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3.4 dose modifications

- In the case of toxicity, appropriate medical treatment should be used (including anti-emetics, anti-diarrheals, etc.).  
- Once a patient has a dose reduction for toxicity, the dose will not be increased.  
- Any of the trial drugs may be stopped for toxicity per the discretion of the primary investigator if is it felt that the toxicity can be directly linked to a specific drug or drugs. However, if 2 drugs or more must be stopped, the patients will be taken off treatment.  
- Participants continuing to experience toxicity at the off-treatment visit will be contacted for additional assessments until the toxicity has resolved or is deemed irreversible. Patients must remain on the study to have additional assessments completed.  
- For AEs that are unrelated to the study drugs, study drug may be held for up to 28 days at the discretion of the PI. Any dose that would have occurred within this window will be skipped and the dose not made up.  
- If any treatment related AE observed was grade > 2, treatment should be delayed by 1 week
(except for hyperbilirubinemia where drug will be held for grade 2 - see **Table 7**; except for electrolyte abnormalities that can be easily replenished or other lab abnormalities that have no clinical consequence) for up to 4 consecutive weeks and possibly patients should be reduced by 1 dose level (See Table 3, Table 4, Table 5, Table 6, **Table 7**).  
- If, after 4 weeks of delay, all treatment related AEs have still not resolved (to grade ≤ 1), then any further treatment should be stopped.  
- If any treatment-related AE observed is grade 4, then all treatments should be stopped. The one exception is grade 4 thrombocytopenia in which the drug will be held and then reduce one dose level.  
| Dose Level   | Dose Reduction Schedule   | T-DM1 Dose            |
|--------------|---------------------------|-----------------------|
| 1            | Starting dose             | 3.6 mg/kg IV q3 weeks |
| -1           | First dose reduction      | 3.0 mg/kg IV q3 weeks |
| -2           | Second dose reduction     | 2.4mg/kg IV q3 weeks  |
| -3           | Third dose reduction      | Discontinue treatment |  
* There will be no dose reductions of the BN-Brachyury vaccines. ** M7824 may be reduced at any time after the DLT period in the study from 2,400mg to 1,800mg at discretion of the PI for any side effects possibly attributed to the TGF-beta component of the drug (i.e., mucosal bleeding, excessive keratoacanthomas).  
| Table 3B: Recommended Dose Reduction Schedule for Adverse Events for Arm 3 Dose Level Dose Reduction Schedule T-DM1 Dose Entinostat 1 Starting dose 3.6mg/kg IV q3 weeks 5mg po q7 days -1 First dose reduction 3.6mg/kg IV q3 weeks 3mg po q7 days -2 Second dose reduction 3.0mg/kg IV q3 weeks 3mg po q7 days -3 Third dose reduction 2.4mg/kg IV q3 weeks 2mg po q7 days -4 Requirement for further  Discontinue treatmentdose reduction   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
* There will be no dose reductions of the BN-Brachyury vaccines. ** M7824 may be reduced at any time after the DLT period in the study from 2,400mg to 1,800mg at the discretion of the PI for any side effects possibly attributed to the TGF-beta component of the drug (i.e., mucosal bleeding, excessive keratoacanthomas).  
3.4.1 Dose Modification of Entinostat  
| Table 4: Non-hematologic Toxicity Toxicity Dose Modifications Grade 4 Administer symptomatic remedies/ start prophylaxis. Hold dose until recovery to Grade 1 or baseline under the following directions: 1. If recovered within 4 weeks of onset (i.e., ≤3 missed doses), resume study drug as follows: - If receiving 5 mg, restart study drug at 3 mg. - If receiving 3 mg, restart study drug at same dose or reduce to 2 mg (see Table 3B). - If receiving 2 mg, discontinue study treatment. 2. If not recovered within 4 weeks, permanently discontinue study drug. Grade 3 Administer symptomatic remedies / start prophylaxis. Hold dose until  recovery to Grade 1 or baseline under the following directions: 1. If recovered within 1 week, resume study drug at prior dose. If not  recovered within 1 week, continue to hold dose. 2. If recovered within 2-4 weeks, resume study drug as follows: - If receiving 5 mg, restart study drug at 3 mg. - If receiving 3 mg, restart study drug at same dose or reduce to 2 mg (see Table 3B). - If receiving 2 mg, permanently discontinue study drug. 3. If not recovered within 4 weeks, permanently discontinue study drug. Recurrence of  the same ≥ Grade 3 toxicity despite  dose reduction If the same ≥ Grade 3 event recurs: 1. Administer symptomatic remedies/ start prophylaxis. Hold1 dose until  recovery to Grade 1 or baseline. 2. If recovered within 2 weeks, resume study drug as follows: - If receiving 5 mg, restart study drug at 3 mg. - If receiving 3 mg, restart study drug at same dose or reduce to 2 mg (see Table 3B). - If receiving 2 mg, permanently discontinue study drug. 3. If the same ≥ Grade 3 event recurs (i.e., third occurrence) despite  entinostat dose reduction to 2 mg, as described above, discontinue study  drug. ≤Grade 2 Administer symptomatic remedies / start prophylaxis. Dosing of study drug may be interrupted at the Investigator's discretion. - If dose is held for 4 consecutive weeks, permanently discontinue study drug. - If toxicity resolves, resume entinostat at the original dose.   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
PIf greater than 50% of doses are missed during any 6 week period, discontinue from study drug *treatment.*  
| Table 5: Hematologic Toxicity Toxicity Dose Modifications ≥ Grade 3 neutropenia, ≥ Grade 3 uncomplicated thrombocytopenia,  or Grade 2 complicated thrombocytopenia Administer symptomatic remedies / start prophylaxis. Hold dose1 until recovery to Grade 1 or study baseline under the following  direction: 1. If not recovered by next scheduled dose, skip the dose. If recovered  by next scheduled dose, resume study drug at prior dose. 2. If receiving 2 mg dose, and not recovered by either of the next 2  scheduled doses, permanently discontinue study treatment. Otherwise, skip each dose. If recovered for either of these doses,  resume study drug as follows:  - If receiving 5 mg, restart study drug at 3 mg. - If receiving 3 mg, restart study drug at 2 mg (see Table 3B). 3. If not recovered within 4 weeks, permanently discontinue study drug. If the same hematologic toxicity recurs: 1. Administer symptomatic remedies/ start prophylaxis. Hold1 dose until  recovery to Grade 1 or baseline. 2. If recovered within 2 weeks, resume study drug as follows: - If receiving 5 mg, restart study drug at 3 mg - If receiving 3 mg, restart study drug at 2 mg. (See Table 3B) - If receiving 2 mg, permanently discontinue study drug 3. If the same ≥ Grade 3 event recurs (i.e., third occurrence) despite  entinostat dose reduction to 2 mg, as described above, permanently  discontinue study drug. Recurrence of the same hematologic  toxicity PIf greater than 50% of doses are missed during any 6 week period, discontinue from study drug treatment. 3.4.2 Dose Modification of T-DM1   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Dose modifications for T-DM1 according to published FDA recommendations. Please see https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/125427lbl.pdf for full details. Once the dose of T-DM1 is reduced, the dose should not be re-escalated. If a planned dose is delayed or missed, it should be administered at the next planned cycle. The infusion may be administered at the dose and rate the patient tolerated in the most recent infusion.  
The infusion rate of T-DM1 should be slowed or interrupted if the patient develops an infusionrelated reaction. Permanently discontinue T-DM1 for life-threatening infusion-related reactions.  
Management of increased serum transaminases, hyperbilirubinemia, left ventricular dysfunction, thrombocytopenia, pulmonary toxicity or peripheral neuropathy may require temporary interruption, dose reduction or treatment discontinuation of T-DM1 as per guidelines provided in 6**, Table 7, Table 8, Table 9**.  
Hepatotoxicity  
| Grade 2                   | Grade 3                                                                                  | Grade 4      |
|---------------------------|------------------------------------------------------------------------------------------|--------------|
| (> 2.5 to < 5x ULN)       | (> 5 to < 20x ULN)                                                                       | (> 20 x ULN) |
| Treat at same dose level. | Do not administer T-DM1 until  AST/ALT recovers to Grade < 2  and then reduce one level. | Permanently discontinue TDM1.              |  
ALT = alanine transaminase; AST = aspartate transaminase; ULN = upper limit of normal.  
Table 7: Dose Modification for Hyperbilirubinemia  
Grade 2  
(> 1.5 to < **3x ULN)**  
Grade 3  
(> 3 to < 10x ULN)  
Grade 4  
(> 10 x ULN)  
Do not administer T-DM1 until total bilirubin recovers to Grade <1 and then treat at same dose level.  
| Grade 3                                                                                          | Grade 4      |
|--------------------------------------------------------------------------------------------------|--------------|
| (> 3 to < 10x ULN)                                                                               | (> 10 x ULN) |
| Do not administer T-DM1 until  total bilirubin recovers to Grade  < 1 and then reduce one level. | Permanently discontinue TDM1.              |  
Permanently discontinue T-DM1 treatment in patients with serum transaminases > 3x ULN and concomitant total bilirubin > 2x ULN. Permanently discontinue T-DM1 in patients diagnosed with nodular regenerative hyperplasia (NRH). Left Ventricular Dysfunction CHF = congestive heart failure; LVEF = left ventricular ejection fraction  
| Table 8: Dose Modification for Left Ventricular Dysfunction  Symptomatic  LVEF < 40% LVEF 40% to  CHF ≤45% AND  decrease is > 10% points from  baseline LVEF 40% to  ≤45% AND  decrease is <  10% points from  baseline   | LVEF > 45%                |                                          |    |    |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------|------------------------------------------|----|----|
| Discontinue TDM1.                                                                                                                                                                                                                           | Do not administer  T-DM1. | Do not administer  T-DM1.                | Continue with TDM1.    | Continue with TDM1.    |
| Repeat LVEF  assessment within  3 weeks. If LVEF  < 40% is  confirmed, then  discontinue TDM1.                                                                                                                                                                                                                           | Repeat LVEF  assessment within  3 weeks. If the  LVEF has  recovered to  within 10% points  from baseline,  discontinue TDM1.                           | Repeat LVEF  assessment within  3 weeks. |    |    |
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thrombocytopenia

A reduction in dose is recommended in the case of Grade 4 thrombocytopenia (platelets <
25,000/mm3).  
| Grade 3                                                                                                      | Grade 4                                                                                                 |
|--------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------|
| PLT 25,000/mm3 to 50,000/mm3                                                                                 | PLT < 25,000/mm3                                                                                        |
| Do not administer T-DM1 until PLT recovers  to Grade < 1 (> 75,000/mm3 ) and then treat at  same dose level. | Do not administer T-DM1 until PLT recovers to Grade <1(> 75,000/mm3 ), and then reduce  one dose level. |
| PLT = platelets                                                                                              |                                                                                                         |
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pulmonary toxicity

T-DM1 should be permanently discontinued in patients with interstitial lung disease (ILD) or pneumonitis.
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peripheral neuropathy

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

Guidelines below are merely suggestions. If an immune-mediated reaction occurs, the subject should be treated according to the best available medical practice.  
| Infusion-Related Reactions (IRR) are an important risk for M7824. NCI-CTCAE Grade Treatment Modification for M7824 Grade 1 - mild - Increase monitoring of vital signs as medically  indicated as participants are deemed medically  stable by the attending Investigator. Mild transient reaction; in general,  infusion interruption not indicated;  intervention not indicated. - Hold infusion if deemed necessary by the  investigator. Grade 2 - moderate - Stop the infusion of the study intervention.  Therapy or infusion interruption  indicated but responds promptly to  symptomatic treatment (for example,  antihistamines, nonsteroidal anti  inflammatory drugs, narcotics, IV  fluids); prophylactic medications  indicated for  24 h. - Increase monitoring of vital signs as medically  indicated as participants are deemed medically  stable by the attending Investigator. - If symptoms resolve quickly, resume infusion at  50% of original rate with close monitoring of any  worsening signs and symptoms, otherwise dosing  held until resolution of symptoms with mandated  premedication for the next scheduled visit. - If not improving, consider administration of  glucocorticoids and stop the infusion for that day. - If the participant has a second IRR Grade ≥ 2 on  the slower infusion rate despite premedication, the  infusion should be stopped, and the investigator  may consider withdrawal of this participant from  the study.   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| NCI-CTCAE Grade                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Treatment Modification for M7824                                                     |                                                                                                                                                                                                                                                                                                    |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 3 or Grade 4 - severe or  life-threatening                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | -                                                                                    | Stop the infusion of study intervention immediately  and disconnect infusion tubing from the participant  with additional appropriate medical measures and  closely monitor until deemed medically stable by  the attending Investigator. Hospitalization and/or  close monitoring is recommended. |
| Grade 3: Prolonged (for example,  not rapidly responsive to  symptomatic medication and/or brief  interruption of infusion); recurrence  of symptoms following initial  improvement; hospitalization  indicated for clinical sequelae.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | -                                                                                    | Administration of glucocorticoids may be required.                                                                                                                                                                                                                                                 |
| -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | For Grade 3 or 4 IRRs, permanent discontinuation  of study intervention is mandated. |                                                                                                                                                                                                                                                                                                    |
| Grade 4: Life-threatening  consequences; urgent intervention  indicated. Once the infusion is interrupted or rate reduced to 50% of previous infusion rate, it must  remain decreased for all subsequent infusions.  For all types and grades of infusion reactions, details about drug physical constitution,  method of preparation, and infusion must be recorded. Participants should be instructed to report any delayed reaction immediately. Once the infusion is interrupted or rate reduced to 50% of previous infusion rate, it must  remain decreased for all subsequent infusions.  For all types and grades of infusion reactions, details about drug physical constitution,  method of preparation, and infusion must be recorded. Participants should be instructed to report any delayed reaction immediately. |                                                                                      |                                                                                                                                                                                                                                                                                                    |
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table 11 immune-related adverse events

Immune-related AEs are specific to immunotherapies and vary by organ system. The following immune-related AEs are important identified risks for M7824:  
| -                                                                         | Immune-related pneumonitis                                                                |
|---------------------------------------------------------------------------|-------------------------------------------------------------------------------------------|
| -                                                                         | Immune-related hepatitis                                                                  |
| -                                                                         | Immune-related colitis                                                                    |
| -                                                                         | Immune-related nephritis and renal dysfunction                                            |
| -                                                                         | Immune-related endocrinopathies                                                           |
| -                                                                         | (thyroid disorders, adrenal insufficiency, type 1 diabetes mellitus, pituitary disorders) |
| -                                                                         | Immune related rash                                                                       |
| -                                                                         | Other immune-related events (myositis, myocarditis, encephalitis)                         |
| The following immune-related AEs are important potential risks for M7824: |                                                                                           |  
- Guillain-Barré syndrome
- Uveitis
- Pancreatitis
- Myasthenia gravis/myasthenic syndrome
Recommended guidance and management for specific irAEs are provided in the current NCCN (guideline available at http://www.nccn.org). Requirements in addition to NCCN guidelines:  
- Permanent treatment discontinuation is required in case of immune-related Grade 4 rash/inflammatory dermatitis, nephritis, autoimmune hemolytic anemia, hemolytic uremic syndrome, aplastic anemia, immune thrombocytopenia, acquired thrombotic thrombocytopenic purpura inflammatory arthritis, myositis and polymyalgia-like syndrome.  
- For Grade 4 immune-related lymphopenia, permanent treatment discontinuation will be required, if lymphopenia is considered immune-related in nature, no clear alternative explanation exists for the event, and it does not resolve within 14 days. Permanent treatment discontinuation is not required when the AE is manifested by a single laboratory value out of normal range without any clinical correlates. In this case, treatment should be held until the etiology is determined. If the event is not considered immune-related and resolves to Grade ≤ 1, restarting treatment may be considered.  
- For Grade 1 immune-related pneumonitis: continue treatment. If clinically indicated, monitor participants weekly or more frequently as needed with history, physical examination and pulse oximetry. If symptoms appear and/or changes in the physical exam are noted, treat as Grade 2.  
- For myositis: in case of management with rituximab, treatment should be discontinued.  
- For Grade 3 or 4 endocrinopathies: withhold until clinically stable or permanently discontinue depending on severity.  
- For hepatitis with no tumor involvement of the liver: withhold if total bilirubin increases to more than 1.5 and up to 3 times ULN, permanently discontinue if more than 3 times ULN.  
- Hepatitis with tumor involvement of the liver: permanently discontinue if total bilirubin increases to more than 3 times ULN.
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table 12 management of m7824

Skin reactions are considered important identified risk for M7824.  
- Hyperkeratosis
- Keratoacanthoma
- Cutaneous squamous cell carcinoma (cSCC)
- Basal cell carcinoma
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- actinic keratosis

Management
- Discontinuation or termination not required in most cases. Continuation of treatment should be evaluated by the Investigator.  
- Emollients may be used - Develop diagnostic and treatment plan in collaboration with Investigator and dermatologist
- Treatment follow-up will depend on number and localization of lesions.  
o Single lesion: full excision may be recommended o Multiple lesion or location not suitable for full excision: Mohrs surgery, cryotherapy or other standard treatment options depending on pathology. Retinoids may be used after discussion with Investigator.  
- Close clinical follow-up for re-evaluation, resolution and potential recurrence should be implemented
- In general, treatment of skin lesions should be based on local guidelines/standard of care.
Additional consideration: Keratoacanthoma lesions may resolve spontaneously without surgical intervention within weeks after discontinuing M7824. Consult with Medical Monitor as needed for management of skin lesions.
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table 13 management of treatment-related

Anemia is considered an important identified risk for M7824.  
- All relevant hematological testing for treatment-related anemias should be done prior to a blood transfusion, if clinically feasible
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basic anemia evaluation

- CBC with emphasis on red cell indices
- If indicated and at clinical discretion, the following should be considered:
o Iron studies o Serum Folate and Vit B12 values o Coagulation factors o Fecal occult blood o Urinalysis o Hormone panel: TSH, Erythropoietin o Peripheral blood smear
Further Recommendation Based on Suspected Etiology (in Addition to Basic Anemia
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testing)

- Suspected Hemolysis o bilirubin, LDH, Coombs test, haptoglobin
- Suspected bleeding:  
| o                                                                                                                              | Consider imaging/interventional radiology consultation as indicated                                           |
|--------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------|
| o                                                                                                                              | Consider imaging and/or endoscopy as clinically indicated                                                     |
| -                                                                                                                              | Suspected aplastic anemia: o Hematology consultation o Consider bone marrow aspiration/morphologic evaluation |
| Additional consideration:  In general, blood transfusions and erythroid growth factors are permitted as clinically  indicated. |                                                                                                               |  
| Table 14 Management of Bleeding Adverse Events Bleeding Adverse Events - Bleeding adverse events are considered important identified risk for M7824. - In general, mild and moderate mucosal bleedings resolve without discontinuation of  treatment. - These events may include, but are not limited to the following: o Epistaxis o Hemoptysis  o Gingival bleeding o Hematuria Non-tumor Bleeding Grading Management Grade 2 - If resolves to Grade ≤ 1 by the day  before the next infusion, study  intervention may be continued - If not resolved to Grade ≤ 1 by the day  before the next infusion, but is  manageable and /or not clinically  relevant, assessment if clinically  reasonable to administer the following  infusion will be per PI discretion. Grade 3 - Permanently discontinue treatment  unless an alternative explanation can be  identified (such as concomitant use of  antithrombotic agents, traumatic events,  etc.)  - In case of alternative explanations, hold  study treatment until the event recovers  to Grade ≤ 1 Grade 4 - Treatment must be permanently  discontinued if no alternative  explanation is identified. Tumor Bleeding   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Grade ≥ 2   | -                                                                                                                               | Study treatment must be held till the  event recovers to Grade ≤ 1   |
|-------------|---------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------|
| -           | Permanently discontinue treatment if the  Investigator considers the participant to  be at risk for additional severe bleeding. |                                                                      |
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table 15 impaired wound healing

- Impaired wound healing is considered important potential risk for M7824.  
- Elective surgery on study will be allowed per PI discretion. - It is recommended to hold study intervention for approximately 4 weeks post major surgery for observation.  
Post-operative wound healing should be closely monitored.
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3.5 study calendar 1 cycle

| Procedure                                                                          | Screening          | Cycle 1        | Cycle 2                | Cycle 3 Cycle 5 Cycle 7   | Cycle 4 Cycle 6 Cycle 8                      | Cycle 9   | End of     |    |    |
|------------------------------------------------------------------------------------|--------------------|----------------|------------------------|---------------------------|----------------------------------------------|-----------|------------|----|----|
| And beyond                                                                         | treatment  visit13 | Safety visit13 | Long term  follow up14 |                           |                                              |           |            |    |    |
| Day 1                                                                              | Day 8              | Day 15         | Day 1                  | Day 1                     | Day 1                                        | Day 1     | (≤ 30 days |    |    |
| (-1/+3days)                                                                        | (-1/+3days)        | (-1/+3days)    | (-1/+3days)            | from last  dose)          | (28 days after  removal from  study therapy) |           |            |    |    |
| Informed consent                                                                   | X                  | X1             |                        |                           |                                              |           |            |    |    |
| History and PE2                                                                    | X                  | X              | X                      | X                         | X                                            | X         | X          | X  | X  |
| (< 3 days)                                                                         |                    |                |                        |                           |                                              |           |            |    |    |
| Vital signs15                                                                      | X                  | X              | X                      | X                         | X                                            | X         | X          | X  | X  |
| Performance Score                                                                  | X                  |                |                        |                           |                                              |           |            |    |    |
| Skin assessment16                                                                  | X                  | X              | X                      | X                         | X                                            | X         | X          | X  |    |
| Clinical Labs                                                                      | X                  | X4 (< 7 days)  | X4                     | X4                        | X4                                           | X4        | X4         | X4 | X  |
| Pathology                                                                          | X                  |                |                        |                           |                                              |           |            |    |    |
| confirmation5 Tumor block                                                          | X6                 |                |                        |                           |                                              |           |            |    |    |
| Tumor biopsy7                                                                      | X                  | X              |                        |                           |                                              |           |            |    |    |
| (< 7 days)                                                                         | (Cycle 3  only)    | X              |                        |                           |                                              |           |            |    |    |
| (optional)                                                                         |                    |                |                        |                           |                                              |           |            |    |    |
| Research Blood8                                                                    | See Table below    |                |                        |                           |                                              |           |            |    |    |
| Urine or serum  pregnancy testing in  women of  childbearing  potential9 (2.4.2.2) | X                  | X              | X                      | X                         | X                                            | X         | X          | X  |    |
| Advance Directives                                                                 | X                  |                |                        |                           |                                              |           |            |    |    |
| Form                                                                               | (optional)         |                |                        |                           |                                              |           |            |    |    |
| Radiological                                                                       | X                  | X              | X                      | X                         | X                                            |           |            |    |    |
| Assessments10                                                                      | (< 28days)         | (q9 wks)       | (q9 wks)               |                           |                                              |           |            |    |    |
| Adverse Events                                                                     | X                  | X              | X                      | X                         | X                                            | X         | X          | X  | X  |
| Concomitant                                                                        | X                  | X              | X                      | X                         | X                                            | X         | X          | X  |    |
| Medications Electrocardiogram11                                                    | X                  | X              | X                      |                           |                                              |           |            |    |    |
| (< 7 days)                                                                         |                    |                |                        |                           |                                              |           |            |    |    |  
| Procedure                                 | Screening                      | Cycle 1                           | Cycle 2                | Cycle 3 Cycle 5 Cycle 7                  | Cycle 4 Cycle 6 Cycle 8                      | Cycle 9   | End of     |
|-------------------------------------------|--------------------------------|-----------------------------------|------------------------|------------------------------------------|----------------------------------------------|-----------|------------|
| And beyond                                | treatment  visit13             | Safety visit13                    | Long term  follow up14 |                                          |                                              |           |            |
| Day 1                                     | Day 8                          | Day 15                            | Day 1                  | Day 1                                    | Day 1                                        | Day 1     | (≤ 30 days |
| (-1/+3days)                               | (-1/+3days)                    | (-1/+3days)                       | (-1/+3days)            | from last  dose)                         | (28 days after  removal from  study therapy) |           |            |
| 2D Echo every ~84  days Arms 2 and 3 only | X  (< 28  days)                | X every 84 days or 4 cycles → → → |                        |                                          |                                              |           |            |
| MVA-BNBrachyury12                                           | X                              | X                                 |                        |                                          |                                              |           |            |
| FPV-Brachyury12                           | X                              | X                                 | X                      |                                          |                                              |           |            |
| Every 12  wks                             |                                |                                   |                        |                                          |                                              |           |            |
| Entinostat weekly12                       | X                              | X                                 | X                      | Take on Days 1, 8, and 15 of each cycle. |                                              |           |            |
| Arm 3 only                                | Can be taken up to 1 day late. |                                   |                        |                                          |                                              |           |            |
| T-DM112                                   | X                              | X                                 | X                      | X                                        | X                                            |           |            |
| Arms 2 and 3 only M782412                 | X                              | X                                 | X                      | X                                        | X                                            |           |            |  
1 Registration must be completed within 24 hours of signing consent.  
2 History including prior treatments and review of baseline adverse events and concurrent medications. Physical examination includes breast and lymph node exam. Height (at screening only) and weight are also included. Full history and physical to be performed at initial clinical visit. On subsequent visits, abbreviated exam, review of symptoms and review of medications.  
3 CBC with differential, PT/INR/PTT, comprehensive metabolic panel (see **2.4.2.2**), and anti-HIV, anti-HCV, and Hepatitis B Surface Ag (within 3 months prior to enrollment). If applicable, CD4 T cell count within 3 months.  
4 Labs must be obtained < 7 days of initiating treatment: CBC with differential, PT/INR/PTT (will be repeated if ≥ 72 hours prior to biopsy for Arms 2 and 3), sodium, potassium, chloride, carbon dioxide, BUN, creatinine or measured creatinine clearance, glucose, AST, ALT, bilirubin, calcium, total protein, albumin, alkaline phosphatase, phosphorus, magnesium, urinalysis. Troponin-I and CK, CK-MB will be evaluated at, C1D1, C1D8, C2D1 and C3D1. Tumor Markers (CEA, CA15-3, CA27.29). TSH and reflexed Free T4 (if TSH abnormal) will be performed at screening and at restaging visits (approximately every 9 weeks) unless there is a clinical concern that warrants evaluation on a more frequent basis.  
5 Pathological confirmation of diagnosis of either TNBC (Arm 1) or ER-/PR-/HER2+ (Arms 2 and 3). Patients may enroll with a pathologist's report showing a histologic diagnosis of TNBC or HER+BC in a College of American Pathologists (CAP) accredited laboratory and a clinical course consistent with the disease. If report not available, disease will be confirmed at the local site.  
6 Primary tumor paraffin block will be requested at C1D1 to perform comparative genomic analysis as part of correlative studies planned for this clinical trial.  
7 Planned biopsy after consent but within 7 days prior to first dose of drug on Cohort 3 as well as a planned biopsy on C3D1. There is also an optional biopsy at the time of progression on all arms. Patients in Cohorts 1 and 2 may also have optional biopsies at baseline and at C3D1.  
8 Where feasible, research blood for all study assessments will be collected as per **Table 16**. The exact collection time for PK samples will be recorded.  
9 For female subjects of childbearing potential, urine or serum beta-HCG will be performed on initial screening. A urine or serum beta-HCG will be performed before each administration of the trial drugs during the treatment phase (must be performed on C1D1), at the end-of-treatment visit, and at the post-treatment follow-up visit.  
10 Baseline imaging at screening (or within 28 days prior to enrollment): CT of chest/abdomen /pelvis (MRI may be used when CT
scan is not an option to follow the disease clinically). Brain MRI may be ordered in patients with known CNS disease or if there is concern for CNS metastasis. Gadolinium will be used with MRI. Head CT with contrast may be done instead of MRI. Other imaging on study: CT of chest/abdomen/pelvis to be repeated every 9 weeks +/- 3 days after start of treatment. Bone scan and additional imaging assessments may also be performed as clinically indicated at screening or during the study.  
11 Baseline12-lead EKG at screening (within 28 days prior to enrollment), at baseline, and repeat EKG on Cycle 1 Day 8 with repeats only for abnormal baseline EKG, Cycle 1 Day 8 EKG or if patient is symptomatic.  
12 Day 1 Dosing Schedule for each Cycle: BN-Brachyury vaccine is preferred to be given prior to the M7824 infusion, which will be followed by T-DM1 administration. Patients will be provided with a take-home supply of entinostat and will self-administer entinostat after the completion of the T-DM1 infusion on day 1 of each cycle, preferably prior to bed. On C1D8, patients will take entinostat at the time of their clinical appointment. If patients return for the optional C1D15 labs, patients will self-administer entinostat upon arrival at the clinical center. Patients will self-administer entinostat on Days 1, 8, and 15 of each subsequent cycle. Allowance for scheduling changes: brief interruption and delay in the 21-day cycle may occasionally be required due to travel delays, airport closure, inclement weather, family responsibilities, security alerts, and government holidays, etc. This can also extend to complications of disease not attributable to disease progression or protocol therapy. These delays will not be considered protocol deviation.  
13 End of treatment visit: Where feasible or logistically possible, on the day of or within 30 days of the decision to discontinue treatment prematurely before completion of one year of treatment. Does not need to be completed if drug is withheld after one year of treatment.  
14 Long term follow up: Every 3 months for a year followed by every 6 months for an additional year. During this time: patients who have progressed on treatment will be followed by phone or email for survival, adverse events and further tumor therapy. Patients who have not progressed on treatment will be scanned until progression.  
15 Vital signs will be monitored every 15 minutes (Cycle 1) and every 30 minutes (Cycle 2 and beyond) during M7824 infusion and until completion of the observation period in all Arms. Vital signs should also be recorded immediately prior to the start of T-DM1 in Arms 2 and 3. (See **3.2.2** and **3.2.3** for more detail.)
16 Skin assessment must be performed at baseline, every 6 weeks, and at the end of treatment or at the 28 (±7 days) day safety followup (if not performed in the previous 6 weeks), if feasible.  
Table 16 Research Blood Draws  
| Week (Day)                                                   | Time     | M7824        | Entinostat                              | Immune and Other Analyses   |    |    |    |    |
|--------------------------------------------------------------|----------|--------------|-----------------------------------------|-----------------------------|----|----|----|----|
| Immune                                                       |          |              |                                         |                             |    |    |    |    |
| phenotyping,  Antigen  specific  responses,  immune  subsets |          |              |                                         |                             |    |    |    |    |
| Histone                                                      |          |              |                                         |                             |    |    |    |    |
| Deacetylation                                                | T cell   |              |                                         |                             |    |    |    |    |
| clonality Soluble  Factors                                   | CfDNA    |              |                                         |                             |    |    |    |    |
| PK                                                           | ADA      | TGFβ1 levels | Accounted for in the 60-80ml for  PBMCs |                             |    |    |    |    |
| Cycle 1 Day 1 predose                                        | X        | X            | X                                       | X                           | X  | X  | X  | X  |
| EOI                                                          | X        |              |                                         |                             |    |    |    |    |
| Cycle 1 Day 8                                                | X        | X            | X                                       | X                           | X  |    |    |    |
| Cycle 1  Day15                                               | Optional | X            | X                                       | X                           | X  | X  |    |    |
| Cycle 2 Day 1 predose                                        | X        | X1           | X1                                      | X1                          | X1 | X1 |    |    |
| Cycle 3 Day 1 predose                                        | X        | X            | X                                       | X                           | X  | X  | X  |    |
| EOI                                                          | X        |              |                                         |                             |    |    |    |    |
| Cycle 6 Day 1 predose                                        | X        | X            |                                         |                             |    |    |    |    |
| End of  Treatment                                            | X        | X            | X                                       | X                           | X  | X  | X  |    |  
1Cycle 2 Day 1 labs to be drawn only if patient did not do Cycle 1 Day 15 optional labs. Do not need both timepoints
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3.6 cost and compensation 3.6.1

Subjects costs will be based on local guidelines as described in the site-specific consent.  
3.6.2 Compensation Participants will not receive compensation on this study. 3.6.3 Reimbursement Reimbursement will be provided per local guidelines as indicated in the site consent document.
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3.7 criteria for removal from

Regardless of reason for removal from study therapy, patients will be asked to have a 28 day follow-up safety visit. Patients who refuse to return for this visit will be asked to review any safety concerns by phone within this time period. 3.7.1 Criteria for removal from protocol therapy
- Clinical or radiographic progression of disease except when the investigator feels the subject is still benefiting from treatment. (It is generally preferable for patients to remain on treatment past initial radiographic progression in case there is pseudo - progression, except when the investigator feels that the clinical picture warrants changing therapy at initial progression.);
- Participant requests to be withdrawn from active therapy;
- Unacceptable toxicity as defined in Sections **3.1.2** and 3.3;
- Start of another systemic anticancer treatment or participation in another investigational therapeutic trial;
- Use of restricted medication (Section 4.3);
- Investigator discretion;
- Positive pregnancy test;
- Permanent loss of capacity to give consent.  
3.7.2 Off-Study Criteria
- Screen failure;
- PI decision to end the study;
- Participant requests to be withdrawn from study;
- Completion of follow up period;
- Participant lost to follow-up; - Investigator discretion;
- Disease progression;
- Permanent loss of capacity to give consent;
- Death.
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3.7.3 lost to follow-up

A participant will be considered lost to follow-up if he or she fails to return for 4 clinic visits scheduled visits and is unable to be contacted by the study site staff.  
The following actions must be taken if a participant fails to return to the clinic for a required study visit:
- The site will attempt to contact the participant and reschedule the missed visit within the next 4 weeks and counsel the participant on the importance of maintaining the assigned visit schedule and ascertain if the participant wishes to and/or should continue in the study.  
- Before a participant is deemed lost to follow-up, the investigator or designee will make every effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, a certified letter to the participant's last known mailing address or local equivalent methods). These contact attempts should be documented in the participant's medical record or study file.  
- Should the participant continue to be unreachable, he or she will be considered to have withdrawn from the study with a primary reason of lost to follow-up.
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4 concomitant medications / measures

Any medications (other than those excluded by the clinical trial protocol) that are considered necessary to protect subject welfare or alleviate symptoms and will not interfere with the trial medications may be given at the Investigator's discretion.
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4.1 antiemetics

Due to low rates of nausea with M7824, BN-Brachyury vaccine and entinostat, no antiemetic prophylaxis will be given. However, if a patient does experience nausea/vomiting, antiemetics may be used. Since T-DM1 is considered a drug with low emetic risk by both ASCO and NCCN, the use of a single dose of a 5-HT3 antagonist or dopamine blocker (i.e., prochlorperazine) is encouraged. Due to the effect of CYP3A4 inhibitors on T-DM1, avoid aprepitant, fosaprepitant, and netupitant, as well as other CYP3A4 inhibitors. The use of steroids for antiemetic effect is allowed per the discretion of the primary investigator. The antiemetic regimen may be modified based on patient specific factors or standard institutional practices.
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4.2 growth factor support

Significant neutropenia is uncommon with single agent T-DM1, M7824 or entinostat. However, growth factor support is allowed for grade 3 neutropenia or at the discretion of the primary investigator after the completion of the DLT period for each trial arm. If neutropenia or a neutropenic fever requiring growth factor support occurs during the DLT period, this will be counted as a DLT.
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4.3 restricted medications

The following medications should NOT be administered during the trial:  
- Other immunotherapies or immunosuppressive drugs for example, chemotherapy or systemic corticosteroids **except** for prophylaxis or treatment of allergic reactions, endocrine replacement therapy at low dose prednisone [≤ 10 mg daily] or equivalent, for the treatment of irAEs, or for short courses (≤ 14 days) as appropriate medical therapy for
unrelated medical conditions (e.g., asthma). Steroids with no or minimal systemic effect (topical, inhalation) are allowed.  
- Prophylactic use of corticosteroids for infusion related reactions. Corticosteroid administration prior to CT scans in patients with intravenous contrast allergy is allowed.  
- Any live vaccine therapies for the prevention of infectious disease. Administration of inactivated vaccines is allowed (for example, inactivated influenza vaccines or locally approved COVID vaccines).  
- Systemic anticancer treatment other than the investigational agents in this trial. - Herbal supplements.
No formal drug-drug interaction studies with the BN-Brachyury-vaccines, M7824 or entinostat have been performed. Use caution when administering entinostat concurrently with medications that have a narrow therapeutic index. No formal drug-drug interaction studies with T-DM1 have been conducted. *In vitro* studies indicate that DM1, the cytotoxic component of T-DM1, is metabolized mainly by CYP3A4 and to a lesser extent by CYP3A5. Concomitant use of strong CYP3A4 inhibitors (e.g., ketoconazole, itraconazole, clarithromycin, atazanavir, indinavir, nefazodone, nelfinavir, ritonavir, saquinavir, telithromycin, and voriconazole) with T-DM1 should be avoided due to the potential for an increase in DM1 exposure and toxicity. Consider an alternate medication with no or minimal potential to inhibit CYP3A4. If concomitant use of strong CYP3A4 inhibitors is unavoidable, consider delaying T-DM1 treatment until the strong CYP3A4 inhibitors have cleared from the circulation (approximately 5 elimination half-lives of the inhibitors) when possible. If a strong CYP3A4 inhibitor is co-administered and T-DM1 treatment cannot be delayed, patients should be closely monitored for adverse reactions (https://www.accessdata.fda.gov).
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4.4 contraception

The effects of BN-Brachyury, entinostat, M7824 and T-DM1 on the developing human fetus are unknown. However, both components of T-DM1 are known to cause fetal defects including oligohydramnios and oligohydramnios syndromes. For this reason, women of childbearing potential must agree to use two methods of adequate contraception (see **Table 17**) at study entry, for the duration of study participation and for at least 7 months for women and 4 months for men after the final dose of any study-related medications (T-DM1 has a prolonged half-life). Cessation of birth control after this point should be discussed with a responsible physician. Periodic abstinence, the rhythm method and the withdrawal method are not acceptable methods of birth control. Women with hormone receptor positive breast cancer should avoid hormonal contraceptive agents.  
Table 17: Effective methods of contraception (two methods must be used for men and women of child-bearing potential)  
Barrier Methods *Intrauterine Device*  
Methods  
Hormonal Methods*  
| Barrier Methods                                             | Intrauterine Device                                           | Hormonal Methods*            |
|-------------------------------------------------------------|---------------------------------------------------------------|------------------------------|
| Methods                                                     |                                                               |                              |
| Male condom plus spermicide                                 | Copper T                                                      | Implants                     |
| Cap plus spermicide                                         | Progesterone Ta                                               | Hormone shot or injection    |
| Diaphragm plus spermicide                                   | Levonorgestrel-releasing  intrauterine system  (e.g., Mirena) | Combined pill Minipill Patch |
| aThis is also considered a hormonal method of birth control |                                                               |                              |  
Minipill Patch aThis is also considered a hormonal method of birth control
*Women with hormone receptor positive breast cancer should avoid hormonal agents; however, since all participants in this study will have hormone receptor negative (or low hormone receptor positivity) breast cancer, these agents can be used.
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4.5 breast feeding

It is not known whether BN-Brachyury, entinostat, M7824 or T-DM1 is excreted in breast milk. It is recommended that women do not breastfeed during treatment on trial and for at least 7 months after the last dose due to the prolonged half-life of T-DM1 and M7824.
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4.6 blood donation

Patients should not donate blood while participating in this study or for 7 months following the last treatment on trial due to the prolonged half-life of T-DM1 and M7824.
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5 correlative studies for research/pharmacokinetic

| 5.1                                            | BIOSPECIMEN COLLECTION                    |                               |                  |                      |
|------------------------------------------------|-------------------------------------------|-------------------------------|------------------|----------------------|
| Test/assay                                     | Volume  (approx.)  per  timepoint         | Type of tube                  | Collection       | Location of specimen |
| point                                          | analysis*  (processing/storage  location) |                               |                  |                      |
| M7824                                          | 4 mL blood                                |                               |                  |                      |
| Pharmacokinetics                               | for serum                                 | Serum  Separator tubes  (SST) | EMD Serono (BPC) |                      |
| ADA by ELISA                                   | 4mL blood  for serum                      |                               |                  |                      |
| Standard and 123  immune cell subsets by  FACS |                                           |                               |                  |                      |  
| Version Date: 07.06.2021 Test/assay                                                                                                                                                          | Volume  (approx.)  per  timepoint                | Type of tube                   | Collection                | Location of specimen   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------|--------------------------------|---------------------------|------------------------|
| point                                                                                                                                                                                        | analysis*  (processing/storage  location)        |                                |                           |                        |
| Functional Analysis of  immune cell subsets by  FACS                                                                                                                                         | 60-80 mL  blood for  PBMCs                       | LTIB (CSP)                     |                           |                        |
| Sodium heparin  (green top)  tubes                                                                                                                                                           | See Section  3.5                                 |                                |                           |                        |
| Antigen Specific  Immune Response by  cytokine staining assay Histone acetylation T cell clonality by  immunoSeq platform                                                                    | NCI Frederick Genomic  Core Facility, Nanostring |                                |                           |                        |
| RNA expression by                                                                                                                                                                            | (CSP)                                            |                                |                           |                        |
| Nanostring Soluble Factors by                                                                                                                                                                | 8 mL blood                                       | Serum                          |                           |                        |
| ELISA                                                                                                                                                                                        | for serum                                        | Separator tubes  (SST)         | LTIB (CSP)                |                        |
| TGFβ1-3 levels by  ELISA or bead based  multiplex assays                                                                                                                                     | 6mL blood                                        | EDTA                           |                           |                        |
| for plasma                                                                                                                                                                                   | (lavender top)                                   | LTIB, Dr. Liang Cao's Lab      |                           |                        |
| tubes                                                                                                                                                                                        | (CSP)                                            |                                |                           |                        |
| Circulating free tumor  DNA (cfDNA) by PCR  system                                                                                                                                           | 10 mL  blood for  plasma                         | EDTA  (lavender top)  tubes    | Dr. Liang Cao's Lab (CSP) |                        |
| Immune Markers by                                                                                                                                                                            | Tumor                                            | N/A                            | NCI - GMB TIME Lab        |                        |
| IHC                                                                                                                                                                                          | samples                                          | (LP)                           |                           |                        |
| RNA expression by                                                                                                                                                                            | Tumor                                            | N/A                            | NCI Frederick Genomic     |                        |
| Nanostring                                                                                                                                                                                   | samples                                          | Core Facility, Nanostring (LP) |                           |                        |
| T cell clonality by                                                                                                                                                                          | Tumor                                            | N/A                            | LTIB and NCI Frederick    |                        |
| immunoSeq platform                                                                                                                                                                           | samples                                          | Genomic Core Facility (LP)     |                           |                        |
| *Research samples will be sent to the Clinical Services Program - Leidos Biomedical Research,  Inc. (CSP) (Section 5.2.1), the Blood Processing Core (BPC) (Section 5.2.2), or Laboratory of |                                                  |                                |                           |                        |  
*Research samples will be sent to the Clinical Services Program - Leidos Biomedical Research, Inc. (CSP) (Section **5.2.1**), the Blood Processing Core (BPC) (Section **5.2.2**), or Laboratory of Pathology (LP) (Section **5.2.3**) for barcoding, initial processing and storage. From these facilities, coded, linked samples will be sent to the designated labs for analysis in batch shipments or upon request.
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5.1.1 peripheral blood collection and

The following tests may be performed on selected patients' peripheral blood samples if there are adequate samples. If collected samples are not sufficient, the correlative studies will be performed in order of priority as detailed below. These correlative studies will be done in an exploratory fashion. The amount of blood that may be drawn from adult patients and volunteers
(i.e., those persons 18 years of age or older) for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any eight week period.
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pharmacokinetics

To evaluate M7824 PK, 4ml of serum will be collected as per Section 3.5. Serum samples will be analyzed by a validated immunoassay to quantitate M7824 concentration. Samples may be further tested in qualified or validated PK characterization assays. Coded, linked samples will be shipped to EMD Serono for analysis.
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ada

To evaluate ADA (anti-drug antibody), 4ml of blood will be collected as per Section **5.1.1**.  
Serum samples will be analyzed by a validated electrochemiluminescence immunoassay (ECLA) to detect the presence of anti-M7824. Samples that screen positive will be subsequently tested in a confirmatory assay. Those confirmed positive will be titered for a quasi-quantitative result. Samples may be further tested in qualified or validated immunogenicity characterization methods. The investigation will be done by EMD Serono using ELISA. Coded, linked samples will be shipped to EMD Serono for analysis.
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immune phenotyping

Exploratory immunologic studies will be conducted to evaluate the study drug's effect on the immune response before and after treatment, to gain insight into potential biomarkers, and help improve the administered therapy. Blood will be collected as per Section **5.1.1**. The following immune assays may be performed at the Laboratory of Tumor Immunology and Biology (LTIB) at the NCI's Center for Cancer Research (CCR) in select patients where adequate samples are available:  
1. Pre- and post-therapy PBMCs, separated by Ficoll-Hypaque density gradient separation, will be analyzed for antigen-specific immune responses using an intracellular cytokine staining assay. PBMCs will be stimulated in vitro with overlapping 15-mer peptide pools encoding the tumor-associated antigen Brachyury; if sufficient PBMCs are available, the cascade antigens CEA, and MUC1 will be included. Control peptide pools will involve the use of human leukocyte antigen peptide as a negative control and CEFT peptide mix as a positive control. CEFT is a mixture of peptides of CMV, Epstein-Barr virus, influenza, and tetanus toxin. Post-stimulation analyses of CD4 and CD8 T cells will involve the production of IFN-γ, IL-2, tumor necrosis factor, and CD107a. If sufficient PBMCs are available, assays may also be performed for the development of T cells to other tumorassociated antigens. A detailed description of this assay has been previously reported. (119)
2. If sufficient PBMCs are available, PBMCs from selected subjects may be analyzed for changes in standard immune cell types (CD4 and CD8 T cells, NK cells, Tregs, myeloidderived suppressor cells [MDSCs], and dendritic cells) as well as 123 immune cell subsets, as described elsewhere (120) and for function of specific immune cell subsets, including CD4 and CD8 T cells, NK cells, Tregs, and MDSCs.
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analyses of soluble factors

1. Sera may be analyzed pre- and post-therapy for the following soluble factors: sCD27, sCD40 ligand, and selected patients may be analyzed for cytokines and antibodies to human tumor antigens such as Brachyury, CEA, or MUC1.
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additional assays

Blood samples may be used for additional research studies, which may include phenotypic and functional analysis of immune-cell subsets, TCR clonality, and analyses for cytokines (IFN-γ, IL10, IL-12, IL-2, IL-4, etc.), chemokines, antibodies, tumor-associated antigens, and/or other markers.
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plasma analysis for tgfb1 levels

Plasma may be analyzed pre- and post-therapy in Dr. Liang Cao's lab for TGFβ1-3 levels using ELISA or bead based multiplex assays and circulating free tumor DNA using PCR-based techniques.
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exploratory pharmacodynamics for entinostat (arm

1. Histone acetylation has been evaluated as a surrogate for entinostat pharmacodynamics.  
Protein lysine acetylation will be measured by multiparameter flow cytometry in PBMCs (CD19 B-cells, CD3T-cells, and CD14 monocytes) to explore the association with PFS. (121) This will be collected at baseline, Cycle 1 Day 8 (within 4-6 hours of taking entinostat) and Cycle 1 Day 15 (optional; within 4-6 hours of taking entinostat). This will be performed on PBMCs collected for the immune analyses. No additional blood collection is needed.  
5.1.2 Tumor Biopsy Collection and Assays
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biopsy acquisition

Biopsies will be performed as per the Study Calendar 3.5. Attempts will be made to obtain up to four cores (18G or 20G preferred) if safe and feasible. These tumor core biopsies will be obtained percutaneously by interventional radiology as long as considered minimal surgical risk. Two 3-millimeter punch biopsies of skin will be acceptable in lieu of 18-gauge core biopsies for patients with skin involvement. Inability to get tissue with a reasonable attempt will not preclude treatment and the patient will remain eligible for all other translational components.  
- Cores 1 and 2: Formalin-Fixed Paraffin-Embedded (FFPE)
- Cores 3 and 4: Fresh frozen Tissue samples will be sent to Laboratory of Pathology for disease evaluation first, remaining samples will be processed and stored for research (Section **5.2.3**). If fresh-frozen cores are obtained, they will be processed and stored in CSP (Section **5.2.1**). See site specific instructions for collection shipping procedures.
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immune markers by immunohistochemistry

Sections of formalin-fixed, paraffin-embedded (FFPE) tissues from before and after therapy may be stained using antibodies for multiple immune cell and tumor cell markers (e.g. CD4, CD8, FOXP3, PD-1, PD-L1) and analyzed using the Vectra Polaris platform/multispectral imaging in selected patients where tumor samples are adequate.
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her2/brachyury expression

HER2 testing may be performed and if so, will be performed according to the recommendations made by the College of American Pathologists (CAP). (122) FFPE samples may be evaluated for brachyury expression by using a rabbit monoclonal anti-brachyury antibody MAb 54-1 at a 1:500-1:2000 dilution, as previously described. (123, 124) Nuclear, cytoplasmic and total brachyury staining will be independently scored, with brachyury being observed either in the nucleus, the cytosol or both compartments of the tumor cells. The relative staining intensity will be scored as weak (+) for pale intensity, moderate (++) for intermediate intensity, and strong (+++) for intense, dark immunoprecipitate. Immunoreactivity index will be calculated by multiplying the percentage of positive cells by the staining intensity.
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5.1.3 genetic assays t-cell clonality

DNA may be extracted from cryopreserved PBMC or tumor tissue. TCR Vb CDR3 sequencing will be performed at the CCR Genomics Core using the Immunoseq kit from Adaptive Biotechnologies at the survey (tumor) or deep (PBMC) resolution. In this assay, a multiplex PCR system amplifies and quantifies the rearranged CDR3b sequences from sample DNA.
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rna expression

RNA may be extracted from cryopreserved PBMC or tumor tissue. Sequencing of select gene panels may be performed at the CCR Genomics Core in collaboration with Nanostring.
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5.2 sample storage tracking and

See site specific instructions for collection and shipping procedures for research blood samples.  
5.2.1 Sample Management and Storage at Clinical Services Program - Leidos Biomedical Research, Inc. (CSP)
All data associated with the patient samples is protected by using a secure database. All Clinical Support Laboratory Staff receive annual training in maintaining records' confidentiality. All samples drawn at the NIH Clinical Center will be transported to the Clinical Support Laboratory at the Frederick National Laboratory for Cancer Research by couriers. Samples will be tracked and managed by Central Repository database, where there is no link to personal identifiable information. All samples will be stored in either a -80ºC freezer or vapor phase liquid nitrogen. These freezers are located at NCI Frederick Central Repository in Frederick, Maryland. NCI Frederick Central Repositories (managed under a subcontract) store, among other things, biological specimens in support of NIH clinical studies. All specimens are stored in secure, limited-access facilities with sufficient security, backup, and emergency support capability and monitoring to ensure long-term integrity of the specimens for research. Specimens are stored in accordance with applicable HHS and FDA Protection of Human Subjects Regulations in accordance with the subcontractor's Federal-wide Assurance. The subcontractor's role limited to clinical research databases and repositories containing patient specimens. The subcontractor does not conduct or have any vested interest in research on human subjects, but does provide services and support the efforts of its customers, many of which are involved in research on human subjects. It is the intent and purpose of the subcontractor to accept only coded, linked samples and sample information. To the limit of our ability, every effort will be made to ensure that protected information is not sent electronically or by hard copy or on vial labels. Sample data is stored in the BioSpecimen Inventory System II (BSI). This inventory tracking system is used to manage the storage and retrieval of specimens as well as to maintain specimen data. BSI is designed for controlled, concurrent access. It provides a real-time, multi-user environment for tracking millions of specimens. The system controls how and in what order database updates and searches are performed. This control prevents deadlocks and race conditions. For security, BSI has user password access, 3 types of user access levels, and 36 user permissions (levels of access) that can be set to control access to the system functions. BSI provides audit tracking for processes that are done to specimens including shipping, returning to inventory, aliquoting, thawing, additives, and other processes. BSI tracks the ancestry of specimens as they are aliquoted, as well as discrepancies and discrepancy resolution for specimens received by the repository. If a specimen goes out of the inventory, the system maintains data associated with the withdrawal request. Vials are labeled with a unique BSI ID which is printed in both eye-readable and bar-coded format. No patient-specific information is encoded in this ID. Investigators are granted view, input, and withdrawal authority only for their specimens. They may not view specimen data or access specimens for which they have not been authorized. Access to specimen storage is confined to repository staff. Visitors to the repositories are escorted by repository staff at all times. 5.2.2 Samples Managed by Dr. Figg's Blood Processing Core (BPC)
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5.2.2.2 sample data collection

All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and stored in the Labmatrix utilized by the BPC. This is a secure program, with access to Labmatrix limited to defined Figg lab personnel, who are issued individual user accounts. Installation of Labmatrix is limited to computers specified by Dr. Figg. These computers all have a password restricted login screen. Labmatrix creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without Labmatrix access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Patient demographics associated with the clinical center patient number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.). 5.2.2.3 Sample Storage Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80 C according to stability requirements. These freezers are located onsite in the BPC and offsite at NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory staff at all times. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in Labmatrix. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol) and that any unused samples must be returned to the BPC. It is the responsibility of the NCI Principal Investigator to ensure that the samples requested are being used in a manner consistent with IRB approval. 5.2.3 Procedures for Storage of Tissue Specimens in the Laboratory of Pathology Tissues designated for clinical diagnostics are transported to the Laboratory of Pathology (LP) where they are examined grossly and relevant portions are fixed, embedded in paraffin and sectioned and stained for diagnostic interpretation. Following completion of the diagnostic workup, the slides and tissue blocks are stored indefinitely in the LP's clinical archives. All specimens are catalogued and retrieved utilizing the clinical laboratory information systems, in accordance with CAP/JCAHO regulations. The use of any stored specimens for research purposes is only allowed when the appropriate IRB approval has been obtained. Specimens/recuts can then be requested with the permission of the appropriate Principal Investigator using the Tissue Resource Committee request for Human Biological Materials for Research form. 5.2.4 Protocol Completion/Sample Destruction All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are remaining at the completion of the protocol will be stored in the conditions described above. The study will remain open so long as sample or data analysis continues. Samples from consenting subjects will be stored until they are no longer of scientific value or if a subject withdraws consent for their continued use, at which time they will be destroyed. If the patient withdraws consent the participant's data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved.  
The site PI will record any loss or unanticipated destruction of samples as a deviation. Reports will be made per the requirements of Section 7.2.
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6 data collection and evaluation

The CCR PI and all site PIs will be responsible for data collection. Site PIs will be trained on use of the CCR remote data capture system C3D. The CCR PI and the site PIs will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D) and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Document AEs from the first study intervention, Study Day 1, through 28 days after the study intervention was last administered. Beyond 28 days after the last intervention, only adverse events which are serious and related to the study intervention need to be recorded.  
An abnormal laboratory value will be recorded in the database as an AE **only** if the laboratory abnormality is characterized by any of the following:  
- Results in discontinuation from the study. - Is associated with clinical signs or symptoms. - Requires treatment or any other therapeutic intervention. - Is associated with death or another serious adverse event, including hospitalization.  
- Is judged by the Investigator to be of significant clinical impact.  
- If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH
Intramural Records Retention Schedule as applicable.  
Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, this will be reported expeditiously per Section 7.2.1.
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what data will be shared?

I will share human data generated in this research for future research as follows:
- Coded, linked data in an NIH-funded or approved public repository.  
- Coded, linked data in BTRIS (automatic for activities in the Clinical Center)
- Identified or coded, linked data with approved outside collaborators under appropriate agreements.
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how and where will the

Data will be shared through:
- An NIH-funded or approved public repository. Clinicaltrials.gov  
- BTRIS (automatic for activities in the Clinical Center).  
- Approved outside collaborators under appropriate individual agreements. - Publication and/or public presentations.
When will the data be shared?  
- Before publication.  
- At the time of publication or shortly thereafter.  
6.2.2 Genomic Data Sharing Plan Unlinked genomic data will be deposited in public genomic databases such as dbGaP in compliance with the NIH Genomic Data Sharing Policy.
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6.3 response criteria 6.3.1 antitumor

For the purposes of this study, patients should be re-evaluated for response every 9 weeks +/- 3 days. In addition to a baseline scan, confirmatory scans can also be obtained 3 to 6 weeks following initial documentation of objective response per the discretion of the primary investigator.  
At baseline, tumor lesions will be selected and categorized as target or non-target lesions. Target lesions include those lesions that can be accurately measured in at least 1 dimension as ≥ 20 mm with conventional techniques or ≥ 10 mm with CT scan. Malignant lymph nodes with a short axis diameter ≥ 15 mm can be considered target lesions. Up to a maximum of 2 target lesions per organ and 5 target lesions in total will be identified at baseline. These lesions should be representative of all involved organs and selected based on their size (those with the longest diameter) and their suitability for accurate repeated measurements. A sum of the longest lesion diameter (LLD) for all target lesions will be calculated and reported as the baseline sum LLD. For malignant lymph nodes identified as target lesions, the short axis diameter will be used in the sum of LLD calculation. All other lesions (or sites of disease) should be identified as non-target lesions (including bone lesions). All post-baseline response assessments should follow the same lesions identified at baseline. The same mode of assessment (e.g., CT) used to identify/evaluate lesions at baseline should be used throughout the course of the study unless subject safety necessitates a change (e.g., allergic reaction to contrast media). For the primary endpoint antitumor activity will be evaluated with target and/or non-target lesions according to the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline (Version 1.1). (125)
6.3.2 Disease Parameters Measurable disease: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as:
- By chest x-ray: >20 mm;
- By CT scan:
o Scan slice thickness 5mm or under: as >10 mm; o Scan slice thickness ˃5mm: double the slice thickness
- With calipers on clinical exam: >10 mm.  
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). Malignant lymph nodes. To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed. Non-measurable disease. All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, these are preferred for selection as target lesions. Target lesions. All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as **target lesions** and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Non-target lesions. All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as **non-target lesions** and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up.
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6.3.3 methods for evaluation of

All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam.  
Clinical lesions: Clinical lesions will only be considered measurable when they are superficial
(e.g., skin nodules and palpable lymph nodes) and 10 mm diameter as assessed using calipers
(e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.  
Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable. Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease.  
Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible. PET-CT: At present, the low dose or attenuation correction CT portion of a combined PET-CT is not always of optimal diagnostic CT quality for use with RECIST measurements. However, if the site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for RECIST measurements and can be used interchangeably with conventional CT in accurately measuring cancer lesions over time. Note, however, that the PET portion of the CT introduces additional data which may bias an investigator if it is not routinely or serially performed. Ultrasound: Ultrasound is not useful in assessment of lesion size and should not be used as a method of measurement. Tumor markers: Tumor markers alone cannot be used to assess response. Cytology, Histology: These techniques can be used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germ cell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease. FDG-PET: While FDG-PET response assessments need additional study, it is sometimes reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in assessment of progression (particularly possible 'new' disease). New lesions on the basis of FDG-PET imaging can be identified according to the following algorithm:  
a. Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD based on a new lesion.  
b. No FDG-PET at baseline and a positive FDG-PET at follow-up: If the positive FDGPET at follow-up corresponds to a new site of disease confirmed by CT, this is PD. If the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, additional follow-up CT scans are needed to determine if there is truly progression occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-PET scan). If the positive FDG-PET at follow-up corresponds to a pre-existing site of disease on CT that is not progressing on the basis of the anatomic images, this is not PD.  
c. FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in cases where a residual radiographic abnormality is thought to represent fibrosis or scarring. The use of FDG-PET in this circumstance should be prospectively described in the protocol and supported by disease-specific medical literature for the indication. However, it must be acknowledged that both approaches may lead to false positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity.
Note: A 'positive' FDG-PET scan lesion means one which is FDG avid with an uptake greater than twice that of the surrounding tissue on the attenuation corrected image. 6.3.4 Response Criteria 6.3.4.1 Evaluation of Target Lesions Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum of diameters. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study. 6.3.4.2 Evaluation of Non-Target Lesions Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis). Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response. Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.  
Progressive Disease (PD): Appearance of one or more new lesions and/or *unequivocal* progression of existing non-target lesions. *Unequivocal progression* should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator).
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6.3.4.3 evaluation of best overall

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| Target    | Non-Target                                                                                                           | New     | Overall                                           | Best Overall Response when   |
|-----------|----------------------------------------------------------------------------------------------------------------------|---------|---------------------------------------------------|------------------------------|
| Lesions   | Lesions                                                                                                              | Lesions | Response                                          | Confirmation is Required*    |
| CR        | CR                                                                                                                   | No      | CR                                                | > 3 to 6 wks. Confirmation** |
| CR        | Non                                                                                                                      |         |                                                   |                              |
| CR/Non-PD | No                                                                                                                   | PR      |                                                   |                              |
| CR        | Not                                                                                                                  | No      | PR                                                |                              |
| evaluated | >3 to 6 wks. Confirmation**                                                                                          |         |                                                   |                              |
| PR        | Non                                                                                                                      |         |                                                   |                              |
| CR/NonPD/not  evaluated           | No                                                                                                                   | PR      |                                                   |                              |
| SD        | Non                                                                                                                      |         |                                                   |                              |
| CR/NonPD/not  evaluated           | No                                                                                                                   | SD      | Documented at least once >4  wks. from baseline** |                              |
| PD        | Any                                                                                                                  | Yes or  | PD                                                |                              |
| No        |                                                                                                                      |         |                                                   |                              |
| Any       | PD***                                                                                                                | Yes or  | no prior SD, PR or CR                             |                              |
| PD        |                                                                                                                      |         |                                                   |                              |
| No        |                                                                                                                      |         |                                                   |                              |
| Any       | Any                                                                                                                  | Yes     | PD                                                |                              |
| *         | See RECIST 1.1 manuscript for further details on what is evidence of a  new lesion.                                  |         |                                                   |                              |
| **        | Only for non-randomized trials with response as primary endpoint.                                                    |         |                                                   |                              |
| ***       | In exceptional circumstances, unequivocal progression in non-target  lesions may be accepted as disease progression. |         |                                                   |                              |  
Note: Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be reported as "symptomatic deterioration." Every effort should be made to document the objective progression even after discontinuation of treatment.  
As an exploratory endpoint antitumor activity will also be evaluated according to iRECIST (REF). Using iRECIST criteria the following will be incorporated into the assessment:
- An increase in the sum of target lesions of more than 20%, unequivocal increase in the non-target lesions or new lesions result in iUPD (unconfirmed progression disease); iUPD can be assigned multiple ties as long as iCPD (confirmed progressive disease) is not confirmed at the next assessment.  
- Progression is confirmed in the target lesion category if the next imaging assessment after iUPD (4-8 weeks later) confirms a further increase in sum of measures of target disease from iUPD, with an increase of at least 5mm. Progression is confirmed in the non-target lesion category if subsequent imaging, done every 4-8 weeks after iUPD shows a further unequivocal increase in non-target lesions. Progression is confirmed in the new lesions category if at next assessment additional new lesions appear or an increase in the size of previously seen new lesions is seen (>5 mm for sum of new target lesion).  
- However, the criteria for iCPD (after iUPD) are not considered to have been met if complete response, partial response or stable disease criteria (compared with baseline and as defined by RECIST 1.1) are met at the next assessment after iUPD. The status is reset (unlike RECIST 1.1, in which any progression precludes later complete response, partial response, or stable disease). iCR, iPR, or iSD should then be assigned; and if no change is detected, then the timepoint response is iUPD.  
| Non-Target Lesions                                                                                                                                           | New Lesions   | Overall Response   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|--------------------|
| CR                                                                                                                                                           | No            | CR                 |
| Non-CR/non-PD                                                                                                                                                | No            | Non-CR/non-PD*     |
| Not all evaluated                                                                                                                                            | No            | not evaluated      |
| Unequivocal PD                                                                                                                                               | Yes or No     | PD                 |
| Any                                                                                                                                                          | Yes           | PD                 |
| * 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease  since SD is increasingly used as an endpoint for assessment of efficacy in some |               |                    |
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for patients with non-measurable disease

6.3.4.4 Duration of Response Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements. 6.3.4.5 Progression-Free Survival Progression Free Survival (PFS) is defined as the duration of time from start of treatment to time of progression or death, whichever occurs first. In the absence of progression or death, PFS is censored at the date of last disease evaluation 6.3.5 Response Review All imaging studies will be reviewed by an independent radiologist and will be assess according to RECIST 1.1.
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6.4 toxicity criteria

The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc).
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7 nih reporting requirements /

Please refer to definitions provided in Policy 801: Reporting Research Events found at: https://irbo.nih.gov/confluence/display/IRBO/Policies+and+SOPs.
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7.2 ohsrp office of compliance

Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802 Non-Compliance Human Subjects Research found at: https://irbo.nih.gov/confluence/display/IRBO/Policies+and+SOPs. Note: Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported per these policies. 7.2.2 IRB Requirements for PI Reporting at Continuing Review Please refer to the reporting requirements in Policy 801: Reporting Research Events found at: https://irbo.nih.gov/confluence/display/IRBO/Policies+and+SOPs.
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7.3 nci guidance for reporting

Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802: Non-compliance in Human Subjects Research, found here. Until such time as direct electronic reporting mechanisms are available to participating sites, the site PI must immediately report to the coordinating center PI any deaths possibly related to the research within 24 hours of PI awareness of the event. The Site PI must also report any other events required by Policy 801 to the coordinating center PI within 7 days of PI awareness.  
The participating site Reportable Event Form is attached as **Appendix** E: CCR Reportable Event Form (REF). Once direct electronic reporting mechanisms are available, these will be utilized. Please also notify the coordinating center PI and study coordinator of your submission at the time you make it. For IND studies, the site PI will also directly submit reports to the CCR as IND sponsor per Section 8.3, 8.6 and 8.8.
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7.4 nih required data and

The clinical research team will meet on a regular basis (approximately weekly) when patients are being actively treated on the trial to discuss each patient. Decisions about dose level enrollment and dose escalation if applicable will be made based on the toxicity data from prior patients. All data will be collected in a timely manner and reviewed by the principal investigator or a lead associate investigator. Events meeting requirements for expedited reporting as described in Section **7.2.1** will be submitted within the appropriate timeline.  
The principal investigator will review adverse event and response data on each patient to ensure safety and data accuracy. The principal investigator will personally conduct or supervise the investigation and provide appropriate delegation of responsibilities to other members of the research staff. 7.4.2 Safety Monitoring Committee (SMC) This protocol will be periodically reviewed by an intramural Safety Monitoring Committee comprised of physicians, biostatisticians and a lay member selected based on experience, area of expertise, reputation for objectivity, absence of conflicts of interest and knowledge of or experience with clinical trial research. Initial review will occur as soon as possible after the annual NIH Intramural IRB continuing review date. Subsequently, each protocol will be reviewed as close to annually as the quarterly meeting schedule permits or more frequently as may be required by the SMC based on the risks presented in the study. For initial and subsequent reviews, protocols will not be reviewed if there is no accrual within the review period. The SMC will operate under the rules of an approved charter that will be written and reviewed at the organization meeting of the SMC. Each review will focus on unexpected protocol safety issues that are identified during the conduct of the clinical trial. Written outcome letters will be generated in response to the monitoring activities and submitted to the Principal investigator and Clinical Director or Deputy Clinical Director, CCR, NCI.
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8 sponsor protocol safety reporting

Any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product (ICH E6 (R2)).
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8.1.2 serious adverse event (sae)

An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following:
- Death;  
- A life-threatening adverse event (see Section **8.1.3**);
- Inpatient hospitalization or prolongation of existing hospitalization; o A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery arranged prior to the start of the study), a planned hospitalization for pre-existing condition, or a procedure required by the protocol, without a serious deterioration in health, is not considered a serious adverse event.  
o A hospitalization/admission that is solely driven by non-medical reasons (e.g.,
hospitalization for patient or subject convenience) is not considered a serious adverse event.  
o Emergency room visits or stays in observation units that do not result in admission to the hospital would not be considered a serious adverse event. The reason for seeking medical care should be evaluated for meeting one of the other serious criteria.  
- Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions;
- A congenital anomaly/birth defect; - Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.  
8.1.3 Life-threatening
An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death (21CFR312.32). 8.1.4 Severity The severity of each Adverse Event will be assessed utilizing the CTCAE version 5.0. 8.1.5 Relationship to Study Product All AEs will have their relationship to study product assessed using the terms: related or not related.  
- Related - There is a reasonable possibility that the study product caused the adverse event. Reasonable possibility means that there is evidence to suggest a causal relationship between the study product and the adverse event.  
- Not Related - There is not a reasonable possibility that the administration of the study product caused the event.  
8.1.6 Adverse Events of Special Interest (AESI)
Adverse events of special interest (AESIs) are serious or nonserious AEs that are of clinical interest and should be closely followed. AESIs include following:
- Infusion-related reactions including immediate hypersensitivity.  
- Immune-related adverse events. - TGFβ inhibition/M7824 mediated skin reactions.  
- Anemia.  
- Bleeding AEs.
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8.2 assessment of safety events

AE information collected will include event description, date of onset, assessment of severity and relationship to study product and alternate etiology (if not related to study product), date of resolution of the event, seriousness and outcome. The assessment of severity and relationship to the study product will be done only by those with the training and authority to make a diagnosis and listed on the Form FDA 1572 as the site principal investigator or sub-investigator. AEs occurring during the collection and reporting period will be documented appropriately regardless of relationship. AEs will be followed through resolution.
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saes will be

- Assessed for severity and relationship to study product and alternate etiology (if not related to study product) by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  
- Recorded on the appropriate SAE report form, the medical record and captured in the clinical database.  
- Followed through resolution by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to Section 6.1. All serious adverse events recorded from the time of first investigational product administration must be reported to the sponsor.
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8.3 reporting of serious adverse

Any AE that meets a protocol-defined serious criteria or meets the definition of Adverse Event of Special Interest that require expedited reporting must be submitted immediately (within 24 hours of awareness) to OSRO Safety using the CCR SAE report form. Any exceptions to the expedited reporting requirements are found in Section 8.4 All SAE reporting must include the elements described in Section **8.1.6**.  
SAE reports will be submitted to the Center for Cancer Research (CCR) at: OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. CCR SAE report form and instructions can be found at: https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions Following the assessment of the SAE by OSRO, other supporting documentation of the event may be requested by the OSRO Safety and should be provided as soon as possible.
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8.4 waiver of expedited reporting

As death due to disease progression is part of the study objectives, and captured as an endpoint in this study, they will not be reported in expedited manner to the sponsor. However, if there is evidence suggesting a causal relationship between the study drug and the event, report the event in an expedited manner according to Section 8.3.
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8.6 reporting pregnancy

All required pregnancy reports/follow-up to OSRO will be submitted to: OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. Forms and instructions can be found here: https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions
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8.6.1 maternal exposure

If a patient becomes pregnant during the course of the study, the study treatment should be discontinued immediately, and the pregnancy reported to the Sponsor no later than 24 hours of when the Investigator becomes aware of it. The Investigator should notify the Sponsor no later than 24 hours of when the outcome of the Pregnancy become known. Pregnancy itself is not regarded as an SAE. However, congenital abnormalities or birth defects and spontaneous miscarriages that meet serious criteria (Section **8.1.2**) should be reported as SAEs.  
The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) should be followed up and documented. 8.6.2 Paternal Exposure Male patients should refrain from fathering a child or donating sperm during the study and for 4 months after the last dose of study drugs. Pregnancy of the patient's partner is not considered to be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) occurring from the date of the first dose until 4 months after the last dose should, if possible, be followed up and documented.
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8.7 regulatory reporting for studies

Following notification from the investigator, CCR, the IND sponsor, will report any suspected adverse reaction that is both serious and unexpected in expedited manner to the FDA in accordance to 21 CFR 31.2.32. CCR will report an AE as a suspected adverse reaction only if there is evidence to suggest a causal relationship between the study product and the adverse event. CCR will notify FDA and all participating investigators (i.e., all investigators to whom the sponsor is providing drug under its INDs or under any investigator's IND) in an IND safety report of potential serious risks from clinical trials or any other source, as soon as possible, in accordance to 21 CFR Part 312.32.  
All serious events will be reported to the FDA at least annually in a summary format.
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8.8 sponsor protocol non-adherence reporting

Protocol non-adherence is defined as any noncompliance with the clinical trial protocol, GCP, or protocol-specific procedural requirements on the part of the participant, the Investigator, or the study site staff inclusive of site personnel performing procedures or providing services in support of the clinical trial. It is the responsibility of the study Staff to document any protocol non-adherence identified by the Staff or the site Monitor on the OSRO Site Protocol Non-Adherence Log. The protocolspecific, cumulative non-adherence log should be maintained in the site essential documents file and submitted to OSRO via OSROMonitoring@mail.NIH.gov on the **first business day of each** month over the duration of the study. In addition, any non-adherence to the protocol should be documented in the participant's source records and reported to the local IRB per their guidelines. OSRO required protocol non-adherence reporting is consistent with E6(R2) GCP: Integrated Addendum to ICH E6(R1): 4.5 Compliance with Protocol; 5.18.3 (a), and 5.20 Noncompliance; and ICH E3 16.2.2 Protocol deviations.
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9 clinical monitoring

Clinical site monitoring is conducted to ensure that the rights of the participants are protected, that the study is implemented per the approved protocol, Good Clinical Practice and standard operating procedures, and that the quality and integrity of study data and data collection methods are maintained. Monitoring for this study will be performed by NCI CCR Office of Sponsor and Regulatory Oversight (OSRO) Monitoring based on OSRO standards, FDA Guidance E6(R2) Good Clinical Practice: Integrated Addendum to ICH E6(R1) March 2018, and applicable regulatory requirements. Details of clinical site monitoring will be documented in a Clinical Monitoring Plan (CMP) developed by OSRO. CMPs will be protocol-specific, risk-based and tailored to address human subject protections and integrity of the study data. The intensity and frequency of monitoring will be based on several factors, including study type, phase, risk, complexity, expected enrollment rate, and any unique attributes of the study and the site. OSRO Monitoring visits and related activities will be conducted throughout the life cycle of each protocol, with the first activity being before study start to conduct a Site Assessment Visit (SAV) (as warranted),
followed by a Site Initiation Visit (SIV), Interim Monitoring Visit(s) (IMVs), and a study CloseOut Visit (COV).  
Some monitoring activities may be performed remotely, while others will take place at the study site(s). Monitoring visit reports will describe visit activities, observations, findings of protocol non-adherence and associated action items or follow-up required for resolution of findings. Monitoring reports will be distributed to the study PI, NCI CCR QA, coordinating center (if applicable) and the OSRO regulatory file. If protocol non-adherence is identified by the Monitor (i.e., any noncompliance with the clinical trial protocol, GCP, or protocol-specific procedural requirements on the part of the participant, the Investigator, or the site Staff) the Monitor will note the observation, review with site Staff and if unresolved, request that the Staff document the non-adherence on the protocol-specific OSRO Site Protocol Non-Adherence Log (see Section 8.8).
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10 statistical considerations 10.1 statistical

We hypothesize that sequentially evaluated combinations of BN-Brachyury, M7824, T-DM1 and entinostat will enhance the clinical and immunologic response in patients with advanced breast cancer, specifically for HER2+BC patients who previously progressed after treatment (or were intolerant to) with a taxane, trastuzumab and pertuzumab. The trial will enroll patients initially to BN-Brachyury + M7824, and then add TDM-1 and finally entinostat.  
- **Primary Endpoints:**
o Objective response rate (ORR; PR+CR), as defined in Section **6.3.3**, for each of the three treatment arms enrolled sequentially as described below.  
o Safety for the combinations explored in the arms.  
- Secondary **Endpoints:**
o Progression free survival, as designed in Section **6.3.4.5.** o Change in absolute percentage of stromal TILs quantified according to the Salgado method in serial tumor biopsies collected at baseline and on C3D1 (Arms 2 and 3 only).
- **Exploratory Endpoints** (studies may be performed in selected patients if adequate samples are available):
o Peripheral blood mononuclear cells (PBMCs):
➢ Changes in immune cell subsets. ➢ Changes in brachyury-specific, MUC1-specific, and CEA-specific T cells measured by intracellular cytokine staining (IFN, IL2, TNF, CD107a) .  
➢ Histone acetylation as surrogate for entinostat pharmacodynamics (Arm 3 only).  
o Plasma/Serum:
➢ Changes in brachyury-specific , MUC1-specific, and CEA-specific antibodies.  
➢ Changes in soluble factors like sCD27 and sCD40.  
➢ Changes in cell free DNA. ➢ Changes in TGF-beta levels. ➢ Changes in pharmacokinetics of M7824.  
o Tissue Biopsy (Cohort 3, Arms 2 and 3):
➢ Brachyury expression on tumor. ➢ T-cell clonality score. ➢ Immunohistochemistry/multispectral imaging.  
➢ Changes in PD-L1 on tumor.  
➢ Neoepitopes derived from tumor biopsies. ➢ HER2 expression changes. ➢ Tumor mutational burden.
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10.2 sample size determination

The primary objective of this trial , on which the sample size is based, is to determine the overall clinical response rate of BN-Brachyury, entinostat, M7824 and T-DM1 (PR+CR) evaluated in arms with increasing numbers of agents as follows:
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arm 1

In similar subjects with TNBC (unselected for PD-L1 expression) who received a checkpoint inhibitor, the clinical response rate was approximately 8-10%. (43-45) The goal would first be to determine if using BN-Brachyury plus M7824 could be shown in a small pilot arm to improve upon this by a modest amount. Accrual of the first 3 patients on trial in this arm will be slow (no more than 1 patient per 6 days) in order to allow for monitoring of toxicity. If there is one or more DLT in the first 3 patients on this arm, accrual of the next 3 patients on the associated arm will proceed slowly with no more than 1 patient per 21 days (1 cycle) to continue to closely monitor safety. If there are 0-1 patients with DLTs among the 6 patients enrolled on this arm, to do so, accrual to this arm will be continue by using a Simon optimal two-stage phase II trial design (126) to rule out an unacceptably low PR+CR rate of 10% (p0=0.10) in favor of an improved response rate of 35% (p1=0.35). With 1-sided alpha=0.10 (probability of accepting a poor treatment=0.10) and beta=0.20 (probability of rejecting a good treatment=0.20), the first stage will enroll 8 evaluable subjects, and if 0 to 1 of the 8 have a clinical response, then no further subjects will be accrued. If 2 or more of the first 8 subjects have a response, then accrual would continue until a total of 13 evaluable subjects have been enrolled. As it may take 3 to 6 months to determine if a subject has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 2 subjects with a response out of 13 subjects, this would be an uninterestingly low response rate. If there were 3 or more of 13 (23.1%) who experienced a response, this would be sufficiently interesting to warrant further study in later trials. Under the null hypothesis (10% response rate), the probability of early termination is 81.3%.
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arms 2 and 3

Following determination of safety in arm 1 (0-1 with DLT among first 6 patients enrolled), patients who are HER-2 positive will undergo an initial safety evaluation and then will be assigned in an alternating fashion (1:1) to the second and third arms: BN-Brachyury, M7824 and T-DM1, or BNBrachyury, M7824, TDM-1, and entinostat. Provided that there are 0-1 patients with DLTs among the 6 patients enrolled on Arm 2, this arm will be temporarily closed and patients will be enrolled on Arm 3 for a safety evaluation. Provided that there are 0-1 patients with DLTs among the 6 patients enrolled on Arm 3, patients with HER2 positive breast cancer will be directly allocated1:1 to Arms 2 and 3. In Arms 2 and 3, accrual of the first 3 patients in each arm will proceed slowly with no more than 1 patient enrolled every 28 days in order to allow for safety monitoring. If there is one or more DLT in the first 3 patients on any arm, accrual of the next 3 patients on the associated arm will proceed slowly with no more than 1 patient per 21 days (1 cycle) to closely monitor safety. If there are 2 or more patients with a DLT among the first 6 treated on either arm, the trial will halt accrual pending an amendment to detail how the study will proceed from that point forward. The primary objective is to determine if either arm could improve upon the 18% response rate by a modest amount. Each arm will be conducted using a Simon minimax two-stage phase II trial design (126) to rule out an unacceptably low PR+CR rate of 18% (p0=0.18) in favor of an improved response rate of 40% (p1=0.40). With 1-sided alpha=0.10 (probability of accepting a poor treatment=0.10) and beta=0.20 (probability of rejecting a good treatment=0.20), the first stage will enroll 14 evaluable subjects in each arm, and if 0 to 3 of the 14 have a clinical response, then no further subjects will be accrued. If 4 or more of the first 14 subjects have a response in an arm, then accrual would continue until a total of 19 evaluable subjects have been enrolled in that arm. As it may take 3 to 6 months to determine if a subject has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 4 to 5 subjects with a response out of 19 subjects in an arm, this would be an uninterestingly low response rate. If there were 6 or more of 19 (31.6%) who experienced a response, this would be sufficiently interesting to warrant further study that combination in later trials. If accrual ends to one arm because of insufficient activity, the other arm will remain open to enroll patients directly. Under the null hypothesis (18% response rate), the probability of early termination is 76.5%. There will be no adjustment for the multiplicity of the three arms. To allow for a small number of inevaluable patients, up to 42 patients may be enrolled in Cohort 2 (21 per Arm, if necessary) to explore this primary objective. With arms requiring up to 13+19+19=51 evaluable patients and allowing for up to 4 total inevaluable patients and up to 10 screening failures, the accrual ceiling will be set at 65 patients. Safety will also be assessed as a primary objective. All patients will be assessed for safety as described in Sections **10.3.1** and **10.4.4**.  
Secondary Objectives: Progression Free Survival (PFS) will be evaluated as a secondary objective. This will be estimated using a Kaplan-Meier curve and the median PFS will be reported along with a 95% confidence interval, separately by arm, based on the patients who are enrolled in the evaluation of clinical response. Change in TILs will also be evaluated as a secondary objective. Data from 678 patients with evaluable TILs enrolled on the CLEOPATRA trial in HR-/HER2+ breast cancer indicated a mean baseline stromal TIL value of 21% (SD=22.4%). In the present study with HR-/HER2+ patients, it is anticipated that the baseline TIL value will be assumed to be approximately 25%, with the same 22% standard deviation. A very conservative estimate of the SD associated with a change between two time points is the baseline SD x square root (2), or 22 x 1.41 =31%, based on an assumption of no correlation between the values at baseline and at the time of biopsy. An assumption of the correlation=0.5 would result in the SD of the change being baseline SD x 1.0, or 22%, but we will assume SD=31% to be conservative. The goal will be to estimate if the absolute change in TIL percentage from baseline is meaningfully large, as well as noting the magnitude and reporting that with appropriate descriptive statistics and graphical presentation. With 15 evaluable patients for TILs in the two larger arms, there will be 82% power to detect a difference between a mean baseline TIL value of 25% (0.25) and mean post-biopsy TIL value of 50% (0.50), assuming a standard deviation of the change of 31% (effect size= 25/31=0.806), using a paired t-test with a two-sided 0.05 significance level. More conservatively if only 13 or 11 patients have paired samples assessed (from any of the three arms), then there will be 82% and 84% power to detect a mean difference of 27% (effect size=0.871) or 31% (effect size=1.0), respectively. In practice, the changes in the TIL% may not be normally distributed, so the study will plan to test the effect using a Wilcoxon signed rank. In practice, the changes in the TIL% may not be normally distributed, so the study will plan to test the effect using a Wilcoxon signed rank test instead of a paired t-test.  
Progression Free Survival (PFS) will be evaluated as a secondary objective. This will be estimated using a Kaplan-Meier curve and the median PFS will be reported along with a 95% confidence interval, separately by arm. based on the patients who are enrolled in the evaluation of clinical response.
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10.3 populations for analyses 10.3.1

All patients who are treated with any amount of the combination of agents will be evaluable for toxicity from the time of their first treatment; thus, all patients will be evaluable for toxicity from the time of their first treatment with the arm-specific combinations of BN-Brachyury, entinostat, M7824 and T-DM1. Any patient who receives some but not all treatments in each given arm will be summarized separately. 10.3.2 Evaluable for objective response: Only those patients who have measurable disease present at baseline, have received at least two cycles of therapy, and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated below. Patients who have measurable disease at baseline, have received at least 1 cycle of therapy and have clinical/objective disease progression prior to the first restaging will be considered evaluable.
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10.4 statistical analyses 10.4.1 general

Data will be summarized descriptively and graphically, with reporting the outcome measures indicated along with two-tailed 95% confidence intervals (CIs), as well as two-tailed 80% CIs (corresponding to the design with one-sided α=0.10) for median PFS. 10.4.2 Analysis of the Primary Efficacy Endpoint The ORR will be estimated in each arm using two-sided 80% and 95% CI about the observed percentages of response (PR+CR). 10.4.3 Analysis of the Secondary Endpoints Stromal TILs will be summarized descriptively and graphically by time point and as absolute and relative change from baseline. The absolute change in TILs values from baseline following two cycles of treatment will be compared via Wilcoxon signed rank test. The distribution of PFS will be estimated using the Kaplan-Meier (KM) method, among patients evaluable for efficacy, and appropriate confidence intervals (80% as well as 95% two-sided confidence intervals) at the median and selected time points will be provided to help interpret results relative to the expected results. This will be done by arm and may be evaluated by combining patients in arms 2 and 3 if they are sufficiently similar (p>0.30 by global two-tailed log-rank test). The fraction of patients who experience a DLT during the single dose level safety phases of the trial will be reported, along with a summary, confidence interval, and description of any toxicities noted of grade 3 or higher.
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10.4.4 safety analyses

Adverse events (AE) recorded throughout treatment will initially be summarized as the maximum grade, for each AE type. The number and frequency of patients experiencing each AE type and grade will tabulated, among patients evaluable for toxicity. The number (%) of patients experiencing at least one grade 3 to 5 AE will be reported with 95% CI. This will be done separately for the initial safety cohort and for the patients in the primary efficacy cohort. Patients who receive at least 1 dose of all drugs in their cohort will be evaluable for safety analyses. 10.4.5 Baseline Descriptive Statistics Continuous variables will be summarized as mean±SD, median and interquartile range, and range of values. Categorical variables will be summarized as number (%) of patients. 10.4.6 Sub-Group Analyses Analyses will be performed separately by arm. 10.4.7 Tabulation of individual Participant Data Given the sample size, graphical summaries of individual data will often be used, for example scatter plot TILs according to time point with the paired values connected by lines. 10.4.8 Exploratory Analyses Blood sample collection (as specified in Section **5.1.1**), as well as biopsy specimens at baseline and cycle 3 will yield a set of immune subsets. Values will be summarized descriptively and graphically over time. Given the sample size we cannot investigate association of baseline or change from baseline to cycle 1 or cycle 2 in relation to PFS using Cox modeling; therefore, these investigations would focus on KM plots according to dichotomized values of biomarker values, without statistical hypothesis testing. In order to avoid guarantee-time bias, the assessments of changes to cycle 1 or cycle 2 may need to take a landmark analysis approach because patients will need to be alive and progression free at cycle 1 or cycle 2 in order to have a blood sample and be part of the analysis. The changes in the biomarkers will also be summarized descriptively and graphically for responders and non-responders.
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11 collaborative agreements 11.1 multi-institutional

Until an electronic submission system is available to participating sites, documents requiring submission to the reviewing IRB per reliance agreement, including local consent documents generated from an approved model consent, should be provided to the coordinating center for submission to the IRB. Thereafter, consents may be submitted directly to the IRB using iRIS.
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12 human subjects protections 12.1

Both men and women are eligible for this trial if they meet eligibility criteria. Subjects from all racial and ethnic groups are eligible for this trial if they meet the eligibility criteria. Efforts will be made to extend the accrual to a representative population. If differences in outcome that correlate to racial or ethnic identity are noted, accrual may be expanded or additional studies may be performed to investigate those differences more fully. As there is a risk of severe bleeding with this study drug, participants must be willing to receive blood transfusions if medically necessary for their own safety. Participants must be able to receive blood transfusions in order to minimize the risks of receiving M7824. Another reason for this is that including these patients could compromise the scientific validity of the study. For example, death from blood loss could make it difficult to assess other aspects of the investigational immunotherapy's safety—a primary scientific goal in this early-phase immunotherapy trial, which are carried out in small numbers of participants.
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12.2 participation of children

The age group for enrollment on this trial is 18 or more years of age. Because no dosing or adverse event data are currently available on the use of BN-Brachyury, entinostat, M7824 or TDM1 in participants < 18 years of age, children are excluded from this study.
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12.3 evaluation of benefits and

BN-Brachyury, entinostat, and M7824 are investigational agents for use in metastatic breast cancer. Furthermore, BN-Brachyury, entinostat, M7824, and T-DM1 have never been used in combination. The protocol provides for detailed and careful monitoring of all patients to assess for toxicity. Toxicity data will be collected and reviewed to ensure that there were no severe toxicities that would preclude further patient enrollment. Patients will be treated with therapeutic intent and response to the therapy will be closely monitored. Risks include the possible occurrence of any of a range of side effects which are listed in the Consent Document and in Section **12.4.1**. Frequent monitoring for adverse effects will help to minimize the risks associated with administration of the study agents.
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12.4 risk/benefit assessment 12.4.1 known

Some of the procedures performed on this study are not known to be associated with risk. These include urine tests and EKGs. Below are a list of procedures and study interventions that are associated with risk. 12.4.1.1 Risk of MRI If there is concern for CNS metastasis or history of CNS metastasis, the PI may request a brain MRI. People are at risk for injury from the MRI magnet if they have some kinds of metal in their body. People with fear of confined spaces may become anxious during an MRI. Those with back problems may have back pain or discomfort from lying in the scanner. The noise from the scanner is loud enough to damage hearing, especially in people who already have hearing loss.  
There are no known long-term risks of MRI scans. 12.4.1.2 Risk of Gadolinium Enhanced MRI The gadolinium infusion may cause mild symptoms such as coldness in the arm during the injection, a metallic taste, headache, and nausea. There are risks of an IV catheter include bleeding, infection, or inflammation of the skin and vein with pain and swelling. Procedure-related risks from MRI and gadolinium enhanced MRI will be explained fully during informed consent. 12.4.1.3 Risks of Exposure to Ionizing Radiation This research study involves the possibility of 7 CT CAP scans, 4 CT guided biopsies, and 1 head CT collected for research purposes per year. The amount of radiation exposure received from these procedures is equal to approximately 11 rem. The CT scans and CT guided biopsies in this study will expose the research participant to 36.7 years' worth of background radiation. This level of exposure results in an increased risk of cancer.
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12.4.1.4 risk of biopsy

CT-guided biopsies will be performed as clinically indicated. Up to 3 CT-guided biopsies may be performed on this study for research purposes per year. Patients in all cohorts may be required to undergo a biopsy at screening in order to confirm their diagnosis Patients in Cohort 3: 2 required biopsies while on treatment, 1 optional biopsy at progression. Patients in Cohort 1 and 2: 3 optional biopsies (2 while on treatment and 1 at progression). All care will be taken to minimize risks that may be incurred by tumor sampling. However, there are procedure-related risks (such as bleeding, infection and visceral injury) that will be explained fully during informed consent.
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12.4.1.5 additional risks of ct

In addition to the radiation risks addressed above, CT scans that employ contrast may cause allergic reactions, injection site reactions abdominal discomfort and fainting.  
12.4.1.6 Research Blood Collection Risks Risks of blood draws include pain and bruising in the area where the needle is placed, lightheadedness, and rarely, fainting. When large amounts of blood are collected, low red blood cell count (anemia) can develop.
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12.4.1.7 other risks

Risks include the possible occurrence of any of a range of side effects which are listed in the Consent Document or this protocol document. Frequent monitoring for adverse effects will help to minimize the risks associated with administration of the study agents. 12.4.2 Known Potential Benefits The potential benefits from this therapy are stabilization or shrinkage of the tumor and a reduction in chances of developing new lesions with decrease of symptoms caused by progressive disease. 12.4.3 Assessment of Potential Risks and Benefits Metastatic or refractory/recurrent breast cancers are in need of improved therapy options. No current therapies are curative. Preclinical studies suggest that the use of a combination of multiple immunotherapy agents may have improved anti-tumor efficacy. A number of clinically appropriate strategies to minimize risks to patients have been built into the protocol through the means of inclusion/exclusion criteria, monitoring strategies, and management guidelines. Overall, the potential benefits of the combination of a therapeutic vaccine against brachyury (BN-brachyury), a bifunctional fusion protein targeting PD-L1 and TGF beta (M7824), an immune and epigenetic modifier (entinostat) and a standard of care antibody-drug conjugate (T-DM1) in subjects with advanced breast cancer for patients retaining the ability to consent and those who lose capacity to consent during the course of the trial outweigh the risks associated with this combination immunotherapy proposed in this study. The potential benefit to a patient that goes onto this study is a reduction in the bulk of their tumor which may or may not have favorable impact on symptoms and/or survival. Potential adverse reactions attributable to the administration of the study drug utilized in this trial are discussed in Section 14. All care will be taken to minimize side effects, but they can be unpredictable in nature and severity.
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12.5 consent process and documentation

The informed consent document will be provided as a physical or electronic document to the participant for review prior to consenting. A designated study investigator will carefully explain the procedures and tests involved in this study, and the associated risks, discomforts and benefits. In order to minimize potential coercion, as much time as is needed to review the document will be given, including an opportunity to discuss it with friends, family members and/or other advisors, and to ask questions of any designated study investigator. A signed informed consent document will be obtained prior to entry onto the study. The initial consent process as well as re-consent, when required, may take place in person or remotely (e.g., via telephone or other permissible secure remote platforms per policy, including HRPP Policy 303) per discretion of the designated study investigator and with the agreement of the participant. Whether in person or remote, the privacy of the subject will be maintained. Consenting investigators (and participant, when in person) will be located in a private area (e.g., clinic consult room). When consent is conducted remotely, the participant will be informed of the private nature of the discussion and will be encouraged to relocate to a more private setting if needed. Note: When required, witness signature will be obtained similarly as described for the investigator and participant as described below. Consent will be documented with required signatures on the physical document (which includes the printout of an electronic document sent to participant) or on the electronic document. Signatures on electronic documents are described below. Note: FDA only regulates electronic signatures (i.e., an electronic timestamp is generated at the time of signature) in FDA regulated research. Please see site specific supplement for electronic signature requirements for participating site. For the optional biopsies in the protocol, the patient will be asked to sign a separate consent at the time of the procedure. If the patient refuses the optional biopsy at that time, the refusal will be documented in the medical record and in the research record.
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13 regulatory and operational considerations

This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable cause. Written notification, documenting the reason for study suspension or termination, will be provided by the suspending or terminating party to study participants, investigator, funding agency, the Investigational New Drug (IND) sponsor and regulatory authorities. If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination or suspension. Study participants will be contacted, as applicable, and be informed of changes to study visit schedule. Circumstances that may warrant termination or suspension include, but are not limited to: Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA).
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13.2 quality assurance and quality

Each clinical site will perform internal quality management of study conduct, data and biological specimen collection, documentation and completion. An individualized quality management plan will be developed to describe a site's quality management.  
- Determination of unexpected, significant, or unacceptable risk to participants - Demonstration of efficacy that would warrant stopping
- Insufficient compliance to protocol requirements
- Data that are not sufficiently complete and/or evaluable - Determination that the primary endpoint has been met
- Determination of futility
Quality control (QC) procedures will be implemented beginning with the data entry system and data QC checks that will be run on the database will be generated. Any missing data or data anomalies will be communicated to the site(s) for clarification/resolution. Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is conducted and data are generated and biological specimens are collected, documented (recorded), and reported in compliance with the protocol, International Conference on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). The investigational site will provide direct access to all trial related sites, source data/documents, and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and regulatory authorities.
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13.3 conflict of interest policy

The independence of this study from any actual or perceived influence, such as by the pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be required to have such conflicts managed in a way that is appropriate to their participation in the design and conduct of this trial. The study leadership in conjunction with the National Cancer Institute and other participating sites has established policies and procedures for all study group members to disclose all conflicts of interest and will establish a mechanism for the management of all reported dualities of interest.
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13.4 confidentiality and privacy

Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to participants. Therefore, the study protocol, documentation, data, and all other information generated will be held in strict confidence. No information concerning the study or the data will be released to any unauthorized third party without prior written approval of the sponsor. All research activities will be conducted in as private a setting as possible. The study monitor, other authorized representatives of the sponsor, representatives of the Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and records required to be maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy records for the participants in this study. The clinical study site will permit access to such records. The study participant's contact information will be securely stored at each clinical site for internal use during the study. At the end of the study, all records will continue to be kept in a secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. Study participant research data, which is for purposes of statistical analysis and scientific reporting, will be transmitted to and stored at the NCI CCR. This will not include the participant's contact or identifying information. Rather, individual participants and their research data will be identified by a unique study identification number. The study data entry and study management systems used by the clinical site(s) and by NCI CCR research staff will be secured and password protected. At the end of the study, all study databases will be archived at the NIH. To further protect the privacy of study participants, a Certificate of Confidentiality has been issued by the National Institutes of Health (NIH). This certificate protects identifiable research information from forced disclosure. It allows the investigator and others who have access to research records to refuse to disclose identifying information on research participation in any civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or local level. By protecting researchers and institutions from being compelled to disclose information that would identify research participants, Certificates of Confidentiality help achieve the research objectives and promote participation in studies by helping assure confidentiality and privacy to participants.
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14 pharmaceutical information 14.1 bn-brachyury

BN-Brachyury is an active cancer immunotherapy. This phase 1b study is being conducted under an IND held by the Center for Cancer Research (CCR), National Cancer Institute (NCI). Bavarian Nordic, Inc. is the manufacturer of BN-Brachyury and will supply product for this phase 1b study. MVA-brachyury and FPV-brachyury will be provided to each clinical trial site by the manufacturer. The investigator or designee (e.g. pharmacist) will maintain an ongoing inventory of the investigational product supply according to standard site procedures. The investigational product will be dispensed at the direction of an investigator for administration to a study participant enrolled on the clinical trial. Disposal of expired or unused product will be returned to the manufacturer or disposed of according to standard site procedures based on agreement between the site and the manufacturer or IND holder.
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14.1.2 toxicity

MVA-BN-derived vectors that encode heterologous (non-vaccinia virus) antigens are being developed for the treatment of cancer. In GLP studies, MVA-BN and MVA-BN-derived vectors have been administered to 5 different animal species, including primates. In addition, MVA-BN and MVA-BN-derived vectors have been administered in clinical trials to over 7800 human subjects, including immunodeficient individuals. No marked toxicity and no drug-related serious AEs have occurred in any of these studies.
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14.1.3 formulation and preparation

MVA-BN-Brachyury is supplied as a frozen aqueous suspension in 2 mL clear borosilicate glass, single use vials. The closure is a sterile bromobutyl rubber stopper, crimped with an aluminum cap and covered with a polypropylene closure. Each 0.5mL injection will contain a nominal virus titer of 2.0 x 108Inf.U. Each vial must be thawed at room temperature for approximately 10–15 minutes prior to preparation, and should not be re-frozen after thawing. The thawed product will be a slightly turbid to milky suspension which may contain clumps or aggregates. To ensure homogeneity, the vial should be swirled vigorously, but not shaken, for approximately 10–20 seconds immediately (within 3 minutes) prior to use. The thawed drug product is to be drawn into a syringe with an appropriately sized safety-shielded needle suited to patient comfort (e.g., 22- to 28-gauge). FPV-Brachyury is a liquid-frozen, highly attenuated, live recombinant virus. FPV-Brachyury is a clear to milky suspension. The product needs to be free from visible extraneous particles considering that very small particles or clumps/aggregates are product related. A turbid appearance including the tendency towards sedimentation is based on the product-inherent characteristics. Additional details can be found in the Pharmacy Manual.  
Packages and vials are labeled according to the respective product specification. One FPVBrachyury vial has sufficient volume to deliver 0.5 mL and contains a nominal virus titer of 2 x 109Inf.U per mL.  
Detailed trial-specific instructions on the preparation and administration of MVA-BN-Brachyury and FPV-Brachyury are provided in the clinical trial protocol and/or a separate Pharmacy Manual supplied to each clinical trial site. 14.1.4 Stability and Storage MVA-BN-Brachyury and FPV-Brachyury are stable at -80°C ± 10°C and -20°C ± 5°C, respectively. MVA-BN-Brachyury and FPV-Brachyury should be stored frozen and remain frozen until use. After the completion of preparation, the injection must be administered within 9 hours. Avoid exposure to direct light and product may not be refrozen 14.1.5 Administration Procedures Administration of BN-Brachyury is described in Section **3.2.1**.  
14.1.6 Incompatibilities There are no known drug interactions associated with the use of MVA-BN and MVA-BNderived vectors.
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14.2 entinostat (ind # 19165)

Entinostat is a class 1-selective histone deacetylase inhibitor. This phase 1b study is being conducted under an IND held by the CCR, NCI. Syndax is the manufacturer of entinostat and will supply product for this phase 1b study. Entinostat will be provided to each clinical trial site by the manufacturer. The investigator or designee (e.g. pharmacist) will maintain an ongoing inventory of the investigational product supply according to standard site procedures. The investigational product will be dispensed at the direction of an investigator for administration to a study participant enrolled on the clinical trial. Disposal of expired or unused product will be returned to the manufacturer or disposed of according to standard site procedures based on agreement between the site and the manufacturer or IND holder.
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14.2.2 metabolism

Th exact mechanism of metabolism is unknown at this time. However, initially a renal and biliary clearance of entinostat was suspected. Subsequent studies have shown that hepatic metabolism seems to be a minor component of drug elimination in humans. (118, 127) The pharmacokinetics of entinostat were linear over the dose range of 2 to 12 mg/m2 given weekly. Considerable inter-patient variation in absorption was observed. The elimination halflife of entinostat ranged from 54 to 161 hours. The median elimination half-life of entinostat under fasted conditions is 140 hours. Data from *in vitro* experiments showed that, while entinostat inhibited cytochrome P-450 (CYP) enzymes 2B6 and 3A4, the degrees of the inhibition make it unlikely that any *in vivo* systemic interactions would occur. Intestinal CYP 3A4 may be inhibited by entinostat. However, entinostat did not inhibit any tested P glucuronosyltransferase (UGT) enzymes. In addition, entinostat was found to induce CYP 1A2, CYP 2C6, and CYP 2B8 as well as UGT 1A4. Finally, entinostat is a substrate for Pglycoprotein (P-gp) and breast cancer resistance protein (BCRP) transporters.  
In pharmacodynamic studies, entinostat-dependent protein lysine hyperacetylation was observed at all doses tested to date, with preliminary data suggesting that hyperacetylation may be associated with improved clinical outcome when entinostat is administered in combination with exemestane in breast cancer. PK and PD data will be evaluated on cycle 1 Day 8 and cycle 1 Day 15 (*optional*).
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14.2.3 toxicity

The most common AEs (>20%) for entinostat based on 217 patients who received monotherapy were fatigue (48%), nausea (47%), thrombocytopenia (45%), anemia (40%), hypoalbuminemia (37%), hypophosphatemia (32%), neutropenia (32%), vomiting (28%), anorexia (25%), headache (24%), diarrhea (21%), hyponatremia (21%). Grade 3 and 4 events (>10%) included thrombocytopenia (22%), anemia (19%), neutropenia (16%), and hypophosphatemia (15%). Serious adverse events (>5%) included fatigue (9%), dyspnea (7%), anorexia (6%), neutropenic infection (6%) and dehydration (6%). The safety profile of entinostat when given in combination was similar to that seen when given as monotherapy. Overall, 97% of the 740 patients who received entinostat in combination with another agent experienced at least 1 TEAE. In general, the AEs reported most frequently among patients receiving entinostat in combination, regardless of indication, were fatigue, nausea, anemia, thrombocytopenia, leukopenia, diarrhea, vomiting, and neutropenia. As would be expected, the AE profiles of entinostat when given in combination varied somewhat based on the agent with which it is given and the corresponding patient population. Entinostat in combination with 5-azacitidine was generally associated with an increased number of AEs relative to its use in combination with an aromatase inhibitor (AI), erlotinib, or other agents. Consistent with the overall AE profile of entinostat, nausea with or without vomiting, fatigue, and anemia were the most prevalent AEs regardless of the patient population or the agent given in combination. Overall, among the 535 patients with solid tumors receiving entinostat in combination, the AEs reported most frequently were as follows: fatigue (66%); nausea (58%); anemia (44%); vomiting and diarrhea (each 34%); thrombocytopenia and anorexia (each 30%); leukopenia (25%); dyspnea (22%); and injection site reaction (20%). Overall, among the 205 patients with hematologic malignancies receiving entinostat in combination, the SAEs reported most frequently were: fatigue (70%); thrombocytopenia (67%); anemia (65%); neutropenia (62%); leukopenia (60%); nausea (58%); anorexia (41%); diarrhea (29%); hypoalbuminaemia (36%); injection site reaction (35%); febrile neutropenia (34%); vomiting (33%); constipation (32%); hyperglycemia; hypocalcaemia and hyponatraemia (each 31%); edema peripheral and dyspnea (each 30%); and headache and hypophosphataemia (each 20%).
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14.2.4 formulation and preparation

Entinostat is supplied in two strengths of film-coated tablets containing 1 mg (pink to light red) or 5 mg (yellow) of entinostat, produced using the polymorph B form of the drug. The film coating is derived from an aqueous suspension consisting of hypromellose, talc, titanium dioxide, and ferric oxide pigments (red and yellow) as coloring. Each tablet also contains mannitol, sodium starch glycolate, hydroxypropylcellulose, potassium bicarbonate, and magnesium stearate as inert fillers.
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14.2.5 stability and storage

Entinostat tablets are to be stored at up to 25°C (77°F); excursions are permitted from 15°C to 30°C (59°F to 86°F). Entinostat tablets are to be stored in a secure, locked storage area to which access is limited. 14.2.6 Administration Procedures Administration of Entinostat is described in Section **3.2.4.1**.  
14.2.7 Incompatibilities At this time, there are no known drug interactions or incompatibilities with entinostat. It is thought that entinostat does inhibit CYP450 enzymes but at higher concentrations than clinically achievable. (127) However, no formal drug-drug interactions have been completed. It is recommended to use caution when administering entinostat concurrently with medication that have a narrow therapeutic index.
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14.3 m7824 (ind # 19165)

M7824 is a novel bifunctional fusion protein composed of a monoclonal antibody against human PD-L1 fused to the soluble extracellular domain of human TGF-β receptor II (TGF-βRII), which functions as a TGF-β "trap." This phase 1b study is being conducted under an IND held by the CCR, NCI. EMD Serono is the manufacturer of M7824 and will supply product for this phase 1b study. M7824 will be provided to each clinical trial site by the manufacturer. The investigator or designee (e.g., pharmacist) will maintain an ongoing inventory of the investigational product supply according to standard site procedures. The investigational product will be dispensed at the direction of an investigator for administration to a study participant enrolled on the clinical trial. Disposal of expired or unused product will be returned to the manufacturer or disposed of according to standard site procedures based on agreement between the site and the manufacturer or IND holder.
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14.3.2 metabolism

This preliminary PK analysis was based on data from 12 subjects (3 subjects each at 1, 3, 10, and 20 mg/kg) from EMR200647-001 as of the data cutoff date of 04 May 2016. Additional PK data from EMR200647-001 and MS200647-0008 will be reported at a later time. Samples for measurement of M7824 serum concentrations in Study EMR200647-001 were collected and mean M7824 serum concentrations were plotted against time after dosing by dose group. The PK profiles at the 3, 10, 20 mg/kg dose groups indicate a one-phase elimination with no obvious distribution phase after 1 hour iv infusion of M7824. Similar PK profile characteristics were observed at the lowest dose groups (1 mg/kg) with a slightly faster elimination rate. Non-compartmental analyses were performed for each dose group with PK parameters summarized. Based on data from 12 patients in the dose escalation part of EMR200647-001, M7824 demonstrated approximately dose-proportional PK from 3 to 20 mg/kg. The mean apparent terminal half-life (t1/2) after the 1st dose was about 6 to 7 days for dose levels above 3 mg/kg. In the 1 mg/kg dose group, a mean terminal t1/2 of about 5 days was observed. When compared to the exposure at NOAEL in the 13-weeks repeat dose toxicity study in cynomolgus monkeys (140 mg/kg biweekly), the exposure at the highest clinical dose tested to date in humans (20 mg/kg biweekly) is 6.6-fold lower in Cmax and 4.3-fold lower in AUC, which indicate a safety margin of at least 4-fold. As T-DM1 is dosed every 3 weeks, a dosing interval of every 3 weeks was preferred to the standard every 2 week dosing for M7824. The dose-level was informed by extensive population PK modeling performed by the EMD Serono pharmacokinetics team after reviewing data from the ongoing M7824 -001 and -0008 trials. For M7824 monotherapy, 1200 mg every 2 weeks was selected as a recommended phase 2 dose. (128) In combination studies in which concomitant therapies are administered q3w, the same dosing interval for M7824 is preferred for convenience and compliance. The steady-state Ctrough (Ctrough,ss) at 1200 mg q2w is considered the target efficacious Ctrough,ss for q3w dosing of M7824 in combination studies with q3w dosing. Based on population PK modeling, median Ctrough,ss achieved with 2400 mg q3w dosing is similar to that projected with 1200 mg q2w dosing. In addition, for >90% of the participants to be dosed with 2400 mg q3w, Ctrough,ss is estimated to be above the lower bound of 95% CI for Ctrough,ss projected with 1200 mg q2w. Since Ctrough,ss is sim2400 mg q3w is expected to achieve target Ctrough,ss and full PD-L1 target occupancy and TGFβ trapping in blood in > 90% of the participants. Safety data to support 2400 mg q3w dosing: The highest dose for M7824 tested in EMR200647001 was 30 mg/kg once every 2 weeks and the maximum tolerated dose was not reached. Three (3) participants in EMR200647-001 dose escalation cohorts received the actual dose of at least 2400 mg q2w: 2 in 30 mg/kg cohort and 1 in 20 mg/kg. All 3 participants who received ≥ 2400 mg q2w had no DLTs. For the 2400 mg q3w dosing, both the maximum and average concentrations at steady state are not expected to exceed those at 2400 mg q2w. The overall safety profile of M7824 in Phase I was considered well tolerated, can be adequately managed and consistent across various tumor types. Refer to Investigator's Brochure for more details.
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14.3.3 toxicity

In a phase 1, open-label 3+3 dose-escalation study of M7824 in 16 patients, 3 patients experienced grade 3 drug-related adverse events including skin infection secondary to grade 2 bullous pemphigoid, lipase increased, and colitis with associated anemia. There were no grade 4 - 5 treatment related adverse events. Please see table below for details: Treatment-related adverse events  
3 mg/kg (n = 3) 10 mg/kg (n = 3) 20 mg/kg (n = 7) *Total (n = 16)*  
Any  
Grade  
Grade  
3  
Any  
Grade Grade 3 Any  
Grade Grade 3 Any  
Grade Grade 3  
Patients with any  
event**  
2  
(66.7)  
1  
(33.3) 1 (33.3) 0 (0.0) 4 (57.1) 2 (28.6) 7 (43.8) **3 (18.8)**  
Anemia 1 (14.3) 1 (14.3) 1 (6.3) 1 (6.3)  
| Treatment-related adverse events 3 mg/kg (n = 3)                                                                                                                                     | 10 mg/kg (n = 3)   | 20 mg/kg (n = 7)   | Total (n = 16)   |          |          |          |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------|--------------------|------------------|----------|----------|----------|
| Any                                                                                                                                                                                  | Grade              | Grade              | Grade 3          | Any      |          |          |
| Any                                                                                                                                                                                  | Grade              | Grade 3            | Any  Grade       | Grade 3  |          |          |
| Grade                                                                                                                                                                                | 3                  |                    |                  |          |          |          |
| Patients with any                                                                                                                                                                    | 2                  | 1                  |                  |          |          |          |
| (33.3)                                                                                                                                                                               | 1 (33.3)           | 0 (0.0)            | 4 (57.1)         | 2 (28.6) | 7 (43.8) | 3 (18.8) |
| event**                                                                                                                                                                              | (66.7)             |                    |                  |          |          |          |
| Anemia                                                                                                                                                                               | 1 (14.3)           | 1 (14.3)           | 1 (6.3)          | 1 (6.3)  |          |          |
| Bullous pemphigoid                                                                                                                                                                   | 1                  |                    |                  |          |          |          |
| (33.3)                                                                                                                                                                               | 1 (6.3)            |                    |                  |          |          |          |
| Colitis                                                                                                                                                                              | 1 (14.3)           | 1 (14.3)           | 1 (6.3)          | 1 (6.3)  |          |          |
| Dermatitis  acneiform                                                                                                                                                                | 1 (33.3)           | 1 (6.3)            |                  |          |          |          |
| Dyspnea  exertional***                                                                                                                                                               | 1 (14.3)           | 1 (6.3)            |                  |          |          |          |
| Hyperthyroidism                                                                                                                                                                      | 1 (14.3)           | 1 (6.3)            |                  |          |          |          |
| Hypophosphatemia                                                                                                                                                                     | 1 (14.3)           | 1 (6.3)            |                  |          |          |          |
| Hypothyroidism                                                                                                                                                                       | 1 (33.3)           | 1 (14.3)           | 2 (12.5)         |          |          |          |
| Infusion-related  reaction                                                                                                                                                           | 1 (14.3)           | 1 (6.3)            |                  |          |          |          |
| Keratoacanthoma                                                                                                                                                                      | 1 (14.3)           | 1 (6.3)            |                  |          |          |          |
| Lipase increase                                                                                                                                                                      | 1 (14.3)           | 1 (14.3)           | 1 (6.3)          | 1 (6.3)  |          |          |
| Nausea                                                                                                                                                                               | 1                  |                    |                  |          |          |          |
| (33.3)                                                                                                                                                                               | 1 (6.3)            |                    |                  |          |          |          |
| Pruritus                                                                                                                                                                             | 1                  |                    |                  |          |          |          |
| (33.3)                                                                                                                                                                               | 1 (6.3)            |                    |                  |          |          |          |
| Rash maculopapular                                                                                                                                                                                      | 1                  |                    |                  |          |          |          |
| (33.3)                                                                                                                                                                               | 1 (33.3)           | 2 (12.5)           |                  |          |          |          |
| Skin infection                                                                                                                                                                       | 1                  | 1                  |                  |          |          |          |
| (33.3)                                                                                                                                                                               | 1 (6.3)            | 1 (6.3)            |                  |          |          |          |
| (33.3)                                                                                                                                                                               |                    |                    |                  |          |          |          |
| Vomiting                                                                                                                                                                             | 1                  |                    |                  |          |          |          |
| (33.3)                                                                                                                                                                               | 1 (6.3)            |                    |                  |          |          |          |
| **There were no treatment-related AEs in the 3 patients treated with 1 mg/kg M7824. ***The differential for this dyspnea was pneumonitis vs. lymphangitic spread of disease (disease |                    |                    |                  |          |          |          |  
The emergent immune-related AE (irAEs) associated with M7824 in more than 670 treated patients with different tumor types in Phase I studies is consistent with PD-(L)1 agents. The other important identified risks for M7824 are dermatological AEs related to TGFβ inhibition and include keratoacanthomas and cutaneous squamous cell carcinoma. These AEs were similar to those observed in Ferguson-Smith Syndrome, in which TGFβ is genetic mutated, as well as phase I studies with fresolimumab (a pan TGFβ inhibitor). (129, 130) In the EMR200647-001/ MS200647-0008 studies, these skin AEs were observed in approximately 7% of participants and were adequately managed via excision and some spontaneously regressed without requiring treatment discontinuation. In the context of encouraging clinical activity in various advanced cancer types, these skin lesions were overall manageable and no new safety signals emerged in the EMR200647-001/MS200647-0008 studies compared with established therapies targeting PD- (L)1 or TGFβ blockade. These lesions have not been a criterion for treatment discontinuation, but thus far have all spontaneously regressed following treatment discontinuation. After discussion among NCI investigators on multiple protocols using M7824, multiple bleeding events ranging from low grade gingival bleeding and epistaxis to more serious hemoptysis, GI bleeding and hematuria have been observed. Some of these events can be attributed to bleeding events related to cancer directly and others bleeding events can be attributed to colitis or cystitis which is a known toxicity of anti-PD-L1 agents including M7824. However, there remains the possibility that M7824 may increase the overall risk of bleeding in ways that may not be directly related to direct tumor bleeding or inflammatory bleeding events described with checkpoint inhibitors like M7824. It is hypothesized that this possible increased bleeding risk may be due to TGF beta inhibition which has an effect on angiogenesis; bleeding has also been observed in patients receiving M7824 and may be drug-related (e.g., gum bleeding, nose bleeds, coughing up blood, blood in their urine, or blood in the stool). Accordingly, patients will be closely monitored for mucosal bleeding (e.g., gum bleeding, nose bleeds, coughing up blood, blood in their urine, or blood in the stool). A preliminary analysis of QT data from about 130 triplicate ECGs in 19 subjects from EMR200647-001 dose escalation cohorts up to a dose of 20 mg/kg showed that M7824 has no effect on QTcF intervals or heart rate, with QT-concentration slope close to 0 (0.002 ms per μg/mL) with 90% CI including zero (-0.01 to 0.01). The predicted upper bound of the 2-sided 90% CI of delta QTc at the average Cmax after the first 20 mg/kg dose (493 μg/mL) was 1.35 msec. while the predicted upper bound of 90% CI of delta QTcF change from baseline at Cmax value was less than 5 ms. Additionally, nonclinical safety study revealed no identified proarrhythmic risk. 14.3.4 Formulation and Preparation M7824 drug product is provided as a sterile liquid formulation and packaged at a 10mg/ml concentration in USP/Ph Eur type I 50R vials that are filled with drug product solution to allow an extractable volume of 60ml (600mg/60ml). The vials are closed with rubber stoppers in serum format complying with USP and Ph Eur with an aluminum crimp seal closure. Each single-use vial contains 600mg of M7824, formulated as 10mg/mL of active, 6% (w/v) Trehalose, 40mM NaCl, 5mM Methionine, 0.05% (w/v) Tween 20, 10 mM L Histidine at pH 5.5. The liquid formulation is diluted directly with 0.9% sodium chloride solution for injection. The estimated volumes of delivery are anticipate to be no more than 250mL. The verified concentration range in the infusion solution is 0.16mg/mL to 9.6 mg/mL. 14.3.5 Stability and Storage M7824 drug product must be stored at 2°C to 8°C until use. The storage condition is based on data from ongoing long term stability studies with M7824. M7824 drug product stored at room (23°C to 27°C) or higher temperatures for extended periods of time might be subject to degradation. The chemical and physical in-use stability for the infusion solution of M7824 in 0.9% saline solution has been demonstrated for a total of 72 hours at room temperature. However, from a microbiological point of view, the diluted solution should be used immediately and is not intended to be stored unless dilution has taken place in controlled and validated aseptic conditions. If not used immediately, in-use storage times and conditions prior to administration are the responsibility of the user. Do not freeze or shake the diluted solution. No other drugs should be added to the infusion containers containing M7824. 14.3.6 Administration Procedures Administration of M7824 is described in Section **3.2.2**.  
14.3.7 Incompatibilities No formal drug interaction studies have been conducted with M7824 in humans.