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Abbreviated Title: The BrEAsT Trial NIH **Protocol \#:** 20C0056 IBC \#: RD-19-IX-02 RSC \#: 2746 NCT \#: NCT04296942 Version Date: June 01, 2021
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nci supplement

Title: A Phase Ib Trial of Sequential Combinations of BN-Brachyury, Entinostat, adotrastuzumab 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
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investigational agents

| Investigational Agents:   | 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    |
| Drug Name:                | BN-Brachyury                                                    | Entinostat | M7824            |             |
| Manufacturer:             | Bavarian Nordic                                                 | Syndax     | EMD Serono, Inc. | CC Pharmacy |  
Coordinating Center: Center for Cancer Research Responsible Safety Monitoring Committee: NCI Safety Monitoring Committee
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precis

Background:
- Current >2 nd 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-tomesenchymal transition in breast cancer. A recently completed phase 1 study of BNBrachyury 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). TDM1 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
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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
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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.  
- 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 + nabpaclitaxel. 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.
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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 TDM1 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:
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-BNBrachyury consists of 2.0 x 108infectious 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.  
| TABLE OF CONTENTS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |
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| 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

We plan to enroll up to 65 patients on this study. We plan to enroll 35 participants at the NIH.
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2 nci recruitment and screening

Please refer to Section 2.3 of the Sponsor Protocol for recruitment strategies.
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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.
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3 nci registration procedures 3.1

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.
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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 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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4 nci treatment assignment procedures

Please refer to Section 2.5 of the Sponsor Protocol for Cohorts, Arms, and Arm Assignment procedures.
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5 cost and compensation 5.1.1

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.
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6 correlative studies for research

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
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6.2 sample storage tracking and

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.  
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 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.
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7 nih reporting requirements /

Please refer to Section 7 of the Sponsor Protocol for reporting requirements and monitoring plan procedures.
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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.
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7.2 institutional biosafety committee (ibc)

The Principal Investigator (or delegate) will notify IBC of any unexpected fatal or lifethreatening 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 lifethreatening, 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.
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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.
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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
- a brief description of any information obtained that is pertinent to an understanding of the gene transfer product's actions, including, for example, information about dose-response, information from controlled trials, and information about bioavailability.
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7.3 nih required data and

Please refer to Section 7.4 of the Sponsor Protocol for the study team and safety monitoring committee procedures.
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8 collaborative agreements 8.1 cooperative

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.
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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.
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9.1 evaluation of benefits and

Please refer to Section 12.4 of the Sponsor Protocol for the risks and benefits of participation on this study.
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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.  
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.
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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.  
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, adotrastuzumab 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
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investigational agents

| Investigational Agents:   | 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                  |
| Drug Name:                | BN-Brachyury                                                    | Entinostat | M7824            | Will be purchased         |
| Manufacturer:             | Bavarian Nordic                                                 | Syndax     | EMD Serono, Inc. | 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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precis background

- Current >2 nd 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-tomesenchymal transition in breast cancer. A recently completed phase 1 study of BNBrachyury 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). TDM1 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
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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
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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.  
- 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 + nabpaclitaxel. 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.
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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 TDM1 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:
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-BNBrachyury consists of 2.0 x 108infectious 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.  
| TABLE OF CONTENTS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
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| 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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| Dose Modification of T-DM1 .                                                                |                                                                                                                         |
| 3.4.2                                                                                       |                                                                                                                         |
| 3.4.3                                                                                       | Treatment Discontinuation of BN-Brachyury Vaccines                                                                      |
| 3.4.4                                                                                       | Treatment discontinuation of M7824                                                                                      |
| 3.4.5                                                                                       | Suggested Evaluation of Suspected Myocarditis                                                                           |
| 3.4.6                                                                                       | Suggested Evaluation of Suspected Bleeding or Hemorrhage Events and Treatment                                           |
| Modification                                                                                |                                                                                                                         |
| 3.4.7                                                                                       | Toxicity Management                                                                                                     |
| 3.5                                                                                         | Study Calendar .                                                                                                        |
| 3.6                                                                                         | Cost and Compensation                                                                                                   |
| 3.6.1                                                                                       | Costs                                                                                                                   |
| 3.6.2                                                                                       | COMPENSATION .                                                                                                          |
| 3.6.3                                                                                       | Reimbursement                                                                                                           |
| 3.7                                                                                         | Criteria for Removal from Protocol Therapy and Off Study Criteria ................................ 66                   |
| 3.7.1                                                                                       | Criteria for removal from protocol therapy .                                                                            |
| 3.7.2                                                                                       | OFF-Study Criteria .                                                                                                    |
| 3.7.3                                                                                       | Lost to Follow-up.                                                                                                      |
| 4                                                                                           | CONCOMITANT MEDICATIONS / MEASURES .                                                                                    |
| 4.1                                                                                         | Antiemetics                                                                                                             |
| 4.2                                                                                         | Growth Factor Support.                                                                                                  |
| 4.3                                                                                         | Restricted Medications                                                                                                  |
| 4.4                                                                                         | Contraception                                                                                                           |
| 4.5                                                                                         | Breast Feeding                                                                                                          |
| 4.6                                                                                         | Blood Donation .                                                                                                        |
| S                                                                                           | CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES ........ 69                                                    |
| 5.1                                                                                         | Biospecimen Collection .                                                                                                |
| Peripheral Blood Collection and Assays                                                      |                                                                                                                         |
| 5.1.1                                                                                       |                                                                                                                         |
| 5.1.2                                                                                       | Tumor Biopsy Collection and Assays                                                                                      |
| 5.1.3                                                                                       | Genetic Assays                                                                                                          |
| 5.2                                                                                         | Sample Storage, Tracking, and Disposition                                                                               |
| 5.2.1                                                                                       | Sample Management and Storage at Clinical Services Program - Leidos                                                     |
| Biomedical Research, Inc. (CSP).                                                            |                                                                                                                         |
| Samples Managed by Dr. Figg's Blood Processing Core (BPC)............................... 74 |                                                                                                                         |
| 5.2.2                                                                                       |                                                                                                                         |
| 5.2.3                                                                                       | Procedures for Storage of Tissue Specimens in the Laboratory of Pathology ...... 75                                     |
| 5.2.4                                                                                       | Protocol Completion/Sample Destruction .                                                                                |
| 6                                                                                           | DATA COLLECTION AND EVALUATION .                                                                                        |
| DATA COLLECTION                                                                             |                                                                                                                         |
| 6.1                                                                                         |                                                                                                                         |
| 6.2                                                                                         | DATA SHARING Plans .                                                                                                    |
| 6.2.1                                                                                       | Human Data Sharing Plan.                                                                                                |
| 6.2.2                                                                                       | Genomic Data Sharing Plan                                                                                               |
| 6.3                                                                                         | Response Criteria                                                                                                       |
| 6.3.1                                                                                       | Antitumor Response                                                                                                      |
| 6.3.2                                                                                       | Disease Parameters .                                                                                                    |
| 6.3.3                                                                                       | Methods for Evaluation of Measurable Disease ....................................................................... 78 |
| 6.3.4                                                                                       | Response Criteria                                                                                                       |
| 6.3.5                                                                                       | Response Review                                                                                                         |
| 6.4                                                                                         | Toxicity Criteria                                                                                                       |  
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| NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN.. 83 |                                                                                                                                              |                 |
| 7                                                                 | 7.1                                                                                                                                          | Definitions     |
| 7.2                                                               | OHSRP Office of Compliance and Training / IRB Reporting ................................................ 83                                  |                 |
| 7.2.1                                                             | Expedited Reporting                                                                                                                          |                 |
| 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 .                                                                                               |                 |
| 7.4.1                                                             | Principal Investigator/Research Team .................................................................................................... 84 |                 |
| 7.4.2                                                             | Safety Monitoring Committee (SMC)                                                                                                            |                 |
| 8                                                                 | SPONSOR PROTOCOL SAFETY REPORTING                                                                                                            |                 |
| DEFINITIONS                                                       |                                                                                                                                              |                 |
| 8.1                                                               | 8.1.1                                                                                                                                        | A Adverse Event |
| 8.1.2                                                             | Serious Adverse Event (SAE)                                                                                                                  |                 |
| Life-threatening                                                  |                                                                                                                                              |                 |
| 8.1.3                                                             |                                                                                                                                              |                 |
| 8.1.4                                                             | SE SEE                                                                                                                                       |                 |
| 8.1.5                                                             | Relationship to Study Product                                                                                                                |                 |
| Adverse Events of Special Interest (AESI)                         |                                                                                                                                              |                 |
| 8.1.6                                                             |                                                                                                                                              |                 |
| 8.2                                                               | Assessment of Safety Events                                                                                                                  |                 |
| 8.3                                                               | Reporting of Serious Adverse Events                                                                                                          |                 |
| Waiver of Expedited Reporting to CCR .                            |                                                                                                                                              |                 |
| 8.4                                                               |                                                                                                                                              |                 |
| 8.5                                                               | Safety Reporting Criteria to the Pharmaceutical Collaborators .............................................. 87                              |                 |
| 8.6                                                               | Reporting Pregnancy                                                                                                                          |                 |
| 8.6.1                                                             | Maternal Exposure                                                                                                                            |                 |
| 8.6.2                                                             | Paternal Exposure                                                                                                                            |                 |
| 8.7                                                               | Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND .................. 88                                                     |                 |
| 8.8                                                               | Sponsor Protocol Non-Adherence Reporting                                                                                                     |                 |
| 9                                                                 | CLINICAL MONITORING                                                                                                                          |                 |
| 10                                                                | STATISTICAL CONSIDERATIONS                                                                                                                   |                 |
| 10.1                                                              | Statistical Hypothesis .                                                                                                                     |                 |
| 10.2                                                              | Sample Size Determination                                                                                                                    |                 |
| 10.3                                                              | Populations for Analyses .                                                                                                                   |                 |
| 10.3.1                                                            | Evaluable for toxicity:                                                                                                                      |                 |
| 10.3.2                                                            | Evaluable for objective response: .................................................................................................. 92      |                 |
| 10.4                                                              | Statistical Analyses                                                                                                                         |                 |
| General Approach .                                                |                                                                                                                                              |                 |
| 10.4.1                                                            |                                                                                                                                              |                 |
| 10.4.2                                                            | Analysis of the Primary Efficacy Endpoint ............................................................................. 92                   |                 |
| 10.4.3                                                            | Analysis of the Secondary Endpoints .................................................................................................. 93    |                 |
| 10.4.4                                                            | Se                                                                                                                                           |                 |
| 10.4.5                                                            | Baseline Descriptive Statistics                                                                                                              |                 |
| 10.4.6                                                            | Sub-Group Analyses .                                                                                                                         |                 |
| 10.4.7                                                            | Tabulation of individual Participant Data ........................................................................................ 93        |                 |
| Exploratory Analyses                                              |                                                                                                                                              |                 |
| 10.4.8                                                            |                                                                                                                                              |                 |
| 11                                                                | COLLABORATIVE AGREEMENTS .                                                                                                                   |                 |
| 1.1                                                               | Multi-Institutional Guidelines .                                                                                                             |                 |
| 12                                                                | HUMAN SUBJECTS PROTECTIONS .                                                                                                                 |                 |
| 12.1                                                              | Rationale For Subject Selection                                                                                                              |                 |
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abbreviated title the breast trial

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| 12.2                                                                                                                            | Participation of Children                                                                                                                                       |
| 12.3                                                                                                                            | Evaluation of Benefits and Risks/Discomforts                                                                                                                    |
| 12.4                                                                                                                            | Risk/Benefit Assessment                                                                                                                                         |
| 12.4.1                                                                                                                          | Known Potential Risks                                                                                                                                           |
| Known Potential Benefits .                                                                                                      |                                                                                                                                                                 |
| 12.4.2                                                                                                                          |                                                                                                                                                                 |
| 12.4.3                                                                                                                          | Assessment of Potential Risks and Benefits ............................................................................ 96                                      |
| 12.5                                                                                                                            | Consent Process and Documentation                                                                                                                               |
| 13 REGULATORY AND OPERATIONAL CONSIDERATIONS ......................................................... 97                       |                                                                                                                                                                 |
| 13.1                                                                                                                            | Study Discontinuation and Closure ....................................................................................................... 97                    |
| 13.2                                                                                                                            | Quality Assurance and Quality Control                                                                                                                           |
| Conflict of Interest Policy.                                                                                                    |                                                                                                                                                                 |
| 13.3                                                                                                                            |                                                                                                                                                                 |
| 13.4                                                                                                                            | Confidentiality and Privacy                                                                                                                                     |
| 14                                                                                                                              | PHARMACEUTICAL INFORMATION                                                                                                                                      |
| BN-Brachyury (IND # 19165 )                                                                                                     |                                                                                                                                                                 |
| 14.1                                                                                                                            |                                                                                                                                                                 |
| 14.1.1                                                                                                                          | Source / Acquisition and Accountability .................................................................................... 99                                 |
| 14.1.2                                                                                                                          | T Toxicity .                                                                                                                                                    |
| Formulation and Preparation                                                                                                     |                                                                                                                                                                 |
| 14.1.3                                                                                                                          |                                                                                                                                                                 |
| 14.1.4                                                                                                                          | Stability and Storage ............................................................................................................................... 100       |
| 14.1.5                                                                                                                          | Administration Procedures.                                                                                                                                      |
| Incompatibilities                                                                                                               |                                                                                                                                                                 |
| 14.1.6                                                                                                                          |                                                                                                                                                                 |
| 14.2                                                                                                                            | Entinostat (IND # 19165 ) ..................................................................................................................... 100             |
| 14.2.1                                                                                                                          | Source / Acquisition and Accountability .................................................................................... 100                                |
| 14.2.2                                                                                                                          | Metabolism .                                                                                                                                                    |
| 14.2.3                                                                                                                          | TOXICITY                                                                                                                                                        |
| 14.2.4                                                                                                                          | Formulation and Preparation                                                                                                                                     |
| 14.2.5                                                                                                                          | Stability and Storage ...................................................................................................................... 102                |
| 14.2.6                                                                                                                          | Administration Procedures                                                                                                                                       |
| 14.2.7                                                                                                                          | Incompatibilities ......................................................................................................................................... 102 |
| 14.3                                                                                                                            | M7824 (IND # 19165 ) .......................................................................................................................... 102             |
| Source / Acquisition and Accountability .............................................................................. 102      |                                                                                                                                                                 |
| 14.3.1                                                                                                                          |                                                                                                                                                                 |
| 14.3.2                                                                                                                          | Metabolism .                                                                                                                                                    |
| 14.3.3                                                                                                                          | TOXICITY                                                                                                                                                        |
| Formulation and Preparation                                                                                                     |                                                                                                                                                                 |
| 14.3.4                                                                                                                          |                                                                                                                                                                 |
| 14.3.5                                                                                                                          | Stability and Storage .................................................................................................................................... 105  |
| 14.3.6                                                                                                                          | Administration Procedures                                                                                                                                       |
| Incompatibilities                                                                                                               |                                                                                                                                                                 |
| 14.3.7                                                                                                                          |                                                                                                                                                                 |
| 14.4                                                                                                                            | T-DM1                                                                                                                                                           |
| 14.4.1                                                                                                                          | Source / Acquisition and Accountability .................................................................................... 106                                |
| 14.4.2                                                                                                                          | Administration Procedures.                                                                                                                                      |
| 15                                                                                                                              | REFERENCES .                                                                                                                                                    |
| 16                                                                                                                              | APPENDICIES                                                                                                                                                     |
| Appendix A: Performance Status Criteria .................................................................................... 16 |                                                                                                                                                                 |
| 16.1                                                                                                                            |                                                                                                                                                                 |
| 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                                                                                                                             |                                                                                                                                                                 |
untitled h1
abbreviated title the breast trial

| 16.4                                                                                                                                                                                                                                                                                               | Appendix D: Clinical and Safety Profile of MVA-BN and Recombinant MVA-Based   |
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| 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.
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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).
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.
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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.
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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

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.
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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).  
| 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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Table 1: Clinical benefit of current standard of care metastatic HER2+
breast cancer regimens.  
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
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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 nonreducible 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)
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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)
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 doselimiting 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.  
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.  
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-DRpositive monocytes and a decrease in granulocytic and monocytic myeloidderived suppressor cells. (38) Furthermore, in preclinical studies immunogenic modulation was seen against a broad range 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.  
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.
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1.2.3 checkpoint inhibitor experiences in

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 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)  
  
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 TGFβ1, -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. 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)
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.  
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 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)
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 twosided. *Figure from Palena et al. 2014*.
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brachyury immunogenicity

By using a major histocompatibility complex (MHC)-peptide binding prediction algorithm, a 9mer 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 dendritic cells, which in turn activate brachyury-specific CD4+ and CD8+ T-cells. (90) A doseescalation 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+ brachyuryspecific 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.
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poxviral vectors

Vaccinia virus has been used for over 200 years as a vaccine for smallpox and has a wellestablished 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 copresentation 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.  
Figure 7 BN-Brachyury recombinant poxvirus vaccine  
 platform.
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overview of modified vaccinia ankara

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 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.
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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). 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 metaanalyses 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)
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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 BNbrachyury 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 demonstrated generation of a brachyury-specific T cell response in breast cancer patients.  
- 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                | Brachyury-TRICOM Vaccine Trial                                                                                                             |                                                                                                                                           |
|----------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------|
| Heery et al 2015. Cancer Immunol Res.(91)    | 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                                                                                                       | Safety and Brachyury-specific T-cell                                                                                                      |
| responses                                    | responses                                                                                                                                  |                                                                                                                                           |
| Safety                                       | No DLTs. No G3 reactions.                                                                                                                  | No DLTs. G3 diarrhea (n=1)                                                                                                                |
| (G = grade)                                  | G1/2 injection site reactions common                                                                                                       | G1/2 injection site reactions and G1/2 flulike 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 ongoing 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 survival of 10 weeks (2.2 months) in patients with MSS, mCRC who receive an anti-PDL1 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.
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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)  
  
(B)  
  
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 HER2targeted 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 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).
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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 5 th 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.  
  
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.
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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 maytansinecontaining 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. 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.
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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 BNBrachyury 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 (4105, 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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2 eligibility assessment and enrollment

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)
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 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.
- 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.
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2.2 eligibility criteria for arms

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 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  
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 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)
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.
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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.
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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. 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
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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.
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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).
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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  
- 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.
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2.5 participant registration and status

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.
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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.
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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

| 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. |  
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.  
| Arms Number    | Name                                                                                            | Description                                                                                                                                                                                                                                                                                         |
|----------------|-------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1              | M7824                                                                                           | Bifunctional fusion molecule involving PD-L1 with                                                                                                                                                                                                                                                   |
| + BN-Brachyury | 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. |
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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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3 study implementation 3.1 study

- 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.
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.
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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-BNBrachyury 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). 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.
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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.
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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.  
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.
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3.1.1 study stopping rule all

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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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
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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 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
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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 selfadminister 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.
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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 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 108infectious 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 109Inf.U.  
Ideally, the BN-Brachyury vaccines will be administered prior to the start of the M7824 infusion.
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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 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 TDM1, 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. TDM1 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.
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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 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.
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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.
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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.
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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.