untitled h1
untitled h2

Abbreviated Title: Pexa-Vec Colorectal Ca CC **Protocol: 17C0092 H**
IBC \# RD-16-XII-15 OSP \# 1706-1624 Version Date: 06/15/2020 NCT: NCT03206073 Title: A Phase I/II study of Pexa-Vec Oncolytic Virus in Combination with Immune Checkpoint Inhibition in Refractory Colorectal Cancer.  
NCI Principal Investigator: Tim Greten, MD
Thoracic & GI Malignancies Branch National Cancer Institute Building 10, Room 2B38B 9000 Rockville Pike Bethesda, MD 20892 240-760-6114 gretentf@mail.nih.gov
untitled h1
investigational agents

| Drug Name:     | Tremelimumab; Durvalumab; Pexastimogene Devacirepvec (PexaVec)                                                       |
|----------------|-------------------------------------------------------|
| BB IND Number: | 17363                                                 |
| Sponsor:       | Center for Cancer Research, National Cancer Institute |
| Manufacturers: | MedImmune/AstraZeneca Sillajen Inc.                   |  
1
untitled h1
precis

Background:  
- Immune-based approaches in colorectal cancer have unfortunately - with the notable exception of immune checkpoint inhibition in microsatellite instable (MSI-hi) disease - been largely unsuccessful. The reasons for this are unclear but no doubt relate to the fact that in advanced disease colorectal cancer appears to be less immunogenic, as evidenced by the lack of infiltrating lymphocytes with advancing T stage
- Pexa-Vec (JX-594) is a thymidine kinase gene-inactivated oncolytic vaccinia virus engineered for the expression of transgenes encoding human granulocyte- macrophage colony-stimulating factor (GM-CSF) and β-galactosidase. Apart from the direct oncolytic activity, oncolytic viruses such as Pexa-Vec have been shown to mediate tumor cell death via the induction of innate and adaptive immune responses
- Tremelimumab is a fully human monoclonal antibody that binds to CTLA-4 expressed on the surface of activated T lymphocytes and causes inhibition of B7-CTLA-4-mediated downregulation of T-cell activation. Durvalumab is a human monoclonal antibody directed against PD-L1.  
- The aim of the study is to evaluate whether the anti-tumor immunity induced by Pexa-Vec oncolytic viral therapy can be enhanced by immune checkpoint inhibition.
Objective:
- To determine the safety, tolerability and feasibility of Pexa-Vec oncolytic virus in combination with immune checkpoint inhibition in patients with refractory metastatic colorectal cancer.  
Eligibility:
- Histologically confirmed metastatic colorectal cancer.  
- Patients must have progressed on, been intolerant of or refused prior oxaliplatin- and irinotecan-containing, fluorouracil-based, chemotherapeutic regimen and have disease that is not amenable to potentially curative resection. Patients who have a known KRAS wild type tumor must have progressed, been intolerant of or refused cetuximab or panitumumabbased chemotherapy.  
- Patients tumors must be documented to be microsatellite-stable (MSS) either by genetic analysis or immunohistochemistry OR microsatellite-high with documented disease progression following anti-PD1/PDL1 therapy.  
- Patients must have at least one focus of metastatic disease that is amenable to pre- and ontreatment biopsy.  
- Willingness to undergo mandatory tumor biopsy.
untitled h1
design

- The proposed study is Phase I/II study of Pexa-Vec oncolytic virus at two dose levels in combination with immune checkpoint inhibition in patients with metastatic colorectal cancer.  
- Patients will receive Pexa-Vec, administered IV every 2 weeks for 4 doses, in 4 separate arms A1, A2, B1, and B2. The first administration will be on Day –(minus) 12, followed by administration on Days 2, 16 and 30 (i.e. 4 doses in total).  
o Arms A1 and A2: In addition to the oncolytic virus patients will also receive durvalumab at a flat dose of 1500 mg beginning on Day 1 followed by q28days until off-treatment criteria are met.  
o Arms B1 and B2: In addition to the oncolytic virus patients will also receive tremelimumab 300 mg and durvalumab 1500 mg on Day 1 followed by q28days subsequent continuation of the durvalumab alone until off-treatment criteria are met.
- All patients will undergo a baseline tumor biopsy and a post treatment biopsy.  
- Accrual ceiling will be set at 35 to allow for patients replaceable for reasons other than toxicity.  
- Patients will be restaged every 8 weeks +/- 3 days  
| TABLE OF CONTENTS                                                                     |                                                                                                                       |                |
|---------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------|----------------|
| PRÉCIS .                                                                              |                                                                                                                       |                |
| TABLE OF CONTENTS.                                                                    |                                                                                                                       |                |
| l                                                                                     | INTRODUCTION .                                                                                                        |                |
| 1.1                                                                                   | Study Objectives .                                                                                                    |                |
| Primary Objective                                                                     |                                                                                                                       |                |
| 1.1.1                                                                                 |                                                                                                                       |                |
| 1.1.2                                                                                 | Secondary Objectives:                                                                                                 |                |
| 1.1.3                                                                                 | Exploratory Objectives:                                                                                               |                |
| Background and Rationale .                                                            |                                                                                                                       |                |
| 1.2                                                                                   | Metastatic colorectal cancer (CRC) and the current therapeutic paradigm ........... 10                                |                |
| 1.2.1                                                                                 |                                                                                                                       |                |
| 1.2.2                                                                                 | Pexa-Vec (pexastimogene devacirepvec; JX-594) ................................................................ 11     |                |
| 1.2.3                                                                                 | Induction of adaptive immunity                                                                                        |                |
| 1.3                                                                                   | T Tremelimumab .                                                                                                      |                |
| 1.4                                                                                   | D DURVALUMAB                                                                                                          |                |
| 1.5                                                                                   | Anti-CTLA4 in combination with anti-PD1/PD-L1 ............................................................ 18         |                |
| Durvalumab and tremelimumab dose and treatment regimen justification ........... 18   |                                                                                                                       |                |
| 1.5.1                                                                                 |                                                                                                                       |                |
| 1.5.2                                                                                 | Durvalumab monotherapy dose rationale                                                                                 |                |
| 1.5.3                                                                                 | Fixed Dosing for durvalumab and tremelimumab .                                                                        |                |
| 1.6                                                                                   | Rationale for timing and mandatory nature of the biopsies ...................................................... 23   |                |
| Amendment D Rationale                                                                 |                                                                                                                       |                |
| 1.7                                                                                   |                                                                                                                       |                |
| 2                                                                                     | ELIGIBILITY ASSESSMENT AND ENROLLMENT.                                                                                |                |
| 2.1                                                                                   | Eligibility Criteria .                                                                                                |                |
| 2.1.1                                                                                 | Inclusion Criteria .                                                                                                  |                |
| 2.1.2                                                                                 | Exclusion Criteria .                                                                                                  |                |
| 2.1.3                                                                                 | Recruitment Strategies .                                                                                              |                |
| 2.2                                                                                   | Screening Evaluation                                                                                                  |                |
| Screening activities performed after a consent for screening has been signed...... 29 |                                                                                                                       |                |
| 2.2.1                                                                                 |                                                                                                                       |                |
| 2.3                                                                                   | Participant Registration and Status Update Procedures ............................................................ 29 |                |
| 2.4                                                                                   | Treatment Assignment Procedures (for registration purposes only):                                                     |                |
| 2.5                                                                                   | Baseline Evaluation                                                                                                   |                |
| STUDY IMPLEMENTATION .                                                                |                                                                                                                       |                |
| 3                                                                                     | 3.1                                                                                                                   | Study Design . |
| Dose Limiting Toxicity (DLT) .                                                        |                                                                                                                       |                |
| 3.1.1                                                                                 | 4                                                                                                                     |                |  
| Version Date: 06/15/2020 3.1.2 Maximum tolerated dose (MTD) 35 3.1.3 Protocol Stopping Rules 35 3.2 Drug Administration 35 3.2.1 Pexa-Vec Drug Administration 35 3.2.2 Durvalumab Administration 36 3.2.3 Tremelimumab Administration 36 3.2.4 Monitoring of Dose Administration 36 3.3 Dose Modifications 37 3.3.1 Pexa-Vec 37 3.3.2 Tremelimumab and durvalumab 37 3.4 Management of Expected Adverse Events and Events of Special Interest 37 3.4.1 Expected Toxicities of Pexa-Vec Treatment 37 3.4.2 Potential Mechanisms of Pexa-Vec Shedding to the Environment,  Biodissemination and Prevention of Transmission 39 3.4.3 Adverse Events of Special Interest for Durvalumab and Tremelimumab 39 3.5 Follow Up Period 42 3.6 Study Calendar 43 3.7 Criteria for Removal from Protocol Therapy and Off Study Criteria 47 3.7.1 Criteria for removal from protocol therapy: 47 3.7.2 Off Study Criteria 47 4 CONCOMITANT MEDICATIONS/MEASURES 47 4.1 Recommended Medications Related to Flu-like Symptoms related to Pexa-Vec 48 4.2 Management of Hypotension and Contraindicated Treatments 48 4.3 Agents with Potential Inhibitory Activity Against Vaccinia Viruses 48 4.4 Contraindicated Antiviral Therapy Shown to Inhibit Vaccinia Replication 48 4.5 Contraindicated Treatments 49 4.6 Other Authorized Concomitant Treatments 49 4.7 Permitted concomitant medications for any combination of investigational drugs 49 4.8 Excluded Concomitant Medications 50 4.9 Methods of contraception 51 4.9.1 Female patient of child-bearing potential 51 4.9.2 Male patients with a female partner of childbearing potential 51 4.9.3 Highly effective methods of contraception 52   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Version Date: 06/15/2020                                                                                    |                                                                                                            |                                  |
|-------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------|----------------------------------|
| 4.10                                                                                                        | Blood donation .                                                                                           |                                  |
| BIOSPECIMEN COLLECTION .                                                                                    |                                                                                                            |                                  |
| S                                                                                                           | 5.1                                                                                                        | Correlative Studies for Research |
| 5.1.1                                                                                                       | Immune monitoring                                                                                          |                                  |
| 5.1.2                                                                                                       | Mandatory tumor biopsies .                                                                                 |                                  |
| Tumor tissue analysis                                                                                       |                                                                                                            |                                  |
| 5.1.3                                                                                                       |                                                                                                            |                                  |
| 5.2                                                                                                         | Sample Storage, Tracking and Disposition .                                                                 |                                  |
| 5.2.1                                                                                                       | Samples Managed by Dr. Figg's Blood Processing Core (BPC)                                                  |                                  |
| 5.2.2                                                                                                       | Samples Managed by the Laboratory of Dr Greten .                                                           |                                  |
| 5.2.3                                                                                                       | Protocol Completion/Sample Destruction .                                                                   |                                  |
| 5.3                                                                                                         | Samples for Genetic/Genomic Analysis .                                                                     |                                  |
| T TCR beta sequencing                                                                                       |                                                                                                            |                                  |
| 5.3.1                                                                                                       | Nanostring analysis (nCounterPan Cancer Immunology Profile) .............................. 56              |                                  |
| 5.3.2                                                                                                       |                                                                                                            |                                  |
| 5.3.3                                                                                                       | Privacy and Confidentiality of medical information/biological specimens ........... 56                     |                                  |
| 5.3.4                                                                                                       | Management of Results                                                                                      |                                  |
| 6                                                                                                           | DATA COLLECTION AND EVALUATION                                                                             |                                  |
| 6.1                                                                                                         | DATA COLLECTION                                                                                            |                                  |
| 6.2                                                                                                         | DATA SHARING Plans .                                                                                       |                                  |
| 6.2.1                                                                                                       | Human Data Sharing plan                                                                                    |                                  |
| Genomic Data Sharing Plan                                                                                   |                                                                                                            |                                  |
| 6.2.2                                                                                                       |                                                                                                            |                                  |
| 6.3                                                                                                         | Response Criteria                                                                                          |                                  |
| 6.3.1                                                                                                       | Definitions                                                                                                |                                  |
| Disease Parameters .                                                                                        |                                                                                                            |                                  |
| 6.3.2                                                                                                       | Methods for Evaluation of Measurable Disease .                                                             |                                  |
| 6.3.3                                                                                                       |                                                                                                            |                                  |
| 6.3.4                                                                                                       | Response criteria                                                                                          |                                  |
| 6.3.5                                                                                                       | D Duration of Response                                                                                     |                                  |
| Modified Immune-related response criteria (irRC) ....................................................... 62 |                                                                                                            |                                  |
| 6.3.6                                                                                                       |                                                                                                            |                                  |
| 6.3.7                                                                                                       | Progression-Free Survival                                                                                  |                                  |
| 6.4                                                                                                         | TOXICITY Criteria                                                                                          |                                  |
| 7                                                                                                           | NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN.. 62                                          |                                  |
| Definitions                                                                                                 |                                                                                                            |                                  |
| 7.1                                                                                                         |                                                                                                            |                                  |
| 7.2                                                                                                         | OHSRP Office of Compliance and Training / IRB Reporting................................................ 62 |                                  |
| 7.2.1                                                                                                       | Expedited Reporting                                                                                        |                                  |
| 6                                                                                                           |                                                                                                            |                                  |  
| Version Date: 06/15/2020 7.2.2 IRB Requirements for PI Reporting at Continuing Review 62 7.3 NCI Clinical Director Reporting 62 7.4 INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) REPORTING CRITERIA 63 7.4.1 Serious Adverse Event Reports to IBC 63 7.4.2 Annual Reports to IBC 63 7.4.3 Clinical Trial Information 63 7.4.4 Progress Report and Data Analysis 63 7.5 NIH Required Data and Safety Monitoring Plan 64 7.5.1 Principal Investigator/Research Team 64 7.5.2 Safety Monitoring Committee (SMC) 64 8 SPONSOR PROTOCOL/SAFETY REPORTING 64 8.1 Definitions 64 8.1.1 Adverse Event 64 8.1.2 Serious Adverse Event (SAE) 65 8.1.3 Life-threatening 65 8.1.4 Severity 65 8.1.5 Relationship to Study Product 65 8.1.6 Adverse Events of Special Interest (AESI) 65 8.2 Assessment of Safety Events 65 8.3 Reporting of Serious Adverse Events 66 8.4 Waiver of expedited reporting to CCR 66 8.5 Safety Reporting Criteria to the Pharmaceutical Collaborators 67 8.5.1 Reporting of serious adverse events 67 8.5.2 Overdose 67 8.5.3 Hepatic function abnormality 68 8.6 Reporting Pregnancy 68 8.6.1 Maternal exposure 68 8.6.2 Paternal exposure 68 8.7 Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND 69 9 CLINICAL MONITORING 69 10 STATISTICAL CONSIDERATIONS 69 11 COLLABORATIVE AGREEMENT 71 11.1 Cooperative Research and Development Agreement (CRADA) 717   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Version Date: 06/15/2020          |                                                                                                                                                    |
|-----------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------|
| 11.1.1                            | Medimmune/Astrazeneca                                                                                                                              |
| 11.1.2                            | Sillajen Inc.                                                                                                                                      |
| Material Transfer Agreement (MTA) |                                                                                                                                                    |
| 1.2                               |                                                                                                                                                    |
| 12                                | HUMAN SUBJECTS PROTECTIONS .................................................................................................................... 71 |
| 12.1                              | Rationale for Subject Selection                                                                                                                    |
| 12.2                              | Participation of Children                                                                                                                          |
| 12.3                              | Participation of subjects unable to give consent                                                                                                   |
| 12.4                              | Evaluation of Benefits and Risks/Discomforts                                                                                                       |
| RISKS/BENEFITS ANALYSIS           |                                                                                                                                                    |
| 12.5                              |                                                                                                                                                    |
| 12.5.1                            | Risk of Biopsy                                                                                                                                     |
| 12.5.2                            | Risks of exposure to ionizing radiation                                                                                                            |
| 12.5.3                            | Risks of CT Scans                                                                                                                                  |
| 12.5.4                            | Research Blood Collection Risks                                                                                                                    |
| Other Risks/Benefits .            |                                                                                                                                                    |
| 12.5.5                            |                                                                                                                                                    |
| 12.5.6                            | Risks/Benefits Analysis                                                                                                                            |
| Consent Process and Documentation |                                                                                                                                                    |
| 12.6                              |                                                                                                                                                    |
| 13                                | PHARMACEUTICAL INFORMATION                                                                                                                         |
| 13.1                              | Tremelimumab (17363)                                                                                                                               |
| 13.1.1                            | SOURCE .                                                                                                                                           |
| 13.1.2                            | TOXICITY                                                                                                                                           |
| 13.1.3                            | Formulation and preparation                                                                                                                        |
| 13.1.4                            | Stability and Storage                                                                                                                              |
| Administration procedures         |                                                                                                                                                    |
| 13.1.5                            |                                                                                                                                                    |
| 13.2                              | D Durvalumab (17363)                                                                                                                               |
| 13.2.1                            | SOURCE .                                                                                                                                           |
| 13.2.2                            | TOXICITY                                                                                                                                           |
| Formulation and Preparation       |                                                                                                                                                    |
| 13.2.3                            | Stability and Storage                                                                                                                              |
| 13.2.4                            |                                                                                                                                                    |
| 13.2.5                            | Administration procedures                                                                                                                          |
| 13.3                              | Pexa-Vec (17363) .                                                                                                                                 |
| SOURCE .                          |                                                                                                                                                    |
| 13.3.1                            |                                                                                                                                                    |
| 13.3.2                            | Toxicity .                                                                                                                                         |
| 13.3.3                            | Formulation and Preparation                                                                                                                        |
| 8                                 |                                                                                                                                                    |  
| 13.3.4   | Stability and Storage 80                                                         |
|----------|----------------------------------------------------------------------------------|
| 13.3.5   | Handling of Pexa-Vec 80                                                          |
| 13.3.6   | Preparation and administration of Pexa-Vec 81                                    |
| 13.3.7   | Cleaning / Disinfection and Disposal 81                                          |
| 13.3.8   | Spills or Environmental Contamination 81                                         |
| 13.3.9   | Unused Drug Return or Destruction 81                                             |
| 14       | REFERENCES 83                                                                    |
| 15       | APPENDICES 85                                                                    |
| 15.1     | Appendix A-Performance Status Criteria 85                                        |
| 15.2     | Appendix B - Tremelimumab and Durvalumab Dosing Modifications 86                 |
| 15.3     | Appendix C: Modified immune-related response criteria (irRC) 112                 |
| 15.4     | Appendix D: List of Adverse Events related to study treatment before amendment D |
|          | 114                                                                              |
untitled h1
1 introduction 1.1 study objectives

- To determine the safety, tolerability and feasibility of Pexa-Vec oncolytic virus in combination with immune checkpoint inhibition in patients with refractory metastatic colorectal cancer.
untitled h1
1.1.2 secondary objectives

- To determine the response rate (by RECIST 1.1) of Pexa-Vec oncolytic virus in combination with immune checkpoint inhibition in patients with refractory metastatic colorectal cancer.  
- To evaluate the 5-month and overall progression-free survival and overall survival in patients with metastatic colorectal cancer during and following treatment with of Pexa-Vec oncolytic virus in combination with immune checkpoint inhibition in patients with refractory metastatic colorectal cancer.
untitled h1
1.1.3 exploratory objectives

- To measure changes in immune parameters in the peripheral blood and tumors in patients with metastatic colorectal cancer during and following treatment with Pexa-Vec oncolytic virus in combination with immune checkpoint inhibition.
untitled h1
1.2 background and rationale 1.2.1

Colorectal cancer is a highly prevalent cancer, with approximately 300,000 new cases diagnosed annually in the U.S. and Europe. Over the past 10-15 years' new chemotherapeutics (oxaliplatin and irinotecan) in addition to so-called targeted agents (bevacizumab and cetuximab/panitumumab) have resulted in improvements in the median overall survival from 1012 months to approximately 2 years. Once a patient progress after first line treatment however, the average survival is in the 12-14-month range, and a huge unmet need exists to improve options for patients with this disease. Immune-based approaches in colorectal cancer have unfortunately - with the notable exception of immune checkpoint inhibition in MSI-hiI disease - been largely unsuccessful. The reasons for this are unclear but no doubt relate to the fact that in advanced disease colorectal cancer appears to be less immunogenic, as evidenced by the lack of infiltrating lymphocytes with advancing T stage[1, 2]. The prevalence of colorectal cancer and the high proportion of patients with metastatic disease who are microsatellite stable (85-90%) mean that this is a large territory of each oncology clinic for whom any advance in immunotherapy would be extremely significant. Given the apparent lack of intrinsic anti-tumor immunity in these patients, innovative immune-stimulative approaches are needed. Oncolytic virus technology falls into this category, their great potential being their ability to induce immunity in tumors[3] such as microsatellite-stable (MSS) colorectal cancer - where low mutagenic burden result in low endogenous immunity (and lack of single-agent activity to checkpoint inhibitors).
untitled h1
1.2.2 pexa-vec (pexastimogene devacirepvec jx-594)

Oncolytic immunotherapy represents a novel therapeutic platform for the treatment of cancer with unique attributes compared to conventional chemotherapy. Oncolytic viruses are native or engineered viruses that preferentially replicate in and lyse cancer cells. Selective tumor cell replication is thought to depend on infection of neoplastic cells, which harbor low levels of protein kinase R (PKR) and dysfunctional type I IFN signaling elements. These changes allow more efficient viral replication, and with selected deletion of specific viral genes, replication in normal cells with activated PKR may not be possible. The FDA recently approved the first oncolytic virus for cancer [Talimogene laherparepvec (T-VEC)], for melanoma on October 15, 2015. Pexa-Vec (JX-594) is a thymidine kinase gene-inactivated oncolytic vaccinia virus engineered for the expression of transgenes encoding human granulocyte- macrophage colony-stimulating factor (GM-CSF) and β-galactosidase. Most of the oncolytic clinical trials to date have involved intratumoral injection. Whilst Pexa-Vec can (and has been) also administered in this manner, it has the advantage of being able to be administered systemically by intravenous administration. Clinical experience: More than 300 patients have been treated with Pexa-Vec administered either intravenously (IV), intratumorally (IT) or both IV and IT. In total, approximately 1200 patient doses of Pexa-Vec have been administered (IV and IT combined). Pexa-Vec treatment has generally been well-tolerated with main side effects being transient flu-like symptoms and transient hypotension. A randomized Phase 2a trial in HCC demonstrated a significant increase in OS at high dose Pexa-Vec versus low dose Pexa-Vec and a Phase 3 trial in first-line HCC is ongoing[4]. In a phase 1 trial of a single intravenous infusion of Pexa-Vec in 23 patients with advanced solid tumors, dose-related antitumor activity was observed and normal tissues were not affected clinically[5]. Pexa-Vec has been administered safely to patients with colorectal cancer with proof of concept for tumor penetration and selectivity[6]. Thus far, at least 60 patients with colorectal cancer have been treated across several Phase 1/2 studies, again demonstrating that Pexa-Vec therapy was well-tolerated. Reproducible infection of CRC metastases following IV Pexa-Vec was confirmed on post treatment biopsies[5].  
The most common adverse events (AEs) reported with Pexa-Vec have been acute, transient flulike symptoms, including fever and chills. Transient, hypotension responsive to fluid administration was also noted in a subset of patients within 24 hours of treatment. Pexa-Vec related skin pustules were observed in 22% (64/292) of patients receiving IV or IT treatments. Pustules developed after 9% (62/691) of IV infusions. The pustules were self-limited and resolved without sequela within 2-3 weeks of treatment, consistent with the timeline for pustule resolution utilizing wild-type vaccinia vaccine. 1.2.2.1 Phase 1b Study of Patients with Colorectal Cancer Treated Intravenously Every Two Weeks with Pexa-Vec Fifteen patients with treatment- refractory colorectal cancer were enrolled on a phase 1b study of Pexa-Vec (also known as JX-594). Pexa-Vec was administered intravenously every 14 days, at dose levels of 1 × 106, 1 × 107, or 3 × 107 plaque-forming units (pfu)/kg. The primary endpoint was to determine the maximum tolerated dose. Secondary endpoints were pharmacokinetics and pharmacodynamics as well as antitumor activity. All patients received at least two Pexa-Vec doses. No dose-limiting toxicities were reported, and the maximum tolerated dose was not reached. The most common adverse events were grade 1/2 flu-like symptoms, generally lasting <24 hours. There was some preliminary evidence of efficacy with the majority of patients (67%) having radiographically stable disease. 1.2.2.2 Phase 1/2a Study of Patients with Colorectal Carcinoma Treated Intravenously (IV)
Weekly with Pexa-Vec Alone and in Combination with Irinotecan A Phase 1/2a dose-escalation study of Pexa-Vec administered weekly alone or in combination with irinotecan in patients with Stage 4, chemotherapy refractory CRC has completed enrollment.  
A total of 52 patients received 5 IV doses of Pexa-Vec weekly at doses of 3x108 pfu to 1x109 pfu alone or in combination with irinotecan. Pexa-Vec alone and in combination with irinotecan was generally well-tolerated with no dose- limiting toxicities experienced. Pexa-Vec has been well-tolerated in study patients with most AEs being mild-to- moderate in severity. The most common Pexa-Vec treatment related AEs identified as a percent of total patients treated included generally mild-to-moderate: pyrexia (94%), chills (84%), hypotension (62%), nausea (46%), and rash pustular (42%). No Grade 4 or 5 AEs related to treatment were reported. The following Grade 3 AEs related to Pexa-Vec single-agent treatment were experienced by (3.6%, n = 1) of patient experienced each of the following: chills, hypotension, fatigue, hypertension, abdominal pain, blood bilirubin increased, cytokine release syndrome, hypoxia, ALT increased, AST increased, somnolence, and troponin increased. No Grade 4 or Grade 5 AEs related to Pexa-Vec were reported. 1.2.2.3 Other clinical trial data for Pexa-Vec
untitled h1
1.2.2.3.1 phase 1 liver tumor

A Phase 1 dose-escalation trial of Pexa-Vec in patients with primary or metastatic liver tumors (JX594-IT-HEP001) has been completed and the results published[6]. Pexa-Vec was administered by IT injection every 3 weeks in patients with refractory, injectable tumors within the liver at one of 4 dose levels (1 × 108 pfu to 3 × 109 pfu). Patients were scheduled to receive 2–4 treatments, but could subsequently receive an additional 4 treatments if there was evidence response or clinical benefit. Fourteen patients, including 3 HCC patients were enrolled and treated. Pexa-Vec was well-tolerated in study patients. All patients treated with Pexa-Vec experienced mild-to-moderate flu-like symptoms, which included fever, chills, anorexia, aches/pain, fatigue, headache, and/or nausea. No significant organ toxicity was reported. Two patients were treated at the 3 × 109 pfu dose and both patients experienced dose limiting toxicities (DLTs) after a single treatment with Pexa-Vec. The DLTs experienced by these study patients included asymptomatic Grade 3 hyperbilirubinemia (n = 2), and Grade 3 anorexia and right upper quadrant pain (n = 1). Hyperbilirubinemia was apparently due to tumor swelling post-treatment and occlusion of the adjacent bile duct. The maximum-tolerated dose on the study was therefore defined as 1 × 109 pfu.  
Response and/or stable disease were observed in 9 out of the 10 evaluable patients. Target tumor responses were demonstrated in 8 patients by RECIST criteria and/or Choi criteria. Responding patients had various tumor types, including HCC. In 3 cases positron-emission tomography - computed tomography (PET-CT) scans demonstrated a decrease in injected tumor metabolic activity (10–100% decrease standardized uptake value [SUV]). Eight patients (57%) survived for at least 8 months, and up to 72+ months. The median cancer-specific survival was 9 months. 1.2.2.3.2 Phase 2 Liver Cancer (HCC) Trial (JX594-IT-HEP007) A Phase 2 randomized dose-finding trial of Pexa-Vec in patients with liver cancer (HCC) (JX594- IT-HEP007) has been completed and the results published[4]. Pexa-Vec was administered by IT injection every 2 weeks for 3 total doses in patients with injectable tumors within the liver. Study dose levels were 1 x 108 pfu (Arm A) and 1 x 109 pfu (Arm B). Thirty patients were treated (16 patients in Arm A, 14 patients in Arm B). Twenty-nine patients received all 3 planned injections; one low-dose patient received 2 Pexa-Vec treatments. IT injection was well-tolerated at both dose levels in this population of patients with HCC. One treatment-related serious adverse event (SAE) was reported in the high-dose group (nausea and vomiting requiring prolonged hospitalization). Flu-like symptoms (Grade 1–2) occurred in all patients over the first 12–24 hours after treatment, including fever, rigors, nausea or vomiting. Four patients responded to treatment based on mRECIST criteria (1 complete response; 3 partial responses). Responses were observed in injected and non-injected tumors. Further, overall survival was significantly longer in the high-dose arm compared with the low-dose arm (median 14.1 months versus 6.7 months, hazard ratio [HR] 0.39; p-value 0.020, Gehan-Breslow-Wilcoxon test; 1-sided test for superiority of high-dose (**Figure 1**).  
The median overall survival was 9.0 months for the entire population.  
  
Twenty-five patients were enrolled on this Phase 2 study investigating one IV dose of Pexa-Vec, followed by 2 IT injections (one week and 3 weeks after the IV infusion), prior to initiation of standard sorafenib therapy. Twenty patients were resistant to sorafenib therapy before Pexa-Vec treatment. Transient flu-like symptoms following Pexa-Vec treatments were the most common AEs. Subsequent therapy with sorafenib was well-tolerated. The 2 agents were not used simultaneously, as sorafenib inhibits Pexa-Vec replication and can potentially impair its activity. Transient decreases in white blood cell (WBC) counts, in particular neutrophils and lymphocytes, may occur within the first 24 hours following each Pexa-Vec treatment dose. Modified Choi responses and/or disease stabilization were observed following IV and IT Pexa-Vec therapy prior to and following standard sorafenib therapy. One patient exhibited a partial response by RECIST criteria in addition to a response by Choi criteria.  
1.2.2.3.4 Phase 2b Randomized Trial of Pexa-Vec Plus Best Supportive Care Versus Best Supportive Care in Patients with Advanced Hepatocellular Carcinoma Who Have Failed Sorafenib Treatment (JX594-HEP018/TRAVERSE)
One hundred and twenty-nine patients having failed previous sorafenib therapy were enrolled on this Phase 2b study and randomized 2:1 to receive either Pexa-Vec plus best supportive care (BSC) or BSC alone. Eighty-six patients were assigned to the Pexa-Vec arm and were to receive one IV dose followed by up to 5 IT injections (1, 3, 6, 12, and 18 weeks after the IV infusion), and 43 to BSC-only arm. The most common AEs related to Pexa-Vec experienced by patients in Arm A included: pyrexia (78.6%), chills (50.0%), pustular rash (28.6%), hypotension (26.2%), and nausea (25%). These AEs were generally mild-to-moderate in severity and tolerable with the exception of hypotension, which was more severe than previously observed in other trials of Pexa-Vec. Grade 3 AEs possibly or probably related to Pexa-Vec included pyrexia (8.3%), hypotension (8.3%), blood bilirubin increased (4.8%), anemia (3.6%), blood aspartate transaminase (AST) increased (3.6%); fatigue, hepatic encephalopathy, hypertension, platelet count decreased, vomiting, (2.4% for each event); abdominal pain and blood alanine aminotransferase (ALT) increased (1.2% for each event). Two Grade 4 or Grade 5 events were noted: respiratory failure (Grade 4) and hepatic failure (Grade 5). Six patients presented with at least one Grade 3–4 AE possibly or probably related to IT injection procedure (7.1%): these AEs included hypotension (2.4%), upper abdominal pain, acute respiratory failure, anemia, ascites, fluid overload, hepatic hemorrhage, pleural effusion, acute renal failure, staphylococcal sepsis, and troponin increase (1.2% for each event). No procedurerelated AE resulted in the patient's death.  
The most frequent Pexa-Vec-related SAE, which occurred in 6 patients (8 SAEs) was severe hypotension defined as a systolic blood pressure <90 mmHg, lasting and requiring a medical treatment for at least 24 hours after Pexa-Vec administration. Notably, anti-hypertensive medication was ongoing at the time of treatment with Pexa-Vec prior to the development of hypotension in the majority of these patients, thereby possibly exacerbating the potential for hypotension. Treatment with Pexa-Vec did not improve overall survival or other efficacy measures, compared with BSC, in patients with advanced HCC in this open-label study. No significant improvement of overall survival was shown in Arm A compared to Arm B (p = 0.426, stratified log-rank test) for the Intent-to-Treat (ITT) population. Median overall survival was 4.2 (95% confidence interval [CI]: 3.3 to 5.4 months) vs 4.4 months (95% CI: 3.2 to 6.0 months) for Arm A vs Arm B, respectively. HR observed was 1.19 (95% CI: 0.78 to 1.80). The overall disease control rate (proportion of patients with complete response [CR], partial response [PR], or SD) during the study was 13% (95% CI: 7% to 22%) vs 19% (95% CI: 8% to 33%) for Arm A vs Arm B, respectively. Notably, the majority of patients in TRAVERSE did not receive the complete protocol specified treatment regimen of JX-594. In Arm A, 12 of 86 randomized patients (13.9%) received all 6 planned treatments; and only half of the patients (51.2%) received at least 3 IT treatments (1 IV plus 3 IT) as administered in previous JX-594 HCC trials.  
The limited number of patients completing treatment on Arm A in conjunction with the significantly shorter median overall survival (~4.2 vs 4.4 months on Arms A and B, respectively) observed in this study versus studies of other agents for second line HCC (~7–9 months) suggests a relatively more advanced HCC patient population was included in TRAVERSE.
untitled h1
1.2.3 induction of adaptive immunity

The concept of combining oncolytic viruses with immune-modulation - and specifically immune checkpoint inhibition - has two enormous theoretical advantages: tumor selectivity and ability to trigger adaptive immunity. Direct tumor cell lysis, release of soluble tumor antigens, and dangerassociated molecular patterns are all thought to help prime and promote tumor-specific immunity[7]. Apart from the direct oncolytic activity, oncolytic viruses such as Pexa-Vec as well as the herpes virus Talimogene laherparepvec (T-VEC) have been shown to mediate tumor cell death via the induction of innate and adaptive immune responses[8]. Oncolytic viruses can induce immunogenic cell death and promote an antitumor inflammatory response. A recent preclinical study of Newcastle disease virus (NDV) demonstrated the systemic immune effects that can occur even when virus is injected locally[3]. In a murine model with B16 melanomas established on the bilateral flanks, direct unilateral intratumoral injection of NDV produced significant increases in tumor-specific CD4 and CD8 + T cells in the contralateral (noninjected) tumor. Moreover, the combination of local NDV and systemic CTLA-4 blockade led to significant increases in tumor regression systemically. The most advanced - in terms of clinical development - oncolytic virus is Talimogene laherparepvec (T-VEC) which was approved by the FDA on October 15, 2015 for melanoma. T-VEC is a herpes simplex virus-1-based oncolytic immunotherapy. T-VEC is now being tested in combination with both CTLA-4 and PD1 blockade. In a phase 1b trial of T-VEC plus ipilimumab, 18 patients unresectable melanoma were enrolled. Treatment was well tolerated (grade 3–4 immune-related adverse events occurred in only two patients) and efficacy was promising (objective response was seen in 56%, and 33% had complete response)[9]. 1.2.3.2 Preclinical update
untitled h1
1.2.3.2.1 evidence of oncolytic vaccinia

Anti-tumor as well as immune effects following IV oncolytic vaccinia treatment was interrogated in RIP-Tag2 mice, a transgenic model in which mice develop pancreatic neuroendocrine tumors. These tumors are VEGF driven, therefore are also representative of several angiogenic tumors, such as renal cell carcinoma. Experimental models such as this, represent human cancer more accurately than do the commonly used xenograft models -- in which human tumors are transplanted into nude mice. This is because xenografts do not develop in their normal tissue environment, the tumor-stroma interactions that support tumor growth are absent and contributions of adaptive immunity to efficacy cannot be interrogated. Oncolytic vaccinia injected I.V. into RIP-Tag mice bearing tumors, selectively infects tumors, replicates, and causes tumor cell death by apoptosis. Virus spread can be monitored by antivaccinia antibody staining, and tumor cell death by staining for activated caspase-3 in sections of excised tumors. Immunofluorescence based staining of the vasculature (anti-CD31) and lymphocyte infiltrate (CD45), in control and treated tumors, revealed a significant increase in both numbers and distribution of CD45+ leukocytes in vaccinia treated mice. T cell recruitment to tumors was demonstrated 5 days post IV vaccinia administration (**Figure 2**). CD8 T cells recruited to tumors were also shown to be activated (as evidenced by granzyme B positivity, data not shown).  
  
These data support a model in which oncolytic vaccinia virus treatment induces tumor lysis that secondarily leads to infiltration and activation of immune cells in the tumor micro-environment. We then explored whether this immune response could be further activated by combining treatment with vaccinia virus with an immune checkpoint blocking antibody targeting the protein PD-1. 1.2.3.2.2 Combination of oncolytic vaccinia virus with anti-PD-1 antibody in RIP-Tag2 mice Anti-tumor activity of the combination of oncolytic vaccinia and anti-PD-1 antibody was evaluated in RIP-Tag2 mice by quantification of activated caspase 3 (marker of apoptosis).  
As demonstrated in **Figure 3,** while virus alone induced the expected levels of cell death, addition of anti-PD-1 antibody led to significantly enhanced tumor cell death, when used in combination (analysis performed at Day 5 post single agent or combination treatment regimen). No significant effect of the anti-PD-1 antibody alone were seen in this experiment. These results provide for a rational basis for combining Pexa-Vec and an anti-PD1 antibody in  
 clinical trials of a combination treatment for renal cell carcinoma.  
Figure 3. Enhanced tumor cell death induced by combination JX594 and anti-PD-1 treatment as compared with anti-PD-1 alone or oncolytic vaccinia alone
untitled h1
1.3 tremelimumab

Tremelimumab is a human immunoglobulin (Ig)G2 mAb that is directed against CTLA-4 and is being developed by AstraZeneca for use in the treatment of cancer.  
To date tremelimumab has been given to more than 1000 patients as part of ongoing studies either as monotherapy or in combination with other anticancer agents. Refer to the current tremelimumab Investigator's Brochure for a complete summary of non-clinical and clinical information including safety, efficacy and pharmacokinetics.
untitled h1
1.4 durvalumab

Durvalumab is a human monoclonal antibody (mAb) of the immunoglobulin G (IgG) 1 kappa subclass that inhibits binding of PD-L1 to PD-1 and CD80 and is being developed by AstraZeneca/MedImmune for use in the treatment of cancer. (MedImmune is a wholly owned subsidiary of AstraZeneca; AstraZeneca/MedImmune will be referred to as AstraZeneca throughout this document.) As durvalumab is an engineered mAb, it does not induce antibodydependent cellular cytotoxicity or complement-dependent cytotoxicity. The proposed mechanism of action for durvalumab is interference of the interaction of PD-L1 with PD-1 and CD80. To date durvalumab has been given to more than 1800 patients as part of ongoing studies either as monotherapy or in combination with other anti-cancer agents. Refer to the current durvalumab Investigator's Brochure for a complete summary of non-clinical and clinical information including safety, efficacy and pharmacokinetics.
untitled h1
1.5 anti-ctla4 in combination with

Because the mechanisms of action of CTLA-4 and PD-1 are non-redundant targeting both PD-1 and CTLA-4 pathways may have additive or synergistic activity [10]; therefore, in addition to evaluating both agents in the monotherapy setting in a number of cancer indications AstraZeneca is also investigating the use of durvalumab + tremelimumab combination therapy for the treatment of cancer. Study D4190C00006 is a Phase Ib dose-escalation study to establish the safety, PK/pharmacodynamics, and preliminary anti-tumor activity of durvalumab + tremelimumab combination therapy in patients with advanced NSCLC. The dosing schedule utilized is durvalumab every 2 or 4 weeks (Q2W, Q4W) up to 12 months, combined with tremelimumab Q4W up to Week 24 for 7 doses then every 12 weeks (Q12W) for 2 additional doses for up to 12 months. The study is ongoing and continues to accrue. In addition, other clinical studies have since started looking at the combination in both NSCLC and other oncology indications. To date more than 100 patients have received the combination using a number of doses and dosing schedules. Refer to the current editions of the durvalumab and tremelimumab IBs for a complete summary of non-clinical and clinical information including safety, PK and efficacy.
untitled h1
1.5.1 durvalumab and tremelimumab dose

The durvalumab + tremelimumab combination therapy doses and regimen selected for this study are based on the goal of selecting an optimal combination dose of durvalumab and tremelimumab that would yield sustained target suppression (sPD-L1), demonstrate promising efficacy, and have an acceptable safety profile. 1.5.1.1 Pharmacokinetics/Pharmacodynamics data Study D4190C00006 included dose cohorts with both a Q4W and a Q2W schedule of durvalumab in combination with a Q4W schedule of tremelimumab. The Q4W schedule was included to align with the Q4W dosing of tremelimumab. PK simulations from durvalumab monotherapy data indicated that a similar area under the plasma drug concentration-time curve at steady state (AUCss; 4 weeks) was expected following both 10 mg/kg Q2W and 20 mg/kg Q4W dosing with durvalumab. The observed durvalumab PK data from the D4190C00006 study were in line with the predicted monotherapy PK data developed pre-clinically and in line with that seen in the firsttime-in-human (FTIH), single agent study (CD-ON-MEDI4736-1108) in patients with advanced solid tumors. This demonstrates similar exposure of durvalumab 20 mg/kg Q4W and 10 mg/kg Q2W, with no alterations in PK when durvalumab and tremelimumab (doses ranging from 1 to 3 mg/kg) are dosed together. While the median maximum plasma concentration at steady state (Cmax,ss) is expected to be higher with 20 mg/kg Q4W (approximately 1.5-fold) and median trough concentration at steady state (Ctrough,ss) is expected to be higher with 10 mg/kg Q2W (approximately 1.25-fold), this is not expected to impact the overall safety and efficacy profile, based on existing preclinical and clinical data. Monotonic increases in pharmacodynamic activity were observed with increasing doses of tremelimumab relative to the activity observed in patients treated with durvalumab monotherapy. There was evidence of augmented pharmacodynamic activity relative to durvalumab monotherapy with combination doses containing 1 mg/kg tremelimumab, including both the 15 and 20 mg/kg durvalumab plus 1 mg/kg tremelimumab combinations. Recent updated PK and pharmacodynamic data from regimens that used tremelimumab doses of greater than 1 mg/kg from Study D4190C00006 indicated superior efficacy with combination regimen of durvalumab 1500 mg Q4W plus tremelimumab 300 mg × 1 dose. An approximate dose-proportional increases in PK exposure (maximum serum concentration and area under the serum drug concentration-time curve from time 0 to Day 28 post-dose) was observed with increasing doses of tremelimumab (1, 3, and 10 mg/kg). An exploratory pharmacodynamic analysis bioanalytically evaluated the effects of tremelimumab on proliferating T-cells from NSCLC patients who received tremelimumab (1, 3, or 10 mg/kg) and durvalumab (15 or 20 mg/kg) combination treatment. Monotonic increases in pharmacodynamic activity with the combination (increased activation/ proliferation markers on CD4 and CD8 T-cells in periphery) were observed with increasing doses of tremelimumab (1, 3, 10 mg/kg). The peak increase (%) from baseline of CD4+Ki67+ T-cells was observed 8 days post administration, and the peak level was significantly increased (p ≤ 0.05) as increasing dose of tremelimumab in the range of 1 to 10 mg/kg. Study data also suggested that higher peak exposure (maximum serum concentration [Cmax]) of tremelimumab is related to a higher maximum pharmacodynamic effect in the NSCLC patient population. Overall, the PK/pharmacodynamic data suggest that tremelimumab of dose greater than 1 mg/kg with a higher peak exposure may be associated with a higher pharmacodynamic effect.  
Additionally, based on simulation data, the Cmax (78 µg/mL) post single dose administration of tremelimumab 4 mg/kg is approximately 4-fold higher than the predicted Cmax (19 µg/mL) post the first dose of tremelimumab 1 mg/kg, and is 3-fold higher than the predicted Cmax (25 µg/mL)
post the fourth dose of tremelimumab 1 mg/kg in a Q4W×4 doses setting.
untitled h1
1.5.1.2 clinical data

In Study D4190C00006 various dose combinations have been explored, with doses of tremelimumab ranging from 1 to 10 mg/kg and doses of durvalumab ranging from 3 to 20 mg/kg. Tremelimumab was given on a Q4W schedule whilst durvalumab was explored in both a Q4W and Q2W schedule, with the goal of identifying the dose combination that best optimizes the risk: benefit profile in an acceptable range of PK and pharmacodynamic values. Patients treated with doses of tremelimumab above 1 mg/kg had a higher rate of adverse events (AEs), including discontinuations due to AEs, serious AEs (SAEs), and severe AEs. Between the 10 mg/kg durvalumab + 1 mg/kg tremelimumab and 10 mg/kg durvalumab + 3 mg/kg tremelimumab cohorts treated at the Q2W schedule, the number of patients reporting any AE, ≥ Grade 3 AEs, SAEs, and treatment-related AEs was higher in the 10 mg/kg durvalumab + 3 mg/kg tremelimumab cohort than the 10 mg/kg durvalumab + 1 mg/kg tremelimumab cohort. A similar pattern was noted in the Q4W regimens, suggesting that, as the dose of tremelimumab increased above 1 mg/kg, a higher rate of treatment-related events may be anticipated. Further, the SAEs frequently attributed to immunotherapy, pneumonitis, colitis and other immune mediated events, were more commonly seen in cohorts using either 3 mg/kg or 10 mg/kg of tremelimumab compared to the 1-mg/kg dose cohorts. Together, these data suggest that a combination using a tremelimumab dose of 1 mg/kg appeared to minimize the rate of toxicity when combined with durvalumab. As a result, all combination doses utilizing either the 3 or 10-mg/kg doses of tremelimumab were eliminated in the final dose selection. In contrast, cohorts assessing higher doses of durvalumab with a constant dose of tremelimumab did not show an increase in the rate of AEs. The data suggested that increasing doses of durvalumab may not impact the safety of the combination as much as the tremelimumab dose. Further, safety data between the 10-mg/kg and 20-mg/kg cohorts were similar, with no change in safety events with increasing dose of durvalumab. In Study D4190C00006, of all treatment cohorts, the cohort of patients treated in the 20 mg/kg durvalumab + 1 mg/kg tremelimumab group had a tolerable safety profile, but still showed strong evidence of clinical activity. No dose-limiting toxicities (DLTs) were reported in this cohort. Preliminary clinical activity of the durvalumab and tremelimumab combination did not appear to change with increasing doses of tremelimumab. The 15- and 20-mg/kg durvalumab Q4W cohorts demonstrated objective responses at all doses of tremelimumab, and increasing doses of tremelimumab did not provide deeper or more rapid responses. Efficacy data suggested that the 20 mg/kg durvalumab + 1 mg/kg tremelimumab dose cohort may demonstrate equivalent clinical activity to other dose combinations. Of the 14 patients in this cohort, there were 4 patients (29%) with PR, 4 patients (29%) with SD, and 2 patients (14%) with PD. Two patients were not evaluable for response. A single dose of tremelimumab 4 mg/kg, while maintaining a similar overall exposure, has a 3- to 4-fold higher Cmax compared to the 4 doses of tremelimumab 1 mg/kg. Therefore, this single administration of the higher dose of tremelimumab may have the potential for better anti-tumor activity while potentially avoiding any cumulative toxicity associated with repeated dosing of the 1 mg/kg tremelimumab. In the Phase I/II study (Study D4190C00022) evaluating the tolerability and clinical activity of durvalumab and tremelimumab in advanced HCC patients who progressed on, are intolerant of, or refused sorafenib-based therapy, durvalumab (1500 mg Q4W) as a single agent and in combination with two dosing regimens of tremelimumab (75 mg Q4W X 4 doses or 300mg X 1 dose) were evaluated. In the pre-planned interim analysis, safety data showed that both combination dose regimens were tolerable and no new safety signals were identified. While efficacy for durvalumab in combination with tremelimumab at the 75mg dose (Q4W X 4) was not meaningfully better than durvalumab monotherapy, efficacy data supports continued evaluation of durvalumab in combination with tremelimumab at the 300mg dose (X1). Therefore, durvalumab in combination with tremelimumab 300 mg x 1 dose will only be continued evaluation in the phase I/II study (Study D4190C00022) and Phase III study (Study D419CC00002). Altogether, the data suggested that a 20 mg/kg durvalumab + 1 mg/kg tremelimumab dose combination should be selected for further development. Refer to the current durvalumab Investigator's Brochure for a complete summary of non-clinical and clinical information on the durvalumab + tremelimumab combination, including safety, efficacy and pharmacokinetics. 1.5.1.3 Rationale for 4 cycles of combination therapy followed by durvalumab monotherapy Long -term follow up on melanoma patients treated with ipilimumab, an anti-CTLA-4 targeting antibody (dosed every 3 weeks [Q3W] for 4 doses and then discontinued), shows that patients responding to ipilimumab derive long-term benefit, with a 3-year OS rate of approximately 22%. Furthermore, the survival curve in this population reached a plateau at 3 years and was maintained through 10 years of follow up. Similar data have been presented for other anti-PD-1/PD-L1 targeting antibodies: Nivolumab (anti-PD-1) was dosed Q2W for up to 96 weeks in a large Phase I dose escalation and expansion study, and showed responses were maintained for a median of 22.94 months for melanoma (doses 0.1 mg/kg to 10 mg/kg), 17 months for NSCLC (doses 1, 3, and 10 mg/kg), and 12.9 months for renal cell carcinoma patients (doses 1 and 10 mg/kg) at the time of data analysis[11]. Furthermore, responses were maintained beyond treatment discontinuation in the majority of patients who stopped nivolumab treatment (either due to protocol specified end of treatment, CR, or toxicity) for up to 56 weeks at the time of data analysis. Similar long-term results may be expected with use of other immune-mediated cancer therapeutics such as tremelimumab, durvalumab, or the combination of the two agents. The durvalumab + tremelimumab combination regimen will be administered for 4 doses Q4W followed by durvalumab monotherapy Q4W for up to a maximum of 2 years' total.
untitled h1
1.5.2 durvalumab monotherapy dose rationale

A durvalumab dose of 20 mg/kg Q4W is supported by in vitro data, non-clinical activity, clinical PK/pharmacodynamics, biomarkers, and activity data from Study 1108 in patients with advanced solid tumors and from a Phase I trial performed in Japanese patients with advanced solid tumor (D4190C00002).
untitled h1
1.5.2.1 pk/pharmacodynamic data

Based on available PK/pharmacodynamic data from ongoing Study 1108 with doses ranging from 0.1 to 10 mg/kg Q2W or 15 mg/kg Q3W, durvalumab exhibited non-linear (dose dependent) PK consistent with target-mediated drug disposition. The PK approached linearity at ≥3 mg/kg Q2W, suggesting near complete target saturation (membrane-bound and sPD-L1), and further shows that the durvalumab dosing frequency can be adapted to a particular regimen given the linearity seen at doses higher than 3 mg/kg. The expected half-life with doses ≥3 mg/kg Q2W is approximately 21 days. A dose-dependent suppression in peripheral sPD-L1 was observed over the dose range studied, consistent with engagement of durvalumab with PD-L1. A low level of immunogenicity has been observed. No patients have experienced immune-complex disease following exposure to durvalumab (For further information on immunogenicity, please see the current IB). Data from Study D4190C00006 (Phase I trial in NSCLC patients using the combination of durvalumab and tremelimumab) also show an approximately dose-proportional increase in PK exposure for durvalumab over the dose range of 3 to 20 mg/kg durvalumab Q4W or Q2W. (For further information on PK observations in Study 006, please see the current IB). The observed durvalumab PK data from the combination study were well in line with the predicted monotherapy PK data (5th median and 95th percentiles) for a Q4W regimen. A population PK model was developed using the data from Study 1108 (doses=0.1 to 10 mg/kg Q2W or 15 mg/kg Q3W (Fairman et al 2014). Multiple simulations indicate that a similar overall exposure is expected following both 10 mg/kg Q2W and 20 mg/kg Q4W regimens, as represented by AUCss (4 weeks). Median Cmax,ss is expected to be higher with 20 mg/kg Q4W (~1.5 fold) and median Ctrough,ss is expected to be higher with 10 mg/kg Q2W (~1.25 fold). Clinical activity with the 20 mg/kg Q4W dosing regimen is anticipated to be consistent with 10 mg/kg Q2W with the proposed similar dose of 20 mg/kg Q4W expected to (a) achieve complete target saturation in majority of patients; (b) account for anticipated variability in PK, pharmacodynamics, and clinical activity in diverse cancer populations; (c) maintain sufficient PK exposure in case of ADA impact; and (d) achieve PK exposure that yielded maximal antitumor activity in animal models. Given the similar area under the plasma drug concentration-time curve (AUC) and modest differences in median peak and trough levels at steady state, the observation that both regimens maintain complete sPD L1 suppression at trough, and the available clinical data, the 20 mg/kg Q4W and 10 mg/kg Q2W regimens are expected to have similar efficacy and safety profiles, supporting further development with a dose of 20 mg/kg Q4W.
untitled h1
1.5.3 fixed dosing for durvalumab

A population PK model was developed for durvalumab using monotherapy data from a Phase 1 study (study 1108; N=292; doses= 0.1 to 10 mg/kg Q2W or 15 mg/kg Q3W; solid tumors). Population PK analysis indicated only minor impact of body weight (WT) on PK of durvalumab (coefficient of ≤ 0.5). The impact of body WT-based (10 mg/kg Q2W) and fixed dosing (750 mg Q2W) of durvalumab was evaluated by comparing predicted steady state PK concentrations (5th, median and 95th percentiles) using the population PK model. A fixed dose of 750 mg was selected to approximate 10 mg/kg (based on median body WT of ~75 kg). A total of 1000 patients were simulated using body WT distribution of 40–120 kg. Simulation results demonstrate that body WT-based and fixed dosing regimens yield similar median steady state PK concentrations with slightly less overall between-subject variability with fixed dosing regimen. Similarly, a population PK model was developed for tremelimumab using data from Phase 1 through Phase 3 (N=654; doses= 0.01 to 15 mg/kg Q4W or Q90D; metastatic melanoma) [12]. Population PK model indicated minor impact of body WT on PK of tremelimumab (coefficient of ≤ 0.5). The WT-based (1 mg/kg Q4W) and fixed dosing (75 mg/kg Q4W; based on median body WT of ~75 kg) regimens were compared using predicted PK concentrations (5th, median and 95th percentiles) using population PK model in a simulated population of 1000 patients with body weight distribution of 40 to 120 kg. Similar to durvalumab, simulations indicated that both body WT-based and fixed dosing regimens of tremelimumab yield similar median steady state PK concentrations with slightly less between-subject variability with fixed dosing regimen. Similar findings have been reported by others [13][, [14], [15], [16]. Wang and colleagues investigated 12 monoclonal antibodies and found that fixed and body size-based dosing perform similarly, with fixed dosing being better for 7 of 12 antibodies. In addition, they investigated 18 therapeutic proteins and peptides and showed that fixed dosing performed better for 12 of 18 in terms of reducing the between-subject variability in pharmacokinetic/pharmacodynamics parameters [15]. A fixed dosing approach is preferred by the prescribing community due to ease of use and reduced dosing errors. Given expectation of similar pharmacokinetic exposure and variability, we considered it feasible to switch to fixed dosing regimens. Based on average body WT of 75 kg, a fixed dose of 750 mg Q2W MEDI4736 (equivalent to 10 mg/kg Q2W), 1500 mg Q4W durvalumab (equivalent to 20 mg/kg Q4W) and 75 mg Q4W tremelimumab (equivalent to 1 mg/kg Q4W) is included in the current study. Fixed dosing of durvalumab and tremelimumab is recommended only for subjects with > 30kg body weight due to endotoxin exposure. Patients with a body weight less than or equal to 30 kg are not eligible for enrollment in the current study, and if a patient's body weight drops below 30 kg while on the study, the patient should be dosed using a weight-based dosing schedule as long as their body weight remains < 30 kg. For patients treated with tremelimumab in the current study, subjects must have a body weight of
> 35 kg to enroll and they will be treated with a fixed dose of tremelimumab. If a patient's body weight drops below 33 kg while on the study, the patient should be dosed with tremelimumab using a weight-based dosing schedule for as long as their body weight remains < 33 kg and according to the study protocol.
untitled h1
1.6 rationale for timing and

Whilst the preclinical data suggest induction of immune cell infiltration following oncolytic viral therapy, with potential for amplification with immune checkpoint therapy, the effect in colorectal cancer - a relatively non-immunogenic tumor - is really unknown. Given that this is essentially a small pilot study evaluating feasibility and safety whose next step in development - if safe and feasible - will most likely be a larger randomized study, it is scientifically important to obtain as much information about the treatment effect. This may lead to altered and improved design of the next study. The best strategy for doing this is with tumor biopsies in order to evaluate immune cell infiltration (CD4/8 T-cells, MDSC). However, if a patient has technically biopsiable disease but the interventional radiologist has concerns that pursuing the biopsy increases the risk to above average, or if pursuing the biopsy creates additional logistical complications (availability, preanesthesia requirements etc.) which cause delays or inconvenience to an unreasonable degree, we will forgo at investigator discretion. The timing of the biopsies reflects twin aims of evaluating immune infiltration post-virus and also post-combination therapy. The baseline –day 1 paired biopsies will provide data on the immune impact of the virus (within the TME) and the baseline - day 29 paired samples will provide data on the immune impact of the combination of the vaccine and immune checkpoint inhibitor. Given the difficulty in obtaining three separate tumor biopsies we have elected to perform a baseline on all patients and then either the D1 or D29 biopsy.
untitled h1
1.7 amendment d rationale

Considering recent updated PK and pharmacodynamic data from regimens that used tremelimumab (Section **1.5.1.1**) and clinical benefit of increased single tremelimumab dose
(Section **1.5.1.2**) with Amendment D we change study design and replace 4 infusions (75 mg each)
of tremelimumab with single infusion (300 mg). We completed enrollment into Arms A1 and A2 and with approval of this amendment we are proceeding to enrollment to Arms B1 and B2 with addition of tremelimumab to combined treatment. Overall, combination of Pexa-Vec with durvalumab even on dose level 2 of Pexa-Vec showed favorable safety profile. No DLT was developed. All patients who received the treatment tolerated well. There were no drug-related discontinuations or deaths. Drug-related AEs occurred in 100%
of patients and were grade 3-4 in 8.6%. See (**Appendix D)**
untitled h1
2 eligibility assessment and enrollment

2.1.1.1 Patients must have histopathological confirmation of Colorectal Carcinoma (CRC) by the Laboratory of Pathology of the NCI prior to entering this study.  
2.1.1.2 Patients must have progressed on, been intolerant of or refused prior oxaliplatin- and irinotecan-containing, fluorouracil-based, chemotherapeutic regimen and have disease that is not amenable to potentially curative resection. Patients who have a known KRAS wild type tumor must have progressed, been intolerant of or refused cetuximab or panitumumab-based chemotherapy.  
2.1.1.3 Patients tumors must be documented to be microsatellite-stable (MSS) either by genetic analysis or immunohistochemistry OR microsatellite-high with documented disease progression following anti-PD1/PDL1 therapy.  
2.1.1.4 Patients must have at least one focus of metastatic disease that is amenable to pre- and on-treatment biopsy and be willing to undergo this. Ideally the biopsied lesion should not be one of the target measurable lesions, although this can be up to the discretion of the investigators.  
2.1.1.5 All patients enrolled will be required to have measurable disease by RECIST 1.1 criteria. 2.1.1.6 Age ≥18 years. Because no dosing or adverse event data are currently available on the use of Pexa-Vec in combination with tremelimumab and/or durvalumab in patients <18 years of age, children are excluded from this study, but will be eligible for future pediatric trials.  
2.1.1.7 ECOG performance status 0-1 (see **Appendix A**)  
| Leukocytes                                                                                                                                                                                       | >3,000/mcL                              |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------|
| absolute neutrophil count                                                                                                                                                                        | > 1,500/mcL                             |
| Platelets                                                                                                                                                                                        | >100,000/mcL                            |
| total bilirubin                                                                                                                                                                                  | ≤1.5X institution upper limit of normal |
| Hb                                                                                                                                                                                               | > 9g/dl                                 |
| AST (SGOT)/ALT (SGPT) ≤ 2.5 x institutional upper limit of normal  unless liver metastases are present, in which case it must be ≤ 5x ULN Creatinine <1.5X institution upper limit of normal  OR |                                         |  
2.1.1.8 Patients must have acceptable organ and marrow function as defined below:  
| creatinine clearance   | >45 mL/min/1.73 m2 , as calculated  below, for patients with creatinine levels  above institutional normal   |
|------------------------|--------------------------------------------------------------------------------------------------------------|  
  
Estimated creatinine clearance (mL/min)  
  
  
May use a 24-hr. urine collection to determine creatinine clearance. Measured creatinine clearance (mL/min)  
2.1.1.9 Patient must be able to understand and willing to sign a written informed consent document 2.1.1.10 The effects of Pexa-Vec, durvalumab and tremelimumab on the developing human fetus are unknown. For this reason, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry, for the duration of study participation and up to 180 days after the last dose of durvalumab + tremelimumab combination therapy or 90 days after the last dose of durvalumab monotherapy, whichever is the longer time period. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately.  
2.1.1.11 Evidence of post-menopausal status or negative urinary or serum pregnancy test for female pre-menopausal patients. Women will be considered post-menopausal if they have been amenorrheic for 12 months without an alternative medical cause. The following agespecific requirements apply:
o Women <50 years of age would be considered post-menopausal if they have been amenorrheic for 12 months or more following cessation of exogenous hormonal treatments and if they have luteinizing hormone and follicle-stimulating hormone levels in the post-menopausal range for the institution or underwent surgical sterilization (bilateral oophorectomy or hysterectomy).  
o Women ≥50 years of age would be considered post-menopausal if they have been amenorrheic for 12 months or more following cessation of all exogenous hormonal treatments, had radiation-induced menopause with last menses >1 year ago, had chemotherapy-induced menopause with last menses >1 year ago, or underwent surgical sterilization (bilateral oophorectomy, bilateral salpingectomy or hysterectomy. Subject is willing and able to comply with the protocol for the
duration of the study including undergoing treatment and scheduled visits and examinations including follow up.  
2.1.1.12 Body weight >35kg  
2.1.2 Exclusion Criteria 2.1.2.1 Patients who have had anti-cancer therapy (chemotherapy, immunotherapy, endocrine therapy, targeted therapy, biologic therapy, tumor embolization, monoclonal antibodies or other investigation agents), large field radiotherapy, or major surgery must wait 4 weeks after completing treatment prior to entering the study.  
2.1.2.2 No prior exposure to immune-mediated therapy including, but not limited to, other anti CTLA-4, anti-PD-1, anti-PD-L1, and anti-programmed cell death ligand 2 (anti-PD-L2) antibodies, including therapeutic anticancer vaccines. The exception to this is those whose tumors are MSI-hi and are refractory to anti-PD1 monotherapy.  
2.1.2.3 Involvement in the planning and/or conduct of the study 2.1.2.4 Previous IP assignment in the present study 2.1.2.5 Patients who are receiving any other investigational agents. 2.1.2.6 Inability to suspend treatment with anti-hypertensive medication (including but not limited to: diuretics, beta-blockers, angiotensin converting enzyme [ACE] inhibitors, aldosterone antagonists, etc.) for 48 hours pre and post each Pexa-Vec administration.  
2.1.2.7 Any unresolved toxicity NCI CTCAE Grade ≥2 from previous anticancer therapy with the exception of alopecia, vitiligo, and the laboratory values defined in the inclusion criteria 2.1.2.8 Patients with Grade ≥2 neuropathy will be evaluated on a case-by-case basis 2.1.2.9 Patients with known brain metastases will be excluded from this clinical trial because of their poor prognosis and because they often develop progressive neurologic dysfunction that would confound the evaluation of neurologic and other adverse events.  
2.1.2.10 Uncontrolled intercurrent illness including, but not limited to, hypertension (systolic BP
> 160, diastolic BP > 100), ongoing or active systemic infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, uncontrolled diabetes or psychiatric illness/social situations that would limit compliance with study requirements. For patients with a history of cardiovascular disease, cardiology consultation is required. Echocardiogram, troponin and creatinine clearance must be obtained prior to enrollment. NOTE: Patients with active cardiac disease (e.g. myocarditis and myocardial infarction) within 12 months of study entry are excluded from study participation.  
2.1.2.11 HIV-positive patients receiving anti-retroviral therapy are excluded from this study due to the possibility of pharmacokinetic interactions between antiretroviral medications and the investigational agent. HIV positive patients not receiving antiretroviral therapy are excluded due to the possibility that Durvalumab or Tremelimumab may worsen their
condition and the likelihood that the underlying condition may obscure the attribution of adverse events.  
2.1.2.12 Known significant immunodeficiency due to underlying illness (e.g. HIV/AIDS) and/or immune-suppressive medication including high-dose corticosteroids (defined as ≥20 mg/day prednisone or equivalent which is ongoing at the time of enrollment and/or was taken for more than 4 weeks within the preceding 2 months of enrollment)
2.1.2.13 History of chronic autoimmune disease (e.g. systemic lupus erythematosus or Wegener's granulomatosis, Addison's disease, multiple sclerosis, Graves' disease, Hashimoto's thyroiditis, hypophysitis, etc.) with symptomatic disease within the 3 years before enrollment. Note: Active vitiligo or a history of vitiligo will not be a basis for exclusion. In addition, a past history of certain autoimmunity e.g. rheumatoid arthritis or thyroiditis may be allowed per PI discretion provided it has been quiescent for a minimum of three years. The following are exceptions to this criterion:  
a) Patients with vitiligo or alopecia b) Patients with hypothyroidism (e.g. following Hashimoto syndrome) stable on hormone replacement c) Any chronic skin condition that does not require systemic therapy d) Patients without active disease in the last 5 years may be included e) Patients with celiac disease controlled by diet alone f) Active or history of inflammatory bowel disease (colitis, Crohn's), irritable bowel disease, celiac disease, or other serious, chronic, gastrointestinal conditions associated with diarrhea.  
2.1.2.14 History of active primary immunodeficiency
2.1.2.15 Active infection including tuberculosis (clinical evaluation that includes clinical history, physical examination and radiographic findings, and TB testing (if clinically indicated), hepatitis B (known positive HBV surface antigen (HBsAg) result), hepatitis C. Patients with a past or resolved HBV infection (defined as the presence of hepatitis B core antibody [anti-HBc] and absence of HBsAg) are eligible. Patients positive for hepatitis C (HCV) antibody are eligible only if polymerase chain reaction is negative for HCV RNA.  
2.1.2.16 Current or prior use of immunosuppressive medication within 14 days before the first dose of durvalumab or tremelimumab. The following are exceptions to this criterion:
o Intranasal, inhaled, topical steroids, or local steroid injections (e.g. intra articular injection)
o Systemic corticosteroids at physiologic doses not to exceed <<10 mg/day>> of prednisone or its equivalent o Steroids as premedication for hypersensitivity reactions (e.g. CT scan premedication)
o Receipt of live attenuated vaccine within 30 days prior to the first dose of IP. Note:
Patients, if enrolled, should not receive live vaccine whilst receiving IP and up to 30 days after the last dose of IP.  
2.1.2.17 Female patients who are breastfeeding. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with Pexa-Vec,
breastfeeding should be discontinued if the mother is treated with Pexa-Vec. These potential risks may also apply to other agents used in this study.  
2.1.2.18 Known allergy or hypersensitivity to IP  
2.1.2.19 Prior randomization or treatment in a previous durvalumab and/or tremelimumab clinical study regardless of treatment arm assignment.  
2.1.2.20 History of sarcoidosis syndrome 2.1.2.21 Mean QT interval corrected for heart rate (QTc) ≥470 ms calculated from 3 electrocardiograms (ECGs) using Fredericia's Correction 2.1.2.22 Patients with a history of Interstitial lung disease or pneumonitis
2.1.2.23 Subjects with uncontrolled seizures  
2.1.2.24 History of leptomeningeal carcinomatosis
2.1.2.25 History of hypersensitivity reaction to human or mouse antibody products. 2.1.2.26 Patients with unhealed surgical wounds for more than 30 days  
2.1.2.27 Ongoing severe inflammatory skin condition (as determined by the Investigator)
requiring medical treatment
2.1.2.28 History of severe eczema (as determined by the Investigator) requiring medical treatment  
2.1.2.29 Patients with tumor(s) invading a major vascular structure (e.g. carotid artery) or other key anatomical structure (e.g. pulmonary airway) in the event of post treatment tumor swelling and/or necrosis (hepatic and portal vein involvement allowed)
2.1.2.30 Patients with liver tumors in a location that would potentially result in significant clinical adverse effects in the opinion of investigator if post-treatment tumor swelling were to occur, including at the site of the common bile duct 2.1.2.31 Clinically significant and/or rapidly accumulating ascites, pericardial and/or pleural effusions. Mild ascites that does not preclude safe tumor biopsy as protocol specified is allowed at the discretion of the treating physician.  
2.1.2.32 Medical conditions, per the investigator's judgment, that predispose the patient to untoward medical risk in the event of volume loading (e.g. intravenous [IV] fluid bolus infusion), tachycardia, or hypotension during or following treatment with Pexa-Vec 2.1.2.33 Any prior or planned organ transplant (e.g. liver transplant) 2.1.2.34 Patients who experienced a severe systemic reaction or side-effect as a result of a previous vaccination with vaccinia 2.1.2.35 Pulse oximetry O2 saturation <90% at rest on room air 2.1.3 Recruitment Strategies
The study may be abstracted into a plain language announcement posted on NIH websites and on NIH social media platforms.
untitled h1
2.2 screening evaluation

2.2.1 Screening activities performed after a consent for screening has been signed Screening evaluation testing/procedures are conducted under the separate screening protocol, 01-
C-0129 (Eligibility Screening and Tissue Procurement for the NIH Intramural Research Program Clinical Protocols). Assessments performed at outside facilities or on another NIH protocol within the timeframes below may also be used to determine eligibility once a patient has signed the consent. Studies should be done within 28 days prior to enrollment unless otherwise noted below.  
- History and Physical Evaluation Complete medical history and physical examination (including height, weight, vital signs, ECG, and ECOG performance status) will be conducted prior to enrollment.  
- Laboratory Evaluation  
o Hematological Profile: CBC with differential and platelet count. o Biochemical Profile: electrolytes, BUN, creatinine, AST, ALT, total and direct bilirubin, calcium, phosphorus, albumin, magnesium.  
o 24-hour urine collection (if creatinine clearance is measured in this manner) o Serum or urine pregnancy test for female participants of childbearing age and anatomic ability.  
o HIV, Hepatitis B and C serology and/or viral load o TB testing (if clinically indicated)
- Cardiology Consultation for patients with history of cardiovascular disease (see criterion 2.1.2.10)
- Echocardiogram  
- CT scan of chest, abdomen and pelvis (or MRI of abdomen if clinically indicated)
- Histologic confirmation (at any time point prior to enrollment). If there is no available tumor sample, biopsy will be performed to confirm the diagnosis and Microsatellite Stability Status.  
o A block or unstained slides of primary or metastatic tumor tissue will be required from each participant to confirm diagnosis with analysis being performed by the Laboratory of Pathology, NIH.  
o Documentation of microsatellite status (by PCR or IHC for MMR proteins, at any time point prior to enrollment).
untitled h1
2.3 participant registration and status

Registration and status updates (e.g. when a participant is taken off protocol therapy and when a participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant Registration & Status Updates found here..
untitled h1
2.4 treatment assignment procedures (for

| Number   | Name   | Description                                                                                       |
|----------|--------|---------------------------------------------------------------------------------------------------|
| 1        | A1     | Subjects enrolled to Pexa-Vec escalation dose levels +  Durvalumab                                |
| 2        | A2     | Subjects enrolled at the MTD of Pexa-Vec after the  MTD is established + Durvalumab               |
| 3        | B1     | Subjects enrolled to Pexa-Vec escalation dose levels +  Durvalumab + Tremelimumab                 |
| 4        | B2     | Subjects enrolled at the MTD of Pexa-Vec after the  MTD is established+ Durvalumab + Tremelimumab |
untitled h1
arms

| Number   | Name   | Description                                                              |
|----------|--------|--------------------------------------------------------------------------|
| 1        | A1     | Pexa-Vec escalation dose levels + Durvalumab                             |
| 2        | A2     | MTD of Pexa-Vec after the MTD is established +  Durvalumab               |
| 3        | B1     | Pexa-Vec escalation dose levels + Durvalumab +  Tremelimumab             |
| 4        | B2     | MTD of Pexa-Vec after the MTD is established+  Durvalumab + Tremelimumab |
untitled h1
arm assignments

Subjects in cohort A1 will be directly assigned to arm A1. Subjects in cohort A2 will be directly assigned to arm A2 after the MTD for Arm A1 has been determined. Subjects in cohort B1 will be directly assigned to arm B1 once enrollment in Arms A1 and A2 are complete. Subjects in cohort B2 will be directly assigned to arm B2 after the MTD for Arm B1 has been determined.
untitled h1
2.5 baseline evaluation

Tests performed during screening do not need to be repeated if done in designated time frame.  
- Physical exam with vital signs and ECOG performance status (obtained within 1 week prior to first dose of Pexa-Vec).  
- CT scan of chest, abdomen and pelvis (or MRI of abdomen if clinically indicated)
(obtained within 28 days prior to first dose of Pexa-Vec)
- Laboratory evaluation (obtained within 72 hours prior to first dose of Pexa-Vec):
o Hematological profile: CBC with differential and platelet count o PT, INR, aPTT, fibrinogen. o Biochemical profile: electrolytes, BUN, creatinine, AST, ALT, total and direct bilirubin, calcium, phosphorus, albumin, magnesium, uric acid, amylase and lipase o Tumor marker profile: CEA o Serum or urine pregnancy test for female participants of childbearing age and anatomic ability.  
o Urinalysis
o Troponins
- Laboratory evaluation (obtained within 28 days prior to first dose of Pexa-Vec):  
o Thyroid function tests (TSH, T3, T4) o ACTH, morning cortisol.
o HLA subtype
- Electrocardiogram (obtained within 72 hrs. prior to first dose of Pexa-Vec)
untitled h1
3 study implementation 3.1 study

The proposed study is a Phase I/II study of Pexa-Vec oncolytic virus at two dose levels in combination with immune checkpoint inhibition in patients with metastatic colorectal cancer.  
  
- Patients with advanced microsatellite-stable colorectal cancer (or MSI-hi disease that is refractory to PD-1 monotherapy) will receive Pexa-Vec, administered IV at a dose of either 3 x 108 plaque forming units (pfu) (DL1) or 109 pfu (DL2) every 2 weeks for 4 doses, in 4 separate arms: A1, A2, B1 and B2. The first administration will be on Day –(minus) 12, followed by administration on Days 2, 16 and 30 (i.e. 4 doses in total).  
- There will be 4 arms, as summarized in **Table 1**: in Arms A1 and A2 subjects will be treated with Pexa-Vec in combination with Durvalumab and in Arms B1 and B2 subjects will be treated with Pexa-Vec in combination with Durvalumab and Tremelimumab. After enrollment is completed for Arms A1 and A2 and full assessment of safety performed, we will report this data to the NIH Intramural IRB. We will only proceed to Arm B1 (PexaVec + Durvalumab + Tremelimumab) upon NIH Intramural IRB approval of updated safety information of ongoing combination therapy evaluating Arms A1 and A2 (Pexa-Vec in combination with Durvalumab).  
| Tremelimumab   | Durvalumab   | Pexa-Vec/pfu   | N            |         |     |
|----------------|--------------|----------------|--------------|---------|-----|
| Arm A1         | DL 1         | NA             | 1500mg q 28d | 3 x 108 | 3-6 |
| DL 2           | NA           | 1500mg q 28d   | 109          | 6       |     |
| Arm A2         | NA           | 1500mg q 28d   | 109 (MTD)    | 4       |     |  
| Arm B1                    | DL 1                   | 300 mg x 1 at cycle   | 1500mg q 28d   | 3 x 108   | 3-6   |
|---------------------------|------------------------|-----------------------|----------------|-----------|-------|
| 1                         | 1500mg q 28d           | 109                   | 6              |           |       |
| DL 2                      | 300 mg x 1 at cycle  1 |                       |                |           |       |
| Arm B2                    | 300 mg x 1 at cycle    | 1500mg q 28d          | 109 (MTD)      | 4         |       |
| 1                         |                        |                       |                |           |       |
| Table 1: Arms/Dose levels |                        |                       |                |           |       |  
- Enrollment of the first two patients in each dose level will be staggered by 2 weeks in order to observe and treat any unexpected toxicities.  
- If 0/3 patients encounter DLT at DL1 we will proceed to DL2, or if 1/3 patients at Dose Level 1 encounter DLT we will expand DL1 to 6 patients as per standard 3+3 criteria. 1/6 with DLT at DL1 will lead to enrollment onto DL2. If 2 or more of 6 patients have a DLT at Dose Level 1, then we will not proceed with further enrollment or will need to revise the treatment regimen before use in a subsequent study, as appropriate.  
- If only 1/6 patients at Dose Level 2 encounter DLT we will expand DL2 to 10 patients. If 2 or more of 6 patients have a DLT at Dose Level 2, then we will not proceed with further enrollment and will need to revise the treatment regimen before use in a subsequent study, as appropriate.  
o Arms A1 and A2: In addition to the oncolytic virus patients will also receive durvalumab at a flat dose of 1500mg as an intravenous infusion beginning on Day 1 followed by q28days until off-treatment criteria are met.  
o Arms B1 and B2: In addition to the oncolytic virus patients will also receive tremelimumab 300 mg and durvalumab 1500mg as intravenous infusions on Day 1 followed by q28days subsequent continuation of the durvalumab alone until offtreatment criteria are met.  
- Pexa-Vec will commence on Day –(minus) 12 to facilitate tumor biopsies following oncolytic virus alone.
- All patients will undergo a baseline tumor biopsy and a post treatment biopsy. Patients will be assigned to receive the second biopsy on Day 1 (i.e. after one dose of Pexa-Vec alone) or Day 29 (i.e. after the combination of immune checkpoint inhibition and Pexa-Vec). (Specific dates of biopsy will have a window of 72 hrs to allow for logistical issues; however, the biopsy will not be performed within 48 hrs after Pexa-Vec administration). Patients should withhold antihypertensive medication for 48 hours pre and post Pexa-Vec administration. Assignment on a 1:1 basis will be performed to determine timing of the second research biopsy. Research Team will assign patient \#1 to early second biopsy, patient \#2 to late second biopsy and so on.
untitled h1
3.1.1 dose limiting toxicity (dlt)

A DLT is defined as any of the below listed laboratory abnormality or adverse drug reaction (ADR) according to the NCI-CTCAE v 4.3, that is possibly, probably, or definitely related to the combination of Pexa-Vec oncolytic virus with either durvalumab or the combination of durvalumab and tremelimumab, occurring during the DLT evaluation period unless specified as exceptions. ADRs are defined in this trial as any AEs suspected to be related to study treatment by the investigator. The DLT observation period comprises the first 28 days of combination treatment, extending from Day 1 to Day 28, however if later toxicities arise these may also be used to inform DLT determination based on the opinion of the investigator. Eligible patients for DLT evaluation will be those who completed the DLT period and have received at least 60% of the scheduled doses of Pexa-Vec and at least 1 dose of immune checkpoint blockade, or those who discontinue study treatment early due to a DLT. Subjects who are not fully eligible for DLT evaluation will be replaced. 3.1.1.1 Immune-mediated adverse events (imAEs)
- Any Grade 4 imAE not attributed to local tumor response (e.g. inflammatory reaction attributed to local tumor response or inflammatory reaction at sites of metastatic disease or lymph nodes).  
- Any Grade ≥3 colitis. - Any Grade 3 or 4 noninfectious pneumonitis, irrespective of duration or Grade 2 pneumonitis that does not resolve to ≤ Grade 1 within 3 days of the initiation of maximal supportive care.  
- Any Grade 3 imAE, excluding colitis or pneumonitis, that does not downgrade to Grade ≤1 or baseline status (for patients who entered the study with an existing laboratory abnormality) within 14 days.  
- Any > grade 2 cardiac toxicity which does not resolve to grade 1 within 3 days of initiation of maximum supportive care.  
3.1.1.2 Non-Immune-mediated Adverse Event (Non-imAE)
- Any Grade ≥3 non-imAE toxicity that does not downgrade to Grade ≤1 or baseline status (for patients who entered the study with an existing laboratory abnormality) within 14 days 3.1.1.3 Exclusions to Dose Limiting Toxicities
- Grade 3/4 infusion-related reactions including fever, chills lasting less than 72 hours or hypotension responsive to IVF or other medical therapy (e.g. vasopressors) and resolving in < 24 hrs.  
- Vitiligo, alopecia, rash, and Grade 3 electrolyte abnormalities that are reversed with appropriate medical intervention within 7 days or derived from a suboptimal prophylactic and curative therapy
- Grade 4 electrolyte abnormalities lasting less than 72 hours or are clinically irrelevant.  
- Grade 3 and 4 diarrhea lasting less than 72 hours.  
- Grade 3 fatigue.  
- Grade 3 rise in creatinine, not corrected to Grade 1 or less after 2 liters or more of intravenous fluids within 24 hours, will be considered dose limiting.  
- Laboratory values out of normal range that are unlikely related to trial treatment according to the investigator, do not have any clinical correlate, and resolve within 7 days with adequate medical management.  
- Transient (<7days) Grade 3 / 4 fatigue, local reactions, headache, nausea, emesis that resolves to ≤ Grade 1.  
- Grade 3 endocrinopathy that is managed with or without systemic corticosteroid therapy and/or hormone replacement therapy and the following criteria are met:
o The subject's hormone levels are within normal limits o The subject is asymptomatic
- Grade 3 inflammatory reaction attributed to a local antitumor response (e.g. inflammatory reaction at sites of metastatic disease, lymph nodes, etc.). imAEs are defined as AEs of an immune nature (i.e., inflammatory) in the absence of a clear alternative etiology. In the absence of a clinically significant abnormality, repeat laboratory testing will be conducted to confirm significant laboratory findings prior to designation as a DLT. Laboratory abnormalities that are not deemed to be clinical significant will not be considered a DLT.
untitled h1
3.1.2 maximum tolerated dose (mtd)

MTD will be that dose where no greater than 1 of 6 patients have a DLT.
untitled h1
3.1.3 protocol stopping rules

This study will utilize the Common Terminology Criteria for Adverse Events (CTCAE) version 4.3 for grading systemic toxicity. For safety reasons, the protocol will be temporarily halted pending discussions with the NIH Intramural IRB and Sponsor regarding necessary amendment for either of the following events:
- One occurrence of grade 5 toxicity by the NCI-CTCAE version 4.3 attributable to the treatment regimen. - Two occurrences of grade 4 toxicity by the NCI-CTCAE version 4.3 attributable to the treatment regimen.
untitled h1
3.2 drug administration 3.2.1 pexa-vec

The Pexa-Vec dose of 3 x 108 pfu (DL 1) or 1 x 109 pfu (DL 2) will be administered via IV infusion over 60 minutes (±5 minutes). Patients will receive 1 L 0.9% Sodium Chloride (NaCl) prior to infusion. Treatments 2–4 may each be administered in a treatment window of ±72hrs. If a treatment is not given within the ±72hrs window, it will be considered missed (e.g. patient will not receive the treatment, but future treatments will be administered as originally outlined in the protocol). 3.2.1.1 Pre-Medication for Treatment Day To mitigate risk of hypotension following Pexa-Vec treatment, the following are required:
- Prehydration with 1 liter of solute-containing fluid (PO or IV) prior to each treatment  
- Suspension of anti-hypertensive medication (including diuretics, beta-blockers, ACE
inhibitors, aldosterone antagonists, etc.) for 48 hours prior to and 48 hours after each PexaVec injection.
All patients should be pre-medicated with acetaminophen (or equivalent, unless contraindicated) on each treatment day. For acetaminophen, the following regimen may be used:
- 500–1000 mg 2 hours' pre-infusion  
- 500–1000 mg at 4 hours' post-procedure
- 500–1000 mg every 6 hours thereafter, as needed (the total acetaminophen dose should be carefully assessed to avoid cumulative toxicity)
3.2.2 Durvalumab Administration A dose of 1500mg will be administered as an IV infusion over approximately 60 minutes (±5 minutes).  
For details, please, see Section **13.2.5**
Less than 55 minutes is considered a deviation. If there are interruptions during infusion, the total allowed time should not exceed 8 hours at room temperature. 3.2.3 Tremelimumab Administration A dose of 300 mg will be administered as an IV solution at a rate of 250 mL/hr. Standard infusion time is 1 hour. However, if there are interruptions during infusion, the total allowed time should not exceed 8 hours at room temperature.  
For details, please, see Section **13.1.5**
3.2.4 Monitoring of Dose Administration
untitled h1
pexa-vec

A 20-hour observation period is required after the first infusion of Pexa-Vec. Vital signs will be collected before infusion, at least once during the infusion, at the completion of the infusion and every 2 hours approximately until completion of 20-hour observation period. At the discretion of the treating physician patients may be admitted for additional supportive care and observation after the 20-hour period. If no clinically significant infusion reactions are observed during or after the first cycle, subsequent infusion observation periods can be at the Investigator's discretion (suggested 8 hours after Pexa-Vec infusion). Tremelimumab and Durvalumab Vital signs will be collected before investigational product infusion and at the completion of the infusion. A 1-hour observation period is required after the first infusion of durvalumab and tremelimumab. If no clinically significant infusion reactions are observed during or after the first cycle, subsequent infusion observation periods can be at the Investigator's discretion (suggested 30 minutes after each durvalumab infusion). In the event of a ≤Grade 2 infusion-related reaction, the infusion rate of study drug may be decreased by 50% or interrupted until resolution of the event and re-initiated at 50% of the initial rate until completion of the infusion. For patients with a ≤Grade 2 infusion related reaction, subsequent infusions may be administered at 50% of the initial rate. Acetaminophen and/or an antihistamine (e.g. diphenhydramine) or equivalent medications per institutional standard may be administered at the discretion of the investigator. If the infusion related reaction is ≥Grade 3 or higher in severity, study drug will be discontinued. The standard infusion time is one hour, however, if there are interruptions during infusion, the total allowed time from infusion start to completion of infusion should not exceed 8 hours at room temperature (otherwise requires new infusion preparation). As with any antibody, allergic reactions to dose administration are possible. Therefore, appropriate drugs and medical equipment to treat acute anaphylactic reactions must be immediately available, and study personnel must be trained to recognize and treat anaphylaxis, as per local institutional guidelines.
untitled h1
3.3 dose modifications

The following broad guidelines for dose delivery schedule delays and alternations apply to the planned dosages of Pexa-Vec, Tremelimumab and Durvalumab or Durvalumab monotherapy and are dependent on the clinical and laboratory assessment on the day of dosing. Pexa-Vec, Tremelimumab and Durvalumab can be delivered within 72 hours of planned interval to accommodate scheduling issues/logistics.
untitled h1
3.3.1 pexa-vec

If a treatment is not given within the ±72-hour window, it will be considered missed (e.g. patient will not receive the treatment, but future treatments will be administered as originally outlined in the protocol).
untitled h1
3.3.2 tremelimumab and durvalumab

Dose modifications of Durvalumab and tremelimumab may be required in the event of treatmentrelated toxicity. General guidelines regarding dose modification are provided in **Appendix B**.
untitled h1
3.4 management of expected adverse

3.4.1 Expected Toxicities of Pexa-Vec Treatment 3.4.1.1 Flu-Like Symptoms (mild-to-severe fever, rigors, anorexia, aches/pain, fatigue, headache, and/or nausea)
These flu-like symptoms typically peak during the first 12 hours after Pexa-Vec treatment. Acute, fever is expected within 4–12 hours post-treatment and has a typical duration of 18–24 hours, although these can last up to 72 hours.
untitled h1
3.4.1.2 hypotension

Across clinical trials and indications, mild-to-moderate transient hypotension can be observed within 24 hours following treatment with Pexa-Vec. However, in the TRAVERSE trial for patients with advanced 2nd line HCC, 6 patients (8 total SAEs) have experienced severe hypotension following Pexa-Vec treatment, requiring >24 hours of medical treatment, intensive care unit care, and vasopressor treatment. Notably, anti-hypertensive medication was ongoing at the time of treatment with Pexa-Vec prior to the development of hypotension in the majority of these patients, thereby potentially exacerbating the potential for hypotension.  
Risk mitigation strategies for hypotension with Pexa-Vec are addressed in section **3.2.1.1**.
untitled h1
3.4.1.3 hematological events

Transient decreases in WBC counts (including neutrophils and lymphocytes), platelets and hematocrit can occur. Transient thrombocytopenia (including reduced platelet count) occurred within days of treatment with Pexa-Vec and resolved within 1 week of treatment without medical intervention. Transient leukopenia (including reduced neutrophil and lymphocyte counts), has been observed with resolution generally occurring within 7 days of treatment. Leukopenia is a common finding associated with viral infection in general. Conversely, transient leukocytosis (including neutrophil, eosinophil, and monocyte subsets) has also been observed. An elevated WBC count is expected based on a GM CSF mediated effect by Pexa-Vec and the infusion of an attenuated vaccinia virus.
untitled h1
3.4.1.4 chemistry changes

Chemistry changes included hyponatremia and hyperglycemia; the relationship to Pexa-Vec treatment is not yet known. Dose-related direct hyperbilirubinemia (due to tumor swelling and occlusion of biliary tract drainage) has been noted in patients treated with Pexa-Vec whose liver tumors were adjacent to or impeding the biliary tract at baseline. In addition, acute respiratory distress secondary to airway obstruction was observed in one patient with HCC metastases to the lung following treatment. Therefore, patients with liver tumors in a location that would potentially result in significant clinical adverse effects if post-treatment tumor swelling were to occur, including at the site of the common bile duct, should not be included as per Exclusion Criteria. Biliary tract drainage should be considered as clinically indicated if biliary occlusion does not resolve quickly enough clinically. Acute post-treatment tumor swelling can result in hyperbilirubinemia within approximately one week following treatment; tumor swelling may resolve over time.
untitled h1
3.4.1.5 rashes

Rashes related to latent virus reactivation have been reported, both with Varicella-Zoster (i.e.,
"shingles") and herpes simplex virus; whether these rashes are related to Pexa-Vec itself, or due to the fever induced, is unknown. Small (<1 cm) superficial skin or oral mucous membrane pustules containing Pexa-Vec may develop after Pexa-Vec treatment. If they develop, it is typically within 1 week after the first IV infusion only. These pustules have rarely (<10%) been seen following IT injection. These pustules generally resolve within approximately the following 2–3 weeks, a time course that is consistent with the usual course following intentional vaccination with wild-type [non-attenuated] vaccinia vaccine. All pustules to date have been self-limited and resolved without complications or the need for specific anti-viral treatment.
untitled h1
3.4.1.6 myocarditis

Wild type vaccinia has been associated with myocarditis. Around 1 in 175 patients administered with ACAM2000, a wildtype vaccinia (smallpox) vaccine, experienced myocarditis and/or pericarditis. To date, the use of Pexa-Vec (non-wild-type), in over 300 patients, has not been associated with myocarditis. The risk of myocarditis may be increased in the context of concomitant checkpoint inhibitors. For this reason, subjects with cardiovascular disease such as myocarditis and history of myocardial infarction are excluded from the study. Echocardiograms will be performed at the screening and during the study if clinically necessary. Troponins will be monitored monthly during the study and follow up period.
untitled h1
3.4.1.7 additional risks

Additional risks include tachycardia, portal vein thrombosis, cytokine release syndrome (CRS),
viremia, herpes infections and hypertension Further information on these risks (e.g. presenting symptoms) can be found in the current version of the Pexa-Vec Investigator Brochure.  
3.4.2 Potential Mechanisms of Pexa-Vec Shedding to the Environment, Biodissemination and Prevention of Transmission 3.4.2.1 Superficial Skin Pustules
Small (<1 cm) superficial skin or oral mucous membrane pustules containing Pexa-Vec may develop after Pexa-Vec treatment. If pustules develop, they do so typically within 1 week after the first administration. These pustules generally resolve within approximately the following 2–3 weeks, a time course that is consistent with the usual course following intentional vaccination with wild-type (non-attenuated) vaccinia vaccine. All pustules to date have been self-limited and resolved without complications or the need for specific anti-viral treatment. The following steps should be carried out in the event of skin or oral mucosa pustule identification (patient or patient contact, which is unexpected):  
- Record the AE (event term for Pexa-Vec related pustules must be recorded as
"papularpustular rash").  
- Instruct the patient/contact to cover the pustule with non-occlusive dressing (or wear a mask when around other people if oral lesions are present).  
3.4.2.2 Urine, Feces, Blood, Throat and/or Saliva
Testing for the presence of Pexa-Vec after patient treatment has been conducted in several clinical studies in throat swabs, urine, feces, and blood samples. No person-to-person transmission of Pexa-Vec has been reported.
untitled h1
3.4.3 adverse events of special

An adverse event of special interest (AESI) is one of scientific and medical interest specific to understanding of the Investigational Product and may require close monitoring and rapid communication by the investigator to the sponsor. An AESI may be serious or non-serious. The rapid reporting of AESIs allows ongoing surveillance of these events in order to characterize and understand them in association with the use of this investigational product. AESIs for durvalumab and tremelimumab include but are not limited to events with a potential inflammatory or immune-mediated mechanism and which may require more frequent monitoring and/or interventions such as steroids, immunosuppressants and/or hormone replacement therapy. These AESIs are being closely monitored in clinical studies with durvalumab monotherapy and combination therapy. An immune-mediated adverse event (imAE) is defined as an adverse event that is associated with drug exposure and is consistent with an immune-mediated mechanism of action and where there is no clear alternate aetiology. Serologic, immunologic, and histologic (biopsy) data, as appropriate, should be used to support an imAE diagnosis. Appropriate efforts should be made to rule out neoplastic, infectious, metabolic, toxin, or other etiologic causes of the imAE. If the Investigator has any questions in regard to an adverse event (AE) being an imAE, the Investigator should promptly contact MedImmune AESIs observed with durvalumab and tremelimumab include:  
- Diarrhea / Colitis and intestinal perforation - Pneumonitis / ILD
- Hepatitis / transaminase increases
- Endocrinopathies (i.e. events of hypophysitis/hypopituitarism, adrenal insufficiency, hyper- and hypothyroidism and type I diabetes mellitus)
- Rash / Dermatitis
- Nephritis / Blood creatinine increases - Pancreatitis / serum lipase and amylase increases - Myocarditis
- Myositis / Polymyositis
- Neuropathy / neuromuscular toxicity (i.e. Guillain-Barré, and myasthenia gravis)  
- Intestinal Perforation
Other inflammatory responses that are rare / less frequent with a potential immune-mediated aetiology include, but are not limited to:
- pericarditis,  
- sarcoidosis,
- uveitis and other events involving the eye, skin, - hematological and rheumatological events, - vasculitis, - non-infectious meningitis and non-infectious encephalitis
It is possible that events with an inflammatory or immune mediated mechanism could occur in nearly all organs. In addition, infusion-related reactions and hypersensitivity/anaphylactic reactions with a different underlying pharmacological aetiology are also considered AESIs. Further information on these risks (e.g. presenting symptoms) can be found in the current version of the durvalumab and tremelimumab Investigator Brochure. More specific guidelines for their evaluation and treatment are described in detail in the Dosing Modification and Toxicity Management Guidelines (please see **Appendix B**). These guidelines have been prepared by the Manufacturer to assist the Investigator in the exercise of his/her clinical judgment in treating these types of toxicities. These guidelines apply to AEs considered causally related to the study drug/study regimen by the reporting investigator. For durvalumab and tremelimumab, AESIs will comprise the following:  
3.4.3.1 Pneumonitis: AEs of pneumonitis are also of interest, as pneumonitis has been observed with use of anti-PD-1 mAbs (but not with anti-PD-L1 mAbs). Initial work-up should include a high-resolution CT scan, ruling out infection, and pulse oximetry. Pulmonary consultation is highly recommended. Guidelines for the management of patients with immune-mediated AEs (imAEs) including pneumonitis are provided in **Appendix B**.  
3.4.3.2 Infusion reactions: AEs of infusion reactions (also termed infusion-related reactions) are of special interest to AstraZeneca and are defined, for the purpose of this protocol, as all AEs occurring from the start of IP infusion up to 48 hours after the infusion start time. For all infusion reactions, SAEs should be reported to AstraZeneca Patient safety.  
3.4.3.3 Hypersensitivity reactions: Hypersensitivity reactions as well as infusion-related reactions have been reported with anti PD-L1 and anti-PD-1 therapy [17]. As with the administration of any foreign protein and/or other biologic agents, reactions following the infusion of mAbs can be caused by various mechanisms, including acute anaphylactic (IgE-mediated) and anaphylactoid reactions against the mAbs and serum sickness. Acute allergic reactions may occur, may be severe, and may result in death. Acute allergic reactions may include hypotension, dyspnea, cyanosis, respiratory failure, urticaria, pruritus, angioedema, hypotonia, arthralgia, bronchospasm, wheeze, cough, dizziness, fatigue, headache, hypertension, myalgia, vomiting, and unresponsiveness. Guidelines for the management of patients with hypersensitivity (including anaphylactic reaction)
and infusion-related reactions are provided in **Appendix B**.  
3.4.3.4 Hepatic function abnormalities (hepatotoxicity): Hepatic function abnormality is defined as any increase in ALT or AST to greater than 3 × ULN and concurrent increase in total bilirubin to be greater than 2 × ULN. Concurrent findings are those that derive from a single blood draw or from separate blood draws taken within 8 days of each other. Followup investigations and inquiries will be initiated promptly by the investigational site to determine whether the findings are reproducible and/or whether there is objective evidence that clearly supports causation by a disease (e.g. cholelithiasis and bile duct obstruction with distended gallbladder) or an agent other than the IP. Guidelines for management of patients with hepatic function abnormality are provided in **Appendix B**.  
3.4.3.5 Gastrointestinal disorders: Diarrhea/colitis is the most commonly observed treatment emergent SAE when tremelimumab is used as monotherapy. In rare cases, colon perforation may occur that requires surgery (colectomy) or can lead to a fatal outcome if not properly managed. Guidelines on management of diarrhea and colitis in patients receiving tremelimumab are provided in **Appendix B**.  
3.4.3.6 Endocrine disorders: Immune-mediated endocrinopathies include hypophysitis, adrenal insufficiency, and hyper- and hypothyroidism. Guidelines for the management of patients with immune-mediated endocrine events are provided in **Appendix B**.  
3.4.3.7 Pancreatic disorders: Immune-mediated pancreatitis includes autoimmune pancreatitis, and lipase and amylase elevation. Guidelines for the management of patients with immune-mediated pancreatic disorders are provided in Appendix B.  
3.4.3.8 Neurotoxicity: Immune-mediated nervous system events include encephalitis, peripheral motor and sensory neuropathies, Guillain-Barré, and myasthenia gravis. Guidelines for the management of patients with immune-mediated neurotoxic events are provided in Appendix B.  
3.4.3.9 Nephritis: Consult with Nephrologist. Monitor for signs and symptoms that may be related to changes in renal function (e.g. routine urinalysis, elevated serum BUN and creatinine, decreased creatinine clearance, electrolyte imbalance, decrease in urine output, proteinuria, etc.)
Patients should be thoroughly evaluated to rule out any alternative etiology (e.g. disease progression, infections etc.) Steroids should be considered in the absence of clear alternative etiology even for low grade events (Grade 2), in order to prevent potential progression to higher grade event. Guidelines for the management of patients with immune-mediated neurotoxic events are provided in **Appendix B**.
untitled h1
3.5 follow up period

Subjects will be followed up on a monthly basis until off-study criteria are met, with restaging scans being performed every 8 weeks. 3.6 Study Calendar  
| Cycle 1i                                      | Cycle 2i   | Cycle 3-4i      | Cycle 5-   | FU j   |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
|-----------------------------------------------|------------|-----------------|------------|--------|----|----|----|----|----|----|----|----|----|----|----|-----|----|----|
| Scree ning                                    | Baseli ne  | D               | D -        |        |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| -12                                           | 5          | Off Treatment i |            |        |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| D                                             | D          | D               | D          | D      | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  | D   | D  |    |
| 1                                             | 2          | 8               | 15         | 22     | 1  | 2  | 8  | 15 | 22 | 1  | 8  | 15 | 22 | 1  | 8  | 15  | 22 |    |
| Durvalumab a                                  | X          | X               | X          | X      |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Tremelimumab b                                | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Pexa-Vec c                                    | X          | X               | X          | X      |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| NIH Advance                                   | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Directive Form d Medical history              | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Concomitant meds                              | X          | X               | X          | X      | X  |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Formal adverse  event evaluation              | X          | X               | X          | X      | X  | X  | X  |    |    |    |    |    |    |    |    |     |    |    |
| Physical exam and  ECOG                       | X          | X e             | X          | X      | X  | X  | X  | X  |    |    |    |    |    |    |    |     |    |    |
| Vital Signs                                   | X          | X e             | X          | X      | X  | X  | X  | X  | X  | X  | X  |    |    |    |    |     |    |    |
| X                                             | X e        | X               | X          | X      | X  | X  | X  | X  | X  | X  | X  | X  | X  | X  | X  | X X | X  | X  |
| CBC w/differential, Platelets f PT, INR, PTT, | X          | X               | X          | X      | X  |    |    |    |    |    |    |    |    |    |    |     |    |    |
| fibrinogen f Thyroid Panel f                  | X          | X               | X          | X      | X  |    |    |    |    |    |    |    |    |    |    |     |    |    |  
| Cycle 1i                               | Cycle 2i   | Cycle 3-4i      | Cycle 5-   | FU j   |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
|----------------------------------------|------------|-----------------|------------|--------|----|----|----|----|----|----|----|----|----|----|----|----|-----|----|----|
| Scree ning                             | Baseli ne  | D               | D -        |        |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| -12                                    | 5          | Off Treatment i |            |        |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| D                                      | D          | D               | D          | D      | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  | D   |    |    |
| 1                                      | 2          | 8               | 15         | 22     | 1  | 2  | 8  | 15 | 22 | 1  | 8  | 15 | 22 | 1  | 8  | 15 | 22  |    |    |
| Biochemical profile                    | X          | X e             | X          | X      | X  | X  | X  | X  | X  | X  | X  | X  | X  | X  | X  | X  | X X | X  | X  |
| f, n  24-hour urine per  PI discretion | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Urinalysis                             | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| ACTH, morning  cortisol                | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Tumor marker  CEA                      | X          | X               | X          | X      | X  |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| ECG                                    | X          | X e             | X          | X      | X  |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Echocardiogram p                       | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Troponins                              | X          | X               | X          | X      | X  | X  |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Restaging Radiologic  Evaluation g     | X          | X e             | X          | X      |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Tumor biopsy h, m                      | X          | X               | X          | X  k   |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| Immune                                 | X          | X               | X          | X      | X  |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| monitoring l Blood for TCR beta        | X          | X               | X          | X      | X  |    |    |    |    |    |    |    |    |    |    |    |     |    |    |
| sequencing l                           |            |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |     |    |    |  
| Cycle 1i                                            | Cycle 2i   | Cycle 3-4i      | Cycle 5-   | FU j   |    |    |    |    |    |    |    |    |    |    |    |    |    |
|-----------------------------------------------------|------------|-----------------|------------|--------|----|----|----|----|----|----|----|----|----|----|----|----|----|
| Scree ning                                          | Baseli ne  | D               | D -        |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| -12                                                 | 5          | Off Treatment i |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| D                                                   | D          | D               | D          | D      | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  | D  |
| 1                                                   | 2          | 8               | 15         | 22     | 1  | 2  | 8  | 15 | 22 | 1  | 8  | 15 | 22 | 1  | 8  | 15 | 22 |
| HIV, Hepatitis B  and C serology  and/or viral load | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| Serum or urine  pregnancy test                      | X          | X e             |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| HLA                                                 | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| TB testing  (if clinically  indicated)              | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| Histologic  confirmation of dx                      | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| Microsatellite stability status                     | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| Cardiology consult                                  | X          |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |
| as indicated o                                      |            |                 |            |        |    |    |    |    |    |    |    |    |    |    |    |    |    |  
a All patients will receive 1,500 mg of durvalumab via IV infusion on Day 1 of each cycle till patients meet off treatment criteria. A 1hour observation period is required after the first infusion of durvalumab. If no clinically significant infusion reactions are observed during or after the first cycle, subsequent infusion observation periods can be at the Investigator's discretion (suggested 30 minutes after each durvalumab infusion).  
b Additionally, patients in Arms B1 and B2 will receive 300 mg of tremelimumab via IV infusion on Day 1 of cycle 1. Tremelimumab will be administered first. Durvalumab infusion will start approximately 1 hour after the end of tremelimumab infusion. The duration will be approximately 1 hour for each infusion. A 1-hour observation period is required after the first infusion of durvalumab and tremelimumab. If no clinically significant infusion reactions are observed during or after the first cycle, subsequent infusion observation periods can be at the Investigator's discretion (suggested 30 minutes after each durvalumab infusion).  
cPexa-Vec (JX-594) will be administered at a dose of 3 x 108 pfu (DL 1) or 1 x 109 pfu (DL 2) via IV infusion over 60 minutes (±5 minutes) for 4 doses only, beginning on Day - (minus) 12. Patients will be observed in the clinic and/or hospital for a minimum of 20 hours after the infusion. Treatments 2–4 may each be administered in a treatment window of ±3days.  
dAs indicated in section **12.3**, all subjects will be offered the opportunity to complete an NIH Advance Directive 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.  
e Tests done on screening do not need to be repeated on baseline if performed in designated time frame f For all labs a '+/- 72hr' window applies, with the exception of those needed to determine proceeding with treatment. Labs may be performed outside of NIH.  
g Restaging CT scan (or MRI if clinically indicated) every 8 weeks +/- 3 days to evaluate TTP in target lesion. h Mandatory baseline and post-treatment tumor biopsies will be obtained on all patients, with the timing of second biopsy being determined by assignment on 1:1 basis.  
i Cycles will be 28 days in length +/- 3 days. j FU: Follow up visits once a month until patients are off study. If patients are not able to come to NIH, they will be followed by phone contact annually for survival, performance status, new cancer treatment.  
k The biopsy around day 85 (Cycle 4 day 1) is optional l Until PD while on protocol treatment, a '+/- 48hr' window applies m Biopsy will have a window of +/-72 hrs. to allow for logistical issues; however, the biopsy will not be performed within 48 hrs. after Pexa-Vec administration n Electrolytes, BUN, creatinine, AST, ALT, total and direct bilirubin, calcium, phosphorus, albumin, magnesium, uric acid, amylase.  
o Performed in patients with history of cardiovascular disease. See section **2.1.2.10**
p Echocardiogram will be mandatory at the screening and performed during the study if clinically indicated.
untitled h1
3.7 criteria for removal from

Prior to removal from study, effort must be made to have all subjects complete a safety visit approximately 30, 60 and 90 days following the last dose of study therapy. 3.7.1 Criteria for removal from protocol therapy:  
- Participant requests to be withdrawn from active therapy
- Unacceptable Toxicity as defined in section **3.1.1** and **Appendix B**
- Positive pregnancy test or intent to become pregnant
- Investigator discretion
- Initiation of alternative anticancer therapy including another investigational agent
- Progressive Disease. NOTE: While RECIST PD will be noted and recorded the immunerelated RECIST criteria will be applied to determine discontinuation of study treatment.  
- Subject who has received any amount of infliximab or other tumor necrosis factor alpha inhibitor
- Intercurrent illness that prevents further administration of treatment 3.7.2 Off Study Criteria
- Lost to follow-up
Subjects will be considered lost to follow-up only if no contact has been established by the time the study is completed such that there is insufficient information to determine the subject's status at that time. Subjects who refuse continuing participation in the study including telephone contact should be documented as "withdrawal of consent" rather than "lost to follow-up." Investigators should document attempts to re-establish contact with missing subjects throughout the study period. If contact with a missing subject is reestablished, the subject should not be considered lost to follow-up and any evaluations should resume according to the protocol.  
- Death
- Participant requests to be withdrawn from study
- Investigator discretion
- PI decision to end the study
untitled h1
4 concomitant medications/measures

All routine and appropriate supportive care (including blood products) will be provided during this study, as clinically indicated, and in accordance with the standard of care practices. Clinical judgment should be utilized in the treatment of any AE experienced by the patient. Information on all concomitant medications, administered blood products, as well as interventions occurring during the study must be recorded on the patient's eCRF in C3D.
untitled h1
4.1 recommended medications related to

(mild-to-severe fever, rigors, anorexia, aches/pain, fatigue, headache, and/or nausea) Acute Post-Treatment Symptom Management During the post-treatment observation period, patients should receive IV solute-containing fluid and other measures (e.g. vasopressor therapy) per SOC as needed for blood pressure support. Antirigor medication (e.g. meperidine) or support medications may be used as needed.  
Anti-emetics may be used at the Investigator's discretion for treatment of nausea or vomiting; it is noted, however, that corticosteroids should not be used. Analgesics, anti-pyretics (e.g. prophylactic acetaminophen), antidepressants, bisphosphonates, EPO growth factors and other supportive care measures may be used at the Investigator's discretion.
untitled h1
4.2 management of hypotension and

To mitigate risk of hypotension following Pexa-Vec treatment, the following are required:
- Prehydration with 1 liter of solute-containing fluid (p.o. or IV) prior to each treatment  
- Suspension of anti-hypertensive medication for 48 hours prior to and 48 hours after all Pexa-Vec treatments
- Anti-hypertensive medications include, but are not limited to, the following:
- Diuretics
- Beta-blockers
- ACE inhibitors
- Aldosterone agonists
untitled h1
4.3 agents with potential inhibitory

In the extremely unlikely case of generalized vaccinia virus infection, encephalitis or another clinically-significant, progressive toxicity that, in the opinion of the Investigator could be related to Pexa-Vec replication, Investigators may consider the use of agents with vaccinia inhibitory activity, if available in their country for such clinical use. Such agents may include anti-vaccinia immune globulin (VIG), cidofovir, and/or Arestvyr (USAN tecovirimat; ST-246). In addition, based on limited preclinical data, interferon, ribavirin, and sorafenib may have anti-vaccinia activity. For clarity, none of these agents have been used or approved for this purpose to date. Since these agents have not been used in Pexa-Vec treated patients to date, clinical judgment should be used when determining the optimal regimen and duration of treatment. The availability of these or similar antiviral products is country-dependent; options will be investigated and a plan for a response to infection-related toxicities will be documented and communicated to study staff prior to initiation of the clinical trial.
untitled h1
4.4 contraindicated antiviral therapy shown

- Cidofovir (except to treat toxicities potentially related to uncontrolled Pexa-Vec replication)
untitled h1
- interferon/peg-interferon ribavirin 4.5 contraindicated

- High dose systemic corticosteroids (defined as ≥20 mg/day prednisone or equivalent which is ongoing at the time of arm allocation and/or was taken for more than 4 weeks within the preceding 2 months of enrollment) or other immunosuppressive medications  
- Anti-coagulation or anti-platelet medication that cannot be interrupted prior to study specified biopsies injections, including:
- Aspirin that cannot be discontinued for 7 days prior to biopsy
- Coumadin that cannot be discontinued for 7 days prior to biopsy
- LMWH that cannot be discontinued >24 hours prior to biopsy and UFH that cannot be discontinued >4 hours prior to biopsy (LMWH or UFH may be used to transition patients on and off the above anti-coagulants, if deemed appropriate by the treating physician) prior to Pexa-Vec treatments as long as the last dose of LMWH is administered 24 hours prior to biopsy and the last dose of UFH is administered >4 hours prior to treatments)  
- Oral direct thrombin inhibitor (dabigatran) or direct Factor Xa inhibitor (rivaroxaban, apixaban, and edoxaban) that cannot be discontinued for 4 days prior to biopsy
untitled h1
4.6 other authorized concomitant treatments

- thrombopoietin, erythropoietin, G-CSF - antidepressants - steroids (NOTE: oral or parenteral steroids are not allowed during the Pexa-Vec treatment period, and for 1 week prior to and 2 weeks after Pexa-Vec treatment)  
- topical therapies for symptomatic relief of hand-foot skin reaction and rash related to sorafenib
- bisphosphonates, vitamin B12 and vitamin D
- Hepatitis B antivirals are ALLOWED as DO NOT inhibit vaccinia virus replication:
lamivudine, adefovir (and adefovir dipivoxil prodrug), telbivudine, tenofovir, emtricitabine, clevudine and entecavir. The ability of combinations of these agents to inhibit vaccinia virus replication has however not been evaluated. Therefore, combination therapy with anti-viral agents should be avoided if at all possible.  
NOTES:
- Please contact MedImmune for any questions regarding management of immunosuppressive, anticoagulant, antihypertensive, or antiviral medications prior to treatments.  
- Sites will be notified by MedImmune as additional information regarding allowed and contraindicated antivirals becomes available
4.7 Permitted concomitant medications **for any combination of investigational drugs**
Table 2. Supportive medications  
| Supportive medication/class of drug:                                                                                                                                                                                              | Usage:                                                |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|
| Concomitant medications or treatments  (e.g. acetaminophen or diphenhydramine)  deemed necessary to provide adequate  prophylactic or supportive care, except for those  medications identified as "prohibited," as listed  above | To be administered as prescribed by  the Investigator |
| Best supportive care (including antibiotics,  nutritional support, correction of metabolic  disorders, optimal symptom control, and pain  management [including palliative radiotherapy  to non-target lesions, etc.])            | Should be used, when necessary, for  all patients     |
| Inactivated viruses, such as those in the                                                                                                                                                                                         | Permitted                                             |
| influenza vaccine Hormone therapy for non-cancer diagnoses                                                                                                                                                                        | Permitted                                             |
untitled h1
4.8 excluded concomitant medications table

| Prohibited medication/class of drug:                                                                                                                                  | Usage:                                                                                                                                                                                                                                                                                                                                                                  |            |         |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------|---------|
| Any                                                                                                                                                                   | investigational                                                                                                                                                                                                                                                                                                                                                         | anticancer | therapy |
| other than those under investigation in this  study                                                                                                                   | Should not be given concomitantly whilst the  patient is on study treatment                                                                                                                                                                                                                                                                                             |            |         |
| mAbs against CTLA-4, PD-1, or PD-L1  other than those under investigation in this  study                                                                              | Should not be given concomitantly whilst the  patient is on study treatment                                                                                                                                                                                                                                                                                             |            |         |
| Any concurrent chemotherapy,  radiotherapy, immunotherapy, or biologic  or hormonal therapy for cancer treatment  other than those under investigation in this  study | Should not be given concomitantly whilst the  patient is on study treatment. (Concurrent use of  hormones for non-cancer-related conditions  [e.g. insulin for diabetes and hormone  replacement therapy] is acceptable. Local  treatment of isolated lesions, excluding target  lesions, for palliative intent is acceptable [e.g. by  local surgery or radiotherapy]) |            |         |  
| Prohibited medication/class of drug:                                                                                                                                                                                              | Usage:                                                                                                                                                                                |         |     |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------|-----|
| Immunosuppressive medications  including, but not limited to, systemic  corticosteroids at doses exceeding <<10  mg/day>> of prednisone or equivalent,  methotrexate, azathioprine, and tumor  necrosis factor-α blockers         | Should not be given concomitantly.                                                                                                                                                    | (Use of |     |
| immunosuppressive                                                                                                                                                                                                                 | medications                                                                                                                                                                           | for     | the |
| management of IP-related AEs, premedication for  patients who had infusion reaction, or in patients  with contrast allergies is acceptable). In addition,  use of inhaled, topical, and intranasal  corticosteroids is permitted. |                                                                                                                                                                                       |         |     |
| Drugs with laxative properties and herbal                                                                                                                                                                                         | Should be used with caution through to 90 days                                                                                                                                        |         |     |
| or natural remedies for constipation                                                                                                                                                                                              | after the dose of tremelimumab during the study                                                                                                                                       |         |     |
| Sunitinib                                                                                                                                                                                                                         | Should not be given concomitantly or through 90  days after the dose of tremelimumab (acute renal  failure has been reported with combination  therapy of tremelimumab and sunitinib) |         |     |
untitled h1
4.9 methods of contraception

The following restrictions apply while the patient is receiving study treatment and for the specified times before and after:
untitled h1
4.9.1 female patient of child-bearing

Females of childbearing potential who are sexually active with a non-sterilized male partner must use at least 2 **highly** effective method of contraception (**Table** 4. ) from the time of screening and must agree to continue using such precautions for 180 days after the last dose of durvalumab + tremelimumab combination therapy or 90 days after the last dose of durvalumab monotherapy. Non-sterilized male partners of a female patient must use male condom plus spermicide throughout this period. Cessation of birth control after this point should be discussed with a responsible physician. Not engaging in sexual activity for the total duration of the drug treatment and the drug washout period is an acceptable practice; however, periodic abstinence, the rhythm method, and the withdrawal method are not acceptable methods of birth control. Female patients should also refrain from breastfeeding throughout this period.
untitled h1
4.9.2 male patients with a

Non-sterilized males who are sexually active with a female partner of childbearing potential must use a male condom plus spermicide from screening through 180 days after the last dose of durvalumab + tremelimumab combination therapy or 90 days after the last dose of durvalumab monotherapy. Eengaging in sexual activity is an acceptable practice; however, occasional abstinence, the rhythm method, and the withdrawal method are not acceptable methods of contraception. Male patients should refrain from sperm donation throughout this period. Female partners (of childbearing potential) of male patients must also use a highly effective method of contraception throughout this period (Table 4. ).  
N.B Females of childbearing potential are defined as those who are not surgically sterile (i.e. bilateral tubal ligation, bilateral oophorectomy, or complete hysterectomy) or post-menopausal.
untitled h1
4.9.3 highly effective methods of

Highly effective methods of contraception, defined as one that results in a low failure rate (i.e. less than 1% per year) when used consistently and correctly are described in **Table** 4. . Note that some contraception methods are not considered highly effective (e.g. male or female condom with or without spermicide; female cap, diaphragm, or sponge with or without spermicide; non-copper containing intrauterine device; progestogen-only oral hormonal contraceptive pills where inhibition of ovulation is not the primary mode of action [excluding Cerazette/desogestrel which is considered highly effective]; and triphasic combined oral contraceptive pills).  
Table 4. Highly Effective methods of contraception (<1% failure rate)  
| Barrier/Intrauterine methods   | Hormonal Methods                                               |    |                                                              |
|--------------------------------|----------------------------------------------------------------|----|--------------------------------------------------------------|
| -                              | Copper T intrauterine device                                   | -  | Etonogestrel implants: e.g. Implanon  or Norplant            |
| -                              | Levonorgesterel-releasing intrauterine  system (e.g. Mirena®)a | -  | Intravaginal device: e.g.  ethinylestradiol and etonogestrel |
| -                              | Medroxyprogesterone injection: e.g.  Depo-Provera              |    |                                                              |
| -                              | Normal and low dose combined oral  contraceptive pill          |    |                                                              |
| -                              | Norelgestromin/ethinylestradiol  transdermal system            |    |                                                              |
| -                              | Cerazette (desogestrel)                                        |    |                                                              |  
a This is also considered a hormonal method
untitled h1
4.10 blood donation

Subjects should not donate blood while participating in this study, and for 3 months after the last durvalumab infusion or 6 months after the last dose of durvalumab + tremelimumab combination therapy, whichever comes later.
untitled h1
5 biospecimen collection 5.1 correlative

The correlative studies which we wish to perform are outlined below and summarized in the **Table**
5. A description of each test including a brief statement of rationale and processing information is made below.  
Table 5. Biospecimen collection  
| Test/assay                                                                                            | Sample volume   | Type               | of    | Collection point   | Location   | of                         |
|-------------------------------------------------------------------------------------------------------|-----------------|--------------------|-------|--------------------|------------|----------------------------|
| (approx)                                                                                              | tube            | specimen analysis1 |       |                    |            |                            |
| Immunemonitoring                                                                                                       | 120mls          | (for               | EDTA  | See                | Study      | Greten Lab                 |
| PBMC)                                                                                                 | Calendar 3.6    |                    |       |                    |            |                            |
| 5-10mls                                                                                               | (for            | EDTA               |       |                    |            |                            |
| serum)                                                                                                |                 |                    |       |                    |            |                            |
| TCR                                                                                                   | beta            | 1ml (for PBMC)     | EDTA  | Study              | Calendar   | Adaptive                   |
| sequencing                                                                                            | 3.6             | Biotechnologies2   |       |                    |            |                            |
| Immune                                                                                                | cell            |                    |       |                    |            |                            |
| infiltration,                                                                                         | IHC,            |                    |       |                    |            |                            |
| (CD3+ CD4/8 cells  etc), PD-L1                                                                        | tumor biopsy    | NA                 | Study | Calendar           | Greten Lab |                            |
| 3.6                                                                                                   |                 |                    |       |                    |            |                            |
| TCR                                                                                                   | beta            | tumor biopsy       | NA    | Study              | Calendar   | Adaptive  Biotechnologies2 |
| sequencing                                                                                            | 3.6             |                    |       |                    |            |                            |
| RNA                                                                                                   | Nanostring      |                    |       |                    |            |                            |
| analysis  (nCounterPan  Cancer  Immunology  Profile)                                                  | tumor biopsy    | NA                 | Study | Calendar           | Greten Lab |                            |
| 3.6                                                                                                   |                 |                    |       |                    |            |                            |
| 1  Blood samples will initially be sent to the Figg laboratory for barcoding and storage (See Section |                 |                    |       |                    |            |                            |  
1 Blood samples will initially be sent to the Figg laboratory for barcoding and storage (See Section 5.2.1).  
2 Lara Gruye, Adaptive Biotechnologies, 1551 Eastlake Ave E \#200, Seattle, WA 98102 (855)
466-8667
untitled h1
5.1.1 immune monitoring

We will analyze PBMC for quantitative and functional changes of effector cells as well as analyze sera for cytokines and chemokines. The effect on (i) CD4 T cell number and activity, (ii) CD8 T cell number and activity, (iii) NK cell number and activity, (iv) Treg number, (vi) MDSC:
frequency + functional assay, (vii) selected cytokines in serum, and (viii) the detection of tumorassociated antigens using tetramer assay.  
Patients will undergo blood sampling on the time points outlined in the Study Calendar 3.6. Blood will initially be sent to the Figg laboratory for barcoding and processing. On certain occasions the blood may also be brought to the Greten lab for processing and analysis.
untitled h1
5.1.2 mandatory tumor biopsies

Tumor biopsies will be collected at baseline and either day 1 +/- 3 days or day 29+/- 3 days by the Interventional Radiology team by a percutaneous approach (note biopsy may not be collected within 48 hours after Pexa-Vec administration). It is preferred that at least two core biopsies ≥18 gauge in diameter and ≥1 cm in length will be obtained.  
If a site is deemed appropriate for biopsy with minimal risk to the participant by agreement between the investigators and Interventional Radiology team, an attempt for biopsy will be made. The use of imaging to facilitate biopsies will be decided by members of the Interventional Radiology team and may include ultrasound, CT scan, PET scan or MRI. Should a CT scan be needed for biopsy, the number of scans for each procedure will be limited to the minimum number needed to safely obtain a biopsy. Tumor biopsies and local anesthesia will be administered only if they are considered to be of low risk to the participant, as determined by the investigators and Interventional Radiology. If an initial attempt at percutaneous biopsy is unsuccessful, the participant will be given an option to proceed with a repeated attempt at percutaneous biopsy.
untitled h1
5.1.3 tumor tissue analysis

IHC will be performed on tumor tissue for assessment of immune cell infiltration (e.g. CD3+ CD4/8 cells etc.) as well as other surface markers such as PD-L1.
untitled h1
5.1.3.1 optional tumor biopsy

A tumor biopsy may be performed at a subsequent timepoint (D85 approximately) if the patient is willing and the procedure can be performed safely. Tumor Tissue will be processed by the Department of Pathology, NCI, NIH (Dr. David Kleiner). Two core biopsies will be the attempted. Each specimen will be processed and analyzed as stated above.
untitled h1
5.2 sample storage tracking and

Samples will be ordered in CRIS Screens and tracked through a Clinical Trial Data Management system. Should a CRIS screen not be available, the CRIS downtime procedures will be followed. All samples will be sent to Dr. Figg's lab for processing and storage until they are distributed to Dr. Greten's lab for sample analysis as described in the protocol. Samples will not be sent outside the National Institutes for Health (NIH) without appropriate approvals and/or agreements, if required.  
All samples will be barcoded, with data entered and stored in the secure databases. These databases create a unique barcode ID for every sample and sample box, which cannot be traced back to patients without database access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Patient demographics associated with the clinical center patient number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.).  
Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -800C according to stability requirements. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in database. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol) and that any unused samples must be returned. It is the responsibility of the NCI Principal Investigator to ensure that the samples requested are being used in a manner consistent with IRB approval.  
5.2.1 Samples Managed by Dr. Figg's Blood Processing Core (BPC)  
5.2.1.1 BPC contact information
Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number).  
For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov .  
5.2.2 Samples Managed by the Laboratory of Dr Greten Contact information: Sophie Wang Building 10 Rm 3B44 Phone: 240-858-3218 E-mail: sophie.wang@nih.gov 5.2.3 Protocol Completion/Sample Destruction All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are remaining at the completion of the protocol will be stored in the conditions described in sections above. The study will remain open so long as sample or data analysis continues. Samples from consenting subjects will be stored until they are no longer of scientific value or if a subject withdraws consent for their continued use, at which time they will be destroyed. If the patient withdraws consent the participant's data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved. The PI will record any loss or unanticipated destruction of samples as a deviation. Reports will be made per the requirements of section 7.2.  
If the patient withdraws consent the participant's data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved.
untitled h1
5.3 samples for genetic/genomic analysis

TCR (cell receptor) gene usage may be quantitated in samples using conventional sequencing techniques of the T cell receptor variable region of the beta chain. Fewer than 100 genes will be analyzed. For TCR Beta Sequencing the NCI Thoracic and GI Malignancies Branch will release coded, linked tumor and PBMC samples to Adaptive Biotechnologies. Blood and tumor samples will be used. 5.3.2 Nanostring analysis (nCounterPan Cancer Immunology Profile) Tumor samples will also be used for Nanostring analysis (nCounterPan Cancer Immunology Profile) 5.3.3 Privacy and Confidentiality of medical information/biological specimens Fresh tumor and blood samples will be stored in a minus 80-degree freezer. Initially the samples of each patient will be sent to Dr. Figg's lab (**5.2.1**). At no time will patient's names be used on the blood and tissue samples. The molecular studies will be performed at the Adaptive Biotechnologies, 1551 Eastlake Ave E \#200, Seattle, WA 98102 (855) 466-8667. Subject's genetic data will be deposited in a database such as dbGaP. Although there is controlled access to such a database, such a submission carries theoretical risks of revealing the identity of the subject. This is discussed in the consent. 5.3.4 Management of Results The genetic testing performed on this protocol are not of sufficient scope to generate incidental findings and the results of molecular studies will not be communicated to the patient due to their investigational nature.
untitled h1
6 data collection and evaluation

The PI will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D) and Labmatrix 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 Day -12, through 90 days after the subject received the last study drug administration. Beyond 90 days after the last intervention, only adverse events which are serious and related to the study intervention need to be recorded.  
An abnormal laboratory value will be recorded in the database as an AE **only** if the laboratory abnormality is characterized by any of the following:
- Results in discontinuation from the study  
- Is associated with clinical signs or symptoms - Requires treatment or any other therapeutic intervention - Is associated with death or another serious adverse event, including hospitalization. - Is judged by the Investigator to be of significant clinical impact - If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.
Adverse Events of grade 1 will not be collected.  
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**.
untitled h1
6.2 data sharing plans

6.2.1 Human Data Sharing plan I will share coded, linked human data generated in this research for future research  
− in a NIH-funded or approved public repository clinicaltrials.gov, dbGaP
− in BTRIS
− with approved outside collaborators under appropriate agreements
− in publication and/or public presentations
at the time of publication or shortly thereafter. 6.2.2 Genomic Data Sharing Plan Unlinked genomic data will be deposited in public genomic databases such as dbGaP in compliance with the NIH Genomic Data Sharing Policy.
untitled h1
6.3 response criteria

For the purposes of this study, patients should be re-evaluated for response every 8 weeks (+/- 3 days). In addition to a baseline scan, confirmatory scans should also be obtained 4-8 weeks following initial documentation of objective response. Whilst immune-related RECIST criteria will be taken into consideration with regard to continuation of therapy in the event of growth (with a requirement for confirmation of PD), standard RECIST criteria will be the primary method used for evaluation of the primary endpoint. Response and progression will be evaluated in this study using the new international criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline
(version 1.1)[18]. Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria.
untitled h1
6.3.1 definitions

Evaluable for toxicity: All patients will be evaluable for toxicity from the time of their first treatment.
untitled h1
evaluable for objective response

Only those patients who have measurable disease present at baseline, have received at least one dose of each agent, and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated below. (Note: Patients who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) Evaluable Non-Target Disease Response: Patients who have lesions present at baseline that are evaluable but do not meet the definitions of measurable disease, have received at least one cycle of each agent, and have had their disease re-evaluated will be considered evaluable for non-target disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions.
untitled h1
6.3.2 disease parameters

Measurable disease: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as
- By chest x-ray: >20 mm;  
- By CT scan:
o Scan slice thickness 5 mm or under as >10 mm with CT scan o Scan slice thickness >5 mm: double the slice thickness
- With calipers on clinical exam: >10 mm.
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). Malignant lymph nodes: To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed. Non-measurable disease: All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, these are preferred for selection as target lesions. Target lesions: All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as **target lesions** and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Non-target lesions: All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as **non-target lesions** and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up.
untitled h1
6.3.3 methods for evaluation of

All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam. Clinical lesions: Clinical lesions will only be considered measurable when they are superficial
(e.g., skin nodules and palpable lymph nodes) and ≥10 mm diameter as assessed using calipers
(e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended. Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable. Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible. Ultrasound: Ultrasound is not useful in assessment of lesion size and should not be used as a method of measurement. Ultrasound examinations cannot be reproduced in their entirety for independent review at a later date and, because they are operator dependent, it cannot be guaranteed that the same technique and measurements will be taken from one assessment to the next. If new lesions are identified by ultrasound in the course of the study, confirmation by CT or MRI is advised. If there is concern about radiation exposure at CT, MRI may be used instead of CT in selected instances. Endoscopy, Laparoscopy: The utilization of these techniques for objective tumor evaluation is not advised. However, such techniques may be useful to confirm complete pathological response when biopsies are obtained or to determine relapse in trials where recurrence following complete response (CR) or surgical resection is an endpoint. Cytology, Histology: These techniques can be used to differentiate between partial responses (PR)
and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germ cell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease. 6.3.4 Response criteria 6.3.4.1 Evaluation of Target Lesions Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum of diameters. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study. 6.3.4.2 Evaluation of Non-Target Lesions Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis). Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response.  
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits. Progressive Disease (PD): Appearance of one or more new lesions and/or *unequivocal progression* of existing non-target lesions. *Unequivocal progression* should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator). 6.3.4.3 Evaluation of Best Overall Response The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| For Patients with Measurable Disease (i.e., Target Disease)  Target  Non-Target  New  Overall  Lesions Lesions Lesions Respons e                                                                                                                                                                                                   | Best Overall Response when  Confirmation is Required*                                                                |           |                                                   |                        |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|-----------|---------------------------------------------------|------------------------|
| CR                                                                                                                                                                                                                                                                                                                                 | CR                                                                                                                   | No        | CR                                                | >4 wks. Confirmation** |
| CR                                                                                                                                                                                                                                                                                                                                 | Non-CR/Non-PD                                                                                                        | No        | PR                                                | >4 wks. Confirmation** |
| CR                                                                                                                                                                                                                                                                                                                                 | Not evaluated                                                                                                        | No        | PR                                                |                        |
| PR                                                                                                                                                                                                                                                                                                                                 | Non-CR/Non                                                                                                                      |           |                                                   |                        |
| PD/not evaluated                                                                                                                                                                                                                                                                                                                   | No                                                                                                                   | PR        |                                                   |                        |
| SD                                                                                                                                                                                                                                                                                                                                 | Non-CR/Non                                                                                                                      |           |                                                   |                        |
| PD/not evaluated                                                                                                                                                                                                                                                                                                                   | No                                                                                                                   | SD        | Documented at least once >4  wks. from baseline** |                        |
| PD                                                                                                                                                                                                                                                                                                                                 | Any                                                                                                                  | Yes or No | PD                                                |                        |
| Any                                                                                                                                                                                                                                                                                                                                | PD***                                                                                                                | Yes or No | PD                                                | no prior SD, PR or CR  |
| Any                                                                                                                                                                                                                                                                                                                                | Any                                                                                                                  | Yes       | PD                                                |                        |
| *                                                                                                                                                                                                                                                                                                                                  | See RECIST 1.1 manuscript for further details.                                                                       |           |                                                   |                        |
| **                                                                                                                                                                                                                                                                                                                                 | Only for non-randomized trials with response as primary endpoint.                                                    |           |                                                   |                        |
| ***                                                                                                                                                                                                                                                                                                                                | In exceptional circumstances, unequivocal progression in non-target lesions may  be accepted as disease progression. |           |                                                   |                        |
| Note: Patients with a global deterioration of health status requiring discontinuation of  treatment without objective evidence of disease progression at that time should be  reported as "symptomatic deterioration." Every effort should be made to  document the objective progression even after discontinuation of treatment. |                                                                                                                      |           |                                                   |                        |