors: A Meta-
Analysis. PLoS One. 2015;10(6):e0131403. 
66. 
Weber JS, D'Angelo SP, Minor D, Hodi FS, Gutzmer R, Neyns B, et al. Nivolumab 
versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 
treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial. Lancet Oncol. 
2015;16(4):375-84. 
67. 
Postow MA, Chesney J, Pavlick AC, Robert C, Grossmann K, McDermott D, et al. 
Nivolumab and ipilimumab versus ipilimumab in untreated melanoma. N Engl J Med. 
2015;372(21):2006-17. 
68. 
Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, et al. Pembrolizumab 
versus Ipilimumab in Advanced Melanoma. N Engl J Med. 2015;372(26):2521-32. 
69. 
Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab 
versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med. 
2015;373(17):1627-39. 
70. 
Herbst RS, Baas P, Kim DW, Felip E, Perez-Gracia JL, Han JY, et al. Pembrolizumab 
versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer 
(KEYNOTE-010): a randomised controlled trial. Lancet. 2016;387(10027):1540-50. 
71. 
Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, et al. 
Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med. 
2015;373(19):1803-13. 
72. 
Seiwert TY, Burtness B, Mehra R, Weiss J, Berger R, Eder JP, et al. Safety and clinical 
activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of 
the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. Lancet Oncol. 
2016;17(7):956-65. 
73. 
Chow LQ, Haddad R, Gupta S, Mahipal A, Mehra R, Tahara M, et al. Antitumor Activity 
of Pembrolizumab in Biomarker-Unselected Patients With Recurrent and/or Metastatic Head and 
Neck Squamous Cell Carcinoma: Results From the Phase Ib KEYNOTE-012 Expansion Cohort. 
J Clin Oncol. 2016. 
74. 
Ferris RL, Blumenschein G, Jr., Fayette J, Guigay J, Colevas AD, Licitra L, et al. 
Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck. N Engl J Med. 
2016;375(19):1856-67. 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
112 
 
75. 
Kaufman HL, Russell J, Hamid O, Bhatia S, Terheyden P, D'Angelo SP, et al. Avelumab 
in patients with chemotherapy-refractory metastatic Merkel cell carcinoma: a multicentre, single-
group, open-label, phase 2 trial. Lancet Oncol. 2016;17(10):1374-85. 
76. 
Massard C, Gordon MS, Sharma S, Rafii S, Wainberg ZA, Luke J, et al. Safety and 
Efficacy of Durvalumab (MEDI4736), an Anti-Programmed Cell Death Ligand-1 Immune 
Checkpoint Inhibitor, in Patients With Advanced Urothelial Bladder Cancer. J Clin Oncol. 
2016;34(26):3119-25. 
77. 
Strauss J, Madan RA, Gulley JL. Considerations for the combination of anticancer 
vaccines and immune checkpoint inhibitors. Expert Opin Biol Ther. 2016;16(7):895-901. 
78. 
Melero I, Berman DM, Aznar MA, Korman AJ, Perez Gracia JL, Haanen J. Evolving 
synergistic combinations of targeted immunotherapies to combat cancer. Nat Rev Cancer. 
2015;15(8):457-72. 
79. 
Knudson K, Hicks K, Luo X, Chen J, J S, Gameiro S. M7824, a novel bifunctional anti-
PD-L1/TGFb TRAP fusion protein, promotes anti-tumor efficacy as monotherapy and in 
combination with vaccine. OncoImmunology. 2018(Epub ahead of print). 
80. 
Grenga I, Donahue RN, Gargulak ML, Lepone LM, Roselli M, Bilusic M, et al. Anti-PD-
L1/TGFβR2 (M7824) fusion protein induces immunogenic modulation of human urothelial 
carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis. 
Urol Oncol. 2018;36(3):93.e1-.e11. 
81. 
Jochems C, Tritsch SR, Pellom ST, Su Z, Soon-Shiong P, Wong HC, et al. Analyses of 
functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824). Oncotarget. 
2017;8(43):75217-31. 
82. 
Lacouture ME, Morris JC, Lawrence DP, Tan AR, Olencki TE, Shapiro GI, et al. 
Cutaneous keratoacanthomas/squamous cell carcinomas associated with neutralization of 
transforming growth factor beta by the monoclonal antibody fresolimumab (GC1008). Cancer 
Immunol Immunother. 2015;64(4):437-46. 
83. 
Strauss J, Heery C, Schlom J, Madan RA, Cao L, Kang Z, et al. Phase 1 trial of M7824 
(MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGF-β, in advanced solid 
tumors. Clin Cancer Res. 2018. 
84. 
Strauss J, Heery CR, Schlom J, Madan RA, Cao L, Kang Z, et al. Phase I Trial of M7824 
(MSB0011359C), a Bifunctional Fusion Protein Targeting PD-L1 and TGFβ, in Advanced Solid 
Tumors. Clin Cancer Res. 2018;24(6):1287-95. 
85. 
Palena C, Roselli M, Litzinger MT, Ferroni P, Costarelli L, Spila A, et al. Overexpression 
of the EMT driver brachyury in breast carcinomas: association with poor prognosis. J Natl 
Cancer Inst. 2014;106(5). 
86. 
Palena C, Hamilton DH. Immune Targeting of Tumor Epithelial-Mesenchymal 
Transition via Brachyury-Based Vaccines. Adv Cancer Res. 2015;128:69-93. 
87. 
Hamilton DH, Roselli M, Ferroni P, Costarelli L, Cavaliere F, Taffuri M, et al. 
Brachyury, a vaccine target, is overexpressed in triple-negative breast cancer. Endocr Relat 
Cancer. 2016;23(10):783-96. 
88. 
Palena C, Polev DE, Tsang KY, Fernando RI, Litzinger M, Krukovskaya LL, et al. The 
human T-box mesodermal transcription factor Brachyury is a candidate target for T-cell-
mediated cancer immunotherapy. Clin Cancer Res. 2007;13(8):2471-8. 
89. 
Heery CR, Palena C, McMahon S, Donahue RN, Lepone LM, Grenga I, et al. Phase I 
Study of a Poxviral TRICOM-Based Vaccine Directed Against the Transcription Factor 
Brachyury. Clin Cancer Res. 2017. 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
113 
 
90. 
Hamilton DH, Litzinger MT, Jales A, Huang B, Fernando RI, Hodge JW, et al. 
Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a 
recombinant brachyury-yeast vaccine. Oncotarget. 2013;4(10):1777-90. 
91. 
Heery CR, Singh BH, Rauckhorst M, Marte JL, Donahue RN, Grenga I, et al. Phase I 
Trial of a Yeast-Based Therapeutic Cancer Vaccine (GI-6301) Targeting the Transcription 
Factor Brachyury. Cancer Immunol Res. 2015;3(11):1248-56. 
92. 
Madan RA, Mohebtash M, Arlen PM, Vergati M, Rauckhorst M, Steinberg SM, et al. 
Ipilimumab and a poxviral vaccine targeting prostate-specific antigen in metastatic castration-
resistant prostate cancer: a phase 1 dose-escalation trial. Lancet Oncol. 2012;13(5):501-8. 
93. 
Vollmar J, Arndtz N, Eckl KM, Thomsen T, Petzold B, Mateo L, et al. Safety and 
immunogenicity of IMVAMUNE, a promising candidate as a third generation smallpox vaccine. 
Vaccine. 2006;24(12):2065-70. 
94. 
Frey SE, Newman FK, Kennedy JS, Sobek V, Ennis FA, Hill H, et al. Clinical and 
immunologic responses to multiple doses of IMVAMUNE (Modified Vaccinia Ankara) followed 
by Dryvax challenge. Vaccine. 2007;25(51):8562-73. 
95. 
von Krempelhuber A, Vollmar J, Pokorny R, Rapp P, Wulff N, Petzold B, et al. A 
randomized, double-blind, dose-finding Phase II study to evaluate immunogenicity and safety of 
the third generation smallpox vaccine candidate IMVAMUNE. Vaccine. 2010;28(5):1209-16. 
96. 
Beukema EL, Brown MP, Hayball JD. The potential role of fowlpox virus in rational 
vaccine design. Expert Rev Vaccines. 2006;5(4):565-77. 
97. 
Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, et al. The 
evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an 
International TILs Working Group 2014. Ann Oncol. 2015;26(2):259-71. 
98. 
Stanton SE, Adams S, Disis ML. Variation in the Incidence and Magnitude of Tumor-
Infiltrating Lymphocytes in Breast Cancer Subtypes: A Systematic Review. JAMA Oncol. 2016. 
99. 
Loi S, Sirtaine N, Piette F, Salgado R, Viale G, Van Eenoo F, et al. Prognostic and 
predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast 
cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin 
with doxorubicin-based chemotherapy: BIG 02-98. Journal of clinical oncology : official journal 
of the American Society of Clinical Oncology. 2013;31(7):860-7. 
100. 
Dieci MV, Mathieu MC, Guarneri V, Conte P, Delaloge S, Andre F, et al. Prognostic and 
predictive value of tumor-infiltrating lymphocytes in two phase III randomized adjuvant breast 
cancer trials. Ann Oncol. 2015;26(8):1698-704. 
101. 
Loi S, Michiels S, Salgado R, Sirtaine N, Jose V, Fumagalli D, et al. Tumor infiltrating 
lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab 
benefit in early breast cancer: results from the FinHER trial. Ann Oncol. 2014;25(8):1544-50. 
102. 
Adams S, Gray RJ, Demaria S, Goldstein L, Perez EA, Shulman LN, et al. Prognostic 
value of tumor-infiltrating lymphocytes in triple-negative breast cancers from two phase III 
randomized adjuvant breast cancer trials: ECOG 2197 and ECOG 1199. Journal of clinical 
oncology : official journal of the American Society of Clinical Oncology. 2014;32(27):2959-66. 
103. 
Ibrahim EM, Al-Foheidi ME, Al-Mansour MM, Kazkaz GA. The prognostic value of 
tumor-infiltrating lymphocytes in triple-negative breast cancer: a meta-analysis. Breast Cancer 
Res Treat. 2014;148(3):467-76. 
104. 
Mao Y, Qu Q, Chen X, Huang O, Wu J, Shen K. The Prognostic Value of Tumor-
Infiltrating Lymphocytes in Breast Cancer: A Systematic Review and Meta-Analysis. PLoS One. 
2016;11(4):e0152500. 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
114 
 
105. 
García-Teijido P, Cabal ML, Fernández IP, Pérez YF. Tumor-Infiltrating Lymphocytes in 
Triple Negative Breast Cancer: The Future of Immune Targeting. Clin Med Insights Oncol. 
2016;10(Suppl 1):31-9. 
106. 
Luen SJ, Salgado R, Fox S, Savas P, Eng-Wong J, Clark E, et al. Tumour-infiltrating 
lymphocytes in advanced HER2-positive breast cancer treated with pertuzumab or placebo in 
addition to trastuzumab and docetaxel: a retrospective analysis of the CLEOPATRA study. 
Lancet Oncol. 2017;18(1):52-62. 
107. 
Roulois D, Loo Yau H, Singhania R, Wang Y, Danesh A, Shen SY, et al. DNA-
Demethylating Agents Target Colorectal Cancer Cells by Inducing Viral Mimicry by 
Endogenous Transcripts. Cell. 2015;162(5):961-73. 
108. 
Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-Lieskovan S, et 
al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J 
Med. 2016;375(9):819-29. 
109. 
Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. Mismatch repair 
deficiency predicts response of solid tumors to PD-1 blockade. Science. 2017;357(6349):409-13. 
110. 
Li X, Gruosso T, Zuo D, Omeroglu A, Meterissian S, Guiot MC, et al. Infiltration of 
CD8. Proc Natl Acad Sci U S A. 2019;116(9):3678-87. 
111. 
Heery CR, Donahue R, Lepone L, Grenga I, Richards J, Metenou S, et al. Phase I, dose-
escalation, clinical trial of MVA-Brachyury-TRICOM vaccine demonstrating safety and 
brachyury-specific T cell responses.: J ImmunoTherapy of Cancer; 2015. p. P132. 
112. 
Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, et al. PD-1 blockade 
in tumors with mismatch-repair deficiency. N Engl J Med. 2015;372(26):2509-20. 
113. 
Strauss J, Heery C, Schlom J, Madan RA, Cao L, Kang Z, et al. Phase 1 trial of M7824 
(MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGF-beta, in advanced solid 
tumors. Clin Cancer Res. 2018. 
114. 
Hicks KC, Fantini M, Donahue RN, Schwab A, Knudson KM, Tritsch SR, et al. 
Epigenetic priming of both tumor and NK cells augments antibody-dependent cellular 
cytotoxicity elicited by the anti-PD-L1 antibody avelumab against multiple carcinoma cell types. 
Oncoimmunology. 2018;7(11):e1466018. 
115. 
Christmas BJ, Rafie CI, Hopkins AC, Scott BA, Ma HS, Cruz KA, et al. Entinostat 
Converts Immune-Resistant Breast and Pancreatic Cancers into Checkpoint-Responsive Tumors 
by Reprogramming Tumor-Infiltrating MDSCs. Cancer Immunol Res. 2018;6(12):1561-77. 
116. 
Bauzon M, Drake P, Barfield R, Cornali B, Rupniewski I, Rabuka D. Maytansine-bearing 
antibody-drug conjugates induce in vitro hallmarks of immunogenic cell death selectively in 
antigen-positive target cells. OncoImmunology; 2019. 
117. 
Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. 
Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: 
American Society of Clinical Oncology/College of American Pathologists clinical practice 
guideline update. Arch Pathol Lab Med. 2014;138(2):241-56. 
118. 
Ryan QC, Headlee D, Acharya M, Sparreboom A, Trepel JB, Ye J, et al. Phase I and 
pharmacokinetic study of MS-275, a histone deacetylase inhibitor, in patients with advanced and 
refractory solid tumors or lymphoma. J Clin Oncol. 2005;23(17):3912-22. 
119. 
Heery CR, Donahue R, Lepone L, Grenga I, Richards J, Metenou S, et al. Phase I, dose-
escalation, clinical trial of MVA-Brachyury-TRICOM vaccine demonstrating safety and 
brachyury-specific T cell responses. J Immunother Cancer. 2015;3(Supp 2):P132. 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
115 
 
120. 
Lepone LM, Donahue RN, Grenga I, Metenou S, Richards J, Heery CR, et al. Analysis of 
123 peripheral human immune cell subsets: defining differences with age and between healthy 
donors and cancer patients not detected in analysis of standard immune cell types. J Circ 
Biomark. In press. 
121. 
Chung EJ, Lee S, Sausville EA, Ryan Q, Karp JE, Gojo I, et al. Histone deacetylase 
inhibitor pharmacodynamic analysis by multiparameter flow cytometry. Ann Clin Lab Sci. 
2005;35(4):397-406. 
122. 
Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. 
Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: 
American Society of Clinical Oncology/College of American Pathologists clinical practice 
guideline update. J Clin Oncol. 2013;31(31):3997-4013. 
123. 
Hamilton DH, Fernando RI, Schlom J, Palena C. Aberrant expression of the embryonic 
transcription factor brachyury in human tumors detected with a novel rabbit monoclonal 
antibody. Oncotarget. 2015;6(7):4853-62. 
124. 
Miettinen M, Wang Z, Lasota J, Heery C, Schlom J, Palena C. Nuclear Brachyury 
Expression Is Consistent in Chordoma, Common in Germ Cell Tumors and Small Cell 
Carcinomas, and Rare in Other Carcinomas and Sarcomas: An Immunohistochemical Study of 
5229 Cases. Am J Surg Pathol. 2015;39(10):1305-12. 
125. 
Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New 
response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J 
Cancer. 2009;45(2):228-47. 
126. 
Simon R. Controlled Clinical Trials. 1989. p. 1-10. 
127. 
Connolly RM, Rudek MA, Piekarz R. Entinostat: a promising treatment option for 
patients with advanced breast cancer. Future Oncol. 2017;13(13):1137-48. 
128. 
Vugmeyster Y, Wilkins   J, Harrison-Moench   E, Geng   W, Koenig   A, Cao   L, et al., 
editors. Selection of the recommended phase 2 dose (RP2D) for M7824 (MSB0011359C), a 
bifunctional fusion protein targeting TGF-βand PD-L1. . ASCO; 2018; Chicago, IL: J Clin Onc 
36: 15_Suppl: Abstr 2566. 
129. 
Goudie DR, D'Alessandro M, Merriman B, Lee H, Szeverenyi I, Avery S, et al. Multiple 
self-healing squamous epithelioma is caused by a disease-specific spectrum of mutations in 
TGFBR1. Nat Genet. 2011;43(4):365-9. 
130. 
Morris JC, Tan AR, Olencki TE, Shapiro GI, Dezube BJ, Reiss M, et al. Phase I study of 
GC1008 (fresolimumab): a human anti-transforming growth factor-beta (TGFβ) monoclonal 
antibody in patients with advanced malignant melanoma or renal cell carcinoma. PLoS One. 
2014;9(3):e90353. 
 


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


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16.2 APPENDIX B: ORAL MEDICATION DIARY (ARM 3 ONLY) 
Oral Medication Diary for Entinostat 
Patient’s Name: __________________________________  
Date: __________________ 
Patient Study ID: _________________________________  
Cycle #: ________________ 
INSTRUCTIONS TO THE PATIENT:  
1. Complete one form for each cycle (21 days). 
2. You will take Entinostat by mouth each Tuesday (white spaces). You may take the capsules one hour before or 
two hours after eating, as you prefer. If you forget to take the pills on your assigned day, you can take the missed 
dose up to 1 day after (light gray space). Do not take the weekly dose if more than 1 day late after scheduled dose 
(dark gray spaces).   
3. Record the date, the number of pills you took, and when you took them. 
4. If you have any comments or notice any side effects, please record them in the Comments column. 
5. Please bring your pill bottle and this form to your physician when you go for your next appointment.  
General Comments: 
______________________________________________________________________________
______________________________________________________________________________  
Day 
# pills and when taken: 
Entinostat 
 
Comments 
Day 
# pills and when taken: 
Entinostat 
 
Comments 
 Date  # Capsules 
Time  
 Date  # Capsules 
Time  
1  
 
 
 
 
15   
 
 
 
2  
 
 
 
 
16   
 
 
 
3  
 
 
 
 
17   
 
 
 
4  
 
 
 
 
18   
 
 
 
5  
 
 
 
 
19   
 
 
 
6  
 
 
 
 
20   
 
 
 
7  
 
 
 
 
21   
 
 
 
8  
 
 
 
 
22  
Next Cycle 
9  
 
 
 
 
23  
10   
 
 
 
24  
11   
 
 
 
25  
12   
 
 
 
26  
13   
 
 
 
27  
14   
 
 
 
28  
Patient’s Signature: ___________________________________Date: ____________________ 
 
 


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Study Team will complete this section:  
1. Date patient started protocol treatment_____________________  
2. Date patient was removed from study _____________________  
3. Patient’s planned daily dose______________________________  
4. Total number of pills taken this cycle ______________________  
Physician/Nurse’s Signature ____________________________________________________________________   
 
 
 


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16.3 APPENDIX C: LTIB SOP ON HUMAN CORE BIOPSY PROCESSING FOR IHC/IF 
PROCESSING  
1. Using sterile forceps and blade, process the tissue immediately after being collected by 
Interventional Radiology by cutting it in different pieces, as needed. Your time frame 
prior to freezing is maximum of 20 min. 
2. Immediately embed each tissue piece in a labeled & dated plastic capsule containing 
OCT medium (TissueTek #4583), free of air bubbles. Cover the entire tissue with extra 
OCT. 
3. Transfer the OCT/core samples to a liquid nitrogen bath and cover with a lid. After 10-15 
min, transfer the frozen specimens to the vapor phase of a liquid nitrogen tank. 


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16.4 APPENDIX D: CLINICAL AND SAFETY PROFILE OF MVA-BN AND RECOMBINANT MVA-
BASED VACCINES  
MVA-BN Vector Backbone  
To date, 19 clinical trials (13 sponsored by Bavarian Nordic [BN] and 6 sponsored by the 
Division of Microbiology and Infectious Diseases [DMID], National Institute of Allergy and 
Infectious Diseases [NIAID], the National Institutes of Health [NIH]) evaluating the safety and 
immunogenicity of MVA-BN have been completed. Currently 2 BN sponsored clinical trials are 
ongoing.  
In total, for MVA-BN and MVA-BN-based recombinant vaccines, the exposure sums up to more 
than 9,600 subjects (more than 7,700 with MVA-BN, more than 400 with recombinant vaccines 
other than MVA-BN Filo, and more than 1,500 with MVA-BN-Filo), having received more than 
15,000 single doses of vaccine. For more details, refer to the current IB of MVA-BN-
BRACHYURY vaccine.  
16.4.1 Safety Overview of MVA-BN and Recombinant MVA-based Vaccines 
In all completed and ongoing clinical trials, vaccinations with MVA-BN have shown to be 
generally safe and well tolerated. No cases of death, assessed as being even possibly related, 
have been reported for a subject in a clinical trial using MVA-BN. Results obtained from 
completed Phase 1 and 2 trials and ongoing trials with several recombinant MVA-BN based 
vaccines in healthy adults and children, HIV infected, and cancer subjects demonstrate a similar 
safety profile as MVA-BN alone. Additional information on the safety profile of MVA-BN and 
recombinant MVA-based vaccines is provided in the Investigator’s Brochure.  
Adverse Drug Reactions  
The following Table 18 summarizes the pooled Adverse Drug Reaction (ADR) data of all 
completed MVA-BN trials. The safety profile of each of the trials with recombinant MVA-BN- 
based vaccines is comparable to the safety profile observed with MVA-BN trials as displayed in 
Table 19 as the occurrence of the ADRs is considered to be a reaction to the vector rather than 
the insert, based on previous experience with recombinant MVA-BN vaccine candidates.  
Table 18: Suspected Adverse Drug Reactions Reported by ≥ 1% of Subjects in Completed MVA-
BN Clinical Trials* (N = 7,535 subjects) 
Preferred Term (PT)  
No. of reports by subjects 
Frequency (%) 
Injection site pain  
6,201 
82.3 
Injection site erythema  
4,920 
65.3 
Injection site swelling  
3,401 
45.1 
Injection site induration  
3,253 
43.2 
Injection site induration (solicited)  
3,248 out of 7,013 
46.3 
Injection site induration (unsolicited)  
5 out of 521 
1.0. 
Injection site pruritus  
2,856 
37.9 
Injection site pruritus (solicited)  
2,645 out of 6,339 
41.7 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
121 
 
Preferred Term (PT)  
No. of reports by subjects 
Frequency (%) 
Injection site pruritus (unsolicited)  
211 out of 1,213 
17.4 
Fatigue  
2,121 
28.1 
Fatigue (solicited) +  
2,121 out of 6,339 
33.5 
Myalgia  
2,444 
32.4 
Myalgia (solicited)  
2,443 out of 7,448 
32.8 
Myalgia (unsolicited)  
1 out of 86 
1.2 
Headache  
2207 
29.3 
Headache (solicited)  
2,176 out of 7,013 
31.0 
Headache (unsolicited)  
31 out of 521 
6.0 
Nausea  
1,073 
14.2 
Nausea (solicited)  
1,072 out of 7,448 
14.4 
Nausea (unsolicited)  
1 out of 86 
1.2 
Rigors/chills  
664 
8.8 
Rigors/chills (solicited)  
663 out of 6,732 
9.8 
Rigors/chills (unsolicited)  
1 out of 802 
0.1 
Body temperature increased  
269 
3.6 
Injection site discoloration  
190 
2.5 
Appetite disorder  
218 
2.9 
Appetite disorder (solicited)++ 
218 out of 1,801 
12.1 
Injection site nodule  
149 
2.0 
Pain in extremity  
147 
2.0 
Pain in extremity (solicited)  
139 out of 1,346 
10.3 
Pain in extremity (unsolicited)  
8 out of 6,188 
0.1 
Arthralgia  
206 
2.7 
Injection site hematoma 
107 
1.4 
Pyrexia 
94 
1.2 
Axillary pain 
91 
1.2 
* POX-MVA-001, -002, -004, -005, -007, -008, -009, -010, -011, -013, -023, -024, -027, -028, -029, -030, -036, HIV-NEF-004 and HIV- POL-
002; 7 subjects in POX-MVA-009 received Dryvax® either on the same day or within 7 days after MVA-BN administration and were therefore 
not included to avoid a potential bias in the adverse event reporting. + not in POX-MVA-001; ++ only in NIH trials  
Looking only at the events that were reported by at least 1% of subjects, the majority of ADRs 
represented local vaccination site reactions as well as common systemic reactions typical for 
modern injectable vaccines and were classified as being mild to moderate in intensity and 
resolved completely without intervention within the first 7 days following vaccination. To date, 
no trends have been identified suggesting the occurrence of any particular unexpected adverse 
reactions or classes of adverse reactions following vaccinations with MVA-BN.  
Cardiac Signs and Symptoms  
Based on observations with replicating smallpox vaccines particular attention has been placed on 
monitoring for cardiac signs and symptoms in all clinical trials using MVA-BN. Despite close 
cardiac monitoring, no confirmed event indicating a case of myo-/pericarditis has been observed 
in any completed MVA-BN trial.  


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
122 
 
Serious Suspected Adverse Drug Reactions  
As of 31 July 2016, a total of 7 (7 out of 7,758 vaccinated subjects = 0.09%) serious suspected 
ADRs have been reported for MVA-BN smallpox vaccine in completed and ongoing trials 
(Table 18).  
All of them have been thoroughly reviewed by BN and the trial specific Data Safety Monitoring 
Board who concluded that the continued use of MVA-BN in a clinical setting presented no 
special risks to the subjects. No pattern regarding Serious Adverse Drug Reactions (SADRs) 
could be detected.  
Table 19: Serious Suspected Adverse Drug Reactions (Assessed by the Investigator to Be At Least 
Possibly Related to MVA-BN) 
Trial 
Code  
Age/ 
Gender 
Days After 
Vaccination  Event  
Outcome  Underlying Diseases/ 
Circumstances  
PI 
Assess- 
ment  
BN 
Opinion 
POX- 
MVA 
-005  
30/ 
Male  
70 days after 
second 
vaccination  
Sarcoidosis  
Stable and 
asympto- 
matic  
Urinary tract infection with 
Chlamydia trachomatis at time of 
first symptoms (arthralgia)  
Possibly 
related  
Possibly 
related  
POX- 
MVA 
-005  
31/ 
Female  
26 months 
after second 
vaccination  
Crohn’s disease  
Stable and 
asympto- 
matic 
under 
therapy  
Abnormal lab results (elevated 
alkaline phosphatase, absolute 
neutrophils and platelet counts) at 
screening for 2-year follow-up 
POX-MVA-023 (excluded) 
Possibly 
related  
Possibly 
related  
POX- 
MVA 
-008  
28/ 
Female  
8 days after 
second 
vaccination  
Transitory ocular 
muscle paresis  
Resolved 
without 
sequelae  
No relevant medical history  
Probably 
related  
Possibly 
related  
POX- 
MVA 
-010  
30/ 
Female  
133 days 
after second 
vaccination  
Congestive heart 
failure due to cardio- 
myopathy  
Stable 
under 
cardiac 
medi- 
cations  
Surgery for ventricular septal 
defect as child. HIV infection. 
Concomitant (denied, therefore 
previously unknown to BN) 
participation in a Growth-
Hormone Releasing Hormone 
(GH- RH) trial; event also 
assessed as possibly related to 
GH-RH  
Possibly 
related  
Unlikely 
related  
POX- 
MVA 
-011  
39/- 
Female  
1 day after 
second 
vaccination  
Simple pneumonia 
and pleurisy  
Resolved 
without 
sequelae  
HIV infection (CD4 count 4 
weeks prior to second vaccination 
was 299 cells/μL). History of 
chronic obstructive pulmonary 
disease. Acute sinusitis and nasal 
congestion due to swimmer’s ear 
which triggered hospital 
admittance.  
Possibly 
related  
Unlikely 
related  
POX- 
MVA 
-036  
27/ 
Female  
0 days after 
second 
vaccination  
Throat tightness and 
other 
hypersensitivity 
symptoms such as 
hives, pruritus, 
tender vaccination 
site, swollen axilla, 
Resolved 
without 
sequelae  
The subject received her second 
dose of MVA-BN 21 days after 
the first dose and after 2 hours 
developed symptoms such as skin 
reactions and throat tightness 
which was responsive to 
epinephrine treatment. She had 
no wheezing and was not 
Possibly 
related  
Possibly 
related  


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
123 
 
Trial 
Code  
Age/ 
Gender 
Days After 
Vaccination  Event  
Outcome  Underlying Diseases/ 
Circumstances  
PI 
Assess- 
ment  
BN 
Opinion 
angioedema of 
forearms  
hypotensive. Symptoms subsided 
after several days under 
prednisone and diphenhydramine 
treatment. She has a family 
history of allergies and a medical 
history of shingles. She has 
received multiple vaccines before 
but never had previous hives or 
other problems with vaccines. 
POX- 
MVA 
-036  
30/Male 
117 days 
after first 
vaccination  
Non-ST-segment 
elevation myocardial 
infarction  
Resolved 
without 
sequelae  
Positive family history for 
cardiovascular diseases (both 
grandfathers had myocardial 
infarctions in their 50ies, father 
had blood clots), as well as 
overweight with a BMI above 33. 
A few days before event onset, 
subject returned from a trip to 
India with diarrhea and was 
started on ciprofloxacin treatment 
(which per US prescribing 
information is associated with 
angina pectoris and myocardial 
infarction). He showed chest pain 
and increased troponin I, but no 
ST segment changes in the ECG 
and no coronary artery disease in 
cardiac catheterization. A post- 
infectious myocarditis (published 
case reports exist for 
campylobacter, shigella, and 
salmonella) was considered as 
alternative etiology for the 
reported event.  
Possibly 
related  
Unlikely 
related  
16.4.2 Safety Profile of MVA-BN Smallpox Vaccine in Healthy Population Compared to 
Special Populations  
BN has evaluated the safety and immunogenicity of MVA-BN-based recombinant vaccines for 
several indications such as cancer, HIV and measles in more than 1,900 subjects including 
healthy and HIV infected populations. In recombinant MVA vaccine trials, doses up to 5 x 10e8 
TCID50 were administered applying varying schedules of repeated vaccinations, e.g. a 3-dose 
schedule was used for recombinant HIV vaccines and multiple vaccinations have also been 
performed in subjects receiving a recombinant therapeutic breast cancer vaccine (MVA-BN- 
HER2). Results obtained from these Phase 1 and 2 trials demonstrate a similar safety profile and 
vector immunogenicity as compared to MVA-BN alone.  
Two of the recombinant vaccine trials allowed for a direct comparison with MVA-BN as an 
active control. These trials (HIV-POL-002 and HIV-NEF-004) were performed to evaluate 2 
different recombinant MVA-based HIV vaccine candidates in HIV-infected subjects. In both 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
124 
 
trials, a total of 3 vaccinations with either the recombinant HIV vaccine or MVA-BN were 
performed according to a 0, 8 and 16 week regimen.  
16.4.3 Immunogenicity Overview of MVA-BN  
MVA-BN was tested for safety and immunogenicity among healthy volunteers in 3 Phase 1 and 
2 dose finding trials. (93-95) Across these trials a linear dose relationship was observed between 
the vaccine doses and both vaccinia ELISA and Plaque Reduction Neutralization Test (PRNT) 
titers. Maximum ELISA seroconversion rates and peak titers were reached 2 weeks after the 
second vaccination, with 100% seroconversion after the second dose for all dose groups 
receiving at least 2 x 107 TCID50 per 0.5 mL dose of MVA- BN. Statistical analysis indicated 
lower doses to be inferior to the standard dose tested throughout all dose ranging trials, whereas 
the standard dose achieved ELISA seroconversion rates between 81% and 100% already after the 
first dose. For the PRNT, the same trend was observed with about 77% seroconversion rates 2 
weeks after the second MVA-BN administration in all groups receiving the highest dose.  
The early onset of seroconversion and the higher titers of total and neutralizing antibodies 
combined with an excellent safety profile qualified the dose of at least 5 x 107 TCID50 as the most 
suitable human dose. The final optimal (standard) dose and schedule for the general population 
was decided to be 2 doses of at least 5 x 107 TCID50 MVA-BN administered subcutaneously 
(s.c.) 2-4 weeks apart.  
 
 
 
 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
125 
 
16.5 APPENDIX E: CCR REPORTABLE EVENT FORM (REF) 
NCI Protocol #: Click or tap here to enter text. 
 
Protocol Title: Click or tap here to enter text. 
Report version: (select one) 
☐ 
Initial Report  
☐ 
Follow-up  
Site Principal Investigator:  Click or tap here to enter text. 
  
Date site PI was notified of the 
problem: Click or tap to enter a date.    
Date of problem:  
Click or tap to enter a date. 
If delay in reporting to the coordinating center, please explain: 
Click or tap here to enter text. 
Location of problem: (e.g.,  patient’s home, doctor’s office ) 
Click or tap here to enter text. 
 
Description of Subject 
Does this problem apply to a subject? 
☐ yes 
☐ not applicable (more than one subject is involved) 
 
If yes, enter details below: 
Subject ID: Click or tap here to enter text.  (do not use medical record number) 
Sex:  ☐Male  ☐ Female 
 
Age: Click or tap here to enter text.  
Diagnosis: Click or tap here to enter text. 
 
Name the problem: (select all that apply)   
☐ Specimen collection issue   
☐ Informed consent issue   
☐ Ineligible for enrollment   
☐ Breach of PII  
☐ Other, briefly state the nature of the problem: Click or tap here to enter text. 
 
Detailed Description of the problem: (Include any relevant treatment, outcomes or 
pertinent history): Click or tap here to enter text. 
 


Abbreviated Title: The BrEAsT Trial   
Version Date: 07.06.2021 
 
126 
 
What are you reporting? 
☐ 
unanticipated problem 
☐ 
death 
☐ 
non-compliance (other than a protocol deviation) 
☐ 
protocol deviation 
☐ 
new information that might affect the willingness of subjects to enroll or 
continue     participation in this study 
 
If interventional or expanded access study, please answer the following questions 
about your site: 
 
How many participants are still receiving the study intervention?  
Click or tap here to enter text. 
 
How many participants completed study interventions but remain in follow up? 
Click or tap here to enter text. 
 
How many participants are enrolled but not yet receiving study interventions? 
Click or tap here to enter text. 
Have similar problems occurred on this protocol at your site? 
☐ Yes      ☐ No 
Describe what steps you have already taken or will be taking as a result of this 
problem: 
Click or tap here to enter text. 
 
INVESTIGATOR’S SIGNATURE:  
 
DATE:   
 
 
 

ICF_001.pdf: 
PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 1 of 24
CC ICF template v. 12.01.20
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
PRINCIPAL INVESTIGATOR: Fatima Karzai, MD
STUDY TITLE: A Phase Ib Trial of Sequential Combinations of BN-Brachyury, Entinostat, ado-
trastuzumab emtansine and M7824 in Advanced Stage Breast Cancer (BrEAsT)
STUDY SITE: NIH Clinical Center
Cohort: Affected Participants 
Consent Version:  07/06/2021
WHO DO YOU CONTACT ABOUT THIS STUDY? 
Principal Investigator:  Fatima Karzai, MD 
Phone: 301-480-7174
Email: fatima.karzai@nih.gov 
KEY INFORMATION ABOUT THIS RESEARCH 
This consent form describes a research study and is designed to help you decide if you would 
like to be a part of the research study.  
You are being asked to take part in a research study at the National Institutes of Health (NIH). 
This section provides the information we believe is most helpful and important to you in making 
your decision about participating in this study. Additional information that may help you make 
a decision can be found in other sections of the document. Taking part in research at the NIH is 
your choice.
You are being asked to take part in this study because you have been diagnosed with metastatic 
breast cancer, such as Triple Negative Breast Cancer (TNBC) or ER-/PR-/HER2+ Breast Cancer 
(HER2+BC).
The purpose of this research study is to determine if a combination of immunotherapy drugs is 
effective in patients with metastatic breast cancer. Everyone on the study will receive the BN 
Brachyury vaccine and M7824. T-DM1 with or without Entinostat may be added in participants 
with HER2+ BC. These drugs are designed to help the immune system fight cancer.
T-DM1 has been approved by the FDA specifically for the treatment of HER2-positive 
metastatic breast cancer (mBC) in patients who previously received trastuzumab and a taxane 
(paclitaxel or docetaxel), separately or in combination. The use of BN-Brachyury, Entinostat, 
and M7824 in this study is considered investigational, which means that the use of this 
combination has not been approved by the U.S. Food and Drug Administration (FDA) to treat 
metastatic breast cancer. However, the FDA has given us permission to use the combination of 
BN-Brachyury, Entinostat, M7824, and T-DM1 in this study.
There are other drugs that may be used to treat your disease, and these can be prescribed/given 
by your regular cancer doctor, even if you are not in this study. 
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 2 of 24
CC ICF template v. 12.01.20
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
If you decide to join this study, here are some of the most important things that you should know 
that will happen:
•
We will first perform some tests and procedures to find out if you meet the study 
requirements. We will also confirm the status of your disease. You must have already 
received treatment for this cancer, for some groups you may need to have already 
received treatment for this cancer with a taxane (docetaxel or paclitaxel), Herceptin, and 
pertuzumab.
•
Study treatments will be given based on your type of cancer and when you enroll in the 
protocol.  The three possible treatment groups or arms are:
o
Arm 1: (TNBC) BN brachyury + M7824
o
Arm 2 (HER2+ BC): BN brachyury + M7824 + T-DM1
o
Arm 3 (HER2+ BC): BN brachyury + M7824 + T-DM1 + Entinostat
•
The first 6 participants with TNBC will be directly assigned to Arm 1. If there are no 
intolerable side effects in this group, we will begin to enroll patients with HER2+ BC 
for Arm 2. We will continue to enroll TBNC participants into group 1 until 8-13 patients 
are enrolled. After 6 HER2+ patients have been tested for side effects in Arm 2, the next 
6 will be enrolled into Arm 3. Once it is known that the combination in Arm 3 is safe, 
the remaining HER2 patients will be enrolled by direct alternating assignment to Arm 2 
or Arm 3. An equal number of participants will be assigned to Arms 2 and 3. 
•
You will receive treatment for as long as you are benefiting from it. Each treatment will 
be given in the Oncology Outpatient Center and will take approximately 2-4 hours. This 
allows time to give the treatment and monitor you before and after.
o
BN-Brachyury is a combination of MVA-BN Brachyury and FPV-Brachyury 
vaccines. The vaccines will be given once every three weeks, on the same day 
you receive M7824 and, if you are assigned, T-DM1. After cycle 9, BN-
Brachyury vaccines will be given to you every 12 weeks. 
o
MVA-BN Brachyury will be given in four injections under the skin at different 
sites on your body. You will only receive this vaccine during your first two 
treatments. 
o
FPV-Brachyury will be given in a single injection under the skin each cycle, 
starting with cycle 3. 
o
M7824 will be given by intravenous (IV) infusion (a needle inserted into a vein 
allowing the solution to be given over a period of time) once every three weeks 
for up to 12 months. The infusion will take approximately one hour, but could 
take more or less time. 
o
If you are assigned to Arm 1, there will be an observation time period 
where you will be monitored for up to an additional 60 minutes after 
your M7824 infusion is complete. 
o
T-DM1 will be given by IV infusion once every three weeks. The first infusion 
will take approximately 90 minutes and later infusions will take approximately 
30 minutes, but these could take more or less time. 
o
If you are assigned to Arm 2 or 3, there will be an observation time period 
where you will be monitored for up to an additional 90 minutes after your T-
DM1 infusion is complete as recommended by the FDA. 
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 3 of 24
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MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
o
Entinostat will be taken by mouth, in tablet form, on a weekly basis.  On the days 
when you receive infusions, this will be taken around bedtime on the day of the 
infusion.
•
You may experience side effects from taking part in this study. Some can be mild or 
temporary, but others may be serious, long-lasting, or permanent, and may include death. 
Examples of some of the side effects that you may have include: injection site reaction, 
fatigue, headache, nausea, diarrhea, chills, shortness of breath, anemia, high blood sugar, 
decreased sensation of your hands/feet, bleeding of your gums or from your nose. A 
more complete list of possible side effects is described later in this consent. It is 
important that you read and understand the possible risks. 
•
M7824 is a new immunotherapy drug and although it may cause similar side effects of 
other immunotherapy agents, it has also been found to cause additional skin problems, 
different problems with the immune system, low red blood cell count (anemia) and 
bleeding. It is possible that the anemia and/or bleeding may be so severe that you require 
a blood transfusion.  You cannot join this study if you are not willing to have a blood 
transfusion.    
•
You will be seen regularly during the study. You will have clinical, laboratory, and 
imaging tests to see how you are doing and to see if your disease is responding to the 
treatment. We will also collect required samples from you (such as blood and tumor 
biopsies) for both clinical and research purposes.
•
After the study treatment has ended, we will contact you for long term follow-up every 
3 months for one year and then every 6 months for one additional year to ask about any 
adverse events you have experienced and further treatment of your tumor for a total of 2 
years. We will also take scans if your tumors have not grown or spread during this time.  
Just as we do not know what side effects that you might have, we cannot know if you may 
benefit from taking part in this study. If you do not benefit, this study and the results from our 
research may help others in the future.
You are free to stop participating in the trial at any time. If you decide to stop, the study doctor 
may ask you to agree to certain tests to make sure it is safe for you to stop.
The remaining document will now describe more about the research study. This information 
should be considered before you make your choice. Members of the study team will talk with you 
about the information described in this document. Some people have personal, religious, or ethical 
beliefs that may limit the kinds of medical or research interventions in which  they would want to 
participate. Take the time needed to ask any questions and discuss this study with NIH staff, and 
with your family, friends, and personal health care providers.
IT IS YOUR CHOICE TO TAKE PART IN THE STUDY
You may choose not to take part in this study for any reason. If you join this study, you may change 
your mind and stop participating in the study at any time and for any reason. In either case, you 
will not lose any benefits to which you are otherwise entitled. However, to be seen at the NIH, you 
must be taking part in a study or are being considered for a study. If you do choose to leave the 
study, please inform your study team to ensure a safe withdrawal from the research.  
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 4 of 24
CC ICF template v. 12.01.20
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
WHY IS THIS STUDY BEING DONE?
The purpose of this research study is to determine if a new combination immunotherapy is effective 
in shrinking tumors in patients with metastatic breast cancer. 
We are asking you to join this research study because you have been diagnosed with metastatic 
breast cancer, such as Triple Negative Breast Cancer (TNBC) or ER-/PR-/HER2+ Breast Cancer 
(HER2+BC).
T-DM1 has been approved by the FDA specifically for the treatment of HER2-positive metastatic 
breast cancer (mBC) in patients who previously received trastuzumab and a taxane (paclitaxel or 
docetaxel), separately or in combination. BN-Brachyury, Entinostat, M7824 and T-DMI are 
considered investigational, which means that the use of this combination has not been approved 
by the U.S. Food and Drug Administration (FDA) to treat metastatic breast cancer. 
WHAT WILL HAPPEN DURING THE STUDY?
Below is a description of what will be asked of you and what will occur if you decide to take part 
in this study.
Before you begin the study
To determine if you can be a part of this study, you will need to have the following tests and 
procedures: Medical history and physical examination including height, weight, and vital signs;

Routine blood tests;

Hepatitis B and C screening tests; 

HIV screening test; 

Pregnancy test for women who are able to have children; 

CT scan of the chest, abdomen and pelvis and/or MRI ;

MRI/CT of the brain;

Echocardiogram, a test to create images of the heart;

Electrocardiogram (EKG), a test to measure the electrical activity of the heartbeat; and

Confirmation of your disease. If there is no available tumor sample or pathology report, 
a biopsy will be performed to confirm the diagnosis.

Measuring oxygen levels in your body
During the study
Once we know that you are eligible and you sign the consent document agreeing to participate in 
the study, you may need to have some of the tests that were done at screening repeated, depending 
on how much time has passed. Your study team will let you know if any of the above tests need to 
be repeated.
During your participation you will receive a combination of BN-Brachyury, Entinostat, M7824, 
and T-DM1. Your specific combination of these drugs will depend on the group you are assigned 
to as described above in Key Information About This Research.  The study drugs will be given in 
cycles, with each cycle lasting 3 weeks. After one year, M7824 will be discontinued, but the other 
study drugs will be continued per the group in which you are enrolled until you are no longer 
benefiting from study therapy. 
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 5 of 24
CC ICF template v. 12.01.20
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY

BN-Brachyury is a combination of MVA-BN Brachyury and FPV-Brachyury vaccines. 
The vaccines will be given once every three weeks, on the same day you receive M7824 
and, if assigned, T-DM1. After cycle 9, BN-Brachyury vaccines will be given to you every 
12 weeks.
o
You will receive MVA-BN Brachyury in four injections under the skin at different sites 
on your body. The preferred injections sites include the left upper arm, the right upper 
arm, the left outer thigh, and the right outer thigh. MVA-BN-Brachyury is given the 
first and second cycles only.
o
You will receive FPV-Brachyury in a single injection under the skin. FPV-Brachyury 
is given starting the third cycle.

You will receive M7824 by IV infusion once every three weeks for up to 12 months. The 
infusion will take approximately one hour but could take more or less time. Before your 
M7824 infusion you may receive standard pre-medication of an antihistamine like 
Benadryl and acetaminophen (Tylenol). If you have a preexisting titanium central line, then 
M7824 may be able to be administered through the central line in place of an IV infusion.
o
If you are assigned to Arm 1, there will be an observation time period where 
you will be monitored for up to an additional 60 minutes after your M7824 
infusion is complete. 

If assigned to Arm 2 or 3, you will receive T-DM1 by IV infusion once every three weeks. 
The first infusion will take approximately 90 minutes and later infusions will take 
approximately 30 minutes, but these could take more or less time. 
o
If you are assigned to Arm 2 or 3, there will be an observation time period 
where you will be monitored for up to an additional 90 minutes after your T-
DM1 infusion is complete as recommended by the FDA. 

If you are assigned to Arm 3, you will take Entinostat by mouth, in tablet form, on days 1, 
8 and 15 of each cycle. It should be taken on an empty stomach (at least 2 hours after a 
meal and 1 hour before the next meal). If you forget to take a dose on the assigned day, 
you can take it up to 24 hours later. On day 1 of each cycle, you should take Entinostat at 
around bedtime on the day of the infusion.  On day 8 of cycle 1, please take it in the morning 
before you come to the clinic. If you will have bloods drawn on day 15 of cycle 1 (this is 
optional), please take it in the morning on that day as well. 
At the start of each cycle, we will give you enough entinostat for a cycle. We will also give 
you a pill diary so you can keep track of the times you are taking Entinostat. At each clinic 
visit, please bring your pill diary and any pills you might have left over.
Do NOT split, crush or chew entinostat tablets. If you vomit after swallowing the 
entinostat, you should not take the dose again unless you can see the tablet in your vomit 
and the tablet is still in one whole piece. You should discuss with the study team before 
you re-take your dose.
We will continue to take assessments, such as physical exam, weight, vital signs, EKG and/or 
echocardiogram, collection of blood for testing, pregnancy test for women of childbearing 
potential, and scans for evaluation of your tumor during your participation. 
In addition to the tests that we will conduct to determine whether you are having side effects or if 
you are responding to the study therapy, we will also collect samples from you for purposes of 
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research only. The samples are being tested to find out how your body processes the study drugs, 
find out whether or not your body creates antibodies (proteins) that attack the study drug, and look 
at the effects of therapy on your immune system and markers of tumor activity, including collecting 
and testing tumor cells. 
The samples included for these studies include:

Blood Tests, weekly during cycle 1 (day 15 of cycle 1 is optional); day 1 of cycles 2, 3, 5 
and 6 and at the end of your treatment. 

Tumor Biopsy, collected before you have taken any study drug, on day 1 of cycle 3. If you 
are enrolled on Cohort 3 (after it is known that the combination in Arm 3 is safe), the biopsy 
will be required.  It is optional if you are enrolled prior.  Your study doctor will let you 
know which group you are in.  

Tumor Block, collected on C1D1 before you have taken any study drug.
In this study, we will perform genetic testing on your DNA and RNA. DNA (also called 
deoxyribonucleic acid) in the cells carries genetic information and passes it from one generation 
of cells to the next – like an instruction manual. RNA (ribonucleic acid) carries instructions from 
your DNA to the parts of the cells that makes proteins. We will perform limited genetic testing on 
certain immune cells in your blood and tumor samples to characterize changes in the DNA of a 
particular molecule located on the immune cell.  We will perform further genetic testing on your 
blood samples, looking at how much RNA you have for certain panels genes. This helps us to learn 
how active the genes are. 
When you are finished with treatment 
End of Treatment Visit
If you stop participation in the study before the completion of one year of treatment, then you will 
be asked to come into the clinic on the day of or within 30 days of the decision to stop treatment 
for the following tests and procedures: physical examination including weight and vital signs;  
routine blood tests; pregnancy test if you are a woman who can have children; scans if you stop 
study therapy before your disease worsens and an optional tumor biopsy. The optional biopsy will 
only be taken at the time your disease gets worse. You will be asked to sign a separate consent at 
the time of the biopsy if you agree to have it done. It is possible that your disease may not have 
gotten worse at the time of this visit.  If not, the optional biopsy will be requested at a later visit. 
Safety Visit
After your treatment ends or if you stop participation in the study for any reason, you will be asked 
to come into the clinic for a 28-day follow-up visit or telephone call following your last dose of 
study drug(s).
During the visit the following tests and procedures will be given: physical examination including 
weight and vital signs; routine blood tests; pregnancy test if you are a woman who can have 
children; and if your disease has worsened an optional tumor biopsy may be requested if we have 
not already collected one. 
Long Term Follow-up
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We will also contact you for long term follow-up every 3 months for one year and then every 6 
months for one additional year. 
If your disease has progressed while being treated then you will be followed by phone or email for 
any adverse events you may have experienced and further treatment of your tumor for two years. 
If your disease has worsened an optional tumor biopsy may be requested if we have not already 
collected one.
If your disease did not progress while being treated then you will receive tumor scans until 
progression of your disease and will be followed by phone or email for  any adverse events, and 
further treatment for two years. 
HOW LONG WILL THE STUDY TAKE?
If you agree to take part in this study, your involvement is expected to last three years, including 
approximately one year of treatment and two years of follow-up.
You will be seen multiple times during treatment. You will be asked to come to the NIH weekly 
to every three weeks depending on where you are in your treatment schedule. Visits usually take 
about 5-6 hours, but will take no longer than 8 hours.
After the end of treatment, you will start the long-term follow-up part of the study. During this 
part, you will be contacted every 3 months for one year, then every 6 months for one year. 
HOW MANY PEOPLE WILL PARTICIPATE IN THIS STUDY?
We plan to have up to 65 people total participate on this study at all participating sites. 
WE PLAN TO HAVE UP TO 35 PARTICIPATES IN THIS STUDY AT THE NIH. WHAT 
ARE THE RISKS AND DISCOMFORTS OF BEING IN THE STUDY?
Risks from Study Drugs
Side effects of BN-Brachyury Vaccines
Likely side effects: 

Injection-site reaction (pain or discomfort, itching, redness, firmness, swelling, skin 
thickening, or bumps) 
Less likely side effects: 

Acute fever 

Chills 

Flu-like symptoms (fatigue, soreness, general body pain, abdominal pain, cough, fever, 
headache) 

Chest tightness 

Shortness of breath 

Leg pain and/or swelling 

Headache 

Loss of appetite 

Weight loss 
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
Diarrhea 

Constipation 

Rash 

Nausea 

Dizziness 

Mild inflammation of tissue lining the lungs 

Chronic inflammation of the skin 

Difficulty sleeping

Open sores (ulcers) at the injection site 

Enlarged lymph nodes 

Low blood levels of sodium 

Inflammation of the testicles

Inflammation of thyroid tissue
Rare, serious side effects:

Difficulty breathing 

Low blood pressure 

Wheezing 

Clots in the lung 

Clots in the leg 

Kidney damage

Decreased blood oxygen levels

Fluid around the lining of the lungs

Fluid around the lining of the heart
Side effects of Entinostat
Common side effects: 

Fatigue (tiredness)

Nausea

Thrombocytopenia (decrease of the blood platelets)

Anemia (low blood cell count)

Hypoalbuminemia (low level of albumin in the blood)

Hypophosphatemia (low serum phosphate concentration)

Neutropenia / Neutropenic infection (low neutrophils, a type of white blood cells)

Vomiting

Anorexia

Headache

Diarrhea

Hyponatremia (low serum sodium concentration)

Dyspnea (difficulty breathing)

Dehydration

Injection site reaction
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
Leukopenia (low white blood cell count)

Hyperglycemia (high blood sugar)

Hypocalcemia (low calcium levels in the blood)
Side effects of M7824
As a side effect of this experimental therapy, the immune system may target normal tissue. 
Normal endocrine organs (organs that make hormones) may not work as well leading to tiredness 
(from low thyroid hormones or adrenal hormones). This may need to be treated by replacement 
hormones (pills).
Common side effects (occurring in more than 5% of patients):

Fatigue (tiredness and lack of energy)

Nausea

Diarrhea

Constipation

Vomiting

Swelling of you lower legs or hands

Fever 

Decreased appetite

Loss of body fluids (dehydration)

Skin growths called keratoacanthomas that resemble skin cancer. These usually go away 
after treatment and can leave a scar. 

Rash, blisters, skin discoloration and other skin abnormalities.

Bleeding has been frequently observed in patients receiving M7824. Patients may 
experience bleeding in different organs such as gums, nose, ears, eyes, vagina, breast, 
blood in the urine, stool, or bleeding in the internal organs or skill, coughing up or 
vomiting blood. Occasionally, this bleeding can be serious and potentially life threatening 
and require you to receive a blood transfusion. If you experience any bleeding on this 
trial, please tell the study team immediately. Tell your doctor if you've had a life-long 
problem of frequent or excessive bleeding or bruising, or if you take aspirin or 
prescription medication to thin your blood. 

Shortness of breath

Cough

Anemia: low number of red blood cells low that can cause tiredness and shortness of 
breath. May require a transfusion

Abdominal pain

Headache

Itching
Occasional side effects (occurring in less than 5% of patients):

Chills (feeling cold)
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
Blood clots that form throughout the body, blocking small blood vessels. Symptoms may 
include chest pain, shortness of breath, leg pain, problems speaking, or problems moving 
parts of the body.

Easy bruising 

Reaction to other drugs such as rash, anaphylaxis, and changes in blood. 

Infusion-related reaction, including dizziness or fainting (low blood pressure), flushing, 
rash, fever, shortness of breath or sick to your stomach, or pain at the site of infusion. 
Although usually reversible with treatment, it can be severe or life threatening.

Cancerous growth on the skin that can be removed

Stroke

Slow wound healing

Thickening of the skin, nails
Allergic reactions or reactions in the context with the infusions might occur during treatment.
Although M7824 is a fully human protein the risk cannot be completely excluded. In general, 
these reactions are mild to moderate and can be handled with appropriate drugs, but in very rare 
cases severe to life-threatening and even fatal reactions might occur, which require advanced 
cardiac life support. 
In addition, immune-mediated side effects might be possible. These side effects are caused by over 
activity of your body’s immune-system. The immune system normally protects you from things 
that are harmful; such as infections, foreign substances, and sometimes from cancer. If the immune 
system is overactive, it can attack normal parts of the body because it mistakenly recognizes them 
as foreign/harmful.
Examples of these side effects are listed below. In rare cases, immune-related side effects can be 
life-threatening or fatal.
Types of immune related side effects:

Inflammation in the lungs (pneumonitis): symptoms may include but are not limited to a 
new or worsening cough, shortness of breath possibly with fever. Preliminary data 
suggested that there may be the tendency of higher frequency and severity in Japanese 
patients compared with non-Japanese patients: tell your study doctor right away if you 
have any of these symptoms as it may need to be treated urgently.

Hypothyroidism (decreased function of the thyroid gland)

Hyperthyroidism (increased function of thyroid gland)

Thyroiditis (inflammatory disease of the thyroid gland)

You may develop inflammation of the liver called hepatitis. Signs and symptoms of this 
include yellowing of the skin or whites of the eyes, dark urine, severe nausea and 
vomiting, pain in the upper right side of your abdomen, skin itchiness, not feeling hungry 
and bleeding or bruising more easily than normal.

Thrombocytopenia (decrease of the blood platelets)

Uveitis (inflammation in the eye) 

Diabetes mellitus (high blood sugar levels)
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
Problems with your adrenal glands (Adrenal Insufficiency): may cause stomach pains, 
vomiting, muscle weakness and fatigue, depression, low blood pressure, weight loss, 
kidney problems, and changes in mood and personality. These complications may be 
permanent and may require hormone replacement

Myositis (inflammation of the muscles characterized by pain and tenderness)

Myasthenia gravis (weakness in the skeletal muscles)

Inflammation of the intestine (colitis). It may cause abdominal pain and diarrhea with or 
without blood. Fever may be present. It may require you to receive additional fluids. If 
left untreated, in rare occasions this may lead to a tear in the wall of the intestine which 
can be serious and life threatening. 

Autoimmune encephalitis is a type of brain inflammation where the body's immune 
system attacks healthy cells and tissues in the brain or spinal cord.

Myocarditis (inflammation of the heart muscle)

Pemphigoid (fluid-filled blisters that can be itchy)

Kidney problems: you may have an increase of creatinine levels in a blood test 
(creatinine is a protein marker that measures kidney function) but not have any symptoms 
or feel unwell. Uncommonly a patient may experience nephritis which is an inflammation 
of the kidneys that stops the kidneys from working properly.

Problems with the pituitary gland (hypopituitarism): Hypopituitarism refers to decreased 
output of hormones from the pituitary gland in the brain and may be caused by 
inflammation of the pituitary gland (hypophysitis). Symptoms may include headaches, 
thirstiness, and trouble seeing or double vision, leakage of breast milk or irregular periods 
in women. These complications may be permanent and may require hormone 
replacement.

Pancreatitis (inflammation of the pancreas)

Liver problems: Increases in the blood level of substances called enzymes found within 
your liver cells. The enzyme changes are unlikely to make you feel unwell, however, if 
these blood enzyme levels become very high, your study doctor may need to stop the 
study medication. 

Nervous system problems: symptoms can include unusual weakness of legs, arms, or 
face, numbness, or tingling in hands or feet. In rare situations, there is the potential for 
the inflammation of the nervous system to be severe.
There is a risk of tumor lysis syndrome due to tumor shrinkage. This complication is caused by 
the breakdown products of dying cells and includes elevated blood potassium, elevated blood 
phosphorus, elevated blood uric acid and elevated urine uric acid, low blood calcium, and 
consequent acute kidney failure.
If any of these side effects occur, you must inform your study doctor immediately.
Side effects of T-DM1
Common side effects:

Fatigue (tiredness)

Nausea
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
Bone and joint pain

Muscle pain

Low blood platelet count

Headache

Constipation 

Nerve damage

Low red blood cell count

Low potassium levels
Rare, serious side effects (occurring in 1-2% of patients):

Heart problems (such as reduced heart function, congestive heart failure)

Lung problems (such as inflammation of lungs with symptoms of trouble breathing, 
cough, tiredness, and fluid in the lungs)

Liver problems (such as liver failure with symptoms of swelling/pain in the abdomen, 
yellow tint in the eyes/skin, swollen legs/feet/ankles)
Risk from Blood Collection
Risks of blood draws include pain and bruising in the area where the needle is placed, 
lightheadedness, and rarely, fainting. When large amounts of blood are collected, low red blood 
cell count (anemia) can develop. 
Risks from Biopsy 
Care will be taken to minimize risks that may happen during collection of a tumor sample. This 
procedure usually causes only brief discomfort at the site from which the biopsy is taken. You may 
experience some bruising around the biopsy site. Rarely, infection or bleeding may occur at the 
needle site. 
Tumor biopsies will be done by a specialist using the CT scanner or ultrasound machine to guide 
the biopsy needle into the tumor to ensure accuracy. To collect the research biopsies as well as the 
biopsy that may be performed at screening, you may be exposed to up to 4 CT scans.   
See Structural MRI
Magnetic resonance imaging (MRI) uses a strong magnetic field and radio waves to take pictures 
of the body. If you have a history of metastases to your brain or if we are concerned that you may 
have metastases to your brain, we will obtain pictures of your brain for this study. The MRI scanner 
is a metal cylinder surrounded by a strong magnetic field. During the MRI, you will lie on a table 
that can slide in and out of the cylinder. We will place soft padding or a coil around your head. 
You will be in the scanner about 30 minutes. You may be asked to lie still for up to 30 minutes at 
a time. While in the scanner you will hear loud knocking noises, and you will be fitted with 
earplugs or earmuffs to muffle the sound. You will be able to communicate with the MRI staff at 
all times during your scan, and you may ask to be moved out of the machine at anytime. 
It is very important for the experiment that you do not move your head or body inside the scanner. 
We will use padding around your head to help keep it in place.
We may place a bar in your mouth to help keep your head still.
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Risks for MRI
People are at risk for injury from the MRI magnet if they have some kinds of metal in their body.  
It may be unsafe for you to have an MRI scan if you have pacemakers or other implanted electrical 
devices, brain stimulators, some types of dental implants, aneurysm clips (metal clips on the wall 
of a large artery), metal prostheses (including metal pins and rods, heart valves, and cochlear 
implants), permanent eyeliner, tattoos, an implanted delivery pump, or shrapnel fragments. 
Welders and metal workers may have small metal fragments in the eye. You will be screened for 
these conditions before having any MRI scan. If you have a question about metal in your body, 
you should inform the staff. You will be asked to complete an MRI screening form before each 
MRI scan you have. 
In addition, all magnetic objects (like watches, coins, jewelry, and credit cards) must be removed 
before entering the MRI scan room.
People with fear of confined spaces may become anxious during an MRI. Those with back 
problems may have back pain or discomfort from lying in the scanner.  The noise from the scanner 
is loud enough to damage hearing, especially in people who already have hearing loss. Everyone 
having a research MRI scan will be fitted with hearing protection. If the hearing protection comes 
loose during the scan, you should let us know right away.
There are no known long-term risks of MRI scans.
Gadolinium enhanced MRI
During part of the MRI you will receive gadolinium, a contrast agent, through an intravenous (iv) 
catheter. It will be done for research and medical purposes. 
It is not known if MRI with contrast is completely safe for a developing fetus. Therefore, all 
women of childbearing potential will have a pregnancy test performed no more than 24 hours 
before each MRI scan with contrast. The scan will not be done if the pregnancy test is positive. 
The risks of an IV catheter include bleeding, infection, or inflammation of the skin and vein with 
pain and swelling.  
Mild symptoms from gadolinium infusion occur in fewer than 1% of those who receive it and 
usually go away quickly. Mild symptoms may include coldness in the arm during the injection, a 
metallic taste, headache, and nausea. In an extremely small number, fewer than one in 300,000 
people, more severe symptoms have been reported including shortness of breath, wheezing, hives, 
and lowering of blood pressure. You should not receive gadolinium if you previously had an 
allergic reaction to it. You will be asked about such allergic reactions before gadolinium is given.   
People with kidney disease are at risk for a serious reaction to gadolinium contrast called 
“nephrogenic systemic fibrosis (NSF)”. This condition always involves the skin and can also 
involve the muscles, joints and internal organs. NSF has resulted in a very small number of deaths.  
A blood test of your kidney function may be done within the month before an MRI scan with 
gadolinium contrast. You will not receive gadolinium for a research MRI scan if your kidney 
function is below the safe level. 
Most of the gadolinium contrast leaves the body in the urine. However, the FDA has issued a safety 
alert that indicates small amounts of gadolinium may remain in the body for months to years. The 
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effects of the retained gadolinium are not clear. At this time, retained gadolinium has not been 
linked to health risks in people whose kidneys work well. Some types of gadolinium contrast drugs 
are less likely to remain in the body than others. In this study, we will use the gadolinium contrast 
drugs that are less likely to remain in the body. We will also give you additional information called 
a “Medication Guide.” Upon request, we will give you individual information about retained 
gadolinium we see on your studies.
What are the risks of radiation from being in the study? below for more details. 
What are the risks related to pregnancy?
If you are able to become pregnant, we will ask you to have a pregnancy test before starting this 
study. You will need to practice an effective form of birth control when starting study treatment, 
during study treatment, and for seven (7) months after you finish study treatment (the restricted 
period). 
If you become pregnant, there may be unknown risks to the fetus or unborn child, or risks that we 
did not anticipate. There may be long-term effects of the treatment being studied that could 
increase the risk of harm to a fetus. You must tell the study doctor if your birth control method 
fails during the restricted period. If you think or know you have become pregnant while 
participating in this research study, please contact the study team as soon as possible. 
If you are a sexually active person with a partner able to become pregnant, it is important that your 
partner not become pregnant during the restricted period. For males the restricted period is four 
(4) months. There may be unknown risks to a fetus or risks we did not anticipate. You and your 
partner must agree to use birth control if you want to take part in this study. If you think your 
partner has become pregnant during the restricted period, please contact the study team as soon as 
possible. If you and your partner plan for your partner to become pregnant after the restricted 
period, please discuss this with the study team.
Two methods of birth control must be used. Acceptable birth control options for you and your 
partner include:

Abstinence;

hormonal contraceptives or therapies (such as birth control pills, injections, patch or 
implants);

barrier methods (such as a condom, cap, or diaphragm) used with a spermicide;

an intrauterine device (IUD), such as Copper T, Progesterone T, Levonorgestrel-releasing 
intrauterine system (e.g., Mirena);

surgical sterilization (tubal ligation or vasectomy).
Men should refrain from donating sperm during the study and for 4 months after the last dose of 
study medications. 
Structural MRI
Magnetic resonance imaging (MRI) uses a strong magnetic field and radio waves to take pictures 
of the body. If you have a history of metastases to your brain or if we are concerned that you may 
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have metastases to your brain, we will obtain pictures of your brain for this study. The MRI scanner 
is a metal cylinder surrounded by a strong magnetic field. During the MRI, you will lie on a table 
that can slide in and out of the cylinder. We will place soft padding or a coil around your head. 
You will be in the scanner about 30 minutes. You may be asked to lie still for up to 30 minutes at 
a time. While in the scanner you will hear loud knocking noises, and you will be fitted with 
earplugs or earmuffs to muffle the sound. You will be able to communicate with the MRI staff at 
all times during your scan, and you may ask to be moved out of the machine at anytime. 
It is very important for the experiment that you do not move your head or body inside the scanner. 
We will use padding around your head to help keep it in place.
We may place a bar in your mouth to help keep your head still.
Risks for MRI
People are at risk for injury from the MRI magnet if they have some kinds of metal in their body.  
It may be unsafe for you to have an MRI scan if you have pacemakers or other implanted electrical 
devices, brain stimulators, some types of dental implants, aneurysm clips (metal clips on the wall 
of a large artery), metal prostheses (including metal pins and rods, heart valves, and cochlear 
implants), permanent eyeliner, tattoos, an implanted delivery pump, or shrapnel fragments. 
Welders and metal workers may have small metal fragments in the eye. You will be screened for 
these conditions before having any MRI scan. If you have a question about metal in your body, 
you should inform the staff. You will be asked to complete an MRI screening form before each 
MRI scan you have. 
In addition, all magnetic objects (like watches, coins, jewelry, and credit cards) must be removed 
before entering the MRI scan room.
People with fear of confined spaces may become anxious during an MRI. Those with back 
problems may have back pain or discomfort from lying in the scanner.  The noise from the scanner 
is loud enough to damage hearing, especially in people who already have hearing loss. Everyone 
having a research MRI scan will be fitted with hearing protection. If the hearing protection comes 
loose during the scan, you should let us know right away.
There are no known long-term risks of MRI scans.
Gadolinium enhanced MRI
During part of the MRI you will receive gadolinium, a contrast agent, through an intravenous (iv) 
catheter. It will be done for research and medical purposes. 
It is not known if MRI with contrast is completely safe for a developing fetus. Therefore, all 
women of childbearing potential will have a pregnancy test performed no more than 24 hours 
before each MRI scan with contrast. The scan will not be done if the pregnancy test is positive. 
The risks of an IV catheter include bleeding, infection, or inflammation of the skin and vein with 
pain and swelling.  
Mild symptoms from gadolinium infusion occur in fewer than 1% of those who receive it and 
usually go away quickly. Mild symptoms may include coldness in the arm during the injection, a 
metallic taste, headache, and nausea. In an extremely small number, fewer than one in 300,000 
people, more severe symptoms have been reported including shortness of breath, wheezing, hives, 
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
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CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
and lowering of blood pressure. You should not receive gadolinium if you previously had an 
allergic reaction to it. You will be asked about such allergic reactions before gadolinium is given.   
People with kidney disease are at risk for a serious reaction to gadolinium contrast called 
“nephrogenic systemic fibrosis (NSF)”. This condition always involves the skin and can also 
involve the muscles, joints and internal organs. NSF has resulted in a very small number of deaths.  
A blood test of your kidney function may be done within the month before an MRI scan with 
gadolinium contrast. You will not receive gadolinium for a research MRI scan if your kidney 
function is below the safe level. 
Most of the gadolinium contrast leaves the body in the urine. However, the FDA has issued a safety 
alert that indicates small amounts of gadolinium may remain in the body for months to years. The 
effects of the retained gadolinium are not clear. At this time, retained gadolinium has not been 
linked to health risks in people whose kidneys work well. Some types of gadolinium contrast drugs 
are less likely to remain in the body than others. In this study, we will use the gadolinium contrast 
drugs that are less likely to remain in the body. We will also give you additional information called 
a “Medication Guide.” Upon request, we will give you individual information about retained 
gadolinium we see on your studies.
What are the risks of radiation from being in the study? 
During your participation in this research study, you will be exposed to radiation from CT scans 
and CT guided biopsies. The amount of radiation exposure you will receive from these procedures 
is equal to approximately 11 rem. A rem is a unit of absorbed radiation. 
Every day, people are exposed to low levels of radiation that come from the sun and the 
environment around them. The average person in the United States receives a radiation exposure 
of 0.3 rem per year from these sources. This type of radiation is called “background radiation.”  
This study will expose you to more radiation than you get from everyday background radiation. 
No one knows for sure whether exposure to these low amounts of radiation is harmful to your 
body. 
The CT scans and CT guided biopsies that you get in this study will expose you to the roughly the 
same amount of radiation as 36.7 years’ worth of background radiation. Being exposed to too much 
radiation can cause harmful side effects such as an increase in the risk of cancer.  The risk depends 
on how much radiation you are exposed to. Please be aware that about 40 out of 100 people (40%) 
will get cancer during their lifetime, and 20 out of 100 (20%) will die from cancer. The risk of 
getting cancer from the radiation exposure in this study is 1.1 out of 100 (1.1%) and of getting a 
fatal cancer is 0.6 out of 100 (0.6%). 
You may not participate in this study if you are pregnant or breast feeding. If you are capable of 
becoming pregnant, we will perform a pregnancy test before exposing you to radiation. You must 
tell us if you may have become pregnant within the previous 14 days because the pregnancy test 
is unreliable during that time.
Non Radiation Risks of CT Scans
In addition to the radiation risks from CT scans discussed above, you may experience an allergic 
reaction to the dye we inject into your veins to help us view the scan better. You might experience 
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
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CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
hives, itching, headache. More serious reactions that would include difficulty breathing, increased 
heart rate and swelling of your throat or other body parts. 
WHAT ARE THE BENEFITS OF BEING IN THE STUDY?
You might not benefit from being in this study. 
However, the potential benefit to you might be stabilization or shrinkage of your tumor and a 
reduction in chances of developing new lesions with a decrease in symptoms caused by progressive 
disease. 
Are there any potential benefits to others that might result from the study?
Despite advances in early detection and effective treatments, there is still a high incidence of 
invasive, metastatic breast cancer and therefore, there is a need for new beneficial treatments. In 
the future, other people might benefit from this study because of the knowledge gained from the 
outcome of this trial using a new combination immunotherapy.   
WHAT OTHER OPTIONS ARE THERE FOR YOU?
Before you decide whether or not to be in this study, we will discuss other options that are 
available to you. Instead of being in this study, you could: 

choose to be treated with surgery, radiation or with drugs already approved by the FDA for 
your disease;

choose to take part in a different study, if one is available; or

choose not to be treated for cancer but you may want to receive comfort care to relieve 
symptoms.
You should discuss with your doctor your other choices and their risks and benefits.
DISCUSSION OF FINDINGS
New information about the study
If we find out any new information that may affect your choice to participate in this study, we 
will get in touch with you to explain what we have learned. This may be information we have 
learned while doing this study here at the NIH or information we have learned from other 
scientists doing similar research in other places.
Return of research results
We do not plan to return research findings to you.
EARLY WITHDRAWAL FROM THE STUDY 
Your doctor may decide to stop your therapy for the following reasons:

if she/he believes that it is in your best interest; 

if your disease worsens or comes back during treatment; 

if you have side effects from the treatment that your doctor thinks are too severe; 

if you become pregnant; 
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 18 of 24
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CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY

if BN-Brachyury, Entinostat, M7824, and/or T-DM1 become unavailable; 

if new information shows that another treatment would be better for you; 

if you do not follow the study rules; and/or

if the study is stopped for any reason.
In this case, you will be informed of the reason therapy is being stopped.  
After therapy is stopped we would like to see you for a safety visit 28 days after your last dose.
You can stop taking part in the study at any time. However, if you decide to stop taking part in the 
study, we would like you to talk to the study doctor and your regular doctor first.
If you decide at any time to withdraw your consent to participate in the trial, we will not collect 
any additional medical information about you. However, according to FDA guidelines, 
information collected on you up to that point may still be provided to Bavarian Nordic, Syndax, 
EMD Serono, Inc., or designated representatives.
STORAGE, SHARING AND FUTURE RESEARCH USING YOUR SPECIMENS AND 
DATA
Will your Specimens or Data be Saved for Use in Other Research Studies?
As part of this study, we are obtaining specimens and data from you. We will remove all the 
identifiers, such as your name, date of birth, address, or medical record number and label your 
specimens and data with a code so that you cannot easily be identified. However, the code will 
be linked through a key to information that can identify you. We plan to store and use these 
specimens and data for studies other than the ones described in this consent form that are going 
on right now, as well as studies that may be conducted future. These studies may provide 
additional information that will be helpful in understanding metastatic breast cancer or other 
diseases or conditions. This could include studies to develop other research tests, treatments, 
drugs, or devices, that may lead to the development of a commercial product by the NIH and/or 
its research or commercial partners. There are no plans to provide financial compensation to you 
if this happens. Also, it is unlikely that we will learn anything from these studies that may directly 
benefit you. 
I give permission for my coded specimens and data to be stored and used for future research as 
described above.
_____ Yes
_____ No
Initials
Initials
Will Your Specimens or Data Be Shared for Use in Other Research Studies?
We may share your coded specimens and data with other researchers. If we do, while we will 
maintain the code key, we will not share it, so the other researchers will not be able to identify 
you. They may be doing research in areas similar to this research or in other unrelated areas. 
These researchers may be at NIH, other research centers and institutions, or commercial entities.
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
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CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
I give permission for my coded specimens and data to be shared with other researchers and used 
by these researchers for future research as described above.
_____ Yes 
_____ No
Initials
Initials
If you change your mind and do not want us to store and use your specimens and data for future 
research, you should contact the research team member identified at the top of this document. 
We will do our best to comply with your request but cannot guarantee that we will always be 
able to destroy your samples. For example, if some research with your specimens and data has 
already been completed, the information from that research may still be used. Also, for example, 
if the specimens and data have been shared already with other researchers, it might not be 
possible to withdraw the specimens and data.
In addition to the planned use and sharing described above, we might remove all identifiers and 
codes from your specimens and data and use or share them with other researchers for future 
research at the NIH or other places.  When we or the other researchers access your anonymized 
data, there will be no way to link the specimens or data back to you.  We will not contact you to 
ask your permission or otherwise inform you before we do this.  We might do this even if you 
answered "no" to the above questions.  If we do this, we would not be able to remove your 
specimens or data to prevent their use in future research studies, even if you asked, because we 
will not be able to tell which are your specimens or data.
NIH policies require that your clinical and other study data be placed in an internal NIH database 
that is accessible to other NIH researchers for future research. These researchers will not have 
access to any of your identifiers, such as your name, date of birth, address, or medical record 
number; and your data will be labeled with only a code. We cannot offer you a choice of whether 
your data to be placed in this database or not.  If you do not wish to have your data placed in this 
database, you should not enroll in this study.  
Will Your Genomic Data Be Shared Outside of This Study?
As part of this research, we will put your genomic data in a large database for broad sharing with 
the research community. These databases are commonly called data repositories. The 
information in this database will include but is not limited to genetic information, race and 
ethnicity, and sex. If your individual data are placed in one of these repositories, they will be 
labeled with a code and not with your name or other information that could be used to easily 
identify you, and only qualified researchers will be able to access them. These researchers must 
receive prior approval from individuals or committees with authority to determine whether these 
researchers can access the data.
Summary information about all of the participants included in this study (including you)  is being 
placed in a database and will be available through open access. That means that researchers and 
non-researchers will be able to access summary information about all the participants included in 
the study, or summary information combined from multiple studies, without applying for 
permission. The risk of anyone identifying you with this information is very low.
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
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CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
NIH policies require that genomic data be placed in a repository for sharing. Therefore, we 
cannot offer you a choice of whether your data will be shared. If you do not wish to have your 
data placed in a repository, you should not enroll in this study.  
How Long Will Your Specimens and Data be Stored by the NIH?
Your specimens and data may be stored by the NIH until they are no longer of scientific value 
or if you withdraw consent for their continued use, at which time they will be destroyed. Your 
specimens and data may be stored by the NIH indefinitely.
Risks of Storage and Sharing of Specimens and Data
When we store your specimens and data, we take precautions to protect your information from 
others that should not have access to it. When we share your specimens and data, we will do 
everything we can to protect your identity, for example, when appropriate, we remove information 
that can identify you. Even with the safeguards we put in place, we cannot guarantee that your 
identity will never become known or someone may gain unauthorized access to your information. 
New methods may be created in the future that could make it possible to re-identify your data or 
samples.
PAYMENT
Will you receive any type of payment for taking part in this study?
You will not receive compensation for participation in this study. 
REIMBURSEMENT
Will you receive reimbursement or direct payment by NIH as part of your participation?
On this study, the NCI will cover the cost for some of your expenses such as those for hotel, travel, 
meals.  Some of these costs may be paid directly by the NIH and some may be reimbursed after 
you have paid. Some of these costs may be paid directly by the NIH and some may be reimbursed 
after you have paid. The amount and form of these payments are determined by the NCI Travel 
and Lodging Reimbursement Policy. You will be given a summary of the policy which provides 
more information.
If your travel to the NIH Clinical Center (e.g. flight, hotel) is arranged and paid for by the NIH, 
the agency making the reservations and their representatives will have access to your identifiable 
information. 
COSTS
Will taking part in this research study cost you anything? 
NIH does not bill health insurance companies or participants for any research or related clinical 
care that you receive at the NIH Clinical Center. 

If some tests and procedures are performed outside the NIH Clinical Center, 
you may have to pay for these costs if they are not covered by your insurance 
company.
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 21 of 24
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MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY

Medicines that are not part of the study treatment will not be provided or paid 
for by the NIH Clinical Center.

Once you have completed taking part in the study, medical care will no longer be 
provided by the NIH Clinical Center.
CONFLICT OF INTEREST (COI)
The National Institutes of Health (NIH) reviews NIH staff researchers at least yearly for conflicts 
of interest. This process is detailed in a COI Guide. You may ask your research team for a copy 
of the COI Guide or for more information. Members of the research team who do not work for 
NIH are expected to follow these guidelines or the guidelines of their home institution, but they 
do not need to report their personal finances to the NIH.
The NIH and the research team for this study are using:

BN-Brachyury developed by Bavarian Nordic,

Entinostat developed by Syndax, and

M7824 developed by EMD Serono, Inc.
through a joint study with your study team and the company. The companies also provide financial 
support for this study.
CLINICAL TRIAL REGISTRATION AND RESULTS REPORTING
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by U.S. Law. This Web site will not include information that can identify you. At most, the Web 
site will include a summary of the results. You can search this Web site at any time.
CONFIDENTIALITY PROTECTIONS PROVIDED IN THIS STUDY  
Some of your health information, and/or information about your specimens, from this study will 
be kept in a central database for research. Your name or contact information will not be put in 
the database. Your test results will be identified by a unique code and the list that links the code 
to your name will be kept separate from your sample and health information. Your information 
may be given out if required by law. For example, certain states require doctors to report to health 
boards if they find a disease like tuberculosis. However, the researchers will do their best to 
make sure that any information that is released will not identify you.
Will your medical information be kept private?
We will do our best to make sure that the personal information in your medical record will be kept 
private.  However, we cannot guarantee total privacy. Organizations that may look at and/or copy 
your medical records for research, quality assurance, and data analysis include:

The National Institutes of Health and other government agencies, like the Food and Drug 
Administration (FDA), which are involved in keeping research safe for people.

The National Institutes of Health Intramural Institutional Review Board.

The study Sponsor, Center for Cancer Research, or their agent(s).

Qualified representatives from Bavarian Nordic, the pharmaceutical company who 
produces BN-Brachyury, or their agent(s).
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
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MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY

Qualified representatives from Syndax, the pharmaceutical company who produces 
Entinostat, or their agent(s).

Qualified representatives from EMD Serono, Inc., the pharmaceutical company who 
produces M7824, or their agent(s).
When results of an NIH research study are reported in medical journals or at scientific meetings, 
the people who take part are not named and identified.  In most cases, the NIH will not release any 
information about your research involvement without your written permission.  However, if you 
sign a release of information form, for example, for an insurance company, the NIH will give the 
insurance company information from your medical record.  This information might affect (either 
favorably or unfavorably) the willingness of the insurance company to sell you insurance.
If we share your specimens or data with other researchers, in most circumstances we will remove 
your identifiers before sharing your specimens and data. You should be aware that there is a slight 
possibility that someone could figure out the information is about you. 
Further, the information collected for this study is protected by NIH under a Certificate of 
Confidentiality and the Privacy Act.
Certificate of Confidentiality 
To help us protect your privacy, the NIH Intramural Program has received a Certificate of 
Confidentiality (Certificate). With this certificate, researchers may not release or use data or 
information about you except in certain circumstances.
NIH researchers must not share information that may identify you in any federal, state, or local 
civil, criminal, administrative, legislative, or other proceedings, for example, if requested by a 
court.
The Certificate does not protect your information when it:  
1. is disclosed to people connected with the research, for example, information may be used 
for auditing or program evaluation internally by the NIH; or 
2. is required to be disclosed by Federal, State, or local laws, for example, when information 
must be disclosed to meet the legal requirements of the federal Food and Drug 
Administration (FDA); 
3. is for other research;
4. is disclosed with your consent. 
The Certificate does not prevent you from voluntarily releasing information about yourself or your 
involvement in this research. 
The Certificate will not be used to prevent disclosure to state or local authorities of harm to self or 
others including, for example, child abuse and neglect, and by signing below you consent to those 
disclosures. Other permissions for release may be made by signing NIH forms, such as the Notice 
and Acknowledgement of Information Practices consent.
Privacy Act
The Federal Privacy Act generally protects the confidentiality of your NIH medical information 
that we collect under the authority of the Public Health Service Act. In some cases, the Privacy 
Act protections differ from the Certificate of Confidentiality.  For example, sometimes the Privacy 
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 23 of 24
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MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
Act allows release of information from your record without your permission, for example, if it is 
requested by Congress. Information may also be released for certain research purposes with due 
consideration and protection, to those engaged by the agency for research purposes, to certain 
federal and state agencies, for HIV partner notification, for infectious disease or abuse or neglect 
reporting, to tumor registries, for quality assessment and medical audits, or when the NIH is 
involved in a lawsuit. However, NIH will only release information from your medical record if it 
is permitted by both the Certificate of Confidentiality and the Privacy Act.
Policy Regarding Research-Related Injuries  
The NIH Clinical Center will provide short-term medical care for any injury resulting from your 
participation in research here. In general, no long-term medical care or financial compensation for 
research-related injuries will be provided by the NIH, the NIH Clinical Center, or the Federal 
Government. However, you have the right to pursue legal remedy if you believe that your injury 
justifies such action.
Problems or Questions
If you have any problems or questions about this study, or about your rights as a research 
participant, or about any research-related injury, contact the Principal Investigator, Fatima Karzai, 
MD, fatima.karzai@nih.gov, 301-480-7174. You may also call the NIH Clinical Center Patient 
Representative at 301-496-2626, or the NIH Office of IRB Operations at 301-402-3713, if you 
have a research-related complaint or concern.
Consent Document 
Please keep a copy of this document in case you want to read it again.
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: 07/06/2021
Page 24 of 24
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MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
Adult Research Participant: I have read the explanation about this study and have been given the opportunity 
to discuss it and to ask questions. I consent to participate in this study.
Signature of Research Participant
Print Name of Research Participant
Date
Investigator:
Signature of Investigator
Print Name of Investigator
Date
Witness to the oral short-form consent process only:
Witness:
Signature of Witness*
Print Name of Witness
Date
*NIH ADMINISTRATIVE SECTION TO BE COMPLETED REGARDING THE USE OF AN 
INTERPRETER:
An interpreter, or other individual, who speaks English and the participant’s preferred language facilitated
the administration of informed consent and served as a witness. The investigator obtaining consent may not 
also serve as the witness.
An interpreter, or other individual, who speaks English and the participant’s preferred language facilitated
the administration of informed consent but did not serve as a witness. The name or ID code of the person 
providing interpretive support is:
.
IRB NUMBER: 20C0056
IRB APPROVAL DATE: 07/14/2021