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Abbreviated Title: AdHER2/neu DC Vaccine CC Protocol \#: 13-C-0016 O Version Date: July 22, 2019 NCT Number: NCT01730118 OSP \#: 1207-1179 IBC \#: RD-12-VIII-07 Title: A Phase I Study of an Adenoviral Transduced Autologous Dendritic Cell Vaccine Expressing Human HER2/neu ECTM in Adults with Tumors with 1-3+ HER2/neu Expression Principal Investigator: Hoyoung Maeng, M.D., Vaccine Branch, CCR, NCI
Building 10, Room 3B37 10 Center Drive Bethesda, MD 20892 Phone: 240-781-3253 Email: hoyoung.maeng@nih.gov
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investigational agent

Agent: Autologous dendritic cells transduced with an adenoviral vector: Ad5f35 human HER2/neu ECTM (AdHER2)  
| Drug Name:    | Ad5f35HER2ECTM transduced autologous dendritic cell vaccine   |
|---------------|---------------------------------------------------------------|
| IND Number:   | 15212                                                         |
| Sponsor:      | Center for Cancer Research                                    |
| Manufacturer: | Department of Transfusion Medicine, NIH Clinical Center       |  
Commercial Agents: None Identifying words: Human epidermal growth factor receptor 2 (HER2) expression, dendritic cell vaccine breast cancer, stomach cancer, metastatic tumors 1
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precis

Background:  
- Human epidermal growth factor receptor 2 (HER2, also known as c-erbB2 or neu) is a proto-oncogene that encodes a 185-kd transmembrane (TM) tyrosine kinase receptor that participates in receptor-receptor interactions that regulate cell growth, differentiation and proliferation. Its over-expression contributes to neoplastic transformation.  
- HER2 is over-expressed in up to 25-30% of node-positive or node-negative primary breast cancers and is associated with clinically aggressive breast cancer, a high recurrence rate and reduced survival.  
- Trastuzumab (Herceptin) is a recombinant humanized mouse monoclonal antibody
(MAb) that binds to the extracellular (EC) domain of the HER2 receptor. Its clinical efficacy is limited to patients with 3+ HER2 tumor expression documented by immunohistochemistry (IHC) or a HER2 amplified tumor by Vysis fluorescent *in situ* hybridization (FISH). IHC is a subjective measurement of HER2/neu protein while FISH
is an objective measurement of amplification of the HER2 oncogene.  
- Although the use of trastuzumab has been associated with improved clinical outcomes, a significant number of patients are unresponsive to therapy and most eventually experience clinical progression. At present, no vaccine is available that induces patients to make their own anti-HER2 antibodies.  
- We propose to investigate the use of an adenoviral vector (Ad5f35) expressing *human* HER2ECTM (Ad5f35HER2ECTM- AdHER2) to transduce autologous dendritic cells for therapeutic vaccination in patients with HER2 expressing solid tumors.
Objectives:
- To determine the safety and toxicity of autologous AdHER2 dendritic cell vaccination.  
- Specifically, to determine if the fraction of patients with cancer therapeutics-related cardiac dysfunction (CTRCD), defined as a decrease in LVEF ≥10 percentage points, to a value LVEF to ≥53% (normal reference value for two-dimensional (2-D)
echocardiography), is sufficiently low to warrant further development in subsequent trials.  
- To determine the immunogenicity of autologous AdHER2 dendritic cell vaccination as measured by a 3-fold increase in anti-HER2/neu antibody concentration or a 4-fold increase in antibody dilution titers over baseline.
Eligibility:  
- Adults ≥ 18 of age
- Patients with 1+ to 3+ HER2/neu expression by IHC or a Vysis FISH result equivocal or positive.  
- Part I: Naïve to trastuzumab (Herceptin™), pertuzumab (Perjeta™) and lapatinib
(Tykerb™), ado-trastuzumab emtansine (Kadcyla™) or other HER2-directed therapies.  
- Part II: Recurrent or progressive metastatic disease after standard of care HER2- targeted therapies with known clinical benefit; i.e. trastuzumab (Herceptin™), pertuzumab
(Perjeta™), lapatinib (Tykerb™), ado-trastuzumab emtansine (TDM1) (Kadcyla™) or other HER2-directed therapies.  
- Eligible malignancies:
o Parts I and II: Malignant soft tissue and bone tumors and recurrent or progressive, metastatic solid tumors who have progressed on standard therapies (Note: Patients with metastatic cancer must have measurable disease per RECIST 1.1); or, o Part I only: Bladder cancer in the adjuvant setting (adjuvant bladder cancer patients):
o Tumor stage T3a, T3b, T4a, T4b and any node positive disease regardless of tumor stage.  
o Status-post primary cystectomy with curative intent. o May or may not have received neoadjuvant cisplatin-based combination chemotherapy per NCCN guidelines.  
o May or may not have received adjuvant radiotherapy or chemotherapy based on pathologic risk per NCCN guidelines.  
o Greater than or equal to 6 weeks status post primary surgery with curative intent.  
- Adequate hematologic, metabolic, hepatic, renal, and cardiac function
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study design

Open label, non-randomized, two-part, phase I study of 48 weeks duration for evaluation of primary endpoints with extended follow-up out to 30 months to monitor LVEF cardiac function.  
Part I involves vaccine dose escalation in a population with **no prior exposure** to trastuzumab or other HER2-targeted therapies to determine if there is a significant, adverse safety signal regarding cardiac toxicity, in addition to preliminary assessment of the vaccine's immunogenicity and clinical activity in patients with metastatic solid tumors or high-risk bladder cancer in the adjuvant setting. Response will be evaluated by a Modified Immune-Related Response Criteria (modified irRC) based on Response Evaluation Criteria in Solid Tumors
(RECIST 1.1).  
Part II is identical to part I in the schedule of treatment and response evaluation, but is conducted in a population with **prior exposure** to trastuzumab and other HER2-targeted therapies.  
| TABLE OF CONTENTS PRÉCIS 2 TABLE OF CONTENTS 4 1 INTRODUCTION 9 1.1 Study Objectives 9 1.1.1 Primary Objective 9 1.1.2 Secondary Objectives 9 1.2 Background and Rationale 9 1.2.1 HER2 Directed Therapies 9 1.2.2 Pre-Clinical Animal Studies 12 1.2.3 Rodent AdneuECTM DC Vaccination 15 1.2.4 Central and Peripheral Tolerance in the Transgenic HER2 Mouse Model 16 1.2.5 Pre-Clinical AdHER2ECTM Vector Expression Testing 17 1.2.6 Pre-Clinical HER2 Dendritic Cell Platform Testing 18 1.2.7 Human Experience with Dendritic Cell Vaccines 22 1.2.8 Human Experience with HER2 Therapeutic Vaccines 22 1.2.9 Previous Human Experience with an Extracellular/Transmembrane/Intracellular  HER2/neu Vaccine 23 1.2.10 Exploratory Correlative Studies 24 2 ELIGIBILITY ASSESSMENT AND ENROLLMENT 25 2.1 Eligibility Criteria 25 2.1.1 Common Eligibility for Parts I and II 25 2.1.2 Part I Eligibility 27 2.1.3 Part II Eligibility 27 2.2 Exclusion Criteria 27 2.2.2 Recruitment Strategies 28 2.3 Pre-Screening and Screening Evaluation 29 2.3.1 Pre-Screening 29 2.3.2 Screening Evaluation 29 2.4 Registration Procedures 29 2.4.1 Treatment Assignment Procedures 30 2.5 Baseline Evaluation 31 3 STUDY IMPLEMENTATION 31   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| 3.1   | Study Schema and Design 31                                                              |
|-------|-----------------------------------------------------------------------------------------|
| 3.1.1 | Dose Limiting Toxicity 35                                                               |
| 3.1.2 | Dose Escalation 36                                                                      |
| 3.2   | Drug Administration 37                                                                  |
| 3.3   | Dose Modification and Immunization Stopping Rules 38                                    |
| 3.4   | Study Calendar 39                                                                       |
| 3.5   | Criteria for Removal from Protocol Therapy and Off Study Criteria 41                    |
| 3.5.1 | Criteria for Removal from Protocol Therapy 41                                           |
| 3.5.2 | Continued Monitoring for Potential Cardiac Toxicity Once Off Protocol Therapy.41        |
| 3.5.3 | Off Study Criteria 42                                                                   |
| 3.5.4 | Off Protocol Therapy and Off Study Procedure 42                                         |
| 4     | CONCOMITANT MEDICATIONS/MEASURES 42                                                     |
| 5     | BIOSPECIMEN COLLECTION 44                                                               |
| 5.1   | Correlative Studies for Research/Pharmacokinetic Studies 44                             |
| 5.2   | Sample Storage, Tracking and Disposition 44                                             |
| 5.2.1 | Samples collected by the Blood Processing Core of the Clinical Pharmacology  Program 45 |
| 5.3   | Samples for Genetic/Genomic Analysis 46                                                 |
| 6     | DATA COLLECTION AND EVALUATION 46                                                       |
| 6.1   | Data Collection 46                                                                      |
| 6.2   | Response Criteria 47                                                                    |
| 6.2.1 | Response Definitions 48                                                                 |
| 6.2.2 | Disease Parameters 48                                                                   |
| 6.2.3 | Methods for Evaluation of Measurable Disease 49                                         |
| 6.2.4 | Evaluation of Target Lesions 49                                                         |
| 6.2.5 | Evaluation of Non-Target Lesions 50                                                     |
| 6.2.6 | Evaluation of Best Overall Response 50                                                  |
| 6.2.7 | Confirmatory Measurement/Duration of Response 53                                        |
| 6.2.8 | Progression Free Survival and Overall Survival 54                                       |
| 6.2.9 | Response Review 54                                                                      |
| 7     | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN 54                           |
| 7.1   | Definitions 54                                                                          |
| 7.2   | OHSRP Office of Compliance and Training/ IRB Reporting 54                               |  
| 7.2.1   | Expedited Reporting NIH Intramural IRB and NCI CD Expedited Reporting of  Unanticipated Problems and Deaths 54                                                                                                        |
|---------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 7.2.2   | IRB Requirements for PI Reporting at Continuing Review 55                                                                                                                                                             |
| 7.3     | NCI Clinical Director Reporting 55 To report these deaths, please send an email describing the circumstances of the  death to Dr. Dahut at NCICCRQA@mail.nih.gov within one business day of  learning of the death 55 |
| 7.4     | Institutional Biosafety Committee (IBC) Reporting Criteria 55                                                                                                                                                         |
| 7.4.1   | Serious Adverse Event Reports to IBC 55                                                                                                                                                                               |
| 7.4.2   | Annual Reports to IBC 55                                                                                                                                                                                              |
| 7.5     | NIH Required Data and Safety Monitoring Plan 56                                                                                                                                                                       |
| 7.5.1   | Principal Investigator/Research Team 56                                                                                                                                                                               |
| 7.5.2   | Safety Monitoring Committee (SMC) 56                                                                                                                                                                                  |
| 8       | SPONSOR SAFETY REPORTING 57                                                                                                                                                                                           |
| 8.1     | Definitions 57                                                                                                                                                                                                        |
| 8.1.1   | Adverse Event 57                                                                                                                                                                                                      |
| 8.1.2   | Serious Adverse Event (SAE) 57                                                                                                                                                                                        |
| 8.1.3   | Life-threatening 57                                                                                                                                                                                                   |
| 8.1.4   | Severity 57                                                                                                                                                                                                           |
| 8.1.5   | Relationship to Study Product 57                                                                                                                                                                                      |
| 8.2     | Assessment of Safety Events 58                                                                                                                                                                                        |
| 8.3     | Reporting of Serious Adverse Events 58                                                                                                                                                                                |
| 8.4     | Reporting Pregnancy 58                                                                                                                                                                                                |
| 8.4.1   | Maternal Exposure 58                                                                                                                                                                                                  |
| 8.4.2   | Parental exposure 59                                                                                                                                                                                                  |
| 8.5     | Regulatory Reporting for Studies Conducted Under CCR-Sponsor IND 59                                                                                                                                                   |
| 9       | CLINICAL MONITORING 59                                                                                                                                                                                                |
| 10      | COLLABORATIVE AGREEMENTS 60                                                                                                                                                                                           |
| 10.1    | Tech Transfer Agreements 60                                                                                                                                                                                           |
| 11      | STATISTICAL CONSIDERATIONS 60                                                                                                                                                                                         |
| 11.1    | Part I (maximum of 30 evaluable patients) 60                                                                                                                                                                          |
| 11.2    | Part II (maximum of 30 evaluable patients) 62                                                                                                                                                                         |
| 11.3    | Overview 62                                                                                                                                                                                                           |  
| 11.4   | Study Termination 64                                |
|--------|-----------------------------------------------------|
| 12     | HUMAN SUBJECTS PROTECTIONS 64                       |
| 12.1   | Rationale for Subject Selection 64                  |
| 12.2   | Participation of Children 64                        |
| 12.3   | Participation of Subjects Unable to Give Consent 65 |
| 12.4   | Evaluation of Benefits and Risks/Discomforts 65     |
| 12.5   | Risks/Benefits Analysis 66                          |
| 12.6   | Consent Process and Documentation 67                |
| 12.6.1 | Telephone consent procedure 68                      |
| 13     | PHARMACEUTICAL INFORMATION 68                       |
| 13.1   | Ad5f35HER2ECTM (AdHER2) 68                          |
| 13.1.1 | Source 68                                           |
| 13.1.2 | Toxicity 70                                         |
| 13.1.3 | Formulation and Preparation 70                      |
| 13.1.4 | Stability and Storage 71                            |
| 13.1.5 | Administration Procedures 72                        |
| 13.1.6 | Incompatibilities 72                                |
| 13.2   | Sargramostim 72                                     |
| 13.2.1 | Source: 72                                          |
| 13.2.2 | Formulation and Preparation: 72                     |
| 13.2.3 | Stability and Storage: 72                           |
| 13.2.4 | Administration Procedures: 73                       |
| 13.2.5 | Incompatibilities: 73                               |
| 13.3   | Interleukin-4 CellGenix 73                          |
| 13.3.1 | Source: 73                                          |
| 13.3.2 | Formulation and Preparation: 73                     |
| 13.3.3 | Stability and Storage: 73                           |
| 13.3.4 | Administration Procedures: 73                       |
| 13.3.5 | Incompatibilities: 73                               |
| 13.4   | KLH (Keyhole Limpet Hemocyanin) 73                  |
| 13.4.1 | Source 73                                           |
| 13.4.2 | Formulation and Preparation: 74                     |
| 13.4.3 | Stability and Storage: 747                          |  
| 13.4.4   | Administration Procedures: 74                                                                            |
|----------|----------------------------------------------------------------------------------------------------------|
| 13.4.5   | Incompatibilities: 74                                                                                    |
| 14       | REFERENCES 75                                                                                            |
| 15       | APPENDICES 78                                                                                            |
| 15.1     | Appendix 1: Performance Status Criteria 78                                                               |
| 15.2     | Appendix 2: New York Heart Association(NYHA) Functional Classification 79                                |
| 15.3     | Appendix 3: Standard Operating Procedure for Manufacture of AdHER2  Autologous Dendritic Cell Vaccine 80 |
| 15.4     | Appendix 4: AdHER2 DC Vaccine Manufacturing Schema 81                                                    |
| 15.5     | Appendix 5: AdHER2 DENTRITIC CELL VACCINE REPORT CARD 85                                                 |
| 15.6     | Appendix 6Anti-HER2 Antibody and Cellular Responses 86                                                   |
| 15.7     | Appendix 7Evaluation of Anti-Ad5 Antibody Immunity 88                                                    |
| 15.8     | Appendix 8Characterization of Soluble HER2 and Anti-HER2 Antibodies 89                                   |
| 15.9     | Appendix 9Exploratory Correlative Studies 90                                                             |
| 15.10    | Appendix 10Ad5f35HER2ECTM (AdHER2) Vector Manufacturing 93                                               |
| 15.11    | Appendix 11Map of the Ad5f35HER2ECTM (AdHER2) Vector 95                                                  |
| 15.12    | Appendix 12 AdHER2 Dendritic Cell Vaccine Cancer Treatment History 96                                    |
| 15.13    | Appendix 13 Patient Recruiting Materials 98                                                              |
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1 introduction 1.1 study objectives

- To determine the safety and toxicity of autologous AdHER2/neu dendritic cell vaccination. Specifically, to:  
o Determine if the fraction of patients with cancer therapeutics-related cardiac dysfunction (CTRCD), (if it occurs) is sufficiently low to warrant further development in subsequent trials. CTRCD is defined as a decrease in LVEF ≥ 10 percentage points, to a value ≥ 53% (normal reference value for two-dimensional (2-D) echocardiography)
1.  
- To determine the immunogenicity of autologous AdHER2 dendritic cell vaccination as measured by a 3-fold increase in anti-HER2/neu antibody concentration (measured as mcg/mL) or a 4-fold increase in antibody dilution titers over baseline.  
1.1.2 Secondary Objectives
- To determine the preliminary activity of autologous AdHER2 dendritic cell vaccination as measured by the fraction of subjects who have stable disease, a partial response or better by modified immune-related response criteria.  
- To determine the impact of autologous AdHER2 dendritic cell vaccination on tumor growth rate and regression rate constants.  
- To characterize vaccine-induced antibody profiles using HER2 peptide microarrays, examining reactivity to HER2 extracellular (EC), transmembrane (TM) and intracellular (IC) domains.  
- To characterize and measure function-associated mRNAs in whole blood, circulating tumor cells, myeloid derived suppressor cells (MDSCs) and other potential biologic/immunologic correlates of clinical response throughout treatment.
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1.2 background and rationale 1.2.1

Human epidermal growth factor receptor 2 (HER2, also known as c-erbB2 or neu) is a protooncogene that encodes a 185-kd transmembrane (TM) tyrosine kinase receptor that participates in receptor-receptor interactions that regulate cell growth, differentiation and proliferation. Its over-expression- either HER2 oncogene amplification and/or over-expression of the HER2 protein, contributes to neoplastic transformation2. HER2 is over-expressed in up to 25-30% of node-positive or node–negative primary breast cancers and is associated with clinically aggressive breast cancer, a high recurrence rate and reduced survival3. Development of agents targeting HER2 has resulted in expansion of therapeutic options and improved clinical outcomes for patients with HER2 over-expressing tumors4. Current FDA-approved treatment options include trastuzumab and lapatinib. A third agent, pertuzumab is under priority review by the FDA with a decision expected by June 8th, 2012 for a proposed indication in combination with trastuzumab and docetaxel chemotherapy in patients with HER2-positive metastatic or locally recurrent, unresectable breast cancer, who have not received previous treatment or whose disease has relapsed after adjuvant therapy.
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1.2.1.1 trastuzumab (herceptin)

Trastuzumab (Herceptin) is a recombinant humanized mouse monoclonal antibody (MAb) that binds to the extracellular (EC) domain of the HER2 receptor and was approved by the FDA in September 1998. It is approved for the adjuvant treatment of early-stage breast cancer with nodal spread (or without nodal spread if the tumor is high risk) as well as for first and secondline/salvage therapy for metastatic breast cancer5. In the adjuvant setting trastuzumab is used in several ways:  
- As part of a treatment course including chemotherapy with doxorubicin, cyclophosphamide and either paclitaxel or docetaxel, known as "ACTH".  
- With chemotherapy drugs docetaxel and carboplatin in a regimen known as "TCH".  
- Alone after treatment with multiple other therapies, including anthracycline-based therapy.
Trastuzumab has two approved uses in metastatic breast cancer:  
- As first line treatment in combination with paclitaxel. - As a single agent in patients who have received one or more courses of chemotherapy for metastatic disease.
Therapy typically involves an initial trastuzumab dose of 4mg/kg IV followed by 2mg/kg IV administered weekly for 52 weeks, alone (salvage therapy) or in combination with paclitaxel for the first 12 weeks (adjuvant and first-line therapy). However, clinical efficacy is limited to patients with 3+ HER2 tumor expression documented by immunohistochemistry (IHC) or gene amplification by fluorescent *in situ* hybridization (FISH). IHC is a subjective measurement of HER2/neu protein while FISH is an objective measurement of amplification of the HER2 oncogene6. Despite the high level efficacy in combination with chemotherapy, the overall response rate of women diagnosed with HER2-positive metastatic breast cancer and treated with trastuzumab as a single first-line agent is only 26%7clearly indicating there is room for improvement in therapeutic options. In addition, a significant number of patients are unresponsive to trastuzumab and most eventually experience clinical progression while on therapy, presumably due to trastuzumab resistance, whose mechanism is poorly understood8-10.  
Trastuzumab is also approved for use in combination with cisplatin and either capecitabine or 5fluorouracil in treatment naïve patients with HER2-positive metastatic stomach cancer or cancer of the gastroesophageal junction. Despite the success of trastuzumab, no vaccine is currently available that induces patients to make their own anti-HER2 antibodies. 1.2.1.2 Trastuzumab Cardiac Toxicity Given the well-documented cardiac toxicity associated with the monoclonal antibody trastuzumab 11-13 we believe it is critical to initially assess the safety of the vaccine as a single agent to minimize confounding attributions of toxicity. Specifically, a retrospective review of records for patients enrolled onto any of seven phase II and III trastuzumab clinical trials revealed patients treated with trastuzumab were found to be at increased risk for cardiac dysfunction11. The incidence was greatest in patients receiving concomitant trastuzumab and anthracycline plus cyclophosphamide (27%) but was substantially lower in patients receiving paclitaxel and trastuzumab (13%) or trastuzumab alone (3% to 7%) although most of these patients had received prior anthracycline therapy. Most trastuzumab-treated patients developing cardiac dysfunction were symptomatic (75%), and most improved with standard treatment for congestive heart failure (79%). Consequently, we propose to conduct this study in two parts to identify any cardiac safety issues that may be associated with vaccine-induced antibodies. Part I of the study involves vaccine dose escalation in a population with **no prior exposure** *to trastuzumab* or other HER2 directed agents to determine if there is a significant, adverse safety signal regarding cardiac toxicity. Assessments of cardiac function will include serial echocardiography to determine left ventricular ejection fraction (LVEF) at baseline and at regular monitoring intervals, in addition to LVEF measurements for at least 2 years following receipt of the last dose of vaccine. Once cardiac safety has been established in this population, Part II repeats the vaccine dose escalation in a population with **significant prior exposure** *to trastuzumab* to determine whether there is an adverse safety signal regarding cardiac toxicity using identical monitoring and follow-up criteria. Please note for patients in parts I and II with taking known CT agents will no longer be on study. This study design and monitoring plan for cardiac toxicity as well as thresholds for discontinuation of vaccine treatment were developed based on FDA recommendations from the Pre-IND meeting review on September 6th, 2011.
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1.2.1.3 lapatinib (tykerb™️)

Lapatinib is an orally administered tyrosine kinase inhibitor of both the HER2/neu protein and the epidermal growth factor receptor. It has shown activity in combination with capecitabine in patients with metastatic HER2 positive breast cancer that have progressed following treatment with trastuzumab14. Common toxicities specific to lapatinib include diarrhea and rash; cardiac toxicity is rarely seen. Like trastuzumab, resistance to lapatinib has been described and it appears that multiple molecular mechanisms underly resistance to both of these drugs15.
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1.2.1.4 pertuzumab (perjeta™️)

Pertuzumab is a humanized monoclonal antibody being studied in early and advanced states of HER2-positive breast cancer and advanced HER2-positive gastric cancer. It is a dimerization inhibitor designed to specifically prevent the HER2 receptor from pairing (dimerizing) with other HER receptors (EFGR/HER1, HER3 and HER4), a process that is believed to play a critical role in the growth and formation of several different cancer types. Through prevention of receptor pairing, pertuzumab is believed to block cell signaling, which may inhibit cancer cell growth or lead to the death of the cancer cell. Binding of pertuzumab to HER2 may also signal the body's immune system to destroy cancer cells. The mechanisms of action of pertuzumab and trastuzumab are thought to complement each other, as both bind to the HER2 receptor but on different regions. The goal of combination pertuzumab/trastuzumab therapy and chemotherapy is to determine if the combination provides a more comprehensive blockade of HER signaling pathways. The pertuzumab application before the FDA is based on results from the pivotal phase III
CLEOPATRA (Clinical Evaluation Of Pertuzumab And TRAstuzumab) study16. CLEOPATRA
is an international, phase III, randomized, double-blind, placebo-controlled study. The study evaluated the efficacy and safety profile of the pertuzumab-based regimen compared to trastuzumab and chemotherapy plus placebo in 808 people with previously untreated HER2positive metastatic breast cancer. The primary endpoint of the study was progression free survival (PFS) with secondary endpoints of overall survival (OS), PFS by investigator assessment, safety profile, overall response rate (ORR), duration of response, time to symptom progression and correlation of biomarkers with clinical outcomes.  
The CLEOPATRA study demonstrated a 6.1-month improvement in median PFS for patients receiving a pertuzumab-based regimen (pertuzumab combined with trastuzumab and docetaxel) compared to those who received trastuzumab and docetaxel alone: median PFS 18.5 vs. 12.4 months. People who received the combination also experienced a 38% reduction in the risk of disease worsening or death (HR=0.62, p-value <0.0001). The pertuzumab regimen was not associated with a higher incidence of cardiac AEs: left ventricular dysfunction was 4.4% in the pertuzumab-containing regimen compared to 8.3% with trastuzumab and docetaxel alone. Final OS analysis from the CLEOPATRA study is expected in 2013. 1.2.1.5 Trastuzumab Emtansine (T-DM1) (Kadcyla™) Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of trastuzumab linked via a non-reducible thiotether to the cytotoxic, anti-tubulin agent maytansine (DM1)17. It is under global development by Roche in collaboration with Genentech and Immunogen for second-line, first-line and third-line treatment of HER2+ metastatic breast cancer in the following phase III trials, respectively:
- The EMILIA study will compare T-DM1 versus lapatinib plus capecitabine in 980 patients (NCT00829166.)  
- The MARIANNE study will compare docetaxel/trastuzumab vs. T-DM1 vs. T-DM1 plus pertuzumab (NCT01120184).  
- The TH3RESA study is comparing T-DM1 versus best supportive care in the salvage setting (NCT01419197).
The results of a 137 patient, randomized phase II study of T-DM1 in untreated HER2+ metastatic breast cancer patients comparing T-DM1 versus trastuzumab plus docetaxel chemotherapy presented in September 2011 at the European Multidisciplinary Cancer Congress in Stockholm Sweden (Abstract \#5001) demonstrated improved median PFS of T-DM1 compared to trastuzumab/docetaxel: median PFS of 14.2 months vs. 9.2 months, respectively; p=0.035. T-DM1 achieved a 64.2% objective response rate vs. 58% for the trastuzumab/docetaxel combination and also had a more favorable side effect profile, with a lower frequency of severe adverse events (SAEs): 46.4% vs. 89.4% respectively. The most frequent SAEs reported with T-DM1 therapy were thrombocytopenia (8.7%) and elevated liver transaminases (8.7%). Data from the EMILIA trial is expected in 2012 and is anticipated to be the basis for seeking FDA marketing approval for second-line treatment of HER2+ cancer in the U.S. and Europe. Ado-trastuzumab emtansine (Kadcyla) was approved by the FDA on February 22, 2013.
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1.2.2 pre-clinical animal studies

We have recently published dramatic results documenting the regression and cure of large established tumors in syngeneic BALB/c mice using a therapeutic adenoviral vector vaccine expressing the extracellular and transmembrane (ECTM) domains of rodent HER2neu18. This anti-tumor activity is mediated by polyclonal vaccine-induced *antibodies* that inhibit HER2 phosphorylation and unlike trastuzumab, are Fc receptor *independent*. Although many mouse tumor vaccine studies have prevented growth of tumors, it has generally not been possible to achieve cure of large established tumors > 1 cm in diameter with cancer vaccines. Using an adenoviral vector vaccine expressing the extracellular and transmembrane (ECTM) domains of rodent HER2/neu, we have cured TUBO mammary gland carcinomas up to 2 cm in diameter in syngeneic BALB/c mice (**Figure 1**) and multiple large established lung metastases (**Figure 2**). Virtually 100% of mice are cured from both subcutaneous and lung tumors. The vaccine also prevents spontaneous orthotopic breast carcinomas in BALB/c mice transgenic for the same rodent neu oncogene18,19.  
  
  
  
30 200 100
Figure 3: AdneuECTM Causes Regression of Large Tumors Figure 4: Large Breast Tumors are Cured with a Single Injection of AdneuECTM  
  
  
Figure 5 : AdneuECTM Vaccine Therapy is Antibody Dependent As shown above in Figure 5, the vaccine's mechanism of action is purely antibody-mediated, with no involvement of CD4 or CD8 T cells in the effector function (only CD4 help at the time of vaccination to induce antibodies).  Neither depletion of CD8 + T cells with antibody nor use of beta-2 microglobulin knockout mice that lack CD8+ T cells affects vaccine-induced protection.
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1.2.3 rodent adneuectm dc vaccination

Similar induction of therapeutically effective antibodies could be obtained by immunizing with 1 million syngeneic dendritic cells transduced with the Ad-neuECTM vector as shown below in Figure 6.  Mice given TUBO cells subcutaneously and then vaccinated on days 5 and 7 when the tumors were 55-250 mm³ showed complete regression of tumors exactly analogous to the regressions induced by the free AdHER2 vector (Steele, Morris et al, unpublished observations).  
Tumors continued to grow to a peak around Day 12, when they reached a maximum of 756 mm 3 ,
and then started to regress, reaching baseline levels after Day 22.  This therapeutic effect was observed whether AdneuECTM-transduced DCs were given alone or with IL-15 and was also antibody dependent.  Thus, immunization with AdneuECTM-transduced DCs and with free AdneuECTM both produced comparable antibody-mediated tumor regression.  Also, sera from such AdneuECTM-transduced DC-immunized mice inhibited growth of TUBO cells in vitro and inhibited phosphorylation of the ErbB2-neu oncoprotein.  
Figure 6: Rodent Adneu**ECTM DC Vaccination**  
  
It should be noted that HER2/neu-transgenic mice have central or thymic tolerance to the oncogene, which is expressed from before birth (unlike some models in which an oncogene is not expressed until sexual maturity). They express a smaller, more limited and different T cell repertoire from that in non-transgenic mice, reflecting the deletion of many T cell clones by central self-tolerance20,21. Thus, it is not surprising that it has been harder to treat established spontaneous tumors in the neu-transgenic mice19,22. Further, in the BALB-neu transgenic model, the oncogene is expressed in all mammary cells of all mammary glands. As a consequence, relentless malignant transformation of mammary gland cells is induced with subsequent generation of independent primary tumors in all 10 mouse mammary glands, making it extremely difficult to suppress all tumors. In contrast, it is still possible to virtually completely suppress the development of tumors in neu-transgenic mice if vaccination is started at a young age, about 7 weeks, when histopathology already shows development of abnormal mammary gland dysplasia (beginning even before 4 weeks) due to the oncogene. Presumably the vaccineinduced antibodies are eradicating or inhibiting the dysplastic neu-expressing cells. Thus, this is really a therapeutic model, not just prophylactic. This situation contrasts with the human breast cancer situation, in which the HER2/neu oncogene is not expressed until malignant transformation in adulthood, so there is no opportunity to develop central tolerance. Indeed, Disis and coworkers23,24 have demonstrated that many patients with HER2+ breast cancers express spontaneous antibodies and T cell responses to the HER2 oncoprotein, demonstrating the absence of central tolerance. Further, humans with breast cancer generally do not have such an oncogene expressed in 100% of mammary gland ductal epithelial cells as in the transgenic mice, leading to the relentless and continuous generation of new independent tumors. Therefore, it is expected that the vaccine will be more effective at inducing therapeutically effective antibodies to HER2 in patients with HER2+breast cancers than it can in centrally tolerant neu-transgenic mice. We have also shown (unpublished data) that vaccine-induced antibodies inhibit the growth of the N202-1A tumor line, another mouse mammary carcinoma expressing the same oncogene. The vaccine's mechanism of action is purely antibody-mediated, with no involvement of CD4 or CD8 T cells in the effector function (only CD4 help at the time of vaccination to induce antibodies). Importantly, the vaccine-induced antibodies work differently from trastuzumab: they are Fc receptor *independent*, kill tumor cells directly without the aid of other cells, and inhibit HER2 phosphorylation and cause it to be modulated off the cell surface. Thus, this vaccine may work in patients in whom trastuzumab or lapatinib is ineffective. In addition, vaccine-induced antibodies are *polyclonal* and may be more resistant to escape mutations than a monoclonal antibody (MAb) like trastuzumab or pertuzumab. Our pre-clinical animal data suggest the potential for even greater therapeutic efficacy than trastuzumab and would eliminate the need for costly, frequent infusions currently associated with this treatment.
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1.2.5 pre-clinical adher2ectm vector expression

Several rounds of pre-clinical experiments have been conducted to determine the optimal conditions for generation of AdHER2 transfected dendritic cells (AdHER2 DC) for autologous therapeutic vaccination. Healthy normal donor cells were used to generate dendritic cells under different conditions to confirm HER2neu expression by FACS or ELISA in multiple experiments and functional assays which were performed by Drs. Luciano Castiello and Marianna Sabatino in the Department of Transfusion Medicine (DTM) and by Dr. Masaki Terabe of the Vaccine Branch. The key outcomes from these experiments are documented below:  
- Equivalent HER2neu expression of DCs transfected under bag or flask conditions. The DTM
currently uses bags to generate DCs from thawed monocytes for autologous DC vaccines as part of their cGMP standard operating procedures and this is the methodology that will be used in our study.  
- A viral particle (VP): DC ratio of 3000:1 was chosen as the ratio for transfection for this clinical trial since high level expression was documented at reasonable MOIs based on data highlighted in the tables below.  
| VP:DC Ratio   | Population   | Expression Marker   | # Events   | % Parent   |
|---------------|--------------|---------------------|------------|------------|
| 1000:1        | FITC G1 HER2 | HER2                | 6,947      | 26.8       |
| 2000:1        | FITC G1 HER2 | HER2                | 20,571     | 79.4       |
| 3000:1        | FITC G1 HER2 | HER2                | 24,657     | 95.0       |
| 5000:1        | FITC G1 HER2 | HER2                | 24,073     | 99.5       |  
- HER2neu mean fluorescent intensity and the percent of cells staining positive for HER2neu was acceptable at the two highest ratios examined (M. Terabe FACS data):  
| VP:DC Ratio   | Mean Fluorescent Intensity (MFI) % Cells Positive (DY and N87) for HER2neu   |       |
|---------------|------------------------------------------------------------------------------|-------|
| 3000:1        | 103                                                                          | 84.5% |
| 5000:1        | 432                                                                          | 97.8% |  
- Although MFI and percent positive cells were even greater at 5000:1, there were concerns about potential subsequent issues of cell viability at this highest ratio. In addition, the efficient vialing of vector for planned MOIs of 3000:1 will allow an adequate supply of virus for up to 66% more patients than if the 5000:1 ratio were to be used.
The above data were reviewed by and discussed with the FDA in a Pre-IND meeting conference call held September 6th, 2011 and were determined to be acceptable.  
1.2.6 Pre-Clinical HER2 Dendritic Cell Platform Testing Studies were conducted utilizing cells from three healthy, normal human donors (11FC00039, 11FC00043 and 11FC00055) to determine the best sequence of AdHER2 vector transfection and dendritic cell maturation for optimal viability and HER2neu expression in the final DC vaccine product. AdHER2 transfected DCs at a ratio of 3000 VP: DC were generated under the following conditions as summarized below and outlined in **Figure 7:**  
➢ Transfection of immature dendritic cells (iDC) for 24 hours: **iDC with (24h)virus** ➢ Immature dendritic cells with no viral transfection: iDC
➢ Maturation of DCs for 24 hours with *no viral transfection*: (24h)mDC **no virus** ➢ Maturation of DCs for 24 hours with transfection at 20 hours: **(24h)mDC with(20h)virus** ➢ Transfection of DCs for 4 hours followed by maturation of DCs: (20h)mDC with(24h)virus
Figure 7: DC Transfection and Maturation Experiment
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1.2.5 pre-clinical adher2ectm vector expression

Goal: Compare HER2neu expression of transfected DCs with or without maturation cocktail (MC)  
  
Figure 8 **Figure 9**  
  
As depicted in **Figure 8**, the post transfection viability of matured DCs (mDCs) ranged from 6082% (mean 70-75%). *Since this post maturation/transfection DC viability is donor dependent* and highly variable, we requested and received FDA approval allowing a release viability criteria of > 60% rather than > 70% for the final AdHER2 DC vaccine product to be used in this study. Historically, DTM reports that the product yield for transfected DC vaccine products is usually between 10-40% of the starting monocyte population. Note in **Figure 9** for an unknown reason, healthy donor 11FC00055 had an extremely poor yield of DCs post maturation/transfection. We have observed similar variations in vaccine viability and yields within and between donors in the manufacture of autologous TARP peptide DC vaccines on NCI protocol 09-C-0139.  
HER2neu expression was also assessed under the different maturation conditions. There was significant variability of expression within and between donors under the different maturation and transfection conditions and immature DCs were observed to have greater variable expression of HER2neu i.e. the mean fluorescent intensity per cell of HER2 expression was more inconsistent on immature DCs *versus* mature DCs (personal communication L. Castiello and M. Sabatino of DTM, data not shown).  
However, as documented below in Figure 10, HER2neu expression (CD340) was 100% on transfected immature and mature DCs, regardless of transfection conditions.
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figure 10 dc her2neu expression

Additional characterizations and assessments were also performed as part of these DC maturation/transfection experiments in an attempt to better characterize the qualitative, functional aspects of the final HER2neu DC vaccine product, including:
- Cluster of Differentiation (CD) antigens on the final DC vaccine product (in order of relative scientific priority): CD86, CD83, HLA-DR, CD38, CD54, CD14, and CCR7 (data not shown).  
- Expression of cytokines and chemokines IL-6, IL-8, IL-12p70, TNF-, IP10, MDC and MIP3 (expressed as a ratio that normalizes expression for the number of cells). Interferon-
, IL-10, IL-13 and IL-15 were at very low concentrations, usually < 5pg/mL (data not shown). Proteomic analysis of dendritic cell supernatants under the different transfection/maturation conditions is in process for the cytokines and chemokines mentioned above.  
Based on these studies, we are planning to manufacture AdHER2 DC vaccine for this study using a 4 hour transfection of the AdHER2 vector first followed by the DC maturation cocktail (denoted as (20)mDC with (24) virus in the previous figures). This decision is based on the following theoretical grounds as well as the observations from the in vitro studies documented above:
➢ It is generally easier to transfect immature DCs i.e. before maturation cocktail is added and ensure optimal antigen processing.  
➢ There was more consistent HER2neu expression on mature DCs compared to immature DCs as measured by MFI, which may lead to greater uniformity of the final product.  
The Certificate of Analysis for the final manufactured AdHER2 DC vaccine product will include documentation on the percentage of HER2neu expressing cells (%HER2neu) in the product and the HER2neu Mean Fluorescent Intensity (HER2neu MFI) of cells in the product to permit preliminary assessment of their correlation with clinical outcomes. *The above outlined* transfection/maturation process for AdHER2 DC vaccine manufacture was approved by the FDA following their review of the data during the Pre-IND meeting held September 6th*, 2011.*
1.2.7 Human Experience with Dendritic Cell Vaccines There are a wide variety of platforms and immunogens that have been extensively studied in therapeutic cancer vaccine platforms including: a) whole tumor cell vaccines, b) dendritic cell (DC)-based platforms, c) peptide and fusion proteins co-delivered with adjuvants and c) viral vectors that serve as delivery vehicles for tumor-associated antigens (TAAs). In addition to classic peptide and protein immunogens, tumor lysates, tumor mRNA, and DNA have all been utilized. DCs serve as a bridge between the innate and adaptive immune system and in turn play a critical role in the activation of naïve CD4+ and CD8+ T cells. As a consequence, researchers in cancer immunotherapy have focused a tremendous amount of investigative effort to delineate and understand DC biology, activation, maturation, and antigen presentation. Because of the pervasive dysfunction of DCs *in vivo* as a consequence of the immune dysregulation associated with high tumor burdens25, they have been the platform of choice for delivery of many tumor cell lines (LNCaP, PC3), peptides, proteins, lysates, mRNAs and viral vectors expressing TAAs26,27.  
Sipuleucel-T is a prototype of this platform approach: even though it is labeled as an autologous cellular immunotherapy, its generation utilizes similar maturation agents and pathways common to dendritic cell vaccines28.  
A better understanding of basic immunologic principles has led to a variety of techniques for enhancing tumor-specific immunity and their potential subsequent translation into improved clinical outcomes. This is exemplified by the FDA approval on April 29th, 2010 of sipuleucel-T
(Provenge), the first licensed therapeutic cellular immunotherapy and a major milestone in the field of cancer immunotherapy. Importantly, approval of sipuleucel-T was based upon a statistically persuasive and clinically meaningful 4.1-month *improvement in median overall* survival (OS) in the IMPACT phase III trial29. The improvement in OS associated with sipuleucel-T has been reported to correlate with CD54 upregulation, a measure of the product's potency30 as well as the development of antibody titers exceeding 400 at any time against the immunizing antigen PA2024 (a fusion protein) or prostatic acid phosphatase (PAP)29.  
Interestingly, although very strong T cell proliferative responses to PA2024 and PAP were also observed in patients receiving sipuleucel-T, no difference or association in survival was documented between patients who exhibited T cell response to either antigen and those who did not. DC vaccine products have been delivered intravenously (as is sipuleucel-T), intramuscularly, intranodally, intradermally and subcutaneously. In our pre-clinical model of rodent Adneu ECTM DC vaccination, the vaccine was given subcutaneously. We are planning to deliver the human autologous HER2 DC vaccine equivalent used in this study intradermally, since it is in keeping with this model and there is evidence to suggest that in humans intradermal delivery may be more optimal than subcutaneous delivery (C. Mackall, personal communication).
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1.2.8 human experience with her2

The clinical efficacy of trastuzumab-based anti-HER2 passive immunotherapy and other HER2targeted therapy has led to the development of vaccination strategies against HER2 that would ideally result in strong immunity producing immune memory associated with anti-tumor activity as well as prevention of tumor recurrence. Different anti-HER2 vaccine strategies currently under investigation and in various stages of clinical development include DNA, peptide, whole tumor cell, and dendritic cell vaccines and combinations of the same e.g. peptide-loaded DC
vaccines31. Peptide vaccines have included monovalent and as well as polyvalent HER2 peptides and been associated with the development of humoral as well as cellular immune responses. We believe the use of our proposed AdHER2 DC vaccine will induce polyclonal antibody responses directed towards both the extracellular (EC) and transmembrane (TM) components of HER2 as well as potentially the intracellular domain (IC) as a result of epitope spreading. The multiple, unique HER2 epitopes targeted by this autologous vaccine platform are likely to differ from trastuzumab binding epitopes and may result in greater affinity and clinical activity than that seen with trastuzumab, especially in patients with lower levels of HER2 expression i.e. <3+ by IHC. In addition, based on our pre-clinical data, the anti-HER2 antibodies induced by vaccination appear to have functional activity that, unlike trastuzumab, is *Fc receptor* independent and that interfere with HER2 expression and phosphorylation. Hence, vaccineinduced anti-HER2 antibodies may also have clinically relevant anti-tumor activity in patients with resistance to trastuzumab, lapitinib or other HER2-directed therapies. 1.2.9 Previous Human Experience with an Extracellular/Transmembrane/Intracellular
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her2/neu vaccine

Our collaborator Rolf Kiessling, and his colleagues at the Karolinska Institute in Sweden have conducted a pilot clinical trial of a plasmid DNA human HER2/neu vaccine encoding the extracellular (EC), transmembrane (TM) and mutated intracellular (ICmutated) domains
(ECTMICmutated) given with GM-CSF and IL-2 32. As a safety measure, a two base pair mutation was introduced in the IC domain to remove an autophosphorylation site that confers oncogenicity and maximize safety. The vaccine was administered to a total of eight patients and no clinical issues of acute toxicity, autoimmunity or cardio toxicity were identified. As might be expected with a DNA plasmid construct, the vaccine demonstrated limited immunogenicity: no specific T-cell proliferation following *in vitro* stimulation of *ex vivo* PBMCs with recombinant human HER2 protein was induced by vaccination. In fact, immediately following three complete cycles of vaccination, no or even decreased HER2-specific CD4+ T-cell responses were observed, but a significant increase in MHC class II restricted HER2-T-cell responses was detected at long term follow-up. Because concurrent trastuzumab therapy was permitted during the study, -subclass specific ELISAs were performed to measure endogenous antibody production without trastuzumab interference. Importantly, in a subgroup of patients, HER2pDNA vaccination induced and boosted HER2-specific antibodies that could be detected for several years following the last vaccine administration. We propose to carry out this phase I and pilot study of vaccination with AdHER2 transduced autologous dendritic cells in patients with 1+ to 3+ HER2neu expressing tumors. Part I involves vaccine dose escalation in a population with **no prior exposure** to trastuzumab *or other HER2-*
directed agents to determine if there is a significant, adverse safety signal regarding cardiac toxicity, in addition to preliminary assessment of the vaccine's immunogenicity and clinical activity. Five doses of 5, 10, 20 or 40 x10^6 viable cells/AdHER2 vaccine will be given intradermally at Weeks 0, 4, 8, 16 and 24 in patients with metastatic solid tumors and adjuvant bladder cancer patients characterized by some HER2/neu expression. While these patients may be less likely to respond due to lower expression levels of the HER2 target, it is equally possible that they may respond better because they will have less peripheral tolerance to HER2. In addition, AdHER2 DC vaccination is likely to induce a polyclonal antibody response to multiple HER2 epitopes that differ from the EC epitope targeted by trastuzumab. These differing polyclonal, multiple-epitope responses may be associated with anti-tumor activity. Once preliminary cardiac safety is demonstrated, Part II of the study will repeat the vaccine dose escalation in a population with **prior exposure** to trastuzumab and other HER2 directed therapies (as required by the FDA) to again determine whether there is an adverse safety signal regarding cardiac toxicity, in addition to assessment of the vaccine's immunogenicity and clinical activity. Vaccine dose escalation, administration and re-staging assessment is identical to that conducted in Part I. 1.2.10 Exploratory Correlative Studies 1.2.10.1 Anti-HER2 Antibody Detection and Characterization Dr. Henriette Hansen Group Leader Sales & Customer Communication Sales & Customer Support T +49-30-6392-7826 X +49-30-6392-7888 X 888-578-2666 (available in the US only) hansen@jpt.com JPT Peptide Technologies GmbH - Volmerstrasse 5 - *12489 Berlin, Germany* Please visit us at www.jpt.com 1.2.10.2 JPT Peptide Technologies, Midissia Therapeutics and NCI A major objective of this study is to determine the immunogenicity of the AdHER2 DC vaccine. Specifically, we seek to examine whether AdHER2 DC vaccination is associated with a 3-fold increase in anti-HER2/neu antibody concentration (measured as mcg/mL) or a 4-fold increase in antibody dilution titers over baseline. Overlapping HER2 peptide microarrays will also be utilized to characterize both vaccine-induced anti-HER2 antibody and cellular responses. Anti-HER2 cellular response and clinical response relationship with anti-Ad5 Ab.
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1.2.10.3 anti-her2 cellular response

The Berzofsky lab, through support from the NCI Frederick Clinical Support Laboratory will assess vaccine-induced HER2-specific responses as measured by IFN-, Granzyme A and Granzyme B ELISPOT assay and/or intracellular cytokine staining (ICS). The Berzofsky lab, through support from the NCI Frederick Clinical Support Laboratory will check humoral immunity i.e. anti-Ad5 antibody titers at baseline as well as post vaccination, to determine if autologous AdHER2 DC vaccination results in a change in these anti-Ad5 titers over time, or is correlated with the increases in anti-HER2 antibody titers post vaccination. 1.2.10.4 Circulating Tumor Cells (CTCs): Jane Trepel, DTB, CCR, NCI Analysis will be performed by the Trepel laboratory utilizing ferrofluidic enrichment and multiparameter flow cytometric detection to study CTCs from a broad range of cancer types. CTCs will be identified as viable, nucleated cells, that positively express one or more epithelial or tumor markers and are negative for expression of hematopoietic markers. CTCs will be further characterized for markers for ICD such as calreticulin, Hsp70 and Hsp90.
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1.2.10.5 immune cell subsets jane

Peripheral blood mononuclear cells (PBMC) will be isolated from patient blood before and after treatment, labeled with a unique 2D barcode and viably stored. it will be thawed and assessed using multiparameter flow cytometry for immune subsets including but not necessarily limited to Tregs, myeloid-derived suppressor cells (MDSC), NK, NKT, CD4+ and CD8+ T-cells, and CD14+ monocyte subsets. Assessment will include functional markers, i.e. PD-1, Tim-3, CTLA4, CD40, HLA-DR and PD-L1.  
1.2.10.6 Function-Associated mRNAs in Whole Blood: Dr. Cindy Yamamoto, Hitachi Chemical Although there have been major recent advances in the field of cancer immunotherapy with the approval of sipuleucel-T (Provenge) and ipilimumab (YERVOY), a significant challenge that remains is identification of immunologic responses that correlate with beneficial clinical outcomes such as tumor regression or survival. In collaboration with Dr. Cindy Yamamoto of Hitachi chemical, we will seek to explore whether characterization of a patient's baseline immune status using assessment of functional mRNAs can be utilized to predict who will respond to vaccination. The assay is a novel high throughput real time PCR system for quantitatively assaying mRNA
for any gene that is activated in cells33. Specifically, the methodology allows for quantification of 17 different leukocyte-function–associated mRNAs in whole blood after *ex vivo* stimulation with up to 8 different antigens (e.g. rIL2, PHA, Anti-TCR, HER2, heat aggregated IgG (HAG, immune complex to activate Fc receptor), LPS, Zymosan, and PBS control) on pre-manufactured strips. Since the volume blood needed for this test is very small and cell isolation and culture are not necessary, this exploratory methodology may serve as a model of personalized medical diagnostics for patient-specific cancer vaccine immunotherapy.
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2 eligibility assessment and enrollment

2.1.1 Common Eligibility for Parts I and II 2.1.1.1 Adults ≥ 18 with malignant soft tissue and bone tumors and recurrent or progressive, metastatic solid tumors who have progressed on standard therapies except in adjuvant for high risk bladder cancer in Part I.  
2.1.1.2 Recurrent or progressive disease on prior standard therapies with known clinical benefit with the exception of adjuvant bladder cancer population.  
2.1.1.3 Performance Status: ECOG 0-1. (Refer to Appendix 1) 2.1.1.4 Baseline LVEF by 2D Echocardiogram ≥ 53%. 2.1.1.5 Greater than or equal to 1 week since standard or investigational treatment for metastatic disease.  
2.1.1.6 Stable, concurrent use of hormone therapy for hormone receptor positive breast cancer is permitted.
2.1.1.7 Hematologic parameters: ANC ≥ 1000 cells/mm3, ALC ≥ 300 cells/mm3, Hemoglobin
≥ 9.0 gm/dL, WBC ≥ 2,500 cells/mm3, platelet count ≥ 75,000/mm3, PT/PTT ≤1.5X the upper limits of normal.  
2.1.1.8 Chemistry parameters: Creatinine ≤ 1.5 mg/dL, SGOT and SGPT ≤ 3X the upper limits of normal and total bilirubin ≤ 1.5 mg/dl, Alk PO4 ≤3X the upper limits of normal (except for patients with documented metastatic disease to bone and/or liver).  
2.1.1.9 Negative serum beta HCG if female and of childbearing potential. 2.1.1.10 Negative HIV 1/2 serology and sample drawn for HTLV. Patients with HIV are excluded from participating on this clinical trial because their immunodeficiency would confound the evaluation of adverse events which would hinder meeting the primary objective.  
2.1.1.11 Negative serology for hepatitis B and C unless the result is consistent with prior vaccination or prior infection with full recovery.  
2.1.1.12 Willingness of female and male subjects to use effective contraception e.g. oral contraceptives, barrier device, intrauterine device, or condoms, during the study and for three months following the last dose of study vaccine. We suggest that subjects do not become pregnant or father a child during the study, and for 3 months following receipt of the investigational AdHER2 DC vaccine.  
2.1.1.13 Able to understand and provide Informed Consent. 2.1.1.14 Patients with 1+ to 3+ HER2/neu expression by IHC or an equivocal or positive FISH
result by 2013 ASCO/CAP guideline.  
HER2 Detection Method Negative Equivocal Positive  
| result by 2013 ASCO/CAP guideline.                                                      |                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                              |          |
|-----------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------|
| HER2 Detection                                                                          | Negative                                                                                                                                                                                                                                                                                                                              | Equivocal                                                                                                                                                                    | Positive |
| Method Vysis FISH Ratio (Measures HER2  oncogene)                                       | -Single-probe average HER2  copy number ≥ 6.0  signals/cell -Dual-probe HER2/CEP17  ratio ≥ 2.0 with an average  HER2 copy number <4.0 signals per cell -Dual-probe HER2/CEP17  ratio ≥2.0 with an average  HER2 copy number <4.0 signals/cell -Dual-probe HER2/CEP17  ratio <2.0 with an average  HER2 copy number ≥6.0 signals/cell |                                                                                                                                                                              |          |
| IHC  (Measures HER2  protein)                                                           | -Single-probe  average HER2  copy number <4.0  signals/cell -Dual-probe  HER2/CEP17  ratio <2.0 with an  average HER2  copy number <4.0 signals/cell                                                                                                                                                                                  | -Single-probe ISH  average HER2  copy number ≥4.0  and <6.0  signals/cell -Dual-probe  HER2/CEP17  ratio <2.0 with an  average HER2  copy number ≥4.0  and <6.0 signals/cell |          |
| 0-1+                                                                                    | 2+                                                                                                                                                                                                                                                                                                                                    | 3+                                                                                                                                                                           |          |
| 2.1.1.15 Patients must have measurable disease, per RECIST 1.1. See Section 6.2 for the |                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                              |          |  
2.1.1.15 Patients must have measurable disease, per RECIST 1.1. See Section 6.2 for the  
evaluation of measurable disease except adjuvant high risk bladder cancer.
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2.1.2 part i eligibility

2.1.2.1 Naïve to trastuzumab (Herceptin™), pertuzumab (Perjeta™) and lapatinib (Tykerb™),
ado-trastuzumab emtansine (Kadcyla™) or other HER2-directed therapies.  
2.1.2.2 Malignancy as follows:
- Malignant soft tissue and bone tumors and recurrent or progressive, metastatic solid tumors who have progressed on standard therapies; or,
- Bladder cancer in the adjuvant setting (adjuvant bladder cancer patients):
o Tumor stage T3a, T3b, T4a, T4b and any node positive disease regardless of tumor stage.  
o Status-post primary cystectomy with curative intent. o May or may not have received neoadjuvant cisplatin-based combination chemotherapy per NCCN guidelines.  
o May or may not have received adjuvant radiotherapy or chemotherapy based on pathologic risk per NCCN guidelines.  
o Greater than or equal to 6 weeks s/p primary surgery with curative intent.  
- NOTE: Patients with breast, ovarian, cervical, colon, gastric/gastroesophageal junction, non-small cell lung, renal cell, bladder, malignant soft tissue and bone tumor, prostate cancer or other solid tumors.
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2.1.3 part ii eligibility

2.1.3.1 Malignant soft tissue and bone tumors and recurrent or progressive, metastatic solid tumors who have progressed on standard therapies.  
2.1.3.2 Recurrent or progressive metastatic disease after standard of care HER2-targeted therapies; i.e. trastuzumab (Herceptin™), pertuzumab (Perjeta™), lapatinib (Tykerb™), ado-trastuzumab emtansine (TDM1) (Kadcyla™) or other HER2-directed therapies.  
2.1.3.3 Stable, concurrent use of tamoxifen or aromatase inhibitors for hormone receptor positive breast cancer is permitted.
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2.2 exclusion criteria

2.2.1.1 Pregnant women are excluded from this study because AdHER DC vaccine may have the potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with AdHER DC vaccine, breastfeeding should be discontinued if the mother is treated with AdHER DC vaccine.  
2.2.1.2 Patients with active CNS metastases or leptomeningeal involvement by tumor (patients with a history of brain metastases who have successfully treated for brain metastasis by surgery or radiation and who have not had any evidence of the new or progressive CNS disease for more than 12 months are eligible).  
2.2.1.3 Patients with rapidly progressing disease in the opinion of the Principal Investigator. 2.2.1.4 Patients with inadequate bilateral peripheral venous or central venous catheter access for the required apheresis to allow generation of the autologous AdHER2 DC vaccine product.  
2.2.1.5 Clinically significant cardiac dysfunction defined as a history of > NYHA Class II
symptoms, angina, congestive heart failure, myocardial infarction, arrhythmias or cardiac dysfunction requiring treatment or discontinuation of chemotherapy.  
2.2.1.6 History of changes in baseline LVEF that occurred during prior treatment with antiHER2 treatment.  
2.2.1.7 Cumulative doxorubicin dose > 400mg/m2 (>450 mg/m2for malignant soft tissue and bone tumor patients) or cumulative epirubicin dose > 800mg/m2.  
2.2.1.8 Use of any standard chemotherapy or other investigational agent(s) within 1 week of study enrollment.  
2.2.1.9 Use of systemic corticosteroid therapy within 2 weeks of study enrollment, including patients receiving replacement corticosteroid therapy. Note: *only* topical, inhaled and intranasal steroid therapy is permitted.  
2.2.1.10 Active systemic viral, bacterial or fungal infection requiring treatment. 2.2.1.11 A medical history which the treating physician believes causes the patient to be excluded. This includes a remote history of cancer. Please note: squamous cell carcinoma, basal cell carcinoma and remote history of cancer with no evidence of recurrence for the past 5 years are eligible.  
2.2.2 Recruitment Strategies
This protocol is being conducted in collaboration with Dr. Henriette Hansen of JPT Peptide Technologies as well as Dr. John Morris of the University of Cincinnati, who was also significantly involved in the pre-clinical animal model studies of the AdHER2 vaccine platform before he left the NCI. We anticipate that a significant number of HER2 positive patients will be referred from these colleagues in addition to patients identified through affiliates at Walter Reed National Military Medical Center. We will submit approved protocol to Dr. Susan Love Research Foundation's Army of Women Program (www.armyofwomen.org/) and ResearchMatch (https://www.researchmatch.org) for review to allow contact with and recruitment through their volunteers. We also made contact with Office of Patient Recruitment, National Institutes of Health Clinical Center to update our contact information, generate materials including flyers, posting for social media and posters. Additionally, this study will be posted on NIH websites and on NIH social media forums. NCI Vaccine Branch Clinical Trials team also opened a twitter account (@NCICCR_VB) for the patient recruitment and sharing the updated information on cancer vaccine and related topics with other professionals and general public. This account is officially registered to Office of Communications and Public Liaison at NCI. In addition to the standard electronic mailings and postings by the NCI CCR Office of Patient Outreach and Recruitment, the former Principal Investigator, now a special volunteer at Vaccine Branch, CCR, NCI, Dr. Wood developed a brief video (no more than 5 minutes in length) that provides an informational overview to both patients and health care providers describing the scientific rationale for and design of the clinical study. This video was posted to the YouTube channels of the NCI Office of Communications and the NIH Clinical Center. Given that so much of consumer media information is now provided in video format that can easily be accessed via the web using computers and smart phone operating systems and applications platforms, we believe that this could serve as a novel recruitment tool for patients interested in participating in clinical research studies. See **Appendix 13**: Patient Recruitment Materials.
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2.3 pre-screening and screening evaluation

All potential patients must provide written documentation of the HER2 status of their primary tumor and/or a recent tumor biopsy specimen, along with availability of a paraffin tissue block. For those with incomplete HER2 characterization as defined by the IHC or FISH, HER2 characterization will be performed by the NCI Laboratory of Pathology. 2.3.2 Screening Evaluation Patients will undergo a medical history and physical examination, review of concomitant medications, including review of symptoms, performance status, and life expectancy and apheresis clinic access assessment at screening. History will also specifically document:
- Prior exposure to anthracycline chemotherapy agents and total cumulative dose.  
- Prior exposure to HER2-directed therapies: agent, duration and number of rounds of treatment.  
- Documentation of New York Heart Association (NYHA) functional classification. (Refer to **Appendix 2**)
- Imaging studies (obtained within 30 days prior to enrollment). Note: if the patient had scans at an outside institution the patient will not be required a screening scan to consent. Instead, the baseline scan will be performed at NIH prior to treatment and used to identify target lesions.  
o CT scan of neck (optional), chest, abdomen and pelvis o MRI of the brain (in select clinically indicated patients) o Technetium 99 bone scan (if clinically indicated, for patients with bone metastases)
- 2D Echocardiogram with documentation of LVEF (obtained within 30 days prior to enrollment). Note: if the patient had an echo at an outside institution the patient will not be required a screening echo to consent. Instead, the baseline echo will be performed at NIH prior to treatment)
- Urinalysis
- Laboratory evaluation (obtained within 30 days prior to enrollment) - CBC with differential counts, PT/PTT, acute care panel, hepatic panel, serum beta-HCG
(for females with childbearing potential only), anti-HIV-1/2 Ab, anti-HCV Ab, HBsAg, HBs Ab, anti-HTLV-1/2 Ab, anti-CMV IgG/IgM and RPR
Refer to Section 3.4 for the Study Calendar schedule of on study clinical, laboratory, radiographic and research evaluations.
pre-clinical testingher2neu dc
2.4 registration procedures

Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) within 24 hours of signing consent. A registration Eligibility Checklist from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov. After confirmation of eligibility at Central Registration Office, CRO staff will call pharmacy to advise them of the acceptance of the patient on the protocol prior to the release of any investigational agents. Verification of Registration will be forwarded electronically via e-mail to the research team.  
| Cohorts Number   | Name                                                                     | Description                                                                                                                                                                                                                                                                 |
|------------------|--------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1                | Solid Tumors HER2- directed therapies naive dose escalation cohort       | Patients with ovarian, cervical, colon, non-small cell lung,  renal cell, bladder, malignant soft tissue and bone tumor,  prostate cancer or other solid tumors as well patients with  breast cancer to be treated with AdHER DC vaccine at  escalating doses.              |
| 2                | Solid Tumors HER2- directed therapies naïve  expansion cohort            | Patients with ovarian, cervical, colon, non-small cell lung,  renal cell, bladder, malignant soft tissue and bone tumor,  prostate cancer or other solid tumors as well patients with  breast cancer to be treated with AdHER DC vaccine at  40x10e6 dendric cells maximum. |
| 3                | Solid Tumors with prior  HER2-directed therapies  dose escalation cohort | Patients with recurrent or progressive, metastatic breast  cancer, gastric/gastroesophageal or other cancers treated with  AdHER DC vaccine at Dose Level 1.                                                                                                                |
| 4                | Solid Tumors with prior  HER2-directed therapies expansion cohort        | Patients with recurrent or progressive, metastatic breast  cancer, gastric/gastroesophageal or other cancers enrolled in  dose expansion cohort.                                                                                                                            |
| Arms Number      | Name                                                                     | Description                                                                                                                                                                                                                                                                 |
| 1                | Part I dose escalation                                                   | AdHER DC vaccine administered at escalating doses.                                                                                                                                                                                                                          |
| 2                | Part I dose expansion                                                    | AdHER DC vaccine administered at 40x10e6 dendric cells  maximum.                                                                                                                                                                                                            |
| 3                | Part II dose escalation                                                  | AdHER DC vaccine administered at Dose Level 1.                                                                                                                                                                                                                              |
| 4                | Part II dose expansion                                                   | AdHER DC vaccine administered at 40x10e6 dendric cells  maximum.                                                                                                                                                                                                            |  
2.4.1 Treatment Assignment Procedures Randomization and Arm Assignment There will be no randomization on this study. Up to 18 patients in Cohort 1 will be assigned to Arm 1 Up to 12 patients in Cohort 2 will be assigned to Arm 2 Up to 6 patients in Cohort 3 will be assigned to Arm 3 Up to 24 patients in Cohort 4 will be assigned to Arm 4
pre-clinical testingher2neu dc
2.5 baseline evaluation

Below studies will be obtained within 30 days prior to the administration of the first dose of vaccine. If any of the studies were performed on another protocol, they may not need to be repeated.  
- CBC with Differential
- Acute care panel, hepatic panel, mineral panel, total protein, amylase, lipase, uric acid
- PT/PTT
- Lipid panel - Immunoglobulin quantification, IgE
- LDH
- CPK, Troponin I
- Thyroid screen (TSH, Free T4)
- 25-OH-vitamin D - Lymphocyte subsets
- Tumor markers (optional) - Urinalysis
- ABO typing and HLA A, B, C and DR (if not done in NIH Clinical Center in the past) - CT CAP (to be done only if screening scans were performed at an outside institution and not repeated at the NCI).  
- MRI Brain (to be done only if clinically indicated and screening scans were performed at an outside institution and not repeated at the NCI).  
- CT Neck (to be done only if clinically indicated and screening scans were performed at an outside institution and not repeated at the NCI).  
- Bone Scan (to be done only if patient has bone metastasis and screening scans were performed at an outside institution and not repeated at the NCI).  
- Echocardiogram (to be done only if screening echo(s) were performed at an outside institution and not repeated at the NCI).
pre-clinical testingher2neu dc
3 study implementation 3.1 study

This is an open label, single site, non-randomized, two-part, phase I study of an autologous dendritic cell (DC) vaccine transduced with AdHER2 to determine vaccine safety, immunogenicity and preliminary activity. The study design schema is outlined in **Figure 11**.  
Once enrollment eligibility is confirmed and informed consent is obtained, the patient will be scheduled for an apheresis of up to 18 liters to collect mononuclear cells by counter-flow elutriation, aliquoted into minimum of 6 vials with up to 333 x 106 cells/vial that will be cryopreserved for future preparation of the autologous AdHER2 DC vaccine product. If the collected apheresis products are not adequate for 5 doses of vaccine, the patient may undergo additional apheresis per investigator's discretion. Co-enrollment in the 111Indium trastuzumab imaging protocol 07-C-0101 will be offered to those patients that are interested and eligible. Note: In order to be cleared for the next treatment, a safety visit (i.e., physical and clinical laboratory assessments, review of toxicities/adverse events) must occur within 7 days prior to the treatment. Treatment visits can also be safety visits for previous injections. This is so the treating physician will evaluate the patient for toxicities potentially caused by previous injections.
pre-clinical testingher2neu dc
part i n = 30

Part I of the study will investigate vaccine dose escalation in adults with recurrent or progressive, metastatic solid tumors as well as adjuvant bladder cancer patients whose tumors are characterized by some HER2/neu expression but for whom trastuzumab is not clinically indicated:  
o Patients with ovarian, cervical, colon, non-small cell lung, renal cell, bladder, malignant soft tissue and bone tumor, prostate cancer or other solid tumors that is known to be HER2 1+, 2+ or 3+ by IHC OR have a Vysis FISH result ≥ 1.8.  
o Patients with breast cancer that is known to be HER2 2+ or 3+ or with an equivocal or positive FISH result.
NOTE: Adjuvant bladder cancer patients will not be enrolled in Part I of the study until AdHER2 DC vaccine safety has been demonstrated out to 12 Weeks in at least 10 patients. The goal of Part I is to determine whether there is a preliminary significant, adverse safety signal regarding cardiac toxicity in this patient population that is naive to *HER2-directed therapies*, in addition to preliminary assessment of the vaccine's immunogenicity and clinical activity. Allowable tumors include breast, ovarian, colon, non-small cell lung, renal cell, bladder and prostate cancer. Inclusion of a broad spectrum of tumors that express HER2 will facilitate more rapid accrual and allow prompt determination of the vaccine's safety and immunogenicity. However, the inclusion of subjects with multiple tumor types that have varying levels of HER2 expression may preclude identification of an optimal biologically active dose. Proposed Dosage and Administration This part of the study will enroll patients in the vaccine dose levels outlined below:
Dose Level 1 (N = 6 patients): 5 x 106*viable* cells/vaccine Dose Level 2 (N = 6 patients): 10 x 106*viable* cells/vaccine Dose Level 3 (N = 6 patients): 20 x 106*viable* cells/vaccine Expansion Cohort (N = 12 patients): 40 x 106*viable* cells/vaccine Autologous AdHER2 DC vaccine will be administered intradermally at Weeks 0, 4, 8, 16 and 24.  
Response will be measured as outlined in section 6.2. Vaccine dose escalation will follow the procedures specified in Section 3.1.2.
pre-clinical testingher2neu dc
part ii n = 30

Part II of the study will be conducted in adults with recurrent or progressive, metastatic breast cancer, gastric/gastroesophageal or other cancers with 1+ to 3+ HER2/neu expression by IHC or an equivocal or positive FISH result. Vaccine dose escalation (as required by the FDA) will be repeated in an identical manner in a population with **prior exposure** to trastuzumab, pertuzumab, lapatinib, ado-trastuzumab emtansine (TDM1) and other HER2-directed therapies) to again determine whether there is an adverse safety signal regarding cardiac toxicity, in addition to assessment of the vaccine's immunogenicity and clinical activity. Vaccine administration and re-staging assessment is identical to that conducted in Part I with revised vaccine dosing as outlined below and in Section **3.1.2**.
pre-clinical testingher2neu dc
proposed dosage and administration

Part II of the study will enroll 30 breast, gastric/gastroesophageal or other cancer patients as outlined in the vaccine dose cohorts outlined below:
Dose Level 1 (N = 6 patients): 20 x 106*viable* cells/vaccine Expansion Cohort (N = 24 patients): 40 x 106*viable* cells/vaccine Autologous AdHER2 DC vaccine will be administered intradermally at Weeks 0, 4, 8, 16 and 24. Re-staging will be performed as outlined in Section 6.2. Vaccine dose escalation will follow the revised procedures specified in Section 3.1.2.  
Abbreviated Title:  AdHER2/neu DC Vaccine Version Date: 07/22/2019 Figure 11: AdHER2 Autologous DC Vaccine Study Schema  
  
| t t t p e w   |        |          |               |             |               |            |    |    |    |    |    |
|---------------|--------|----------|---------------|-------------|---------------|------------|----|----|----|----|----|
| 0T X 9 W      |        |          |               |             |               |            |    |    |    |    |    |
| Screen        | X99W   | X99W     | t t t i e e w | 87 X 9 9 W  | TE X 9 9 W    | d s        |    |    |    |    |    |
| ijaaW         | X 99 W | 07 p 99W | 95 199W       | 0 X X 9 9 W | 8 1 1 1 2 2 2 | 09 $ 9 9 W |    |    |    |    |    |
| ve M          |        |          |               |             |               |            |    |    |    |    |    |
| Consent       | T      | 9T       | 9L            |             |               |            |    |    |    |    |    |
| 0             | 4      | 8        |               |             |               |            |    |    |    |    |    |
| Baseline      |        |          |               |             |               |            |    |    |    |    |    |
| Vaccine       | V      | V        | V             | V           | V             |            |    |    |    |    |    |
| Echo          | v      | V        | V             | V           | V             | V          | v  | V  | V  | V  | V  |
| V             | V      | v        | V             | V           | V             | V          | V  | V  | V  | V  |    |
| Scans         |        |          |               |             |               |            |    |    |    |    |    |
| Correlatives  | V      | V        | V             | V           | V             | V          | V  | V  | V  | V  | V  |
pre-clinical testingher2neu dc
3.1.1 dose limiting toxicity

The following assessment guidelines for the management of dose limiting toxicity (DLT) 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 4.0 will be utilized for AE and DLT reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc_40). All adverse events will be classified as follows for determination of relatedness to AdHER2 vaccination:
- Unrelated
- Unlikely
- Possibly related
- Probably related
- Definitely related For the purposes of this study, the definitions of dose limiting-toxicities include:
- CTCAE Grade 2 and greater allergic reactions
- CTCAE Grade 2 and greater autoimmune disorders
- CTRCD, defined as a decrease in the LVEF of > 10 percentage points, to a value in <
53%1. Documentation of the above specified change in LVEF will require cardiac consultation for determination of clinical significance. Cardiac function deterioration clinically associated with apparent cardiotoxic agent use after off-therapy will be censored at the point of cardiotoxic agent use.  
- CTCAE Grade 3 or greater organ toxicity (dermatologic, gastrointestinal, hepatic, pulmonary, renal/genitourinary, or neurologic) occurring within 30 days of vaccine administration that are at least possibly related to research in.
pre-clinical testingher2neu dc
immunization-related dose limiting toxicity

- CTCAE Grade 3 and greater anaphylaxis: symptomatic bronchospasm with or without urticaria; parenteral intervention indicated; allergy-related edema/angioedma; hypotension (Grade 3). Life threatening consequences; urgent intervention indicated (Grade 4).  
- CTCAE Grade 3 and greater injection site reactions: ulceration or necrosis; severe tissue damage; operative intervention indicated (Grade 3). Life threatening consequences; urgent intervention indicated (Grade 4).
pre-clinical testingher2neu dc
early stopping rules for accrual

- Two or more of 6 patients in a given dose level develop evidence of cardiac toxicity, accrual will be stopped to that dose cohort. Accrual expansion of an additional 12 patients will occur in the next lower dose level to further evaluate for cardiac toxicity.  
- Any death (unrelated to underlying primary disease) that occurs and is attributed as being possibly, probably or definitely related to the investigational AdHER2 DC vaccine (FDA requirement)
pre-clinical testingher2neu dc
3.1.2 dose escalation

It has been reported that up to 7% of patients with HER2/neu expressing tumors treated with trastuzumab monotherapy for metastatic breast cancer developed clinically significant cardiac dysfunction including congestive heart failure. It would be considered acceptable if the therapeutic AdHER2 vaccine proposed for this study would produce no greater than that level of cardiac toxicity, but not if it were to be exceeded. In order to evaluate this using a limited number of patients, a two-part design, but incorporating an early stopping rule, will be implemented. The goal is to accrue 30 evaluable patients in each of Part I and Part II of the study and treat them with the experimental AdHER2 vaccine. Unless an early stopping rule is invoked, the number of patients who have experienced a clinically significant cardiac toxicity will be determined to estimate the frequency of cardiac toxicity. In addition, the dose level(s) at which toxicity is observed will also be examined. The statistical rationale for the targeted accrual of 30 patients in each part of the study is outlined in the statistical Section 10.  
Dose escalation will proceed in groups of 6 patients. *Dose escalation will proceed as long as 0* to1 of 6 patients at given dose level *have no evidence of toxicity and at least 3 of the 6 patients at* the dose level *have reached Week 12 on study.* If 2 of 6 patients on any given dose level develop cardiac toxicity, that dose cohort will be closed to further accrual, no higher doses will be explored, and dose expansion of an additional 12 patients will be enrolled in the next lower dose level to further assess for cardiac toxicity. The vaccine dose at which 0 to 1 of 18 *treated* patients in Dose Levels 1 to 3 develop cardiac toxicity will be defined as the *maximum* tolerated dose (MTD). If two or more patients out of 18 at a given dose level have developed cardiac toxicity, then the MTD will have been exceeded at that dose, and the dose *level* below that one will be considered the *MTD.* Since enrollment in Dose Level 3 has been reached and 0 of 6 patients has toxicity, then an expansion cohort of 12 patients, will be added for a maximum targeted accrual of 30 patients in Part 1. The 12 patients will be accrued at the 40x10^6 dose level without any planned interim stop, as cardiac toxicity is not presently anticipated given the results as of 12/11/15. With all 12 enrolled as a single group, there is a 4.9% probability of having 2 or more of 12 patients with cardiac toxicity if the true probability for a given patient is 3%, and 55.7% of having 2 or more of 12 with cardiac toxicity if the true probability for a given patient is 15%. Therefore, observing 0-1 of 12 with toxicity would be acceptable, and 40 x10^6 would be considered the maximum evaluated and tolerated dose. If we observe 2 or more with cardiac toxicity at or before accruing the 12th patient, this exceeds a safe level. Further patients would be added subsequently, by amendment, to the next lower dose, to confirm its safety. If dose escalation has proceeded in the first three dose levels in Part I and 0 of 20 patients have experienced cardiac toxicity, Part II of the study in patients with metastatic breast, gastric, gastroesophageal junction or other cancers and prior exposure to HER2-directed therapies will proceed with vaccine dosing based on the safety confirmed in Part I and outlined in Section 3.1.2. A maximum accrual of 30 patients will be enrolled in Part II.  
If no DLTs are observed, then dose levels in Part I and Part II will be assessed for the fraction of patients with immunogenicity and clinical outcomes to determine the biologically optimal vaccine dose that would be used in any future phase II studies involving those respective patient populations. To allow for the possibility of inevaluable patients, an accrual ceiling of 65 will be used.
pre-clinical testingher2neu dc
3.2 drug administration

All patients will undergo up to18-liter apheresis to remove peripheral blood monocytes for dendritic cell preparation. Cells used for subsequent dendritic cell maturation will be derived from monocytes frozen during the initial apheresis. Autologous AdHER2 dendritic cell vaccine will be manufactured under cGMP conditions by the NIH Clinical Center Department of Transfusion Medicine as outlined in **Appendix 3.** If the mononuclear cells or plasma collected are not adequate for minimum of 5 doses of vaccine, the patient will undergo optional additional leukapheresis or plasma collection per investigator's discretion.  
- Autologous AdHER2 DC vaccine preparations will be assessed for release standards
(nucleated cell content and concentration, appearance, flow cytometric verification of DC validation markers, viability > 60%, and product sterility and safety testing) as well as for HER2 expression (percent of cells expressing HER2/neu and the ratio of geometric mean fluorescent intensity (MFI) of HER2/neu expression to isotype control).  
- For patients in both Part I and Part II of the study, autologous AdHER2 DC vaccinations will be dosed according to enrollment dose levels (5, 10, 20, or 40 x 10^6 *viable* cells/vaccine)
and administered intradermally in up to two injection sites at Weeks 0, 4, 8, 16 and 24 for a total of five vaccinations. Vaccine will be administered in the 3SE Day Hospital and patients will be monitored for immediate adverse event vaccine reactions (VS, clinical assessment) for 1 hour following their first HER2 DC vaccine dose. If no adverse reactions are observed with the first vaccination, patients will be monitored for 15 minutes for all subsequent vaccinations.  
- If an adverse reaction is observed following the first vaccine, the reaction will be characterized and a determination made as to whether it is considered a dose limiting toxicity
(DLT) as outlined in Section **3.1.1**. If the adverse reaction is determined not to be a DLT, the duration of post-vaccination monitoring for subsequent vaccinations will be determined by the Principal Investigator and Lead Associate Investigator as clinically indicated depending on the severity of the initial vaccine reaction.  
- All patients will be given an AdHER2 DC Vaccine Report Card (refer to **Appendix 5**) and instructed on how to complete it, following each AdHER2 DC vaccine dose.  
- Continued injection of the same forearm is permissible for patients who have one functional forearm or a medical condition that does not allow for continued injections of both forearms.  
Since this protocol involves AdHER2 DC vaccination in humans using this vector for the first time, enrollment into subsequent dose level for both Part I and Part II patients will be staggered to allow for safety monitoring as outlined in Section **3.1.2**: dose escalation will proceed as long as 0 of 6 or 1 of 6 patients within a dose level have no evidence of toxicity and at least 3 of the 6 patients within the same dose level *have reached Week 12 on study* before enrollment in the next vaccine dose level can begin. If no dose limiting toxicities as defined in Section **3.1.1** are observed in the twelve-week window inclusive of the first three vaccinations, enrollment of additional patients may proceed as quickly as is logistically feasible as outlined above and in accordance with the dose escalation parameters outlined in Section **3.1.2**.
pre-clinical testingher2neu dc
3.3 dose modification and immunization

Unlike trastuzumab, the AdHER2 vaccine being given in this study targets both the extracellular (EC) and transmembrane (TM) domains of HER2. Use of trastuzumab in women with breast cancer has been shown to be associated with a small, but significant risk (about 7%) for developing cardiac dysfunction. *Exactly how trastuzumab causes cardiac toxicity is unknown.* The goal of vaccination with this product is to stimulate the patient's own immune system to make antibodies (and also potentially killer cells) that recognize HER2. It is not known whether or not antibodies made by a person's own immune system will cause cardiac dysfunction. In addition, if antibodies made by the immune system following AdHER2 DC vaccination *were* to cause cardiac toxicity or other significant clinical adverse events, it would be very challenging because the immune system cannot simply be "turned off". Thus, no dose modifications will be made in patients receiving AdHER2 DC vaccination. Subjects will cease to receive immunization if they experience dose-limiting toxicity (DLT) as outlined in Section 3.1.1.
pre-clinical testingher2neu dc
supportive management of patients experiencing

- For patients experiencing Grade 2 or greater allergic or autoimmune DLT, supportive clinical intervention will be provided until the DLT resolves to < Grade 1.  
- For patients experiencing Grade 2 or greater cardiac DLT, supportive clinical intervention will be provided until the DLT resolves to < Grade 1.  
o Patients will also undergo repeat LVEF measurements at 4-week intervals until LVEF
returns to baseline function. Thereafter, cardiac function will continue to be monitored according to the study schema outlined in **Figure 11** until study Week 124.  
o Patients who experience Grade 2 or greater cardiac DLT that does *not resolve* to <
Grade 1 will continue to receive clinical support and be monitored according to the study schema outlined in **Figure 11**.  
o until study Week 124.  
- For patients experiencing Grade 3 or greater organ (dermatologic, gastrointestinal, hepatic, pulmonary, renal/genitourinary or neurologic) DLT supportive clinical intervention will be provided until the DLT resolves to < Grade 1.  
- For patients experiencing Grade 3 or greater anaphylaxis DLT, supportive clinical intervention will be provided until the DLT resolves to < Grade 2.  
- For patients experiencing Grade 3 or greater local injection site reactions, supportive clinical intervention will be provided until the DLT resolves to < Grade 1.  
- **NOTE:** Once patients have completed participation in this study, there is no provision for long term medical care by the Clinical Center, NIH or the NCI.
pre-clinical testingher2neu dc
3.4 study calendar

Study Procedures *Screen*  
Wk  
0  
Wk  
4  
Wk  
8  
Wk  
12  
Wk  
16  
Wk  
20  
Wk  
24  
Wk  
28  
Wk  
32  
Wk  
36  
Wk  
40  
Wk  
4817  
Wk  
6018  
Wk  
7618  
Wk  
10018  
Wk  
12418  
NIH Advanced Directives1 √  
Informed Consent √ Height/Wt/NYHA √  
Screening labs/HLA2 √ Physical Exam/ECOG3 √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √  
Concomitant Medications √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √  
Clinical labs4 √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ Tumor Markers5 √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ LDH/CK/Troponin6 √ √ √ √ √ √ √ √ √ √ √  
TSH/25-OH Vit D √ √ √ √ Lymphocyte Subsets/  
Ig QA/IgE √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √  
Serum beta HCG7 √ √ √ √ √ √ √ √ √ √ √  
Urinalysis √ √ √ √  
Anti-HER2 Ab/ Profile8 √ √ √ √ √ √ √ √ √ √ √ Anti-Ad5 Antibody9 √ √ √ √ √ √ √ √ √ √ √  
Cellular HER2 Response10 √ √ √ √ √ √ √ √ √ √ √ Immune Subset/CTCs11 √ √ √ √ √ √ √ √ √ √ √  
Hitachi mRNA √  
MRI Brain12, CT Neck12,  
bone scan13 √ √ √ √ √ √ √ √ √ √ √  
CT Chest/Abd/Pelvis14 √ √ √ √ √ √ √ √ √ √ √ 2-D ECHO15 √ √ √ √ √ √ √ √ √ √ √ AdHER2 DC vaccine16 *Apheresis* √ √ √ √ √  
Vaccine Report Card √ √ √ √ √  
Adverse Events √ √ √ √ √ √ √ √  
1 As indicated in Section **12.3**, all subjects ≥ age 18 will be offered the opportunity to complete an NIH advanced directives form. This should be done preferably at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is strongly recommended, but is not required 2 Screening labs: CBC diff, PT/PTT, acute care panel, hepatic panel, serum beta HCG, urinalysis, and the following DTM labs: Anti-HIV 1/2 Ab, Anti-HCV Ab, HBsAg, Anti-HBs Ab, anti-HTLV 1/2 Ab (pending result is acceptable for eligibility), anti-CMV IgG/IgM, and RPR. If DTM labs are performed > 30 days prior to apheresis, DTM labs must be repeated. HLA will not determine eligibility and if it was done previously at the NIH it does not need to be repeated.  
3 Patients should be evaluated within 7 days prior to upcoming vaccination as a clearance for treatment. 4 Clinical labs CBC with differential, PT/PTT, acute care panel, hepatic panel, mineral panel, total protein, amylase/lipase, uric acid, lipid panel.  
5 Tumor markers are optional and will be tested dependent on the primary sites. Patients with primary breast, ovarian and GI tract cancer might be tested for CA 125, CA 15-3, CA19-9, CA 27-29, and CEA. Other organ specific tumor markers can be tested 6,7 Only until 12 weeks after the last dose of vaccination.  
8-11 Research bloods will not be used in data analysis if the patient has started on any treatment that can affect the data of the study. Week 40 and after samples will only be collected if the patient has not progressed (PD).  
12 This is optional and to be performed if clinically indicated. If the patient received scans within 30 days of enrollment at an outside institution the patient will not be required to have a second scan performed. Instead, the baseline scan will be used to collect target lesions.  
13 If the patient received scans within 30 days of enrollment at an outside institution the patient will not be required a screening scan to consent.  
Instead, the baseline scan will be used to identify target lesions. If the patient has bone metastasis and clinically indicated, a bone scan will be done at restaging.  
14 If the patient received scans within 30 days of enrollment at an outside institution the patient will not be required to have a second scan performed. Instead, the baseline scan will be used to collect target lesions. If a confirmatory scan is performed in less than 8 weeks from prior CT,
the interval of CT follow-up will be adjusted to maintain 8 weeks interval to avoid unnecessary radiation exposure. For bladder cancer adjuvant indication, CT scans every 12 weeks during first 12 month, every 16 weeks for the next 12 months and at the end of study if no recurrence.  
15 Optional documentation on cardiac function will be collected once a patient is on study but off treatment for the purposes of long-term assessment of the impact of AdHER2 vaccination on cardiac function. Note: if the patient had an echo within 30 days of enrollment at an outside institution the patient will not be required a screening echo to consent. Instead, the baseline echo will be performed at NIH prior to treatment and used to identify target lesions.  
16 Dose can be delayed up to 8 weeks. The study will reset at the time of study resumption. For example, if dose at week 8 is delayed and resumed at original week 11, that week 11 will now be considered week 8.  
17 If there is a need for confirmatory scans based on the result of Week 48 evaluation, additional visit will occur at week 52 which will include clinical assessment with physical exam and laboratory assessment.  
18 Patients who progress do not require a full evaluation. Only physical exam, clinical labs, echocardiogram and AE reports will be required.
pre-clinical testingher2neu dc
3.5 criteria for removal from

Prior to removal from study, effort must be made to have all subjects complete a safety visit approximately 30 days following the last dose of study therapy. 3.5.1 Criteria for Removal from Protocol Therapy  
3.5.1.1 Completion of protocol therapy (receipt of 5 doses of autologous AdHER2 DC
vaccine).  
3.5.1.2 Disease progression per 6.2, **Response Criteria**. Note Exception: Clinically stable patients with PD at Weeks 8, 16 or 24 re-staging will be allowed to remain on protocol therapy per PI's discretion.  
3.5.1.3 Patient experiences a dose limiting toxicity as outlined in Section **3.1.1**.  
3.5.1.4 Patient experiences a Grade 3 or greater adverse event/toxicity not described in Section 3.1.1 that is deemed by the Principal Investigator as possibly, probably or definitely related to AdHER2 DC vaccination.  
3.5.1.5 Patient experiences unresolved treatment-related toxicity. 3.5.1.6 Diagnosis of a secondary malignancy other than superficial basal cell or squamous cell carcinoma of the skin.  
3.5.1.7 The patient experiences a treatment delay of more than 8 weeks from the scheduled target date for receipt of AdHER2 DC vaccine.  
3.5.1.8 Inter-current illness or medical circumstance that in the opinion of the Principal Investigator unacceptably increases the risk of treatment. This applies to the conditions related to the cancer diagnosis that led to the enrollment in current study.  
3.5.1.9 The patient is non-compliant with study requirements. 3.5.1.10 The patient becomes too ill to return for protocol specified study visits. 3.5.1.11 The patient becomes pregnant during the study. 3.5.1.12 The patient requests to be withdrawn and refuses further therapy. 3.5.1.13 The patient requires a prohibited concomitant medication. 3.5.1.14 Death
3.5.2 Continued Monitoring for Potential Cardiac Toxicity Once Off Protocol Therapy  
3.5.2.1 Patient is removed from therapy for reasons outlined in Section **3.5.1**.  
3.5.2.2 Patient will continue to undergo designated cardiac monitoring studies at subsequent study weeks as outlined in **Figure 11**.  
3.5.2.3 Cardiac troponin levels (Weeks 0, 4, 8, 12, 16, 20, 24, 28, 32, 40 and 48) and echocardiograms (Weeks 4, 12, 20, 28, 32, 40, 48, 60. 76, 100 and 124) including HPE and ECOG assessments.  
3.5.2.4 Cardiac monitoring will be performed either at the NIH or through *the patient's LMD*.  
For patients who come off-treatment for disease progression or other reasons, every effort will be made to capture ongoing documentation on cardiac function as is reasonably practicable, to allow long-term assessment of the impact of AdHER2 vaccination on cardiac function.
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3.5.3 off study criteria

3.5.3.1 Completion of scheduled study visits. 3.5.3.2 PI decision to end the study.  
3.5.3.3 Patient requests to be withdrawn and refuses further study participation.  
3.5.3.4 The patient is non-compliant with study requirements. 3.5.3.5 The patient becomes too ill to return for protocol specified study visits. 3.5.3.6 Documentation of rapid disease progression or clinical deterioration between the screening (including outside information) and baseline evaluations. These patients will be removed from study and precluded from receiving AdHER2 DC vaccination.  
3.5.3.7 If a patient begins to take a known cardiotoxic agent the patient will come off study and we will not use data after that point.  
3.5.3.8 Death 3.5.4 Off Protocol Therapy and Off Study Procedure All subjects must be taken off study through the NCI Central Registration Office CRO). Authorized staff must notify CRO when a subject is being taken off protocol therapy and when a subject is taken off-study. A Participant Status Updates Form the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistrationl@mail.nih.gov.
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4 concomitant medications/measures

- Patients are allowed to be on concomitant therapy with tamoxifen or aromatase inhibitors for ER+ tumor status.  
- Patients may be on concomitant drugs to prevent skeletal related events (SRE), including bisphosphonates and denosumab.  
- Study subjects will be allowed to take multivitamins, analgesics (NSAIDS or acetaminophen), antipyretics, and antihistamines for symptomatic relief of local or systemic injection site reactions.  
- Patients are allowed to continue on medications as clinically indicated for treatment of chronic medical conditions e.g. hypertension, diabetes, hypercholesterolemia, etc.  
- *Excluded* Therapy:
o Trastuzumab, pertuzumab, lapatinib, ado-trastuzumab emtansine (TDM1) or other HER2-directed therapies within 1 *week* of study enrollment.  
o Chemotherapy: Concomitant use of chemotherapy is not allowed during this trial. o Anti-Cancer Radionuclides: Concomitant use of anti-cancer radionuclides is not allowed during this trial. Patients will be allowed to co-enroll in studies of novel radionuclide imaging agents e.g. 111Indium CHX-A DTPA trastuzumab.  
o Secondary Hormonal Therapies: Concomitant use of supplementary hormonal treatments other than tamoxifen or aromatase inhibitors is not allowed.  
o Corticosteroids: Concomitant, chronic systemic corticosteroids are not *allowed* during this trial (excepting emergent use for clinical indications). However, the use of inhaled corticosteroids, intranasal sprays and topical creams on limited body areas is *allowed*.
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vitamin d3 (cholecalciferol) supplementation

Vitamin D when ingested is metabolized in the liver to 25-OH vitamin D. Inside cells, it is metabolized further by 1-hydroxylase where it is transformed into a seco-steroid hormone that is important to a host of critical cellular and immune functions within the body. Within cells is a second enzyme 24-hydroxylase whose function is to decrease vitamin D, thereby maintaining intracellular vitamin D homeostasis. The classic function of vitamin D is to regulate calcium homeostasis and in turn, bone formation and resorption. However, additional functions of vitamin D have been demonstrated and include effects on immune response by promoting cellular apoptosis and differentiation. The exact role of vitamin D deficiency in prostate, breast, colon and other cancers has been controversial, with some laboratory studies suggesting there is a role and other epidemiological studies suggesting that there is no role or even possibly that supplementation should be avoided. In a recent study by Marshall and colleagues34 vitamin D supplementation of 4.000 IU per day for one year was examined in men with low risk prostate cancer (Gleason score of 6, 1-6 cores positive out of 12 possible and a PSA <10) under active surveillance. After one year upon re-biopsy, 60% showed a decrease in the number of positive cores, Gleason score or both and in 6% these factors remained unchanged. In addition, PSA levels did not rise. In another study reported by Vieth and colleagues at AACR in April 201235, 66 men scheduled to undergo radical prostatectomy were randomly assigned to receive a daily vitamin D dose of 400, 10,000 or 40,000 IU daily for 3 to 8 weeks prior to surgery. Calcitriol levels in the prostate increased progressively with increasing vitamin D dosing and corresponded with lower levels of Ki67 as well as higher levels of specific growth-inhibitor microRNAs. Several studies have shown that women with low vitamin D levels have an increased risk of breast cancer incidence and mortality, but research is lacking investigating vitamin D levels and prognostic variables e.g. hormone receptor status, Oncotype DX etc. in this patient population. In a case control study of 194 women s/p breast cancer surgery and 194 cancer-free controls conducted by Peppone and colleagues at the University of Rochester36, women with breast cancer were found to have significantly lower 25-OH
vitamin levels than controls (32.7ng/mL vs. 37.4 ng/mL respectively, P=0.02). Importantly, women with suboptimal 25-OH vitamin D levels (<32 ng/mL) had significantly increased odds of having ER-negative (OR = 2.59, 95% confidence interval [95% CI] = 1.08-6.23) and triple-negative cancer (OR = 3.15, 95% CI = 1.05-9.49) than those with optimal 25-OH D concentrations. In addition, women with basal-like phenotype had lower 25-OH vitamin D levels than women luminal A phenotype (24.2 ng/mL vs. 32.8 ng/mL, respectively P = 0.04). In summary, women with a more aggressive breast cancer molecular phenotype (basal-like) and worse prognostic indicators (ER- and triple-negative) had lower mean 25-OH vitamin D levels. Given its critical role in immune function and possible role in cancer pathophysiology, all patients will have 25-OH vitamin D levels obtained at baseline. Although there is debate about the target level of 25-OH vitamin D for optimum health, most vitamin D experts agree that it should be greater than 40 ng/mL.  
- All patients with 25-OH vitamin D levels < 40 ng/mL will be initiated on oral supplements of Vitamin D3 (cholecalciferol) per standard clinical care guidelines. 25-OH vitamin D levels will continue to be monitored at study Weeks 8, 16, 28 and 60.
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5 biospecimen collection 5.1 correlative

There are no pharmacokinetic studies that will be performed as part of this clinical investigation. Evaluation of anti-HER2 antibody and cellular HER2 responses Tetramer and IFN-γ ELISPOT
Assays will be performed as outlined in **Appendix 6**. If the patient does not need a confirmatory scan at Week 52, the same set of evaluations will be done at Week 60 instead of Week 52. Characterization of vaccine-induced antibody profiles (anti-HER2 Antibody Profile) will be performed as outlined in **Appendix 6**. No additional blood to be drawn: serum will be aliquoted from blood drawn for anti-HER2 antibody studies.  
Evaluation of anti-Ad5 antibody immunity will be performed as outlined in **Appendix 7**.  
Characterization of soluble HER2 and anti-HER2 antibodies will be performed on the stored samples as outlined in **Appendix 8Characterization** of Soluble HER2 and Anti-HER2 Antibodies. Exploratory correlative studies (Week 0) measuring function-associated mRNAs in whole blood to be performed by Dr. Cindy Yamamoto and circulating tumor cells (CTCs) and immune subset analysis will be performed by Jane Trepel as outlined in Appendix 9.
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5.2 sample storage tracking and

For research samples obtained for investigation, the Clinical Support Laboratory, Leidos Biomedical Research, Inc. processes and cryopreserves samples in support of IRB-approved, NCI clinical trials. The laboratory is located in a controlled-access building and laboratory doors are kept locked at all times. Upon specimen receipt each sample is assigned a unique, sequential laboratory accession I.D. number. All products generated by the laboratory that will be stored either in the laboratory freezers or at a central repository facility are identified by this accession I.D. An electronic database is used to store information related to patient samples processed by the laboratory. Vial labels do not contain any personal identifier information. Samples are stored inventoried in locked laboratory freezers and are routinely transferred to the NCIFrederick repository facilities for long-term storage. These facilities are operated by American Type Culture Collection (ATCC), under subcontract to Leidos Biomedical Research, Inc. Access to stored clinical samples is restricted. Investigators establish sample collections under "Source Codes" and the investigator responsible for the collections, typically the protocol Principal Investigator and/or Lead Associate Investigator, specifies who has access to the collection. When requests are submitted by the NCI investigator for shipment of samples outside of the NIH it is the policy of the laboratory to request documentation that a Material Transfer Agreement (MTA) is in place that covers the specimen transfer. The laboratory does not provide patient identifier information as part of the transfer process but may, at the discretion of the NCI investigator, group samples from individual patients when that is critical to the testing process. The NCI investigator responsible for the sample collection is responsible for ensuring appropriate IRB approvals are in place and that an MTA has been executed prior to requesting the laboratory to ship samples outside of the NIH. Blood, urine and tissue specimens collected in the course of this research project may be banked and used in the future to investigate new scientific questions related to this study. However, this research may only be done if the risks of the new questions were covered in the consent document and the proposed research has undergone prospective IRB review and approval. If new risks are associated with the research (e.g., analysis of germ line genetic mutations.) the Principal Investigator must amend the protocol and obtain informed consent from all research subjects.  
Once primary research objectives for the protocol are achieved, intramural researchers can request access to remaining samples provided they have an IRB-approved protocol and patient consent. Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material. If at any time, a patient withdraws from the study and does not wish for their existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed (or returned to the patient, if so requested), and reported as such to the IRB. The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of Section 0. 5.2.1 Samples collected by the Blood Processing Core of the Clinical Pharmacology Program All samples will be bar-coded, with data entered and stored in the Patient Sample Data location.  
Bar coded samples are stored in bar coded boxes in a locked freezer at either -20 or -80C
according to stability requirements. These freezers are located onsite in the CPP and offsite at NCI Frederick Central Repository Services (ATCC) in Frederick, MD. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in the PSDM System. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this specific clinical trial (as per the IRB approved protocol) and that any unused samples must be returned to the CPP. Following completion of this study, samples will remain in storage as detailed above. Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material. Samples will be stored for future use following completion of the study unless a subject has opted out of the future use of data and specimen. If, at any time a patient withdraws from the study and does not wish for his/her existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed (or returned to the patient, if so requested), and reported as such to the IRB. Any samples lost (in transit or by a researcher) or destroyed due to unknown sample integrity (i.e. broken freezer allows for extensive sample thawing, etc.) will be reported as such to the IRB. Sample barcodes are linked to patient demographics and limited clinical information. This information will only be provided to investigators listed on this protocol, via registered use of the PSDMS. It is critical that the sample remains linked to patient information such as race, age, dates of diagnosis and death, and histological information about the tumor, in order to correlate genotype with these variables.
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5.3 samples for genetic/genomic analysis

No genetic/genomic analyses will be performed as part of this clinical investigation.
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6 data collection and evaluation

1. Each patient must meet all eligibility requirements and a completed registration must be sent to the NCI Central Registration Office (CRO).  
2. The Consent Document must be signed prior to registration with the CRO.  
3. Treatment will be given according to protocol (on-study and treatment notes, reports of adverse events and documentation of any deviation from the study protocol).  
4. Data will be entered into a secure software system (C3D Database) produced by Oracle™
Corporation (Redwood Shores, CA). Data will be collected based on protocol-specific requirements, verified for accuracy and completeness. Any hard copy data will be kept in locked secure area in the Vaccine Branch Clinical Trials Offices (Bldg 10, Rm. 4B-54).  
5. Toxicity will be assessed according to the protocol using the CTCAE v4.0 that is available at: http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm 6. Response will be assessed according to the protocol and documentation of response (e.g.  
CT scans, laboratory reports, dated notes on tumor assessment and clinical assessment as appropriate) using Immune Response Related Criteria as outlined in Section **6.2.**
7. Vaccine report cards associated with each AdHER2 DC vaccination will be obtained for each patient.
The PI 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. All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Document AEs from the first study intervention, Study Week 0, through 30 days after the last study intervention. Beyond 30 days after the last study intervention, only adverse events which are serious and related to the study intervention need to be recorded. An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following:  
- Results in discontinuation from the study - Is associated with clinical signs or symptoms - Requires treatment or any other therapeutic intervention - Is associated with death or another serious adverse event, including hospitalization. - Is judged by the Investigator to be of significant clinical impact - If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.
Laboratory and clinical symptom abnormalities less than or equal to Grade 1 will not be graded or assessed for attribution to AdHER2 DC vaccine.  
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 requirements in Section **7.2.1**.  
Dr. Fojo will continue to have access to the data for purposes of data analysis and publication. The necessary data will be sent via encrypted email.
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6.2 response criteria

Tumor measurements for response will be conducted at Weeks 8, 16, 24, 36, 48, 76, 100, and 124 with confirmatory scans obtained not less than 4 weeks following initial documentation of objective response. Adjuvant bladder cancer patients will undergo re-staging at Weeks 8, 16, 24, 36, 48, 76, 100, and 124 with confirmatory scans with confirmatory scans obtained not less than 4 weeks following initial documentation of objective response as outlined in the protocol Study Calendar in Section 3.4 and clinical evaluations will include physical examination, neck
(optional), chest, abdomen and pelvis CT imaging and bone scan when clinically indicated. Dependent on the tumor site, MRI can be performed instead of CT scan. Patients with history of CNS disease will be evaluated for the necessity of follow up brain MRI. Due to the requirement of confirmational scans for the evaluation of the response at least 4 weeks after the initial documentation of the response, subsequent scan schedules can be adjusted to allow adequate interval and to avoid unnecessary radiation exposure from fixed CT scan schedules. After offtherapy or PD documentation, restaging scans are optional.  
A modified Immune-Related Response Criteria (modified irRC) based on RECIST 1.1 will be used to determine the response as outlined in Section 6.2.6 **Table 2**
37. Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes will be used as per RECIST 1.1 criteria. The modified irRC will be based upon RECIST 1.1 criteria (described below) except for 2 major changes: 1) require confirmation of progression by imaging at least 4 weeks after initial imaging and 2) do not necessarily score the appearance of new lesions as progressive disease if the sum of lesion diameters of target lesions (minimum of 10 mm per lesion, maximum of 5 target lesions, maximum of 2 per organ) and measurable new lesions does not increase by ≥ 20%. Efficacy assessments will be made by the Investigator at the investigational site. All objective responses will have confirmatory scans performed at least 4 weeks after the response was first documented per the modified irRC convention (e.g. initial scan at Week 8, confirmatory scan at Week 12 or after etc.). The PI will review and confirm all objective responses.
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6.2.1 response definitions

Response and progression will be evaluated in this study using the new modified irRC37 (Section 6.2.6.) Note: Lesions are either measurable or non-measurable using the criteria for disease parameters outlined in Section **6.2.2** provided below. The term "evaluable" in reference to measurability will not be used because it does not provide additional meaning or accuracy. 6.2.2 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 5 mm or under as >10 mm with CT scan o Scan slice thickness >5 mm: double the slice thickness
- By MRI: >10 mm
- With calipers on clinical exam: >10 mm
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters).  
Note: Tumor lesions that are situated in a previously irradiated area are not to be considered measurable unless they demonstrate progression. 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 that can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Non-target lesions. All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as **non-target lesions** and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up. 6.2.3 Methods for Evaluation of Measurable Disease All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam. 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. For certain body parts or lesions, MRI can replace CT for response evaluation and such an MRI should be optimized for the evaluation of the type and site of the disease.  
Clinical lesions: Clinically visible skin lesions will be utilized to evaluate measurable disease. Chest x-ray: Chest x-ray will not be utilized to evaluate measurable disease.  
Ultrasound: Ultrasound will not be utilized as a method of measurement. 6.2.4 Evaluation of Target Lesions  
o **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.  
o **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.
o **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).  
o **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.
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note

- Confirmation by two consecutive observations not less than 4 weeks apart is required for CR,
PR and PD
- New lesions result in unconfirmed PD. Confirmed PD is assigned only if additional new lesions appear or an increase in size of new lesions is seen (≥5 mm for sum of new lesion target or any increase in new lesion non-target) at next assessment; the appearance of additional new lesions when none have previously been recorded, can also confirm PD
6.2.5 Evaluation of Non-Target Lesions o **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.  
o **Non-CR/Non-PD**: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.  
o **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.  
Note:
- Confirmation by two consecutive observations not less than 4 weeks apart was required for CR, PR and PD
- New lesions result in unconfirmed PD. Confirmed PD is assigned only if additional new lesions appear or an increase in size of new lesions is seen (≥5 mm for sum of new lesion target or any increase in new lesion non-target) at next assessment; the appearance of additional new lesions when none have previously been recorded, can also confirm PD.  
6.2.6 Evaluation of Best Overall Response The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria. For the purposes of this study, best overall response will be defined according to modified irRC. **Table 2** below is modified from Wolchok JD et al, *Clin Canc Res* 2009, Hodi FS et al, J Clin Oncol 2016 and Nishino M et al, Clin Canc Res 201337-39 comparing RECIST 1.1 criteria, modified irRC and iRECIST. iRECIST is similar to modified irRC used in this study except that iRECIST defined unconfirmed PD and confirmed PD separately.  
| Table 2 Comparison of RECIST v1.1, modified irRC and iRECIST Category RECIST v1.1 Modified irRC iRECIST Measurement of  Unidimensional Unidimensional Unidimensional tumor burden Target lesions Maximum 5 Maximum 15 Maximum 5 New lesion Results in progressive  Does not always results in progressive disease at first  disease at first appearance appearance unless the sum of tumor burden fits the criteria  for progressive disease Complete  response Disappearance of all target and nontarget lesions Lymph nodes must regress to <10 mm short axis No new lesions, confirmation required Partial response ≥ 30% decrease in tumor  burden compared with  baseline ≥ 30% decrease in tumor  burden compared with  baseline† Confirmation  required ≥ 30% decrease in tumor  burden compared with  baseline Progressive  ≥ 20% + 5-mm absolute  disease increase in tumor burden  compared with nadir.  Appearance of new lesions  or progression of nontarget lesions ≥ 20% increase in tumor  burden compared with  baseline, with nadir, or reset  baseline. New lesions added to  the tumor burden Confirmation required ≥ 20% + 5-mm absolute  increase in tumor burden  compared with nadir.  Appearance of new lesions  or progression of nontarget lesions.  Confirmation required Stable disease Neither partial response nor progressive disease For Patients with Measurable Disease (i.e., Target Disease) Target  NonTarget  Lesions Lesions New  Overall  Best Overall Response when  Lesions Response Confirmation is Required* CR CR No CR >4 wks. Confirmation** CR NonCR/NonPD No PR CR Not  No PRevaluated >4 wks. Confirmation**   |
|---|  
| PR                                                                                                                                                                                                                                                                                                                                  | NonCR/Non - PD/not  evaluate d                                                                                    | No     | PR   |                                                   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------|--------|------|---------------------------------------------------|
| SD                                                                                                                                                                                                                                                                                                                                  | NonCR/Non - PD/not  evaluate d                                                                                    | No     | SD   | Documented at least once >4  wks. from baseline** |
| PD                                                                                                                                                                                                                                                                                                                                  | Any                                                                                | Yes or | PD   |                                                   |
| No                                                                                                                                                                                                                                                                                                                                  | no prior SD, PR or CR                                                              |        |      |                                                   |
| Any                                                                                                                                                                                                                                                                                                                                 | PD**                                                                               | Yes or | 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 nontarget 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. |                                                                                    |        |      |                                                   |
| For Patients with Non-Measurable Disease (i.e., Non-Target Disease)                                                                                                                                                                                                                                                                 |                                                                                    |        |      |                                                   |  
| Non-Target Lesions   | New Lesions   | Overall Response   |
|----------------------|---------------|--------------------|
| CR                   | No            | CR                 |
| Non-CR/non-PD        | No            | Non-CR/non-PD*     |
| Not all evaluated    | No            | not evaluated      |
| Unequivocal PD       | Yes or No     | PD                 |
| Any                  | Yes           | PD                 |  
* 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease since SD is increasingly used as an endpoint for assessment of efficacy in some trials so to assign this category when no lesions can be measured is not advised Overall responses derived from changes in index, non-index and new lesions as demonstrated in the following table:  
| Measurable response                                   | Non-measurable response   | Overall response using  modified irRC   |      |
|-------------------------------------------------------|---------------------------|-----------------------------------------|------|
| Index and new,  measurable lesions  (tumor burden)* % | Non-index                 | New, non-measurable                     |      |
| lesions                                               | lesions                   |                                         |      |
| Decrease 100                                          | Absent                    | Absent                                  | CR** |
| Decrease 100                                          | Stable                    | Any                                     | PR** |
| Decrease 100                                          | Unequivocal  progression  | Any                                     | PR** |
| Decrease ≥ 30%                                        | Absent / Stable           | Any                                     | PR** |
| Decrease ≥ 30%                                        | Unequivocal  progression  | Any                                     | PR** |
| Decrease < 30 to increase <  20                       | Absent / Stable           | Any                                     | SD   |
| Decrease < 30 to increase <                           | Unequivocal  progression  | Any                                     | SD   |
| 20 Increase ≥ 20                                      | Any                       | Any                                     | PD** |  
* Decreases assessed relative to baseline ** Assuming response (CR and PR) and progression (PD) are confirmed by a second, consecutive assessment at least 4 weeks apart.
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6.2.7 confirmatory measurement/duration of response

Confirmation: To be assigned a status of a response of CR or PR, changes in tumor measurements must be confirmed by repeat assessments that should be performed at least 4 weeks after the criteria for response are first met. PD must be confirmed by a second observation no less than 4 weeks later. 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 PD is objectively documented. The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that recurrent disease is objectively documented. Time to progression (TTP) will also be recorded for patients with CR, PR and SD subsequently developing recurrent or progressive disease. Duration of stable disease: SD is measured from the start of the treatment until the PD criteria for progression are met. 6.2.8 Progression Free Survival and Overall Survival Progression-free survival (PFS) will be assessed *only* in adjuvant bladder cancer patients and in metastatic bladder cancer patients that have completed first line chemotherapy that do not have measurable disease. Overall survival (OS) is not being assessed as part of this clinical investigation.
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6.2.9 response review

Responses will be reviewed by the Principal Investigator, Lead Associate Investigator and their designees. Since the assessment of the preliminary anti-tumor activity of autologous AdHER2 DC vaccination is only a secondary objective of this phase I, two-part dose escalation study, responses will not be reviewed by an expert(s) independent of the study.
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7 nih reporting requirements/data and

The Principal Investigator and Lead Associate Investigator will be responsible for overseeing the Data and Safety Monitoring Plan. As information is gathered from this trial, clinical results will be shared with the patients. Laboratory and clinical data will be gathered on a monthly basis and any new significant observation(s) found during the course of the research that may affect patient safety or a patient's willingness to participate further will be explained at the time patients are consented for the study. Confidentiality of information concerning participants will be maintained including in all publications and presentations resulting from this study. Names of patients or material identifying participants will not be released without permission, except as such release is required by law. Records will be maintained according to current legal requirements, and are made available for review as required by the U.S. Food and Drug Administration (FDA) or other authorized users, only under the guidelines established by the Federal Privacy Act.
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7.1 definitions

Please refer to definitions provided in Policy 801: Reporting Research Events found here.
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7.2 ohsrp office of compliance

7.2.1 Expedited Reporting NIH Intramural IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths
Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802 Non-Compliance Human Subjects Research found here. Note: Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported per these policies
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7.2.2 irb requirements for pi

Please refer to the reporting requirements in Policy 801: Reporting Research Events found here.
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7.3 nci clinical director reporting

Problems expeditiously reported to the OHSRP/IRB in iRIS will also be reported to the NCI Clinical Director. A separate submission is not necessary as reports in iRIS will be available to the Clinical Director. In addition to those reports, all deaths that occur within 30 days after receiving a research intervention should be reported via email to the Clinical Director unless they are due to progressive disease. To report these deaths, please send an email describing the circumstances of the death to Dr.  
Dahut at NCICCRQA@mail.nih.gov within one business day of learning of the death.
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7.4 institutional biosafety committee (ibc)

The Principal Investigator (or delegate) will notify IBC of any unexpected fatal or lifethreatening experience associated with the use of AdHER2 DC vaccine as soon as possible but in no event later than 7 calendar days of initial receipt of the information. Serious adverse events that are unexpected and associated with the use of the AdHER2 DC vaccine, but are not fatal or life-threatening, much be reported to the NIH IBC as soon as possible, but not later than 15 calendar days after the investigator's initial receipt of the information. Adverse events may be reported by using the FDA Form 3500a.
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7.4.2 annual reports to ibc

Within 60 days after the one-year anniversary of the date on which the IBC approved the initial protocol, and after each subsequent anniversary until the trial is completed, the Principal Investigator (or delegate) shall submit the information described below. Alternatively, the IRB continuing review report can be sent to the IBC in lieu of a separate report. Please include the IBC protocol number on the report.  
7.4.2.1 Clinical Trial Information A brief summary of the status of the trial in progress or completed during the previous year. The summary is required to include the following information:
- the title and purpose of the trial  
- clinical site
- the Principal Investigator - clinical protocol identifiers; - participant population (such as disease indication and general age group, e.g., adult or pediatric);
- the total number of participants planned for inclusion in the trial; the number entered into the trial to date whose participation in the trial was completed; and the number who dropped out of the trial with a brief description of the reasons
- the status of the trial, e.g., open to accrual of subjects, closed but data collection ongoing, or fully completed,
- if the trial has been completed, a brief description of any study results.  
7.4.2.2 Progress Report and Data Analysis
Information obtained during the previous year's clinical and non-clinical investigations, including:  
- a narrative or tabular summary showing the most frequent and most serious adverse experiences by body system
- a summary of all serious adverse events submitted during the past year - a summary of serious adverse events that were expected or considered to have causes not associated with the use of the gene transfer product such as disease progression or concurrent medications
- if any deaths have occurred, the number of participants who died during participation in the investigation and causes of death
- a brief description of any information obtained that is pertinent to an understanding of the gene transfer product's actions, including, for example, information about dose-response, information from controlled trials, and information about bioavailability.
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7.5 nih required data and

The clinical research team will meet on a weekly basis when patients are being actively treated on the trial to discuss each patient. 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 timelines.  
The Principal Investigator will review adverse event and response data and make attribution determinations 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. 7.5.2 Safety Monitoring Committee (SMC) This protocol will require oversight from the Safety Monitoring Committee (SMC). Initial review will occur as soon as possible after the annual 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. For initial and subsequent reviews, protocols will not be reviewed if there is no accrual within the review period. Written outcome letters will be generated in response to the monitoring activities and submitted to the Principal investigator and Clinical Director or Deputy Clinical Director, CCR, NCI.
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8 sponsor safety reporting 8.1

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 **8.1.3**)
- Inpatient hospitalization or prolongation of existing hospitalization - Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions  
- A congenital anomaly/birth defect. - Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.  
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 4.03. 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.
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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.
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8.3 reporting of serious adverse

Any AE that meets 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.  
All SAE reporting must include the elements described in Section 9.  
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/pages/viewpage.action?pageId=157942842 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 reporting pregnancy 8.4.1 maternal

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 (**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. 8.4.2 Parental exposure Male patients should refrain from fathering a child or donating sperm during the study and for 90 days after the last dose of AdHER2 DC vaccine. Pregnancy of the patient's partner is not considered to be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) occurring from the date of the first dose until 90 after the last dose should, if possible, be followed up and documented.
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8.5 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. 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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9 clinical monitoring

As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring program. The CCR's program allows for confirmation of: study data, specifically data that could affect the interpretation of primary and secondary study endpoints; adherence to the protocol, regulations, ICH E6, and SOPs; and human subjects protection. This is done through independent verification of study data with source documentation focusing on:  
- Informed consent process - Eligibility confirmation
- Drug administration and accountability
- Adverse events monitoring - Response assessment.
The monitoring program also extends to multi-site research when the CCR is the coordinating center. This trial will be monitored by personnel employed by a CCR contractor. Monitors are qualified by training and experience to monitor the progress of clinical trials. Personnel monitoring this study will not be affiliated in any way with the trial conduct.
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10 collaborative agreements 10.1 tech

Stored samples are being sent to Midissia for testing and characterization of soluble HER2 and anti-HER2 antibodies as a part of this protocol using a Material Transfer Agreement (MTA \#42793) which was executed on 09/17/2017. There is also a CDA between Midissia and the NCI (CDA \#13887-18, executed 11/10/2017). Additional tech transfers include:  
- CRADA \# 03048, executed 03/02/2016
- MTA \#44020-28, executed 01/24/2018
- MTA \#3394-13, executed 11/13/2014
- CRADA \#03225, currently being negotiated
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11 statistical considerations

The primary objectives of this open label, single site, non-randomized, two-part, phase I study of AdHER2 DC vaccination are to determine if the fraction of patients with cardiac toxicity (if it occurs) is sufficiently low to warrant further exploration in subsequent trials and to determine an approximate estimate of the vaccine immunogenicity, as represented by the fraction of patients who develop at least a 3-fold increase in anti-HER2/neu antibody concentration (measured as mcg/mL) or a 4-fold increase in antibody dilution titers over baseline at the time points measured. An important secondary objective is to obtain a preliminary estimate of the proportion of patients who may experience tumor shrinkage or stabilization that is sufficient by modified immune response related criteria (modified irRC) to be considered stable disease, a partial response or better. It has been reported that up to 7% of patients with HER2/neu expressing tumors treated with trastuzumab monotherapy for metastatic breast cancer developed clinically significant cardiac dysfunction including congestive heart failure. It would be considered acceptable if the therapeutic AdHER2 DC vaccine proposed for this study would produce no greater than that level of cardiac toxicity, but not acceptable if it were to be exceeded. In order to evaluate this using a limited number of patients, a two-part design, but incorporating an early stopping rule, will be implemented. Statistical considerations pertaining to each part of the study are as follows:
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11.1 part i (maximum of

The goal of Part I is to investigate vaccine dose escalation and determine whether there is a significant, adverse safety signal regarding cardiac toxicity in patients with recurrent or progressive, metastatic solid tumors as well as adjuvant bladder cancer patients with HER2 expressing tumors that are *naive to trastuzumab, lapatinib, ado-transtuzumab and other HER2-* directed therapies, in addition to preliminary assessment of the vaccine's immunogenicity and clinical activity. Allowable tumors include breast, ovarian, colon, non-small cell lung, renal cell, bladder and prostate cancer with HER2 characterization by IHC and FISH as outlined in Section 2.1.1.14.  
Inclusion of a broad spectrum of tumors that express HER2 will facilitate more rapid accrual and allow prompt determination of the vaccine's safety and immunogenicity. However, the inclusion of subjects with multiple tumor types that have varying levels of HER2 expression may preclude identification of an optimal biologically active dose. During this part, vaccine safety, immunogenicity and preliminary activity will be determined for each dose level given escalating doses of vaccine as follows:
Dose Level 1 (N = 6 patients): 5 x 106 viable cells/vaccine  
$$10\ \mathrm{x}\ 10^{6}\ \mathrm{viable\cells/vacince}$$
$$\mathbb{C V C I}\ \angle\ (1\mathbf{v})$$
Dose Level 2 (N = 6 patients): 10 x 106viable cells/vaccine  
Dose Level 3 (N = 6 patients): 20 x 106 viable cells/vaccine Expansion Cohort (N= 12 patients): 40 x 106viable cells/vaccine Dose escalation will proceed as long as 0 of 6 or 1 of 6 patients within a dose level *have* no evidence of toxicity and at least 3 of the 6 patients within a level have reached Week 12 on study. If 2 of 6 patients on any given dose level develop cardiac toxicity, that dose cohort will be closed to further accrual, no higher doses will be explored, and dose expansion of an additional 12 patients will be enrolled in the next lower dose *level* to further assess for cardiac toxicity. The vaccine dose at which 0 to 1 of 18 treated patients in Dose Levels *1 to 3 develop* cardiac toxicity will be defined as the maximum tolerated dose (MTD). If two or more patients out of 18 at a given dose have developed cardiac toxicity, then the MTD will have been exceeded at that dose, and the dose level below that one will be considered the *MTD.* Since enrollment in Dose Level 3 has been reached and 0 of 6 patients has toxicity, then an expansion cohort of an additional 12 patients will be added, for a maximum targeted accrual of 30 patients in Part 1. The Expansion Cohort will be treated at 40 x 106*viable* cells/vaccine and will be monitored for toxicity in an identical manner as previously identified. The 12 patients will be accrued at the 40x10^6 level without any planned interim stop, as cardiac toxicity is not presently anticipated given the results as of 12/11/15. With all 12 enrolled as a single cohort, there is a 4.9% probability of having 2 or more of 12 patients with cardiac toxicity if the true probability for a given patient is 3%, and 55.7% of having 2 or more of 12 with cardiac toxicity if the true probability for a given patient is 15%. Therefore, observing 0-1 of 12 with toxicity would be acceptable, and 40 x10^6 would be considered the maximum evaluated and tolerated dose. If we observe 2 or more with cardiac toxicity at or before accruing the 12th patient, this exceeds a safe level. Further patients would be added subsequently, by amendment, to the next lower dose, to confirm its safety.  
To ensure that the trial does not continue to accrue patients in the event that a much greater incidence of toxicity than expected would be recognized early, the following early stopping rules for accrual will be invoked in each part of the study if:
- Two or more of 6 patients in a given dose develop evidence of cardiac toxicity, accrual will be stopped to that dose level. Accrual expansion of an additional 12 patients will occur in the next lower dose level *to further evaluate for cardiac toxicity.*  
- *Any death (unrelated to underlying primary disease) that occurs and is attributed as* being possible, probably or definitely related to the investigational AdHER2 DC vaccine (FDA requirement).
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11.2 part ii (maximum of

Since the risk for cardiac toxicity may differ in individuals who have previously received HER2directed therapy, the FDA is requesting that repeat dose escalation of AdHER2 DC vaccination be conducted in Part II of the study in a population with **prior exposure** *to trastuzumab,*
pertuzumab, lapatinib, ado-trastuzumab emtansine (TDM1) and other HER2-directed therapies. This population will include adults with recurrent or progressive, metastatic *breast*, gastric/gastroesophageal or other cancers with 1+ to 3+ HER2/neu expression by IHC or an equivocal or positive FISH result. Vaccine dose escalation will be repeated to again determine whether there is an adverse safety signal regarding cardiac toxicity, in addition to assessment of the vaccine's immunogenicity and clinical activity. Vaccine dose escalation, administration, restaging assessment and early stopping rules that will be invoked that are identical to that conducted in Part I. The goal is to accrue up to 30 evaluable patients in Part II of the study and treat them with the experimental AdHER2 vaccine. Unless an early stopping rule is invoked in either Part I or Part II of the study, the number of patients who have experienced a clinically significant cardiac toxicity will be determined to estimate the frequency of cardiac toxicity. In addition, the dose level(s) at which toxicity is observed will also be examined. If in part I, dose escalations have taken place in the first three dose levels *and 0 of 20 patients* have experienced cardiac toxicity, Part II of the study in patients with metastatic *breast* cancer and prior exposure to HER2-directed therapies will proceed with vaccine dosing based on the safety confirmed in Part I and as outlined in Section *3.1.2*. A maximum accrual of 30 patients will be enrolled in Part II and patients will continue to enroll on that dose unless 3 patients have experienced cardiac toxicity.
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11.3 overview

In addition to reporting the observed fraction with toxicity, the 80% and 95% confidence intervals about this fraction will be reported. At the conclusion of the study, the overall fractions with cardiac toxicity among 18, 24 or 30 patients enrolled at one dose or within the entire cohort will be determined. For illustrative purposes only, under a simplifying assumption that the 18-30 patients were enrolled in a single cohort without any intermediate evaluations, the following probabilities suggest that 2+/18, 2+/24 or 3+/18 with cardiac toxicity are more likely to be associated with 15% toxicity (arbitrarily chosen to substantially exceed 7%) than with 3% (arbitrarily chosen to be well below 7%). In any given 18 patients assessed for toxicity, if two or more of the 18 develop toxicity:  
- There is a 10% probability of this occurring if the true toxicity is 3%.  
- There is a 78% probability of this occurring if the true toxicity is 15% (potentially unacceptable).
In any given 24 patients assessed for toxicity (6 at one dose level and 18 at a second level), if two or more of the 24 develop toxicity:
- There is a 16% probability of this occurring if the true toxicity is 3%.  
- There is an 89% probability of this occurring if the true toxicity is 15% (potentially unacceptable).
If there are 30 patients assessed for toxicity (18 at one dose level plus 6 at one level and 6 at another level), if three or more of the 30 develop toxicity:
- There is a 6% probability of this occurring if the true toxicity is 3%.  
- There is an 85% probability of this occurring if the true toxicity is 15% (potentially unacceptable).
As of 12/11/15, 0 of 20 patients enrolled to date that have received a minimum of 2 doses of AdHER2 DC vaccine have developed cardiac toxicity. Since we have observed 0 of 20 subjects with cardiac toxicity, this would have a 68% chance of occurring if the true probability of cardiac toxicity for a given patients was 2%; similarly, it would be 36% if the probability of cardiac toxicity was 5%, 12.2% if the probability of cardiac toxicity was 10% and 3.8% if the probability of cardiac toxicity was 15%. We are assuming that the probability of cardiac toxicity is no more than 10% since higher probabilities of toxicity are associated with a much lower chance of seeing 0 of 20 patients with cardiac toxicity as has been observed. Since 0 of 20 patients already have been documented to have NO cardiac toxicity, and 0 to 2 patients out of 30 would have been acceptable per the original study design, a statistical estimate was performed to determine what the probability of having 0 to 2 of the next 10 patients in the Part 1 Expansion Cohort having cardiac toxicity would be. If the probability of cardiac toxicity were 2%, there would be 99.91% probability of 0 to 2 of 10 with toxicity; if it were 5%, there would be 98.8% probability; and if it were 10%, there would be 93% probability of 0 to 2 of 10 with toxicity. Thus, assuming the next 10 patients would have the same underlying per-patient probability of developing cardiac toxicity as the first 20 had, and it is likely to truly be at or below 10%, there is between a 93% and 99.9% probability that the next 10 patients enrolled in the Part I Expansion Cohort would have 0 - 2 toxicities and therefore 0 - 2 of 30 would have cardiac toxicity as per the original study design. Consequently, FDA approval was received on 12/14/15 to initiate enrollment in Part II of the study in parallel, while enrollment of the Part I Expansion Cohort is completed. The fraction of subjects with immunogenicity, defined as a 3-fold increase in anti-HER2/neu antibody concentration (measured as mcg/mL) or a 4-fold increase in antibody dilution titers over baseline will be reported, along with 80% and 95% confidence intervals, at the following time points: Weeks 4 (after 1 vaccine dose), 8 (after 2 vaccine doses), 12 (after 3 vaccine doses), 28 (after 5 vaccine doses), 48 and 52 weeks and during extended follow-up for monitoring of cardiac adverse events at weeks 76, 100 and 124. Finally, as a secondary objective, the fraction of subjects who haves stable disease, an objective partial response or better by modified irRC will be determined along with appropriate confidence intervals with exceptions to the PR assessment at Week 12 as noted in Sections 3.5.1, **3.5.2** and 6.2.4. The fraction identified in this trial will potentially be useful information for design of any subsequent trials using this vaccine in both of the patient populations being studied. Part I of the study will accrue both males and females with HER2 expressing metastatic cancers for whom trastuzumab is not indicated and who are naïve to HER2-directed therapies. Accrual to Part II of the study will be restricted to females or males with recurrent or progressive metastatic breast, gastric/gastroesophageal or other cancers that have received clinically indicated prior treatment with trastuzumab, pertuzumab, lapatinib, ado-trastuzumab emtansine or other HER2-directed therapies. The study will seek to accrue racially and ethnically diverse population affected by the cancer types targeted for enrollment in this study.  
We expect to accrue at least 2 patients per month with expected completion of accrual within 2.5 years. If after 2 years, 20 or fewer patients have been accrued to Part I of the study, the study will be terminated for failure to accrue at a viable rate. If accrual to Part I of the study has been completed and enrollment in Part II has begun, if after 1 year, 10 or fewer patients have been accrued to Part II of the study, the study will be terminated for failure to accrue at a viable rate. To allow for the possibility of inevaluable patients, an accrual ceiling of 65 will be used.
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11.4 study termination

This study will be terminated for the following reasons:  
- Completion of enrollment accrual and all scheduled monitoring studies for enrolled subjects.  
- Development of clinically significant CTRCD toxicity (defined as a decrease in the LVEF of > 10 percentage points, to a value < 53%) in two or more of the first 10 patients enrolled in the study or four or more of the first 20 patients enrolled in the study.  
- Failure to accrue study subjects in a timely manner.
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12 human subjects protections 12.1

Subjects targeted for enrollment in this study have tumors that express varying levels of HER2, a well-known oncogene that when present, is characterized by more aggressive disease in certain cancers e.g. breast cancer. Significant improvements in clinical outcomes have been realized through the HER2-directed therapies such as trastuzumab and lapatinib. However, despite these successes, the usefulness of these therapies is mitigated by the requirement for a specific level of HER2 expression i.e. HER2 3+ IHC and an equivocal or positive FISH result for trastuzumab to realize clinical outcomes, the continued need for drug administration, and the ultimate development of resistance to treatment with subsequent disease progression. All of these factors point to a need for more effective therapies targeting HER2 designed to address and overcome these issues. Subjects from both gender groups and all racial/ethnic groups are eligible for this study if they meet the eligibility criteria. Pregnant women are excluded from participation in the trial because AdHER DC vaccine may have the potential for teratogenic or abortifacient effects.
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12.2 participation of children

Since this is the first in human clinical trial of this AdHER2 autologous dendritic cell vaccine and the potential safety risks are unknown, patients < 18 years of age will not be allowed to enroll in this study.
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12.3 participation of subjects unable

Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (Section **12.5**), all subjects ≥ age 18 will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for evaluation as needed for the following: an independent assessment of whether an individual has the capacity to provide consent; assistance in identifying and assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint a surrogate. For those subjects that become incapacitated and do not have pre-determined substitute decision maker, the procedures described in MAS Policy 87-4 and NIH HRPP SOP 14E for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and (b) who do not have a legal guardian or durable power of attorney, will be followed.
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12.4 evaluation of benefits and

The AdHER2 DC vaccine being given in this study targets both the extracellular (EC) and transmembrane ™ domains of the HER2 protein receptor. The goal of therapeutic vaccination with this product is to stimulate the patient's own immune system to make *polyclonal antibodies*
(and also potentially killer cells) that recognize HER2. It is hoped that that these antibodies will have a similar beneficial effect in controlling disease and potentially prolonging survival. Use of trastuzumab in women with breast cancer has been shown to be associated with a small, but significant risk (about 7%) for developing cardiac dysfunction. It is usually observed in patients who have previously received chemotherapy that is known to affect the heart. The risk of cardiac dysfunction is greater (about 27%) when trastuzumab is given in combination with anthracyline chemotherapy drugs such as doxorubicin (Adriamycin) and cyclophosphamide (Cytoxan). Exactly how trastuzumab causes cardiac toxicity is unknown. It is also not known whether antibodies made by a person's own immune system will cause cardiac dysfunction. Because of this, cardiac function will be monitored very closely while receiving the vaccine treatment and for 24 months following the last dose of vaccine. In addition, there are strict stopping rules included in this study for any signals of cardiac toxicity that would appear to be exceeding the 7% frequency observed with trastuzumab monotherapy. There are currently multiple clinical trials with vaccines using different platforms and approaches to target HER2 and to date there have been no reports of significant vaccine-associated cardiac toxicity. The only other likely risk associated with receiving this vaccine is local injection site reactions i.e. discomfort, itching, redness, swelling, or soreness/pain at the site where the vaccine is injected into the skin. In addition, due to the proposed mechanism of action that includes a possible acute inflammatory response at focal sites of tumor, subjects may experience discomfort at sites of metastatic disease and/or acute reactive pleural or peritoneal effusions There is a more remote potential for systemic symptoms such as fatigue, malaise or fever associated with vaccination but these are much less likely because patients are being given their own cells that have been transduced to express HER2. The AdHER2 vector used to transduce the patient's autologous dendritic cells is not replication competent and does not integrate into the human genome. The construct has specifically omitted the oncogenic intracellular domain of HER2 to ensure additional safety.  
Unlike trastuzumab or pertuzumab that are monoclonal antibodies, we anticipate that therapeutic vaccination with the AdHER2 DC vaccine will induce a polyclonal antibody *response* that targets multiple epitopes of HER2, since the immunogen contains both the extracellular (EC) and transmembrane (TM) components of HER2. This could potentially be beneficial in patients who have failed trastuzumab, lapatinib or other HER2-directed therapies due to resistance since these agents only recognize one small component of the outer portion of HER2 and as a result, many tumors become resistant. Because we anticipate the development of polyclonal antibodies that recognize multiple sites in HER2, the vaccine may also be beneficial in patients with HER2+ solid tumors as well as adjuvant bladder cancer patients at high risk of disease recurrence for whom trastuzumab is not clinically approved or for those breast cancer who don't have sufficient expression of HER2 i.e. IHC <3+ or an equivocal or positive FISH result and hence weren't eligible for trastuzumab treatment, thereby addressing an unmet medical need. Both of the currently available HER2 therapeutics, trastuzumab and lapatinib target the extracellular component of HER2. Trastuzumab's biologic activity is believed to work by binding to HER2 receptors and interfering with signaling controlling tumor growth and division, as well as signaling the immune system to destroy the cancer cells. Lapatinib is an orally administered tyrosine kinase inhibitor of both the HER2/neu protein and the epidermal growth factor receptor. A third agent currently being investigated in clinical trials is pertuzumab, which is also a humanized monoclonal antibody. It has a unique function in that it prevents HER2 receptor dimerization and subsequently blocks cell signaling. All of these agents must be administered exogenously and continuously for beneficial effect at great cost and often with significant discomfort to the patient. All care will be taken to minimize side effects. Patients will be examined and evaluated prior to enrollment. All evaluations to monitor the treatment of patients will be recorded in the patient chart. If patients suffer any physical injury as a result of the participation in this study, immediate medical treatment is available at the NIH Clinical Center, Bethesda, Maryland. Although no compensation is available, any injury will be evaluated and treated in keeping with the benefits or care to which patients are entitled under applicable regulations. In all publications and presentations resulting from this trial, patients' anonymity will be protected to the maximum extent possible. Authorized personnel from the National Cancer Institute (NCI) and Food and Drug Administration (FDA) or other regulatory authorities may have access to research files in order to verify that patients' rights have been safeguarded. In addition, patient names will be given to the Central Registration to register and verify patients' eligibility.
pre-clinical testingher2neu dc
12.5 risks/benefits analysis

Subjects (including subjects unable to give consent) undergoing therapeutic autologous AdHER2 DC vaccination are likely to experience local injection site reactions commonly associated with the delivery of all vaccines i.e. discomfort, itching, redness, swelling, and/or soreness/pain at the injection site. The potential for adverse systemic symptoms such as fatigue, malaise, fever or acute allergic reactions is unknown but is less likely because patients are being given their own cells. However, because patients are being given their own cells as well as a "self" tumor antigen (HER2), there is the potential for inducing antibodies that react with the patient's own tissues resulting in autoimmune disease. This has not been described with other HER2 vaccines that have been studied but patients will be monitored for the development of autoimmune disease. The greatest concern associated with this therapeutic vaccine platform is the potential development of vaccine-induced antibodies that result in cardiac dysfunction and toxicity. Unlike trastuzumab, where therapy can be withdrawn, there is no way to "turn off" the immune system without significant adverse consequences. Because of this, cardiac function will be monitored very closely while receiving the vaccine treatment and for 24 months following the last dose of vaccine. In addition, stringent stopping rules are in place in the event of any early (or even late) signals of cardiac toxicity. The patients eligible to enroll in Part I of this study have recurrent or progressive metastatic disease as well as adjuvant bladder cancer patients with HER2 expressing tumors or naïve to HER2 targeted therapies that are not candidates for currently available HER2-directed therapies such as trastuzumab and lapatinib. Thus, therapeutic vaccination with the AdHER2 DC vaccine platform proposed in thus study may result in the development of polyclonal antibodies that successfully target and interfere with HER2 expressed at lower levels and on tumors other than breast cancer and provide a potential direct benefit. The patients eligible to enroll in Part II of this study have recurrent or progressive metastatic breast cancer, gastric or gastroesophageal junction cancer have high HER2 expression and have failed or progressed on prior FDA-approved HER2-directed therapies such as trastuzumab, pertuzumab, lapatinib, ado-trastuzumab emtansine (TDM1) or other HER2- directed agents (e.g. MGAH22). The induction of polyclonal antibodies associated with the AdHER2 DC vaccine platform may provide recognition of new HER2 targets and overcome resistance to trastuzumab and lapatinib with the potential for beneficial clinical outcomes. In addition, the results may help the investigators learn more about the disease and develop new treatments for patients with this disease. Overall, preliminary findings support a favorable benefit:risk ratio for the use of AdHER DC vaccine in these patients with tumors with 1-3+ HER2/neu expression. Several measures, including project-specific safety related inclusion/exclusion criteria, regular physical examinations, laboratory testing, etc., throughout the study and toxicity management guidelines have been incorporated into the study protocol to mitigate any potential or identified risks.  
NOTE: This same risks/benefits analysis applies to all study populations included in this clinical study.
pre-clinical testingher2neu dc
12.6 consent process and documentation

The investigational nature and objectives of this trial, the procedures and treatments involved and their attendant risks and discomforts, and the potential benefits will be carefully explained to the patient and the patient's advocate. It will be stated clearly that participation in the research study is voluntary and that participants can withdraw from the study without losing benefits they would otherwise be entitled to. The patient and family members will be encouraged to ask questions. This process will include a general description of the disease process, as well as a description of the patient's expected clinical course. Enrollment on this study will only occur if the patient meets all eligibility criteria. Eligible patients will be presented with a detailed description of the study protocol plan and treatment and provided with a copy of the IRB-approved Informed Consent to review in advance of any discussions with the clinical trials team. The specific requirements, research objectives, risks, alternatives, time commitments and potential benefits will be reviewed with the patient. The patient will be reassured that participation in this study is entirely voluntary and that they may withdraw or decide against receipt of vaccination at any time without adverse consequences. All questions about the study will be answered and alternatives to participation will also be discussed. The Principal Investigator, Lead Associate Investigator or their designee is responsible for obtaining a signed protocol Informed Consent using the current version approved by the NIH Intramural IRB and posted on the web. The original signed informed consent will be placed in the patient's medical record and a copy will be provided to the patient.
pre-clinical testingher2neu dc
12.6.1 telephone consent procedure

Consent on this study may be obtained via telephone according to the following procedure: the informed consent document will be sent to the subject. An explanation of the study will be provided over the telephone after the subject has had the opportunity to read the consent form. The subject will sign and date the informed consent. The original informed consent document will be sent back to the consenting investigator who will sign and date the consent form with the date the consent was obtained via telephone. A fully executed copy will be returned via mail for the subject's records. The informed consent process will be documented in the medical record.
pre-clinical testingher2neu dc
vector derivation

The Ad5f35HER2ECTM vector was produced under cGMP conditions in the Vector Production Facility (VPF) of the Center for Cell and Gene Therapy (CAGT) at Baylor College of Medicine. The knob and fiber of Ad35 is substituted yielding a recombinant Ad5f35 vector that allows for greater viral tropism and more efficient transduction of human dendritic cells (DC) that are the proposed platform for this vaccine as outlined in Figure 12.
figure 12 ad5f35 human her2/neu
vector derivation

13.1.1.2 Vector Manufacturing Summary The vector was manufactured under cGMP conditions by the Baylor College of Medicine Center for Cell and Gene Therapy (CAGT). The adenoviral vector was initially purified by two rounds of plaque isolation and then serially propagated on human embryonic kidney cells (HEK-293). The final amplified adenoviral vector was purified by ultracentrifugation using double cesium chloride gradients. Residual cesium chloride was removed by dialysis of the solution containing the vector against the formulation buffer. Refer to adenoviral vector cGMP production as outlined in **Appendix 10.**
An initial Master Virus Bank of Ad5f35HER2ECTM was generated (VM1003) and underwent two rounds of plaque purification with ultimate generation of purified Master Virus Bank Bulk Vector (VMB1003) which was subsequently used to produce the first clinical clot of vector that was then vialed for clinical use. The Ad5f35HER2ECTM clinical product is labeled VMC1003 and contains 1x10e12 viral particles/mL and 0.2mL/vial. A map of the Ad5f35HER2ECTM
vector is outlined in **Appendix 11**.  
13.1.1.3 Vector Quality Assurance/Quality Control (QA/QC) Per contractual requirements with the Baylor College of Medicine CAGT, the manufacture and testing of the final clinical Ad5f35HER2ECTM vector product (both bulk and vialed) was conducted under current Good Manufacturing Practice (cGMP) guidelines in accordance with the cGMP Regulations outlined in 21 CFR Parts 210 and 211. CAGT is required to prepare and complete documentation as manufacturing tasks have been performed and this documentation is subsequently reviewed by the CAGT QA division. This documentation includes sequence verification, the manufacturing batch records, certificate of analysis and any other manufacturing related information for CMC documentation required for IND filing.
figure 12 ad5f35 human her2/neu
13.1.2 toxicity

There is no known toxicity of the Ad5f35HER2ECTM vector. It is a replication incompetent vector that does not integrate into the genomic DNA of the host cells it is used to transduce/transfect. Transduction of dendritic cells is associated with some reduction in cell viability that his highly donor dependent based on pre-clinical studies characterizing the proposed transduction process that will be used in this study in normal donors. The Ad5f35HER2ECTM vector has undergone replication capacity assay (RCA) assay testing that has been reviewed by the FDA and determined to be acceptable for its proposed use in this study. 13.1.3 Formulation and Preparation 13.1.3.1 Vector Vialing and Storage The product labeling for the *first* **clinical lot of Ad5f35HER2ECTM vector (VMC1003)** that was vialed on 12/21/10 and **that will be used in the current study** for transduction of autologous dendritic cells to generate vaccines for this study is: