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Abbreviated Title: Nivo, tadalafil, vancomycin NIH Protocol: 19C0033 Version Date: 01/19/2022 NCT: NCT03785210 Title: Phase II Study of Nivolumab (anti-PD1), Tadalafil and Oral Vancomycin in Patients with Refractory Primary Hepatocellular Carcinoma or Liver Dominant Metastatic Cancer from Colorectal or Pancreatic Cancers  
NCI Principal Investigator: Tim F. 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  
1  
| Investigational Agents: Drug Name: Nivolumab   | Tadalafil            | Vancomycin          |                     |
|------------------------------------------------|----------------------|---------------------|---------------------|
| IND Number:                                    | 133973               | 133973              | 133973              |
| Sponsor:                                       | NCI CCR              | NCI CCR             | NCI CCR             |
| Manufacturer:                                  | Bristol Myers Squibb | Generic             | Generic             |
| Supplier:                                      | Bristol Myers Squibb | NIH Clinical Center | NIH Clinical Center |
| Pharmacy                                       | Pharmacy             |                     |                     |  
PRÉCIS Background:
Current treatment options for patients with liver cancers, including hepatocellular carcinoma
(HCC) and advanced liver cancers are limited and take no account of the known biological and genetic heterogeneity in these diseases. Median survival for advanced disease remains poor at approximately 1 year.  
Nivolumab is a fully human monoclonal immunoglobulin G4 (IgG4) antibody that is specific for human programmed death-1 (PD-1, cluster of differentiation 279 [CD279]) cell surface membrane receptor. Nivolumab has been approved by FDA for the treatment of HCC and other solid tumors.  
Tadalafil is a phosphodiesterase type 5 (PDE5) inhibitor which have been approved by the FDA for the treatment of pulmonary arterial hypertension, benign prostatic hyperplasia and erectile dysfunction, with a relative safe clinical profile. PDE5 inhibitors have been examined in multiple malignancies and cancer cell lines for their direct anticancer activities, for their efficacy as chemo-sensitizers and for cancer chemoprevention.  
Oral vancomycin is antibiotic that has effect on altering gut commensal bacteria subsequently inducing a liver-selective anti-tumor effect.  
The aim of the study is to evaluate whether the immunomodulatory effect induced by PDE5 inhibitor and oral vancomycin can be enhanced by immune checkpoint inhibition in advanced liver cancer.  
Objective:
To determine the Best Overall Response (BOR) according to Response Evaluation Criteria
(RECIST 1.1) to combined treatment of nivolumab, oral vancomycin and tadalafil in patients with refractory primary HCC or liver dominant metastatic cancer from colorectal cancer (CRC) or pancreatic adenocarcinoma (PDAC).  
Eligibility:
Histologically confirmed, hepatocellular carcinoma (HCC)
Or Histologically confirmed carcinoma highly suggestive of a diagnosis of HCC
Or Histologically confirmed advanced colorectal or pancreatic malignancy with liver involvement as dominant site of metastasis Measurable lesion, accessible for biopsy.  
Age ≥ 18 years ECOG ≤ 1 Acceptable renal, bone marrow and liver function.  
Willingness to undergo two mandatory tumor biopsies.  
Design:
The proposed study is a phase II study of combined nivolumab, oral vancomycin and tadalafil treatment in patients with HCC or liver dominant metastatic cancer from colorectal or pancreatic cancers.  
Treatment will be delivered in cycles consisting of 4 weeks (+/- 3 days) until progression or unacceptable toxicity.  
Patients will be seen in Clinical Center on monthly basis with disease status evaluation every 8 (+/-1) weeks after start of study therapy.
3  
| TABLE OF CONTENTS                                                                                                   |                                                                                                                                                      |                  |
|---------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|
| PRÉCIS                                                                                                              |                                                                                                                                                      |                  |
| TABLE OF CONTENTS                                                                                                   |                                                                                                                                                      |                  |
| STATEMENT OF COMPLIANCE                                                                                             |                                                                                                                                                      |                  |
| INTRODUCTION .                                                                                                      |                                                                                                                                                      |                  |
| 1                                                                                                                   | 1.1                                                                                                                                                  | Study Objectives |
| 1.1.1                                                                                                               | Primary Objective: .                                                                                                                                 |                  |
| 1.1.2                                                                                                               | Secondary Objectives:                                                                                                                                |                  |
| 1.1.3                                                                                                               | Exploratory Objectives: ...................................................................................................................9         |                  |
| 1.2                                                                                                                 | Background and Rationale........................................................................................................10                   |                  |
| 1.2.1                                                                                                               | HCC And Immune Checkpoint Inhibitors (ICIs) .............................................................10                                          |                  |
| 1.2.2                                                                                                               | Liver Metastases and Current Treatment Options ............................................................12                                        |                  |
| 1.2.3                                                                                                               | T TUMOR MICROENVIRONMENT (TME)  ..............................................................................................................14     |                  |
| 1.2.4                                                                                                               | PDE5 Inhibitors Augment Immunotherapy by Targeting on TME.............................16                                                             |                  |
| Gut Microbiome and Liver Cancer Control..........................................................................23 |                                                                                                                                                      |                  |
| 1.2.5                                                                                                               |                                                                                                                                                      |                  |
| 1.2.6                                                                                                               | Nivolumab                                                                                                                                            |                  |
| 1.2.7                                                                                                               | PDE5 Inhibitor and Oral Vancomycin in Combination with Nivolumab .................27                                                                 |                  |
| 2                                                                                                                   | ELIBILITY ASSESSMENT AND ENROLLMENT ...................................................................28                                            |                  |
| 2.1                                                                                                                 | Eligibility Criteria .                                                                                                                               |                  |
| 2.1.1                                                                                                               | Inclusion Criteria ...............................................................................................................................28 |                  |
| 2.1.2                                                                                                               | Exclusion Criteria ....................................................................................................................30            |                  |
| 2.1.3                                                                                                               | Recruitment Strategies                                                                                                                               |                  |
| 2.2                                                                                                                 | Screening Evaluation ............................................................................................................................31  |                  |
| 2.2.1                                                                                                               | Screening activities performed prior to obtaining informed consent .......................31                                                         |                  |
| 2.2.2                                                                                                               | Screening activities performed after a consent for screening has been signed.........32                                                              |                  |
| 2.3                                                                                                                 | Participant Registration and Status Update Procedures                                                                                                |                  |
| 2.3.1                                                                                                               | Treatment Assignment Procedures (for registration purposes only) ..........................33                                                        |                  |
| 2.4                                                                                                                 | Baseline Evaluation                                                                                                                                  |                  |
| 3                                                                                                                   | STUDY IMPLEMENTATION                                                                                                                                 |                  |
| 3.1                                                                                                                 | Study Design                                                                                                                                         |                  |
| 3.2                                                                                                                 | D Drug Administration                                                                                                                                |                  |
| 3.2.1                                                                                                               | Nivolumab Administration ...................................................................................................34                       |                  |
| 3.2.2                                                                                                               | Tadalafil Administration                                                                                                                             |                  |
| 3.2.3                                                                                                               | Vancomycin Administration                                                                                                                            |                  |
| 3.2.4                                                                                                               | Sequence and Monitoring of Dose Administration .......................................................35                                             |                  |
| Dose Delay or Modifications                                                                                         |                                                                                                                                                      |                  |
| 3.3                                                                                                                 |                                                                                                                                                      |                  |
| 3.3.1                                                                                                               | General Guidance .......................................................................................................................35           |                  |
| 3.3.2                                                                                                               | Nivolumab ...............................................................................................................................36          |                  |
| 3.3.3                                                                                                               | Tadalafil                                                                                                                                            |                  |
| 3.3.4                                                                                                               | Oral Vancomycin                                                                                                                                      |                  |
| 3.4                                                                                                                 | Study Calendar...........................................................................................................................43          |                  |
| 3.5                                                                                                                 | Costs and Compensation                                                                                                                               |                  |  
| Version Date: 01/19/2022                                                                                                         |                                                                                                                                                  |                 |
|----------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------|-----------------|
| 3.5.1                                                                                                                            | Costs .................................................................................................................................46        |                 |
| 3.5.2                                                                                                                            | COMPENSATION ...........................................................................................................................46       |                 |
| 3.5.3                                                                                                                            | Reimbursement ......................................................................................................................46           |                 |
| 3.6                                                                                                                              | Criteria for Removal from Protocol Therapy and Off Study Criteria ............................46                                                 |                 |
| 3.6.1                                                                                                                            | Criteria for Removal from Protocol Therapy ..................................................................46                                  |                 |
| 3.6.2                                                                                                                            | OFF Study Criteria ...................................................................................................................46         |                 |
| 3.6.3                                                                                                                            | Lost to Follow-up .......................................................................................................................47      |                 |
| 4                                                                                                                                | CONCOMITANT MEDICATIONS/MEASURES                                                                                                                 |                 |
| 4.1                                                                                                                              | Excluded Concomitant Medications                                                                                                                 |                 |
| 4.2                                                                                                                              | Methods of Contraception ......................................................................................................48                |                 |
| 4.2.1                                                                                                                            | Female Patient of Child-bearing Potential                                                                                                        |                 |
| 4.2.2                                                                                                                            | Male Patients with a Female Partner of Childbearing Potential ...................................48                                              |                 |
| 4.2.3                                                                                                                            | Highly Effective Methods of Contraception                                                                                                        |                 |
| 4.3                                                                                                                              | Blood Donation                                                                                                                                   |                 |
| s                                                                                                                                | CORRELATIVE STUDIES FOR RESEARCH                                                                                                                 |                 |
| 5.1                                                                                                                              | Biospecimen Collection                                                                                                                           |                 |
| 5.2                                                                                                                              | Correlative Studies of Blood Samples .........................................................................................................51 |                 |
| 5.2.1                                                                                                                            | Immune Monitoring                                                                                                                                |                 |
| 5.2.2                                                                                                                            | Serum Cytokines                                                                                                                                  |                 |
| 5.2.3                                                                                                                            | Tadalafil Pharmacokinetic Analysis .................................................................................................51           |                 |
| 5.2.4                                                                                                                            | Vancomycin Pharmacokinetic Analysis ..............................................................................................51             |                 |
| 5.2.5                                                                                                                            | Bile Acid Analysis                                                                                                                               |                 |
| 5.2.6                                                                                                                            | Food Diary for Mixed Meal Study ....................................................................................................51           |                 |
| 5.3                                                                                                                              | Tumor Studies (Non-genetic Analysis) ..........................................................................................52                |                 |
| 5.3.1                                                                                                                            | Tumor Tissue Analysis                                                                                                                            |                 |
| 5.3.2                                                                                                                            | Stool Collection and Processing ..............................................................................................................52 |                 |
| 5.4                                                                                                                              | Sample Storage, Tracking and Disposition ....................................................................................52                  |                 |
| 5.4.1                                                                                                                            | Samples Managed by Dr. Figg's Blood Processing Core (BPC) ....................................53                                                 |                 |
| 5.4.2                                                                                                                            | Samples Managed by the Laboratory of Dr. Tim Greten .....................................................53                                      |                 |
| 5.4.3                                                                                                                            | Protocol Completion/Sample Destruction ...................................................................................53                     |                 |
| 5.5                                                                                                                              | Samples for Genetic/Genomic Analysis                                                                                                             |                 |
| 5.5.1                                                                                                                            | T TCR Beta Sequencing                                                                                                                            |                 |
| 5.5.2                                                                                                                            | Whole exome/Panel (tumor/normal) and RNAseq (tumor).............................................54                                               |                 |
| 5.5.3                                                                                                                            | Microbial DNA Sequence                                                                                                                           |                 |
| 5.5.4                                                                                                                            | Management of Results                                                                                                                            |                 |
| 5.5.5                                                                                                                            | Genetic Counseling                                                                                                                               |                 |
| DATA COLLECTION AND EVALUATION ...............................................................................................55 |                                                                                                                                                  |                 |
| 6                                                                                                                                | 6.1                                                                                                                                              | DATA COLLECTION |
| 6.2                                                                                                                              | DATA SHARING Plans                                                                                                                               |                 |
| 6.2.1                                                                                                                            | Human Data Sharing Plan                                                                                                                          |                 |
| 6.2.2                                                                                                                            | Genomic Data Sharing Plan                                                                                                                        |                 |
| 6.3                                                                                                                              | Response Criteria                                                                                                                                |                 |
| Disease Parameters .                                                                                                             |                                                                                                                                                  |                 |
| 6.3.1                                                                                                                            |                                                                                                                                                  |                 |
| 6.3.2                                                                                                                            | Methods for Evaluation of Measurable Disease ....................................................................57                              |                 |  
6.3.3 Response Criteria 6.4 Toxicity Criteria  
| 7                                                                                                                                    | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN                                                                                              |                                                                                                                                                                  |
|--------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 60                                                                                                                                   |                                                                                                                                                         |                                                                                                                                                                  |
| 7.1                                                                                                                                  | Definitions .........................................................................................................................................60 |                                                                                                                                                                  |
| OHSRP Office of Compliance and Training / IRB Reporting ..........................................60                                 |                                                                                                                                                         |                                                                                                                                                                  |
| 7.2                                                                                                                                  |                                                                                                                                                         |                                                                                                                                                                  |
| 7.2.1                                                                                                                                | Expedited Reporting .................................................................................................................60                 |                                                                                                                                                                  |
| 7.2.2  IRB Requirements for PI Reporting at Continuing Review .........................................61                            |                                                                                                                                                         |                                                                                                                                                                  |
| 7.3                                                                                                                                  | NCI Clinical Director Reporting ..............................................................................................61                        |                                                                                                                                                                  |
| 7.4                                                                                                                                  | NIH Required Data and Safety Monitoring Plan.................................................................61                                         |                                                                                                                                                                  |
| 8                                                                                                                                    | SPONSOR SAFETY REPORTING .............................................................................................61                                |                                                                                                                                                                  |
| 8.1                                                                                                                                  | DEFINITIONS                                                                                                                                             |                                                                                                                                                                  |
| 8.1.1                                                                                                                                | Adverse Event..............................................................................................................................61           |                                                                                                                                                                  |
| 8.1.2                                                                                                                                | Serious Adverse Event (SAE) ................................................................................................61                          |                                                                                                                                                                  |
| 8.1.3                                                                                                                                | Life-threatening .......................................................................................................................62              |                                                                                                                                                                  |
| 8.1.4                                                                                                                                | Severity .                                                                                                                                              |                                                                                                                                                                  |
| Relationship to Study Product ....................................................................................................62 |                                                                                                                                                         |                                                                                                                                                                  |
| 8.1.5                                                                                                                                |                                                                                                                                                         |                                                                                                                                                                  |
| 8.2                                                                                                                                  | Assessment of Safety Events ........................................................................................................62                  |                                                                                                                                                                  |
| 8.3                                                                                                                                  | Reporting of Serious Adverse Events ..........................................................................................63                        |                                                                                                                                                                  |
| 8.4                                                                                                                                  | Waiver of expedited reporting to CCR....................................................................................63                              |                                                                                                                                                                  |
| 8.5                                                                                                                                  | Safety Reporting to the Manufacturer, Bristol Myers Squibb ..........................................63                                                 |                                                                                                                                                                  |
| 8.6                                                                                                                                  | Reporting Pregnancy ...................................................................................................................65               |                                                                                                                                                                  |
| 8.6.1                                                                                                                                | MATERNAL EXPOSURE                                                                                                                                       |                                                                                                                                                                  |
| 8.6.2                                                                                                                                | Paternal exposure                                                                                                                                       |                                                                                                                                                                  |
| 8.7                                                                                                                                  | Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND .................66                                                                  |                                                                                                                                                                  |
| 9                                                                                                                                    | CLINICAL MONITORING................................................................................................................66                   |                                                                                                                                                                  |
| STATISTICAL CONSIDERATIONS ...........................................................................................66             |                                                                                                                                                         |                                                                                                                                                                  |
| 10                                                                                                                                   | 10.1                                                                                                                                                    | Study Objective ..............................................................................................................................................66 |
| Primary Objective                                                                                                                    |                                                                                                                                                         |                                                                                                                                                                  |
| 10.1.1                                                                                                                               |                                                                                                                                                         |                                                                                                                                                                  |
| 10.1.2                                                                                                                               | Secondary Objectives ...............................................................................................................67                  |                                                                                                                                                                  |
| 10.2                                                                                                                                 | Sample Size Determination ...........................................................................................................67                 |                                                                                                                                                                  |
| 10.3                                                                                                                                 | Populations for Analyses ..........................................................................................................68                   |                                                                                                                                                                  |
| 10.4                                                                                                                                 | Statistical Analyses                                                                                                                                    |                                                                                                                                                                  |
| 10.4.1                                                                                                                               | General Approach                                                                                                                                        |                                                                                                                                                                  |
| 10.4.2                                                                                                                               | Analysis of the Primary Endpoints ................................................................................68                                    |                                                                                                                                                                  |
| 10.4.3                                                                                                                               | Analysis of the Secondary Endpoint...........................................................................68                                         |                                                                                                                                                                  |
| 10.4.4                                                                                                                               | Se                                                                                                                                                      |                                                                                                                                                                  |
| Baseline Descriptive Statistics                                                                                                      |                                                                                                                                                         |                                                                                                                                                                  |
| 10.4.5                                                                                                                               |                                                                                                                                                         |                                                                                                                                                                  |
| 10.4.6                                                                                                                               | Planned Interim Analyses ......................................................................................................68                       |                                                                                                                                                                  |
| 10.4.7                                                                                                                               | Exploratory Analyses                                                                                                                                    |                                                                                                                                                                  |
| 11                                                                                                                                   | COLLABORATIVE AGREEMENT............................................................................................69                                   |                                                                                                                                                                  |
| Cooperative Research and Development Agreement (CRADA) .......................................69                                     |                                                                                                                                                         |                                                                                                                                                                  |
| 1.1                                                                                                                                  |                                                                                                                                                         |                                                                                                                                                                  |  
| Version Date: 01/19/2022                                                                                                      |                                                                                                                                                                        |                                                                                                                                           |
|-------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------|
| 12                                                                                                                            | HUMAN SUBJECTS PROTECTIONS ...........................................................................................69                                               |                                                                                                                                           |
| 12.1                                                                                                                          | Rationale for Subject Selection ...............................................................................................69                                      |                                                                                                                                           |
| 12.2                                                                                                                          | Participation of Children                                                                                                                                              |                                                                                                                                           |
| 12.3                                                                                                                          | Participation of Subjects Unable to Give Consent .............................................................69                                                       |                                                                                                                                           |
| 12.4                                                                                                                          | Evaluation of Benefits and Risks/Discomforts for All Participants .................................70                                                                  |                                                                                                                                           |
| 12.4.1                                                                                                                        | Benefits .                                                                                                                                                             |                                                                                                                                           |
| 12.4.2                                                                                                                        | Risks ...........................................................................................................................................70                    |                                                                                                                                           |
| Risks/Benefits Analysis for All Participants ..............................................................................71 |                                                                                                                                                                        |                                                                                                                                           |
| 12.5                                                                                                                          |                                                                                                                                                                        |                                                                                                                                           |
| 12.6                                                                                                                          | Consent Process and Documentation                                                                                                                                      |                                                                                                                                           |
| 12.6.1                                                                                                                        | Consent Process for Adults Who Lack Capacity to Consent to Research                                                                                                    |                                                                                                                                           |
| Participation                                                                                                                 |                                                                                                                                                                        |                                                                                                                                           |
| REGULATORY AND OPERATIONAL CONSIDERATIONS                                                                                     |                                                                                                                                                                        |                                                                                                                                           |
| 13                                                                                                                            | 13.1                                                                                                                                                                   | Study Discontinuation and Closure .....................................................................................................72 |
| 13.2                                                                                                                          | Quality Assurance and Quality Control                                                                                                                                  |                                                                                                                                           |
| 13.3                                                                                                                          | Conflict of Interest Policy...................................................................................................................73                       |                                                                                                                                           |
| 13.4                                                                                                                          | Confidentiality and Privacy                                                                                                                                            |                                                                                                                                           |
| 14                                                                                                                            | PHARMACEUTICAL INFORMATION ..........................................................................................74                                                |                                                                                                                                           |
| 14.1                                                                                                                          | Nivolumab (133973)                                                                                                                                                     |                                                                                                                                           |
| SOURCE:                                                                                                                       |                                                                                                                                                                        |                                                                                                                                           |
| 14.1.1                                                                                                                        |                                                                                                                                                                        |                                                                                                                                           |
| 14.1.2                                                                                                                        | Toxicity:......................................................................................................................................74                      |                                                                                                                                           |
| 14.1.3                                                                                                                        | Formulation and Preparation ................................................................................................74                                         |                                                                                                                                           |
| 14.1.4                                                                                                                        | Stability and Storage                                                                                                                                                  |                                                                                                                                           |
| 14.1.5                                                                                                                        | Administration Procedures .....................................................................................................75                                      |                                                                                                                                           |
| 14.2                                                                                                                          | T Tadalafil                                                                                                                                                            |                                                                                                                                           |
| 14.2.1                                                                                                                        | SOURCE                                                                                                                                                                 |                                                                                                                                           |
| 14.2.2                                                                                                                        | DESCRIPTION                                                                                                                                                            |                                                                                                                                           |
| 14.2.3                                                                                                                        | How Supplied .................................................................................................................................75                       |                                                                                                                                           |
| 14.2.4                                                                                                                        | TOXICITY                                                                                                                                                               |                                                                                                                                           |
| 14.2.5                                                                                                                        | Administration: .....................................................................................................................76                                |                                                                                                                                           |
| 14.3                                                                                                                          | Oral Vancomycin.............................................................................................................................76                         |                                                                                                                                           |
| 14.3.1                                                                                                                        | SOURCE                                                                                                                                                                 |                                                                                                                                           |
| 14.3.2                                                                                                                        | Mode of Action                                                                                                                                                         |                                                                                                                                           |
| 14.3.3                                                                                                                        | Description                                                                                                                                                            |                                                                                                                                           |
| 14.3.4                                                                                                                        | How Supplied ..............................................................................................................................76                          |                                                                                                                                           |
| 14.3.5                                                                                                                        | Administration: ........................................................................................................................77                             |                                                                                                                                           |
| 14.3.6                                                                                                                        | S Storage                                                                                                                                                              |                                                                                                                                           |
| 15                                                                                                                            | REFERENCES .........................................................................................................................................................78 |                                                                                                                                           |
| APPENDICES                                                                                                                    |                                                                                                                                                                        |                                                                                                                                           |
| 16                                                                                                                            | 16.1                                                                                                                                                                   | Appendix A: Performance Status Criteria ................................................................................84                |
| 16.2                                                                                                                          | Appendix B: Patient's Medication Diary                                                                                                                                 | ...................85                                                                                                                     |
| 16.3                                                                                                                          | Appendix C: Management Algorithms For Endocrinopathy, Gastrointestinal, Hepatic,                                                                                       |                                                                                                                                           |
| Neurological, Pulmonary, Renal, And Skin Adverse Events ..........................................................88          |                                                                                                                                                                        |                                                                                                                                           |
| 16.4                                                                                                                          | Appendix D: Infusion-related Reaction Management Algorithm .................................96                                                                         |                                                                                                                                           |  
| 16.5   | Appendix E: Modified immune-related response criteria (irRC) 98   |
|--------|-------------------------------------------------------------------|
| 16.6   | Appendix F: Child-Pugh Classification System 100                  |
| 16.7   | Appendix G: Pregnancy Form 101                                    |
| 16.8   | Appendix H: Food Diary for Mixed Meal Study 105                   |
| 16.9   | Appendix I : Mixed Meal PK Sheet 106                              |
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statement of compliance

The trial will be carried out in accordance with International Conference on Harmonisation Good Clinical Practice (ICH GCP) and the following:
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are responsible for the conduct, management, or oversight of NIH-funded clinical trials have completed Human Subjects Protection and ICH GCP Training. The protocol, informed consent form(s), recruitment materials, and all participant materials will be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both the protocol and the consent form must be obtained before any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB before the changes are implemented to the study. In addition, all changes to the consent form will be IRB-approved; an IRB determination will be made regarding whether a new consent needs to be obtained from participants who provided consent, using a previously approved consent form
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1 introduction 1.1 study objectives

To determine the Best Overall Response (BOR) according to Response Evaluation Criteria
(RECIST 1.1) to combined treatment of nivolumab, oral vancomycin and tadalafil in patients with refractory primary HCC or liver dominant metastatic cancer from colorectal cancer (CRC) or pancreatic adenocarcinoma (PDAC).
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1.1.2 secondary objectives

To assess the safety and tolerability of nivolumab in combination with oral vancomycin and tadalafil in patients with refractory primary HCC or liver dominant metastatic cancer from CRC or PDAC.  
To assess overall survival (OS) of nivolumab combined with oral vancomycin and tadalafil in patients with refractory HCC or liver dominant metastatic cancer from CRC or PDAC.
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1.1.3 exploratory objectives

To measure changes in immune cell population in the peripheral blood and tumors of patients treated on this study, including CD4+, CD8+, MDSCs, MAITS NKT cells, IFNgamma signaling pathway, perforin and granzyme.  
To measure secreted cytokines in the peripheral blood to monitor the relative level change with the treatment To measure changes in bile acid in the peripheral blood of patients treated in this study.  
To evaluate tadalafil and oral vancomycin pharmacokinetic level to monitor compliance and association between the level and response rate or immune parameters To evaluate TCR beta sequencing to analyze the T cell response from PBMC and tumor samples To assess gene and protein expression and metabolite profile from tumor to evaluate the association of the change immune cell signature, signaling pathways and metabolite with clinical outcome To measure changes in gut microbiome in the stool of patients treated on this study.  
To evaluate the incidence of vancomycin-resistant enterococci (VRE) development during the trial.  
To evaluate the relationship between the response rate, overall survival and immune parameters, gut microbiome, and bile acid component in patients treated on this study.
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1.2 background and rationale 1.2.1

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second leading cause of cancer associated mortality with an average life expectancy of 6-9 months[1]. In the United States, there were 39,230 new cases and 27,170 deaths related to liver cancer in 2016[2]. By 2030, these numbers are expected to rise, primarily due to increase rates of hepatitis C virus (HCV) related cirrhosis[3]. In early stage disease, treatment has traditionally comprised surgery (partial resection or transplantation) or locoregional therapies like ablation or chemoembolization. Survival rates are varied between the different modalities. However, most patients relapse with recurrent and often, metastatic disease. In advanced disease, systemic therapy with sorafenib or lenvatinib is the standard first line treatment. Sorafenib is a potent oral multikinase inhibitor that prevents tumor cell growth and angiogenesis. It is approved for inoperable or metastatic HCC based on two randomized phase III clinical trials. In the SHARP trial, sorafenib improved median overall survival (OS) by 3 months compared to best supportive care (BSC); 10.7 months in the sorafenib arm and 7.9 months in the BSC arm (HR 0.69;95% CI 0.55-0.87, p<0.001)[4]. In a similarly designed study by Cheng, et al, Asian patients were enrolled to receive sorafenib versus placebo. Median OS improved by approximately 2 months, 6.5 months in the sorafenib arm vs 4.2 months with placebo (HR 0.68, 95% CI 0.50-0.93, p=0.014)[5]. Recent FDA-approved lenvatinib for advanced HCC based on REFLECT trial showed non-inferior but not statistically superior to sorafenib for OS (HR 0.92; 95% CI: 0.79, 1.06). Median OS in the lenvatinib arm was 13.6 months and 12.3 months in the sorafenib arm. REFLECT also demonstrated a statistically significant improvement in progression-free survival (PFS) with lenvatinib as compared to sorafenib. Median PFS was 7.3 months in the lenvatinib arm and 3.6 months in the sorafenib arm (HR 0.64; 95% CI: 0.55, 0.75; p<0.001). The overall response rate was higher for the lenvatinib arm as compared to sorafenib (19% vs. 7% per RECIST 1.1).[6] For patients who progress following first line treatment, regorafenib and recently nivolumab, are approved as second line agents. Regorafenib is a multikinase inhibitor targeting tumor growth and angiogenesis. In a study comparing regorafenib versus placebo in patients with advanced HCC who progressed through sorafenib, regorafenib improved OS by approximately 4 months (HR 0.63, 95% CI 0.50-0.79, p<0.001), and progression free survival (HR 0.46, 95% CI 0.37-0.56, p<0.001) [7]. To date, there have been 4 published immunotherapy trials in HCC (**Table 1**) [8-11]. The first was a single-arm phase II trial evaluating anti-CTLA-4 tremelimumab in 21 patients with HCC in the setting of second line treatment[10]. Tremelimumab showed promising antitumor activity and an acceptable safety profile. The partial response rate was 17.6% (3/17), and the disease control rate was 76% (13/17). Duration of response was 3.6, 9.2, and 15.7 months in the 3 responders, respectively. More recently, tremelimumab in combination with loco-reginal therapy has demonstrated activity and feasibility [8]. In this pilot study, 32 patients with HCC received tremelimumab at 2 dose levels (3.5 mg/kg in 6 patients and 10 mg/kg in 26 patients) every 4 weeks for 6 doses, then every 3 months. In conjunction with this, 7 patients with Barcelona Clinic Liver Cancer (BCLC) stage B disease underwent Transarterial chemoembolization, 10 patients with BCLC stage C disease underwent radiofrequency ablation, and 11 patients with BCLC stage C disease underwent cryoablation. Rapid disease progression was observed in 4 patients who were then excluded. Response assessment was performed in the 19 patients with evaluable lesions outside of the ablation zone. The partial response rate was 26% (5/19) and the disease control rate was 84% (16/19). CheckMate-040 was the largest study to evaluate immunotherapy in HCC thus far[9]. This was a multicenter phase I/II dose escalation and expansion study investigating nivolumab in patients with advanced HCC. A total of 214 patients with advanced HCC and ChildPugh score of 6 or less received nivolumab 3 mg/kg every 2 weeks on the dose expansion phase.  
The study included patients with and without chronic viral hepatitis, including active HBC (31%) and HCV (21%) but not those with active co-infection with HBV and HCV or with hepatitis D virus infection. Most patients had advanced progressive disease, with 68% having extrahepatic spread and 76% having progressed despite prior systemic therapy. Partial responses were observed in 15% and 20% of patients in the dose-escalation and dose-expansion phases, respectively. Disease control rates were 64% and 58%, respectively. Responses were durable, averaging 17 months and 10 months, respectively. On the basis of CheckMate-040, FDA granted nivolumab accelerated approval in September 2017 for patients with advanced HCC previously treated with sorafenib. Following the aforementioned strong objective activity of nivolumab in the second-line scenario, phase III trials are testing the effect of nivolumab against sorafenib for patients naive to systemic therapy (NCT02576509). Recently, pembrolizumab (another PD-1–blocking agent) showed clinical activity for sorafenib-exposed patients (NCT02702401, KEYNOTE-240)[11]. In this study, 104 eligible patients were enrolled and treated, and objective response was 17% (18/104). The best overall responses were one (1%) complete and 17 (16%) partial responses; meanwhile, 46 (44%) patients had stable disease, 34 (33%) had progressive disease. Overall, the ORR is still suboptimal from these strategies.  
| Table 1: Key immunotherapy trials in Hepatocellular Carcinoma (HCC)  Drug(s) Target Population No.   | RR     | Reference                                             |     |       |                         |
|------------------------------------------------------------------------------------------------------|--------|-------------------------------------------------------|-----|-------|-------------------------|
| Tremelimumab                                                                                         | CTLA-4 | Advanced HCC                                          | 21  | 17.6% | Sangro et  al.[10]      |
| Tremelimumab +  Ablation                                                                             | CTLA-4 | Advanced HCC                                          | 32  | 26.3% | Duffy et al. [8]        |
| Advanced HCC (naïve to  Sorafenib)                                                                   | 80     | 22.5%                                                 |     |       |                         |
| Nivolumab                                                                                            | PD-1   | Advanced HCC (Intolerant or  progressed to Sorafenib) | 182 | 18.6% | EI-Khoueiry et  al. [9] |
| Pembrolizumab                                                                                        | PD-1   | Advanced HCC (Intolerant or  progressed to Sorafenib) | 104 | 17.3% | Zhu et al. [11]         |
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1.2.2 liver metastases and current

Tumors metastatic to the liver are more common than primary tumors. The most common sites of primary tumor are breast, lung, and colorectal cancer. Some authors have reported hepatic metastases in as many as 40 to 50% of adult patients with extrahepatic primary tumors[12]. Due to the central role in the portal circulation, the liver is a frequent site for metastatic tumor, especially for tumors of the digestive tract such as colon and pancreas [13-16]. The incidence of liver metastases is different among the primary tumor types. Accordingly, liver metastasis affects more than 50% of colorectal cancer (CRC) patients during their lifespan[17] and 60% metastatic pancreatic adenocarcinoma (PDAC) patients[13]. Liver metastases also occur at high frequencies in other solid tumors but are usually detected in the late stage of tumor progression. About 8% of prostate cancer patients develop clinically evident liver metastases during their treatment period [18] and a much higher percentage (25–45%) of prostate cancer patients show liver involvement at autopsy[19, 20]. A similar pattern is also observed in breast cancer patients where less than 5% of the patients have clinically evident liver metastases at the time of diagnosis [21, 22]. Liver metastases are associated with a poor prognosis and remain a major cause of morbidity and mortality in patients with malignant tumors of different origins. The presence of liver metastases still remains a clinically significant and therapeutically frustrating problem in clinical oncology secondary to the sanctuary effect of the liver in relation to systemic chemotherapy. For example, 15 to 25% of patients with CRC present with liver metastases, and another 25 to 50% develop liver metastasis within 3 years following resection of the primary tumor [23, 24].  
| Drug(s)       | Target   | Population     | No.   | Liver   |     |      |
|---------------|----------|----------------|-------|---------|-----|------|
| metastases    | RR       | Refer          |       |         |     |      |
| dMMR CRC      |          |                |       |         |     |      |
| Pembrolizumab | PD-1     | Refractory CRC | 11    | 55%     | 40% |      |
| Nivolumab     | PD-1     | Refractory CRC | 74    | N/A     | 32% | Over |  
| Table 2: Key immunotherapy trials in CRC Drug(s) Target                                                                                                          | Population                      | No.                                            | Liver   |     |                        |                               |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------|------------------------------------------------|---------|-----|------------------------|-------------------------------|
| metastases                                                                                                                                                       | RR                              | Refer ence et al.  [26] Over man  et al.  [27] |         |     |                        |                               |
| pMMR CRC                                                                                                                                                         |                                 |                                                |         |     |                        |                               |
| Nivolumab + Ipilimumab                                                                                                                                           | PD-1 +  CTLA-4                  | Refractory CRC                                 | 119     | N/A | 55%                    | Le et                         |
| Pembrolizumab                                                                                                                                                    | PD-1                            | Refractory CRC                                 | 21      | 52% | 0%                     | al.  [25]                     |
| Unselected CRC                                                                                                                                                   | Chun g et  al.  [28]            |                                                |         |     |                        |                               |
| Tremelimumab                                                                                                                                                     | CTLA-4                          | Refractory CRC                                 | 47      | N/A | 2%                     | Topal ian et  al.  [29]  [30] |
| Nivolumab                                                                                                                                                        | PD-1                            | Refractory CRC                                 | 19      | N/A | 0%                     | Brah mer  et al.  [30]        |
| Atezolizumab +  Bevacizumab                                                                                                                                      | Refractory CRC                  | 14                                             | N/A     | 7%  |                        |                               |
| Atezolizumab +  FOLFOX/bev BMS-936559                                                                                                                            | PD-L1                           | Refractory CRC                                 | 18      | N/A | 0%                     | Bend ell et  al.  [31]        |
| PD-L1                                                                                                                                                            | Metastatic CRC (70% first line) | 30                                             | N/A     | 40% | Bend ell et  al.  [32] |                               |
| Conventional treatment of liver metastases include surgery (hepatic resection), intervention  Atezolizumab +  PD-L1 +  MEK Refractory CRC 23 N/A 17% Cobimetinib |                                 |                                                |         |     |                        |                               |  
Bend ell et al. [32]
Conventional treatment of liver metastases include surgery (hepatic resection), intervention
(embolization and transcatheter arterial chemoembolization), systemic chemotherapy, and radiofrequency ablation. Among them, only hepatic resection for metastatic disease has gained general acceptance as a potentially curative option. The potentially curative treatment of liver metastasis of CRC with surgical resection has been reported for many years, with 5-year survival rates now of 50%-60% [33, 34]. Moreover, if patients are well selected, up to 20% can achieve long-term disease-free survival after metastectomy [33-35]. The role of surgery for metastases from neuroendocrine tumors on long-term outcome is also well-documented [36, 37]. Surgical resection of hepatic metastases from gastric adenocarcinoma was associated with a significantly improved overall survival [38]. It is also noted that surgical resection of hepatic metastases was associated with a significantly improved overall 1-year and 3-year survival with median survival of 9.9 months for all PDAC patients with liver metastases [39]. Surgical resection allows for the maximum response rates, but unfortunately, the number of patients that are eligible for resection and/or ablation based on a finite number of liver metastasis and liver-only disease is small. Only 10%-20% of liver metastases from CRC are amenable to resection. Nevertheless, recurrences occur in two-thirds of patients after resection for liver metastases within the first 2 years. One multi-center study of 1669 patients found that more than half developed recurrences, with median disease-free survival duration of 16.3 months[40].  
| Table 3: Key immunotherapy trials in Pancreatic adenocarcinoma (PDAC) Drug(s) Target Population No.                                                                                                                                                          | Liver                  |                          |     |     |                                    |              |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------|--------------------------|-----|-----|------------------------------------|--------------|
| metastases                                                                                                                                                                                                                                                   | RR                     | Refer ence Royal         |     |     |                                    |              |
| Ipilimumab                                                                                                                                                                                                                                                   | CTLA-4                 | Advanced/Metastatic PDAC | 27  | N/A | 3.7%                               | et al.  [41] |
| Advanced/Metastatic PDAC                                                                                                                                                                                                                                     | Anjali                 |                          |     |     |                                    |              |
| Ipilimumab +  Gemcitabine                                                                                                                                                                                                                                    | CTLA-4                 | 13                       | N/A | 15% | et al.  [42]                       |              |
| Advanced/Metastatic PDAC                                                                                                                                                                                                                                     | Le et                  |                          |     |     |                                    |              |
| Ipilimumab + GVAX                                                                                                                                                                                                                                            | CTLA-4                 | 30                       | N/A | 0%  | al.  [43]                          |              |
| Advanced/Metastatic PDAC                                                                                                                                                                                                                                     | Brah mer  et  al.[30 ] |                          |     |     |                                    |              |
| BMS-936559                                                                                                                                                                                                                                                   | PD-L1                  | 14                       | N/A | 0%  |                                    |              |
| Advanced/Metastatic PDAC                                                                                                                                                                                                                                     | Weiss                  |                          |     |     |                                    |              |
| Pembrolizumab +  Gem/Nab                                                                                                                                                                                                                                     | PD-1                   | 11                       | N/A | 18% | et al.  [44]                       |              |
| Advanced/Metastatic PDAC                                                                                                                                                                                                                                     | Weiss                  |                          |     |     |                                    |              |
| Pembrolizumab +  Gem/Nab                                                                                                                                                                                                                                     | PD-1                   | 15                       | N/A | 20% | et al.  [45]                       |              |
| Advanced/Metastatic PDAC                                                                                                                                                                                                                                     | 10-                    | Segal                    |     |     |                                    |              |
| MEDI4736                                                                                                                                                                                                                                                     | PD-L1                  | 20                       | N/A | ?%  | et al.  [46] Beatt y et  al.  [47] |              |
| Despite the positive and encouraging results in HCC, clinical results appear different in patients  with liver metastases. No single trial specifically targets patients with liver metastatic disease.  Advanced/Metastatic PDAC CP-870,893 CD40 22 82% 18% |                        |                          |     |     |                                    |              |
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1.2.3 tumor microenvironment (tme)

It is critical to understand the mechanism of immune evasion during tumorigenesis in order to develop innovative and effective therapeutics based on cancer immunology. There is emerging data suggesting that the tumor microenvironment (TME) controls anti-tumor immunity by accumulation of regulatory T cells (Tregs), natural killer T cells (NKT), tumor associated macrophages (TAMs), indoleamine 2,3-dioxygenase (IDO) and myeloid derived suppressor cells (MDSCs) [48]. MDSCs form a critical link between the innate and adaptive immune system through their ability to influence the fate of Tregs in antigen specific T cell tolerance through diverse mechanisms [49]. MDSCs are a heterogeneous population of immature and immunosuppressive myeloid cells, which display a variety of pro-tumoral effects. They promote tumor angiogenesis through VEGF production[50] and subvert both innate and adaptive antitumor immunity[51]. MDSCs impair CD4+ and CD8+ T-cell responses via increased arginase activity, leading to arginine depletion[52], and through the production of reactive oxygen and nitrogen species that disrupt TCR signaling[53]. MDSCs abrogate hepatic NK-cell activity via membranebound TGF-β[54] and facilitate the expansion of Tregs cells and the induction of Tregs cells through IL-10 and TGF-β production. Increased numbers of CD14+ HLA-DR−/low MDSCs have been found in both tumor tissue and peripheral blood from patients with HCC, and elevated cell counts were related to tumor progression[50, 52]. Nevertheless, MDSCs affect T-cell function, survival and trafficking. MDSCs express galectin-9 that binds to TIM-3 on T cells, inducing T cell apoptosis [55]. MDSCs also express ADAM17 that down-regulates L-selectin (CD62L) levels on T cells, limiting their homing to lymph nodes and tumors[56]. Additionally, MDSCs interact with liver macrophages and causes their up-regulation of B7-H1, further strengthening the immunosuppressive phenotype[57]. MDSCs can also impair NK cell function. In human HCC,
MDSCs (CD14+HLA-DR−/low) inhibit NK cell cytotoxicity and cytokine release, which is mediated by the NKp30 receptor[52]. Tumor-derived IL-1β induces Ly6C negative MDSCs which also inhibit NK cell development and function[58]. Together, MDSCs contribute to immunosuppressive network through multiple mechanisms and are potential immunotherapy targets for liver cancer. Therefore, the abundance of these immunosuppressive factors in the HCC or metastatic liver cancer TME calls for a multi-targeted approach based on solid rationale for synergy. NKT cells represent another important cell type in the liver cancer TME. The liver has the largest number of NKT cells, which play critical roles in the pathogenesis of liver diseases and liver cancer [59]. NKT cells are a heterogeneous group of nonconventional T lymphocytes expressing both NK and T cell markers that share phenotypic and functional characteristics with NK cells. NKT cells not only recognize malignant cells directly and protect against tumor progression in a CD1ddependent manner[60], but also kill CD1d-expressing tumor-associated macrophages to suppress tumor growth[61]. We recently described that NKT cell activity is regulated by the ratio of primary bile acid to secondary bile acid, which can be modified by the gut microbiome[62]. Nevertheless, clinical evidence has showed high intratumoral NKT cells, high intratumoral IFN-g or combination of both high was associated with prolonged overall and recurrence-free survival in patients with HCC after curative resection[63]. Overall, it is generally believed that NKT cells play anti-tumor roles through the production of IFN-g promoting NK cell activation and killing tumor cells or tumor-associated macrophages. Therefore, we hypothesize that antitumor effect of ICIs will be boosted by augmenting NKT cells  
 and suppressing MDSCs would be a promising synergistic strategy to treat liver cancer (**Figure** 1).  
Figure 1: Tadalafil suppressed tumor growth and reduced tumor MDSC number. (A) RIL-175 hepatoma cells (5x105/ 20μL) were orthotopically implanted into the livers of male B6(Cg)-Tyrc-2J/J mice.  
The establishment and growth of tumors were blindly monitored by BLI with the Xenogen IVIS. BLI represents proliferation rate through luciferase total flux signals. They were followed up for 21 days. Tadalafil (2mg/kg), a PDE5 inhibitor, was daily administered intraperitoneally in tumor bearing mice. (B) Analysis of BLI images was carried out by Living Image 2.50 software (PerkinElmer, Waltham, MA, USA). Calculated ROIs are graphed. (C) Accumulation of intrahepatic tumoral MDSCs was determined by flow cytometry.
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1.2.4 pde5 inhibitors augment immunotherapy

Phosphodiesterase type 5 (PDE5) inhibitor, e.g. sildenafil and tadalafil, have been approved by the FDA for the treatment of pulmonary arterial hypertension, benign prostatic hyperplasia and erectile dysfunction, with a relative safe clinical profile. In the last decade, a significant number of studies have reported an increased expression of PDE5 in several human cancers compared to normal or surrounding non-neoplastic tissues[64-67]. Concomitantly, PDE5 inhibitors have been examined in multiple malignancies and cancer cell lines for their direct anticancer activities, for their efficacy as chemo-sensitizers and for cancer chemoprevention [68, 69]. Interestingly, the suppression of MDSCs by PDE5 inhibitor was well documented recently. In preclinical mouse models, it has been demonstrated that PDE5 inhibitor, sildenafil, and tadalafil significantly inhibited the MDSC functions by the downregulation of iNOS and ARG1 activities, leading to the activation of antitumor immunity and the prolongation of survival of tumor-bearing mice [70-72]. Recently, we found the treatment with a PDE5 inhibitor, tadalafil, caused suppression of tumor growth by reversing MDSC suppressor function (**Figure 1**). In 2015, two randomized, prospective, double blinded, placebo controlled, phase II clinical trials to determine the activity of PDE5 inhibitors on immune function in head and neck squamous cell carcinoma (HNSCC) patients were conducted[70, 73] (**Figure 2**). Results showed that tadalafil can reverse tumor-specific immune suppression in these patients by lowering MDSCs and Tregs and increasing tumor-specific CD8+ T cells in a dose-dependent fashion, and augments general and tumor-specific immunity in HNSCC patients. In an open-labeled pilot study, the biologic effects, safety and efficacy of palliative treatment with tadalafil was assessed in patients with advanced metastatic melanoma [74]. Stable disease was achieved in 3/12 patients (25%). Median progression-free survival was 4.6 months, median overall survival 8.5 months. Stable patients displayed significantly higher numbers of CD8+ tumor infiltrating lymphocytes (TILs) in the center of metastases before treatment as compared with progressive patients. Upon therapy, they showed increased expression of ζ-chain (used as a marker of T cell activation) in CD8+and CD4+TILs and CD8+T cells in the peripheral blood as compared with baseline. Tadalafil improved clinical outcome of advanced melanoma patients by enhancing antitumor immunity. It was also demonstrated that the addition of tadalafil in a patient with endstage relapsed/refractory multiple myeloma demonstrated reduced MDSC function and generated a dramatic and durable anti-myeloma immune and clinical response[75]. All of these clinical studies elaborate the decreased amounts of MDSC by PDE5 inhibitor and their immunosuppressive pattern correlated with an increased T cell reactivity and improved clinical outcome of advanced cancer patients. Currently, there are two clinical trials ongoing to evaluate the efficacy of combination of PDE5 inhibitor with regorafenib on advanced solid tumor (NCT02466802) and with vaccine on HNSCC (NCT02544880), respectively. No clinical trial is evaluating the therapeutic effects of PDE5 inhibitor on liver cancer or liver metastases with ICIs.  
Based on the available pre-clinical and clinical trial data, it is highly suggested PDE5 inhibitor  
 likely has synergistic antitumor efficacy when combined with ICIs (**Figure 1**).  
Figure 2: Tadalafil suppressed tumor growth by modulating tumor microenvironment. (A-C): tadalafil modulates tumor microenvironment. CD33/IL4Rα (A), CD4/FoxP3 (B), or CD8/CD69 (C) intratumoral concentration was evaluated by immune-fluorescence microscopy in HNSCC. D. Representative image of PET-CT scans from metastatic melanoma patient prior (1), after 4 cycles of ipilimumab (2), and 6 weeks later to exclude pseudo-progression (3); here progressive disease was confirmed, and the patient included into the TaMe trial; after 8 weeks of treatment with tadalafil 5 mg daily PO (4) and after 5 months (5). The scans show progressive disease under ipilimumab (1–3) and stable disease under tadalafil (3–5) - with a good regression of cutaneous metastases in the leg and a progressing left inguinal lymph node.
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1.2.4.1 tadalafil toxicity

Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and may not reflect the rates observed in practice. Tadalafil was administered to over 9000 men during clinical trials worldwide. In trials of tadalafil for once daily use, a total of 1434, 905, and 115 were treated for at least 6 months, 1 year, and 2 years, respectively. For tadalafil for use as needed, over 1300 and 1000 subjects were treated for at least 6 months and 1 year, respectively. In addition, as discussed above, tadalafil has been administrated to study the efficacy to improve antitumor immunity with daily dose for short period times.
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1.2.4.1.1 tadalafil for use as

In eight primary placebo-controlled clinical studies of 12 weeks duration, mean age was 59 years (range 22 to 88) and the discontinuation rate due to adverse events in patients treated with tadalafil 10 or 20 mg was 3.1%, compared to 1.4% in placebo treated patients. When taken as recommended in the placebo-controlled clinical trials, the following adverse reactions were reported (**Table 4**) for tadalafil for use as needed:
Table 4: Treatment-Emergent Adverse Reactions Reported by ≥2% **of Patients Treated with**
Tadalafil (10 or 20 mg) and More Frequent on Drug than Placebo in the Eight Primary PlaceboControlled Clinical Studies (Including a Study in Patients with Diabetes) for tadalafil for Use as Needed for ED  
| Adverse                                                    | Placebo   | Tadalafil   | 5       | mg  Tadalafil   | 10   | mg  Tadalafil   | 20   | mg   |
|------------------------------------------------------------|-----------|-------------|---------|-----------------|------|-----------------|------|------|
| Reaction                                                   | (N=476)   | (N=151)     | (N=394) | (N=635)         |      |                 |      |      |
| Headache                                                   | 5%        | 11%         | 11%     | 15%             |      |                 |      |      |
| Dyspepsia                                                  | 1%        | 4%          | 8%      | 10%             |      |                 |      |      |
| Back pain                                                  | 3%        | 3%          | 5%      | 6%              |      |                 |      |      |
| Myalgia                                                    | 1%        | 1%          | 4%      | 3%              |      |                 |      |      |
| Nasal congestion 1%                                        | 2%        | 3%          | 3%      |                 |      |                 |      |      |
| Flushing a                                                 | 1%        | 2%          | 3%      | 3%              |      |                 |      |      |
| Pain in limb                                               | 1%        | 1%          | 3%      | 3%              |      |                 |      |      |
| a The term flushing includes: facial flushing and flushing |           |             |         |                 |      |                 |      |      |  
1.2.4.1.2 Tadalafil for Once Daily Use for ED In three placebo-controlled clinical trials of 12 or 24 weeks duration, mean age was 58 years (range 21 to 82) and the discontinuation rate due to adverse events in patients treated with tadalafil was 4.1%, compared to 2.8% in placebo-treated patients. The following adverse reactions were reported (**Table 5**) in clinical trials of 12 weeks duration:
Table 5: Treatment-Emergent Adverse Reactions Reported by ≥2% of Patients Treated with tadalafil for Once Daily Use (2.5 or 5 mg) and More Frequent on Drug than Placebo in the Three Primary Placebo-Controlled Phase 3 Studies of 12 weeks Treatment Duration (Including a Study in Patients with Diabetes) for tadalafil for Once Daily Use for ED  
| Adverse Reaction                     | Placebo   | Tadalafil   | 2.5   | mg  Tadalafil   | 5   | mg   |
|--------------------------------------|-----------|-------------|-------|-----------------|-----|------|
| (N=248)                              | (N=196)   | (N=304)     |       |                 |     |      |
| Headache                             | 5%        | 3%          | 6%    |                 |     |      |
| Dyspepsia                            | 2%        | 4%          | 5%    |                 |     |      |
| Nasopharyngitis                      | 4%        | 4%          | 3%    |                 |     |      |
| Back pain                            | 1%        | 3%          | 3%    |                 |     |      |
| Upper respiratory tract infection 1% | 3%        | 3%          |       |                 |     |      |
| Flushing                             | 1%        | 1%          | 3%    |                 |     |      |
| Myalgia                              | 1%        | 2%          | 2%    |                 |     |      |
| Cough                                | 0%        | 4%          | 2%    |                 |     |      |
| Diarrhea                             | 0%        | 1%          | 2%    |                 |     |      |
| Nasal congestion                     | 0%        | 2%          | 2%    |                 |     |      |
| Pain in extremity                    | 0%        | 1%          | 2%    |                 |     |      |
| Urinary tract infection              | 0%        | 2%          | 0%    |                 |     |      |
| Gastroesophageal reflux disease 0%   | 2%        | 1%          |       |                 |     |      |
| Abdominal pain                       | 0%        | 2%          | 1%    |                 |     |      |  
The following adverse reactions were reported (**Table 6**) over 24 weeks treatment duration in one placebo-controlled clinical study:
Table 6: Treatment-Emergent Adverse Reactions Reported by ≥2% of Patients Treated with tadalafil for Once Daily Use (2.5 or 5 mg) and More Frequent on Drug than Placebo in One PlaceboControlled Clinical Study of 24 Weeks Treatment Duration for tadalafil for Once Daily Use for ED  
| Adverse Reaction                     | Placebo   | Tadalafil   | 2.5   | mg  Tadalafil   | 5   | mg   |
|--------------------------------------|-----------|-------------|-------|-----------------|-----|------|
| (N=94)                               | (N=96)    | (N=97)      |       |                 |     |      |
| Nasopharyngitis                      | 5%        | 6%          | 6%    |                 |     |      |
| Gastroenteritis                      | 2%        | 3%          | 5%    |                 |     |      |
| Back pain                            | 3%        | 5%          | 2%    |                 |     |      |
| Upper respiratory tract infection 0% | 3%        | 4%          |       |                 |     |      |
| Dyspepsia                            | 1%        | 4%          | 1%    |                 |     |      |
| Gastroesophageal reflux disease 0%   | 3%        | 2%          |       |                 |     |      |
| Myalgia                              | 2%        | 4%          | 1%    |                 |     |      |
| Hypertension                         | 0%        | 1%          | 3%    |                 |     |      |
| Nasal congestion                     | 0%        | 0%          | 4%    |                 |     |      |  
1.2.4.1.3 Tadalafil for Once Daily Use for Benign Prostate Hyperplasia (BPH) and for ED and
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bph

In three placebo-controlled clinical trials of 12 weeks duration, two in patients with BPH and one in patients with ED and BPH, the mean age was 63 years (range 44 to 93) and the discontinuation rate due to adverse events in patients treated with tadalafil was 3.6% compared to 1.6% in placebotreated patients. Adverse reactions leading to discontinuation reported by at least 2 patients treated with tadalafil included headache, upper abdominal pain, and myalgia. The following adverse reactions were reported (**Table 7**).  
| Adverse Reaction   | Placebo (N=576)   | Tadalafil 5 mg (N=581)   |
|--------------------|-------------------|--------------------------|
| Headache           | 2.3%              | 4.1%                     |
| Dyspepsia          | 0.2%              | 2.4%                     |
| Back pain          | 1.4%              | 2.4%                     |
| Nasopharyngitis    | 1.6%              | 2.1%                     |
| Diarrhea           | 1.0%              | 1.4%                     |
| Pain in extremity  | 0.0%              | 1.4%                     |  
Table 7: Treatment-Emergent Adverse Reactions Reported by ≥1% **of Patients Treated with**
tadalafil for Once Daily Use (5 mg) and More Frequent on Drug than Placebo in Three PlaceboControlled Clinical Studies of 12 Weeks Treatment Duration, including Two Studies for Tadalafil for Once Daily Use for BPH and One Study for ED and BPH Table 7: Treatment-Emergent Adverse Reactions Reported by ≥1% of Patients Treated with tadalafil for Once Daily Use (5 mg) and More Frequent on Drug than Placebo in Three PlaceboControlled Clinical Studies of 12 Weeks Treatment Duration, including Two Studies for Tadalafil for Once Daily Use for BPH and One Study for ED and BPH  
| Adverse Reaction   | Placebo (N=576)   | Tadalafil 5 mg (N=581)   |
|--------------------|-------------------|--------------------------|
| Myalgia            | 0.3%              | 1.2%                     |
| Dizziness          | 0.5%              | 1.0%                     |  
1.2.4.1.4 Tadalafil for Once Daily Use for HNSCC In a study of HNSCC with tadalafil to modify antitumor immunity, the following AEs were reported (**Table 8**) [73].  
Table 8: Treatment-Emergent Adverse Reactions Reported with Tadalafil for Once Daily for at least 20 days  
| Possible                                   | Probable   |    |    |         |    |    |
|--------------------------------------------|------------|----|----|---------|----|----|
| Symptoms                                   | Placebo    | 10 | 20 | Placebo | 10 | 20 |
| Grade 1                                    | Back pain  | 2  |    |         |    |    |
| Constipation                               | 1          |    |    |         |    |    |
| Cough                                      | 1          |    |    |         |    |    |
| Diarrhea                                   | 1          | 1  |    |         |    |    |
| Ear, nose and throat examination  abnormal | 1          |    |    |         |    |    |
| Headache                                   | 1          | 2  | 1  |         |    |    |
| Neck pain                                  | 1          | 2  |    |         |    |    |
| Pain in extremity                          | 1          |    |    |         |    |    |
| Pulmonary                                  | 1          | 1  |    |         |    |    |
| Sexual                                     | 1          |    |    |         |    |    |
| TOTAL AE grade 1                           | 3          | 2  | 9  | 2       | 0  | 2  |
| Total patients with AE grade 1             | 2          | 2  | 4  | 1       | 0  | 2  |
| Grade 2                                    | Back pain  | 1  |    |         |    |    |
| Headache                                   | 1          |    |    |         |    |    |
| Neck pain                                  | 1          |    |    |         |    |    |
| Pain in extremity                          | 1          | 1  |    |         |    |    |
| Total AE grade 2                           | 1          | 3  | 1  |         |    |    |  
| Table 8: Treatment-Emergent Adverse Reactions Reported with Tadalafil for Once Daily for at  least 20 days Possible Probable Symptoms Placebo 10 20 Placebo 10 20 Total patients with AE grade 2 1 1 1 Grade 3 Back pain 1 1 Myalgia 1 Total AE grade 3 1 2 Total patients with AE grade 3 1 2 Note: placebo (n=5); 10 mg arm (n=14); 10 mg arm (n=15). AE= Adverse Events  Additional, less frequent adverse reactions (<1%) reported in the controlled clinical trials of  tadalafil for BPH or ED and BPH included: gastroesophageal reflux disease, upper abdominal   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Additional, less frequent adverse reactions (<1%) reported in the controlled clinical trials of tadalafil for BPH or ED and BPH included: gastroesophageal reflux disease, upper abdominal pain, nausea, vomiting, arthralgia, and muscle spasm.  
Back pain or myalgia was reported at incidence rates described in Table 4, Table 5, Table 6, Table 7, Table 8. In tadalafil clinical pharmacology trials, back pain or myalgia generally occurred 12 to 24 hours after dosing and typically resolved within 48 hours. The back pain/myalgia associated with tadalafil treatment was characterized by diffuse bilateral lower lumbar, gluteal, thigh, or thoracolumbar muscular discomfort and was exacerbated by recumbency. In general, pain was reported as mild or moderate in severity and resolved without medical treatment, but severe back pain was reported with a low frequency (<5% of all reports). When medical treatment was necessary, acetaminophen or non-steroidal anti-inflammatory drugs were generally effective; however, in a small percentage of subjects who required treatment, a mild narcotic (e.g., codeine) was used. Overall, approximately 0.5% of all subjects treated with tadalafil for on demand use discontinued treatment as a consequence of back pain/myalgia. In the 1-year open label extension study, back pain and myalgia were reported in 5.5% and 1.3% of patients, respectively. Diagnostic testing, including measures for inflammation, muscle injury, or renal damage revealed no evidence of medically significant underlying pathology. In studies of tadalafil for once daily use, adverse reactions of back pain and myalgia were generally mild or moderate with a discontinuation rate of <1% across all indications. Across all studies with any tadalafil dose, reports of changes in color vision were rare (<0.1% of patients). The following adverse reactions have been identified during post approval use of tadalafil. Because these reactions are reported voluntarily from a population of uncertain size, it is not always possible to reliably estimate their frequency or establish a causal relationship to drug exposure. These events have been chosen for inclusion either due to their seriousness, reporting frequency, lack of clear alternative causation, or a combination of these factors. Cardiovascular and Cerebrovascular - Serious cardiovascular events, including myocardial infarction, sudden cardiac death, stroke, chest pain, palpitations, and tachycardia, have been reported post marketing in temporal association with the use of tadalafil. Most, but not all, of these patients had preexisting cardiovascular risk factors. Many of these events were reported to occur during or shortly after sexual activity, and a few were reported to occur shortly after the use of tadalafil without sexual activity. Others were reported to have occurred hours to days after the use of tadalafil and sexual activity. It is not possible to determine whether these events are related directly to tadalafil, to sexual activity, to the patient's underlying cardiovascular disease, to a combination of these factors, or to other factors. Body as a Whole - hypersensitivity reactions including urticaria, Stevens-Johnson syndrome, and exfoliative dermatitis *Nervous* - migraine, seizure and seizure recurrence, transient global amnesia Ophthalmologic - visual field defect, retinal vein occlusion, retinal artery occlusion Non-arteritic anterior ischemic optic neuropathy (NAION), a cause of decreased vision including permanent loss of vision, has been reported rarely post marketing in temporal association with the use of phosphodiesterase type 5 (PDE5) inhibitors, including tadalafil. Most, but not all, of these patients had underlying anatomic or vascular risk factors for development of NAION, including but not necessarily limited to: low cup to disc ratio ("crowded disc"), age over 50, diabetes, hypertension, coronary artery disease, hyperlipidemia, and smoking. It is not possible to determine whether these events are related directly to the use of PDE5 inhibitors, to the patient's underlying vascular risk factors or anatomical defects, to a combination of these factors, or to other factors. Otologic - Cases of sudden decrease or loss of hearing have been reported post marketing in temporal association with the use of PDE5 inhibitors, including tadalafil. In some of the cases, medical conditions and other factors were reported that may have also played a role in the otologic adverse events. In many cases, medical follow-up information was limited. It is not possible to determine whether these reported events are related directly to the use of tadalafil, to the patient's underlying risk factors for hearing loss, a combination of these factors, or to other factors
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urogenital - priapism 1.2.5 gut

Various bacteria populating in mammal gastrointestinal tract are an indispensable part in intestine ecosystem and play a pivotal role in gut barrier[76]. Commensal bacteria have been showed to regulate host immune system through the crosstalk with host intestinal epithelial cells and lymphatic cells[77]. In addition, the bacterial metabolism is another factor influencing the host immune homeostasis[77]. It is well known that oral antibiotics results in short- and long-term changes of the intestinal microbiome in humans[78, 79]. For instance, oral vancomycin is noted to reduce fecal microbial diversity with a decrease of gram-positive bacteria (mainly *Firmicutes*) and a compensatory increase in gram-negative bacteria (mainly *Proteobacteria*). Concurrently, vancomycin increases plasma primary bile acids with decreased fecal secondary bile acids that is predominantly associated with altered *Firmicutes*[80]. In line, this change might have implication on host immunity. The gut microbiome has emerged as a critical factor regulating anti-tumor immunity controlling the efficacy of chemo- and immunotherapies[81-85]. Most recently, it was noted that there were significant differences in the diversity and composition of the patient gut microbiome of responders versus non-responders to anti-PD-1 treatment in melanoma [84]. This study further found patient fecal microbiome samples (n = 43, 30 responders, 13 non-responders) significantly higher alpha diversity and relative abundance of bacteria of the *Ruminococcaceae* family in responding patients. The intestinal microbiome change by oral antibiotics may change the clinical outcome to ICIs. It was found that antibiotics inhibited the clinical benefit of anti-PD-1 or antiPD-L1 in patients with advanced cancer. Fecal microbiota transplantation from responder into germ-free or antibiotic-treated mice ameliorated the antitumor effects of PD-1 blockade, whereas fecal microbiota transplantation from non-responder failed to do so. Surprisingly, analysis of patient stool samples at diagnosis revealed correlations between clinical responses to PD-1 blockade and the relative abundance of *Akkermansia muciniphila*. Oral supplementation with A. muciniphila after Fecal microbiota transplantation with no responder feces restored the efficacy of PD-1 blockade in an interleukin-12–dependent manner by increasing the recruitment of CCR9+CXCR3+CD4+ T lymphocytes into mouse tumor beds[85]. These data have important implications for the treatment of cancer patients with ICIs.  
  
Profound effects of the gut microbiome on HCC development have been described[86, 87]. Recently, we demonstrated that altering gut commensal bacteria by oral vancomycin induced a liver-selective anti-tumor effect[62] (**Figure 3**). An increase of hepatic CXCR6+ NKT cells was observed, independent of mouse strain, gender or presence of liver tumor. Hepatic NKT cells showed an activated phenotype and produced more IFNg upon antigen stimulation. In vivo functional studies confirmed that NKT cells mediated the liver-selective tumor inhibition. The data also showed that NKT cell accumulation was regulated by CXCL16 expression of liver sinusoidal endothelial cells, which was controlled by gut microbiome-mediated primary-tosecondary bile acid conversion. Interestingly, previous study on healthy volunteer, treated with oral vancomycin, showed decreased fecal secondary bile acids with a simultaneous postprandial increase in primary bile acids in plasma (**Figure 4**)[80]. This finding indicates there are certain similarity between human and mice regarding the metabolic change of bile acid to oral vancomycin. Feeding secondary bile acids or colonization of bile acidmetabolizing bacteria reversed both NKT cell accumulation and inhibition of liver tumor growth in mice with altered gut commensal bacteria. This study suggests a new link between gut bacteriacontrolled bile acid metabolism and liver anti-tumor immune-surveillance.
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1.2.6 nivolumab

PD-1 is a negative regulatory molecule that is expressed transiently following T-cell activation and on chronically stimulated T cells characterized by an "exhausted" phenotype. Subsequently, PD-1 positive T cells lose function and proliferative capacity while enhancing a suppressive tumor microenvironment. PD-1 may act together with other T-cell modulating molecules, including CTLA-4, TIM-3, lymphocyte-activation gene 3 (LAG-3) as well as indoleamine-pyrrole 2,3dioxygenase 1 (IDO-1), cytokines, and transforming growth factor beta. Two ligands specific for PD-1 have been identified: PD-ligand 1 (PD-L1, also known as B7-H1 or CD274, expressed on tumor, antigen-presenting cells [APCs], and dendritic cells [DCs]) and PD-L2 (also known as B7- DC or CD273, expressed on endothelial cells). The interaction of PD- 1 with PD-L1 and PD-L2 results in negative regulatory stimuli that down-modulate the activated T-cell immune response through SHP-1 phosphatase. PD-L1 expression is found on a number of tumors, and is associated with poor prognoses based on OS in many tumors[88]. Nivolumab (OPDIVO®, BMS-936558, MDX-1106, and ONO-4538) is a fully human monoclonal immunoglobulin G4 (IgG4) antibody (HuMAb) that is specific for human programmed death-1 (PD-1, cluster of differentiation 279 [CD279]) cell surface membrane receptor[88]. Nivolumab binds to cynomolgus monkey PD-1 but not mouse, rat, or rabbit molecules. Clinical activity of nivolumab has been evidenced by the recent FDA approval in patients with melanoma, non-small cell lung cancer (NSCLC), SCLC, renal cell carcinoma (RCC), HCC, dMMR CRC, HNSCC and urothelial cancer. Specifically, on the basis of CheckMate-040, FDA granted nivolumab accelerated approval in September 2017 for patients with advanced HCC previously treated with sorafenib.
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1.2.6.1 pharmacokinetics

Pharmacokinetics (PK) of nivolumab was linear in the range of 0.3 to 10 mg/kg, with doseproportional increases in maximum serum concentration (Cmax) and area under the concentrationtime curve from time zero to infinity (AUC0-∞), with low to moderate inter-subject variability observed at each dose level[88]. Clearance of nivolumab is independent of dose in the dose range (0.1 to 10 mg/kg) and tumor types studied. Body weight normalized dosing showed approximately constant trough concentrations over a wide range of body weights. The mean terminal elimination half-life of BMS-936558 is 17 to 25 days consistent with the half-life of endogenous IgG4.
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1.2.6.2 efficacy

In the multicenter phase I/II dose escalation and expansion study, CheckMate-040, total 214 patients with advanced HCC and Child-Pugh score of 6 or less received nivolumab 3 mg/kg every 2 weeks on the dose expansion phase[9]. Partial responses were observed in 15% and 20% of patients in the dose-escalation and dose-expansion phases, respectively. Disease control rates were 64% and 58%, respectively. Responses were durable, averaging 17 months and 10 months, respectively. In the monotherapy cohort of CheckMate-142, nivolumab provided durable responses with 31% investigator-assessed objective response rate (ORR) (median duration of response not yet reached with median follow-up of 12.0 months), 69% sustained disease control, 54% (9 months) and 50% (12 months) PFS, and 78% (9 months) and 73% (12 months) OS in previously treated patients with dMMR/MSI-H mCRC [26]. In the combination therapy cohort of CheckMate-142, at median follow-up of 13.4 months, nivolumab plus ipilimumab demonstrated 55% investigator-assessed ORR (95% CI, 45.2 to 63.8), and 80% disease control rate in the patients with dMMR/MSI-H mCRC. Median duration of response was not reached and most responses (94%) were ongoing at data cutoff. PFS rates were 76% (9 months) and 71% (12 months); respective OS rates were 87% and 85%. Statistically significant and clinically meaningful improvements were observed in patient-reported outcomes, including functioning, symptoms, and quality of life [27].
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1.2.6.3 toxicology

A maximum tolerated dose (MTD) of nivolumab was not defined[29]. Serious adverse events (SAEs) occurred in 32 of 296 patients (11%) similar to the immune-related inflammatory events seen with ipilimumab: pneumonitis, vitiligo, colitis, hepatitis, hypophysitis, and thyroiditis (with noted pulmonary toxicity resulting in 3 deaths. Renal failure, symptomatic pancreatic and DM, neurologic events, vasculitis, insomnia and hematologic toxicity have also been reported). In combination with ipilimumab in the concurrent-regimen, grade 3 to 4 treatment-related adverse events (AEs) occurred in 32% of patients and were manageable. Patients (13%) who discontinued treatment because of study drug-related AEs had an ORR (63%) consistent with that of the overall population[27].
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1.2.6.4 pharmacodynamics/biomarkers

Tumor-cell expression (melanoma) of PD-L1 was characterized in combination with ipilimumab with the use of IHC staining and pharmacodynamics changes in the peripheral-blood absolute lymphocyte count[89]. With PD-L1 positivity defined as expression in at least 5% of tumor cells, biopsy specimens from 21 of 56 patients (38%) were PD-L1–positive. Among patients treated with the concurrent regimen of nivolumab and ipilimumab, ORs were observed in patients with either PD-L1–positive tumor samples (6 of 13 patients) or PD-L1–negative tumor samples (9 of 22). In the sequenced regimen cohorts, a higher number of overall responses was seen among patients with PD-L1–positive tumor samples (4 of 8 patients) than among patients with PD-L1– negative tumor samples (1 of 13) suggesting the possibility that these tumors have higher response rates to the combination. The relationship between PDL-1 expression and responses may not be present in patients treated with the combination. Tissue expression of PDL-2, interferon-γ (IFN- γ), IDO, and T cell CD8+ are of current interest. Until more reliable data based on standardized procedures for tissue collection and assays are available, PD-L1 status cannot be used to select patients for treatment at this time.
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1.2.7 pde5 inhibitor and oral

Because the mechanisms of action of PDE5 inhibitor, oral vancomycin and PD-1 are nonredundant targeting at MDSCs, NKT cells and PD-1 pathways as abovementioned, the  
  
combination may have additive or synergistic activity (**Figure 5**). There has no such treatment strategy developing or developed.
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1.2.7.1 pde5 inhibitor and oral

Per the FDA approval for HCC treatment with nivolumab, patients can be treated with 240 mg every 2 weeks or 480 mg every 4 weeks for HCC. As discussed above, patients with HNSCC received tadalafil 20 mg once a day (20 mg daily) for at least 10 days demonstrated augmentation of general and tumor-specific immunity[70]. However, in the other study, it seemed that tadalafil immunomodulatory activity was maximized at an intermediate dose (10 mg daily) but not at higher doses (20 mg daily)[73]. The potential mechanism is a possible off-target effect on PDE11 at high dosages that, by increasing intracellular cAMP, may negatively affect antitumor immunity. Vancomycin is poorly absorbed after oral administration. During multiple dosing of 250 mg every 8 hours for 7 doses, fecal concentrations of vancomycin in volunteers exceeded 100 mg/kg in the majority of samples. No blood concentrations were detected, and urinary recovery did not exceed 0.76%. Oral vancomycin remains the mainstay of therapy for severe infections produced by Clostridium difficile, the most prevalent cause of healthcare-associated infectious diarrhea in developed countries. Its short- and long-term effects on the human intestinal microbiota remain largely unknown, however, 125 mg 4 times daily for 10 days has been used as initial standard dose for non-severe Clostridium difficile infection. Since there are no previous reports regarding this combination therapy in cancer patients, we will choose the FDA approved standard dose and previous reported single agent dose as initial one. The dose may need titrate for the future study after the complicate analysis after this trial.
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2 elibility assessment and enrollment

Patients must have histopathological confirmation of HCC (Cohort 1)
OR  
histopathological confirmation of carcinoma in the setting of clinical and radiological characteristics which, together with the pathology, are highly suggestive of a diagnosis of HCC (Cohort 1)
OR  
histopathological confirmation of advanced colorectal or pancreatic malignancy with liver involvement as dominant site of metastasis (Per multidiscipline tumor board review and approval) (Cohort 2).  
Patients must have disease that is not amenable to potentially curative resection, transplantation or ablation.  
Patients must have progressed on, been intolerant to, or refused prior sorafenib/lenvatinib and/or atezolizumab/bevacizumab therapy (Cohort 1 only).  
Subjects must have progressed on or after standard systemic chemotherapy (at least one line of chemotherapy for patients with liver metastasis from PDAC, at least two lines of chemotherapy for patients with liver metastasis from CRC) (Cohort 2 only).  
Patients must have evaluable or measurable disease per RECIST 1.1 (See Section **6.3.1**).  
Patients must have lesion accessible for biopsy and be willing to undergo pre- and posttreatment biopsies.
ECOG performance status of 0 to 1 (**Appendix A**) If liver cirrhosis is present, patient must have a Child-Pugh score 7 (**Appendix F**)  
Active chronic HBV infected subjects must be on antivirals and have HBV DNA
<100IU/mL. HCV infected subjects can be enrolled with close HCV RNA level monitoring.  
Age > 18 years. Because no dosing or adverse event data are currently available on the use of nivolumab in combination with tadalafil and vancomycin in patients <18 years of age, children are excluded from this study, but will be eligible for future pediatric trials Adequate hematological function defined by:
white blood cell (WBC) count ≥ 3 × 109/L
absolute neutrophil count (ANC) ≥ 1.5 ×109/L,
lymphocyte count ≥ 0.5 × 109/L, platelet count ≥ 60 × 109/L, and
Hgb ≥9 g/ dL (more than 48 hours post-completion of blood transfusion))
Adequate hepatic function defined by:
a total bilirubin level ≤ 1.5 × ULN,  
an AST level <5×ULN, an ALT level <5 ×ULN.
Adequate renal function defined by:  
Creatinine clearance (CrCl) >50 mL/min/1.73 m2 by 24 hours urine collection or as predicted by the Cockcroft-Gault formula:
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crcl = (140-age (y)) x

The effects of nivolumab 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 and for the duration of study participation and up to 5 months (women) and 7 months (men) after the last dose of the drug. 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.  
Patients with a history of cardiovascular disease may be enrolled per cardiology consultation and approval with echocardiogram and troponin level in normal range at the time of enrollment.  
Patient must be able to understand and willing to sign a written informed consent document.
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2.1.2 exclusion criteria

Patients who have had standard-of-care anti-cancer therapy or therapy with investigational agents (e.g. chemotherapy, immunotherapy, endocrine therapy, targeted therapy, biologic therapy, tumor embolization, monoclonal antibodies or other investigation agents), large field radiotherapy, or major surgery within 4 weeks prior to enrollment.  
Therapy with antibiotics within 30 days prior to enrollment.  
Therapy with nitrates, alpha-blockers, or cytochrome P450 (CYP3A4) inhibitors within 7days prior to enrollment and for whom stopping is unsafe and/or a safe substitute is not medically recommended. Use of PDE5 inhibitors such as vardenafil (Levitra®), tadalafil (Cialis®), and sildenafil citrate (Viagra®) ≤15-days prior to enrollment.  
The patient must not be currently on a corticosteroid dose greater than physiologic replacement dosing defined as 10 mg of cortisone per day or its equivalent.  
For PDAC patients with liver metastases, primary PDAC has not been resected (unless the primary is in the tail of the pancreas).  
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.  
Have signs of liver failure, e.g. clinically significant ascites, encephalopathy, or variceal bleeding within 6 months prior to enrollment.  
Prior major liver resection: remnant liver <50% of the initial liver volume. Patients with a biliary stent can be included.
Patients with active autoimmune disease or history of autoimmune disease that might recur, which may affect vital organ function or require immune suppressive treatment including systemic corticosteroids. These include but are not limited to patients with a history of immune related neurologic disease, multiple sclerosis, autoimmune (demyelinating) neuropathy, Guillain-Barre syndrome or CIDP, myasthenia gravis; systemic autoimmune disease such as SLE, connective tissue diseases, scleroderma, inflammatory bowel disease (IBD), Crohn's, ulcerative colitis, hepatitis; and patients with a history of toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome, or phospholipid syndrome. Such diseases should be excluded because of the risk of recurrence or exacerbation of disease.  
Of note, patients with vitiligo, endocrine deficiencies including thyroiditis managed with replacement hormones including physiologic corticosteroids are eligible. Patients with rheumatoid arthritis and other arthropathies, Sjogren's syndrome and psoriasis controlled with topical medication and patients with positive serology, such as antinuclear antibodies (ANA), anti-thyroid antibodies should be evaluated for the presence of target organ involvement and potential need for systemic treatment but should otherwise be eligible.  
Have history of idiopathic pulmonary fibrosis (including bronchiolitis obliterans with organizing pneumonia) or evidence of active pneumonitis on screening chest CT scan.  
Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements.  
Patients with myocardial infarction or myocarditis within 12 months prior to enrollment. History of severe or unstable cerebrovascular disease. HIV-positive patients are excluded because HIV causes complicated immune deficiency and study treatment can possess more risks for these patients.  
Have had prior transplant of any kind.  
Have ascites. History of allergic reactions attributed to compounds of similar chemical or biologic composition to nivolumab, tadalafil or vancomycin.  
History of severe hypersensitivity reaction to any monoclonal antibody.  
Prior invasive malignancy (except non-melanomatous skin cancer) unless disease free for a minimum of 3 years prior to enrollment.  
Pregnant women are excluded from this study because nivolumab's potential for teratogenic or abortifacient effects is unknown. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with nivolumab, breastfeeding should be discontinued if the mother is treated with nivolumab
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2.1.3 recruitment strategies

The study will be posted on the CCR website and on clinicaltrials.gov. NIH social media platforms may also be used to notify potential subjects of the study. Outside providers and colleagues may directly refer patients for screening into this study.
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2.2 screening evaluation 2.2.1 screening

The following activities will be performed only after the subject has signed the consent for study \#01-C-0129 on which screening activities will be performed. 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.  
Complete Medical History and Physical Evaluation (including height, weight, vital signs, and ECOG performance status).  
Laboratory Evaluation o Hematological Profile: CBC with differential and platelet count.  
Sustained hypotension (<90/50 mmHg) or uncontrolled hypertension (>160/100 mmHg)
Stroke within 6 months prior to enrollment.  
o Biochemical Profile: electrolytes, BUN, creatinine, AST, ALT, total and direct bilirubin, calcium, phosphorus, albumin, magnesium.  
o PT, INR, PTT, fibrinogen (if clinically indicated)
o 24-hour urine collection (if creatinine clearance is tested this way)
o ACTH
o morning cortisol  
o Thyroid tests TSH, T3, T4 o Uric acid, amylase and lipase o Tumor marker CEA, AFP, CA19-9 o Serum or urine pregnancy test for female participants of childbearing potential (within 2 weeks prior to enrollment).  
o HIV, Hepatitis B and C serology and/or viral load o TB testing (if clinically indicated)
o Troponin I (if clinically indicated) o Urinalysis
Cardiology Consultation (if clinically indicated)
Echocardiogram (if clinically indicated)  
EKG CT scan of chest, abdomen and pelvis (or MRI of abdomen if clinically indicated)
Documentation of histologic cancer confirmation (at any time point prior to enrollment; required from each participant to confirm diagnosis). If there is no available documentation, biopsy will be performed to confirm the diagnosis.
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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.
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2.3.1 treatment assignment procedures (for

| Number   | Name     | Description                                                                           |
|----------|----------|---------------------------------------------------------------------------------------|
| 1        | Cohort 1 | Patients with HCC or patients with carcinoma highly suggestive of a  diagnosis of HCC |
| 2        | Cohort 2 | Patients with liver metastases from CRC or PDAC                                       |  
Arms  
| Number   | Name   | Description                              |
|----------|--------|------------------------------------------|
| 1        | Arm 1  | Nivolumab, tadalafil and oral vancomycin |
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treatment assignment

Patients in Cohort 1 and 2 will be directly assigned to treatment in Arm 1
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2.4 baseline evaluation

Tests performed during screening do not need to be repeated if done in designated time frame prior to start of study treatment. Within 28 days prior to first dose:
CT scan of chest, abdomen and pelvis (or MRI of abdomen if clinically indicated)  
HLA subtype- HLA-A, B, C and sequenced based HLA-A-02* (any time prior to treatment)
Baseline research biopsy (does not need to be repeated if biopsy was done during screening)
Within 3 days prior to first dose:
Concomitant medications Baseline signs and symptoms evaluation Stool collection or rectal swab for bile acid Research blood for mixed meal test for bile acid and for whole exome panel
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3 study implementation 3.1 study

The proposed study is an open label, single-arm study of nivolumab, oral vancomycin and tadalafil in patients with refractory primary HCC or liver dominant metastatic cancer from CRC or PDAC, see **Table 9** and **Schema 1**. Treatment will be delivered in cycles consisting of 4 weeks (+/- 3 days). Treatment will continue until off treatment criteria are met (see Section **3.6.1**). If one or two study drugs permanently discontinued because of toxicity, treatment with the other drug/s will continue. Patients will be monitored every 8 (+/- 1) weeks with imaging per **Study Calendar**. Patients will have two mandatory research biopsies: at baseline and anytime during week 3 of Cycle 2 while patient is still on oral vancomycin. (If patient's disease progresses before week 3 of cycle 2, the post-treatment biopsy may be performed per PI discretion at the time of progression).  
Table 9: Treatment regimen  
| Nivolumab   | Tadalafil   | Oral Vancomycin   |
|-------------|-------------|-------------------|  
Table 9: Treatment regimen
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schema 1 treatment and monitoring

Nivolumab will be given on Day 1 of every cycle at a flat dose of 480 mg. Nivolumab will be administered over approximately 30 to 60-minutes via IV infusion (See section **14.1.5**).
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3.2.2 tadalafil administration

Tadalafil will be given orally at 10 mg dose daily. Patients will begin tadalafil dosing on the Day 1 of Cycle 1 and will continue to receive tadalafil on every day of every cycle. In case of missing dose or vomiting after taking tadalafil, patients will be instructed not to increase next day dosage and not to repeat taking tadalafil on this day. Patients will complete and return Patient's Diary (**Appendix B).** Missing of 20% of the doses for any given cycle is allowed.
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3.2.3 vancomycin administration

Vancomycin will be given orally 125 mg every 6 hours at 500 mg total daily dose. Patients will begin vancomycin dosing on the Day 1 of Cycle 1 and will continue to receive vancomycin from week 1 to week 3 with week 4 off treatment on every cycle. If vancomycin administered earlier than scheduled or missed, dose can be taken in the time period of +/- 2 hours of scheduled time. In case of vomiting after taking vancomycin, next administration of vancomycin should proceed as scheduled. Patients will complete and return Patient's Diary (**Appendix B**). Missing of 20% of the doses for any given cycle is allowed.
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3.2.4 sequence and monitoring of

On day 1 of every cycle tadalafil and vancomycin can be administered at any time prior to or after nivolumab injection. Vital signs will be collected within 1 hour before nivolumab infusions, at least once during the infusions, and within 30 minutes after the completion of the 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 must 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. See also **Appendix D** 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.
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3.3 dose delay or modifications

When, at the beginning of a treatment cycle, treatment delay related to either tadalafil or vancomycin treatment alone is indicated, treatment with nivolumab may not be delayed. If treatment delay is due to nivolumab only, tadalafil and vancomycin treatment may be continued without delay. If, in the opinion of the investigator, a toxicity is considered to be due solely to one drug (e.g. flushing by tadalafil, dysgeusia by oral vancomycin), the dose of the other drugs does not require modification.
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3.3.2 nivolumab

Dose modifications of nivolumab may be required in the event of treatment-related toxicity. General guidelines regarding dose modification are provided in **Appendix C**.
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3.3.2.1 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. An AESI may be serious or non-serious. AESIs for nivolumab 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 nivolumab. An immune-mediate 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. AESIs observed with nivolumab include:  
Diarrhea / Colitis Pneumonitis / ILD
ALT/AST increases / hepatitis / hepatotoxicity Neuropathy / neuromuscular toxicity (e.g. Guillain-Barré, and myasthenia gravis)
Endocrinopathies (i.e. events of hypophysitis, hypopituitarism adrenal insufficiency, diabetes insipidus, hyper- and hypothyroidism and type I diabetes mellitus)
Rash / Dermatitis Nephritis / Blood creatinine increases Pancreatitis (or labs suggestive of pancreatitis - increased serum lipase, increased serum amylase)
Other inflammatory responses that are rare with a potential immune-mediated etiology include, but are not limited to, myocarditis, pericarditis, and uveitis.
In addition, infusion-related reactions and hypersensitivity/anaphylactic reactions with a different underlying pharmacological etiology are also considered AESIs. More specific guidelines for AESIs evaluation and treatment are described in detail in the Dosing Modification and Toxicity Management Guidelines (see **Appendix C**). If new or worsening pulmonary symptoms (e.g. dyspnea) or radiological abnormality suggestive of pneumonitis/interstitial lung disease is observed, toxicity management as described in detail in the Dosing Modification and Toxicity Management Guidelines (see **Appendix C**) will be applied. It is strongly recommended to perform a full diagnostic workup, to exclude alternative causes such as lymphangitic carcinomatosis, infection, allergy, cardiogenic edema, or pulmonary hemorrhage. In the presence of confirmatory HRCT scans where other causes of respiratory symptoms have been excluded, a diagnosis of pneumonitis (ILD) should be considered and the Dosing Modification and Toxicity Management Guidelines should be followed.
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for nivolumab aesis will comprise

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 C**.  
Pneumonitis (ILD): The following assessments, and additional assessments if required, will be performed to enhance the investigation and diagnosis of potential cases of pneumonitis. The results of the assessment will be collected. Physical examination: Signs and symptoms (cough, shortness of breath and pyrexia, etc.) including auscultation for lung field will be assessed.  
SpO2: Saturation of peripheral oxygen (SpO2) Other items: When pneumonitis (ILD) is suspected during study treatment, the following markers should be measured where possible:
o ILD Markers (KL-6, SP-D) and β-D-glucan Tumor markers: Particular tumor markers which are related to disease progression.  
Additional Clinical chemistry: CRP, LDH Infusion reactions: AEs of infusion reactions (also termed infusion-related reactions) are of special interest to BMS and are defined, for the purpose of this protocol, as all AEs occurring from the start of nivolumab infusion up to 48 hours after the infusion start time. For all infusion reactions, SAEs should be reported to BMS Patient safety.
Hypersensitivity reactions: Hypersensitivity reactions as well as infusion-related reactions have been reported with anti PD-L1 and anti-PD-1 therapy [90]. 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 D**.  
Hepatic function abnormalities (hepatotoxicity): nivolumab induced liver injury is defined as concurrent ALT 10 × ULN AND total bilirubin 2 × ULN or baseline value (if elevated total bilirubin at study entry), AND no other immediately apparent possible causes of ALT elevation and hyperbilirubinemia, including, but not limited to, tumor progression, acute viral hepatitis, cholestasis, pre-existing hepatic disease or the administration of other drug(s), herbal medications and substance known to be hepatotoxic. Guidelines for management of patients with hepatic function abnormality are provided in **Appendix C**.  
Gastrointestinal disorders: Diarrhea/colitis is one of the most commonly observed treatment emergent SAE when nivolumab is used. 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 nivolumab are provided in **Appendix C**.  
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 C**.  
Pancreatic disorders: Immune-mediated pancreatitis includes autoimmune pancreatitis, and lipase and amylase elevation. Guidelines for the management of patients with immunemediated pancreatic disorders are provided in **Appendix C**.  
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 C.  
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 C**.
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3.3.2.2 nivolumab dose delay criteria

Dose delay criteria apply for all nivolumab-related adverse events. Treatment delay up to 8 weeks from the last dose is allowed. If treatment is not resumed during 8 weeks, patients will be taken off nivolumab treatment. After nivolumab discontinuation, the patient can continue with tadalafil and vancomycin treatment. Nivolumab administration should be delayed for the following:  
Any grade ≥ 2 non-skin, drug-related adverse event, with the following exceptions: Grade 2 drug-related fatigue or laboratory abnormalities do not require a treatment delay Any grade 3 skin, drug-related adverse event, any grade 3 drug-related laboratory abnormality, with the following exceptions for asymptomatic amylase or lipase, ALT, or AST:
Grade 3 amylase or lipase abnormalities that are not associated with symptoms or clinical manifestations of pancreatitis do not require a dose delay.  
If a subject has a baseline AST or ALT that is within normal limits, delay dosing for drug-related Grade ≥ 2 toxicity (2-grade shift).  
If a subject has baseline AST or ALT within the grade 1 toxicity range, delay dosing for drug-related Grade ≥ 3 toxicity (2-grade shift).  
If a subject has baseline AST or ALT within the Grade 2 toxicity range, delay dosing for drug-related increase in AST or ALT at 2x baseline value or when AST or ALT is 8x ULN (whichever is lower).  
Any adverse event, laboratory abnormality, or intercurrent illness which, in the judgment of the investigator, warrants interrupting the dose of study medication. subjects who require delay of nivolumab should be re-evaluated weekly or more frequently if clinically indicated. It is recommended to monitor elevations in AST or ALT approximately every 3 days till levels peak or begin to decline. Nivolumab
dosing can be resumed when re-treatment criteria are met. Tumor assessments for all subjects should continue as per protocol even if study drug dosing is delayed.  
Resumption after delay is discussed in section **3.3.2.4** below.
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3.3.2.3 nivolumab dose discontinuation criteria

Nivolumab administration should be permanently discontinued if at least one of the following drug-related adverse event(s) occurs:  
Any ≥ Grade 2 drug-related uveitis, eye pain, or reduction of visual acuity that does not respond to topical therapy and does not improve to Grade 1 severity within 2 weeks of starting therapy OR requires systemic treatment Any Grade 3 non-skin, drug-related adverse event lasting > 7 days, with the following exceptions:
Grade 3 drug-related uveitis, pneumonitis, bronchospasm, neurological toxicity, hypersensitivity reaction, or infusion reaction of any duration requires discontinuation.  
Grade 3 drug-related endocrinopathies adequately controlled with only physiologic hormone replacement do not require discontinuation.  
Grade 3 drug-related laboratory abnormalities do not require treatment discontinuation except:
Grade 3 drug-related thrombocytopenia > 7 days or associated with bleeding requires discontinuation Any drug-related liver function test (LFT) abnormality that meets the following criteria require discontinuation:
o AST or ALT > 10 x ULN for > 2 weeks, o AST or ALT > 15 x ULN irrespective of duration, o T. bilirubin > 8 x ULN irrespective of duration for subjects with elevated bilirubin at study entry or > 5 x ULN for those with normal T bilirubin at entry, o Concurrent AST or ALT > 3 x ULN and T. bilirubin > 5 x ULN for subjects entering treatment with a normal bilirubin and up to 8 x ULN for subjects with elevated bilirubin Any drug-related Grade 4 drug-related adverse event or laboratory abnormality, except for the following events which do not require discontinuation:
Grade 4 neutropenia < 7 days Grade 4 lymphopenia or leukopenia Grade 4 amylase or lipase abnormalities that are not associated with symptoms or clinical manifestations of pancreatitis.  
Isolated Grade 4 electrolyte imbalances/abnormalities that are not associated with clinical sequelae and are corrected with supplementation/appropriate management within 72 hours of their onset For Grade 4 endocrinopathy adverse events such as hyper- or hypothyroidisms, or glucose intolerance, which resolve or are adequately controlled with physiologic hormone replacement (steroids, thyroid hormones) or glucose controlling agents, respectively Dosing delays to allow for prolonged steroid tapers to manage drug-related adverse events are allowed. Tumor assessments should continue as per protocol even if dosing is delayed. Periodic study visits to assess safety and laboratory studies should also continue every 6 weeks or more frequently if clinically indicated during such dosing delays.  
Dosing delays lasting > 6 weeks from the previous dose that occur for non-drug-related reasons may be allowed. Tumor assessments should continue as per protocol even if dosing is delayed. Periodic study visits to assess safety and laboratory studies should also continue every 6 weeks or more frequently if clinically indicated during such dosing delays.  
Any adverse event, laboratory abnormality or intercurrent illness which, in the judgment of the Investigator, presents a substantial clinical risk to the subject with continued nivolumab dosing.
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3.3.2.4 nivolumab dose resumption criteria

Subjects may resume treatment with study drug when the drug-related AE(s) resolve to Grade ≤ 1 or baseline value, with the following exceptions:
Subjects may resume treatment in the presence of Grade 2 fatigue.  
Subjects who have not experienced a Grade 3 drug-related skin AE may resume treatment in the presence of Grade 2 skin toxicity.  
Subjects with baseline Grade 1 AST, ALT, or total bilirubin who require dose delays for reasons other than a drug-related hepatic event may resume treatment in the presence of Grade 2 AST, ALT, or total bilirubin.  
Subjects who require dose delays for drug-related increased AST, ALT, or bilirubin may resume treatment when hepatic parameters are at baseline or Grade 1.  
Subjects with AST, ALT or bilirubin values meeting discontinuation parameters (Section 3.3.2.3) should have treatment permanently discontinued.  
Drug-related pulmonary toxicity, diarrhea, or colitis must have resolved to baseline before treatment is resumed.  
Drug-related endocrinopathies adequately controlled with only physiologic hormone replacement may resume treatment.  
Subjects who delay study treatment due to any Grade ≥ 3 amylase or lipase abnormality that is not associated with symptoms or clinical manifestations of pancreatitis, and that is assessed by the investigator not to be related to nivolumab, may resume nivolumab when the amylase or lipase abnormality has resolved to Grade < 3.
Resuming treatment after a delay of up to 8 weeks from the previous dose is permitted with the following exceptions:  
Dosing delays to allow for prolonged steroid tapers to manage drug-related adverse events are allowed. Tumor imaging assessments should continue as per protocol even if dosing is interrupted.  
Dosing delays > 8 weeks from the last dose that occur for non-drug-related reasons may be allowed.
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3.3.3 tadalafil

If patient's CrCl is reduced to 30 - 50 mL/minute (as measured by the Cockcroft-Gault formula), the dose of tadalafil will be reduced to 5 mg daily.  
If patient's CrCl is reduced less than 30 mL/minute, tadalafil will be held until the renal function improved:
to 30 - 50 mL/minute, tadalafil will resume at 5 mg daily;  
to > 50 mL/minute, tadalafil will resume at 10 mg daily;
If renal function is not improved after 4 weeks, tadalafil will be permanently discontinued. If patient experiences priapism, hearing loss, vision change, or hypotension that is associated with the use of tadalafil, tadalafil will be permanently discontinued. If tadalafil is permanently discontinued, treatment with nivolumab and vancomycin will continue.
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3.3.4 oral vancomycin

Vancomycin will be held temporally for adverse events > grade 1 confirmed by PI as associated with oral vancomycin:
nephrotoxicity  
ototoxicity uncontrolled nausea, diarrhea or abdominal pain.
Vancomycin can be resumed at full dose after toxicity resolves to baseline. If toxicity is not resolved for 4 weeks, vancomycin will be permanently discontinued. Vancomycin will be discontinued if patient develops anaphylactoid reactions that is confirmed to be associated with oral vancomycin. If vancomycin is permanently discontinued, treatment with nivolumab and tadalafil will continue.  
| 3.4                                                         | Study Calendar       | 28                 |    |    |
|-------------------------------------------------------------|----------------------|--------------------|----|----|
| Days  Safety  FU11,13                                       | Long  Term  FU12, 13 |                    |    |    |
| Screening1                                                  | Baseline1            | All Cycles Day 1 1 |    |    |
| Nivolumab2                                                  | X                    |                    |    |    |
| Tadalafil3                                                  | X                    |                    |    |    |
| Oral vancomycin4                                            | X                    |                    |    |    |
| Medical History                                             | X                    |                    |    |    |
| Height                                                      | X                    |                    |    |    |
| Cardiology consult 5                                        | X                    |                    |    |    |
| Echocardiogram 5                                            | X                    |                    |    |    |
| Histologic confirmation of disease                          | X                    |                    |    |    |
| TB testing 5                                                | X                    |                    |    |    |
| 24-hour urine (if creatinine  clearance is tested this way) | X                    |                    |    |    |
| Troponin I 5                                                | X                    |                    |    |    |
| HIV, Hepatitis B and C serology  and/or viral load 6        | X                    | X                  |    |    |
| EKG                                                         | X                    | X                  |    |    |
| Physical exam, weight and ECOG                              | X                    | X                  | X  | X  |
| Vital Signs                                                 | X                    | X7                 | X  |    |
| CBC w/differential, Platelets                               | X                    | X                  | X  | X  |
| Biochemical profile8                                        | X                    | X                  | X  | X  |
| Radiologic Evaluation9                                      | X                    | X                  | X  |    |
| HLA                                                         | X                    |                    |    |    |
| Urinalysis                                                  | X                    |                    |    |    |
| Thyroid tests TSH, T3, T4                                   | X                    | X                  | X  |    |
| Uric acid, amylase and lipase                               | X                    | X                  | X  |    |
| ACTH and morning cortisol                                   | X                    |                    |    |    |
| PT, INR, PTT, fibrinogen                                    | X5                   | X                  | X  |    |
| Serum or urine pregnancy test                               | X                    | X                  |    |    |  
| 28                                                                                               |                      |                    |    |
|--------------------------------------------------------------------------------------------------|----------------------|--------------------|----|
| Days  Safety  FU11,13                                                                            | Long  Term  FU12, 13 |                    |    |
| Screening1                                                                                       | Baseline1            | All Cycles Day 1 1 |    |
| Tumor marker CEA, AFP, CA19-9                                                                    | X                    | X                  | X  |
| Concomitant medications                                                                          | X                    | X                  | X  |
| Adverse event evaluation                                                                         | X                    | X                  | X  |
| Baseline signs and symptoms                                                                      | X                    |                    |    |
| Tumor biopsy10                                                                                   | X                    | X                  |    |
| Research blood for Immune  monitoring14                                                          | X                    |                    |    |
| Research blood for cytokines 14                                                                  | X                    |                    |    |
| Research blood for tadalafil PK15                                                                | X                    |                    |    |
| Research blood for mixed meal test  for bile acid16                                              | X                    | X                  |    |
| Research blood for vancomycin  level 17                                                          | X                    |                    |    |
| Research blood for TCR beta  sequencing                                                          | X                    |                    |    |
| Research blood for whole exome  panel                                                            | X                    |                    |    |
| Stool collection or rectal swab for  bile acid19                                                 | X                    | X                  |    |
| Stool collection or rectal swab for  Vancomycin-resistant enterococci                            | X                    |                    |    |
| (VRE) development 18 Stool collection or rectal swab for  microbial DNA sequences 18             | X                    |                    |    |
| Nutrition consult and completion of  food diary (Appendix H) 19                                  | X                    | X                  |    |
| Phone call or e-mail for survival  every 6 month                                                 | X                    |                    |    |
| 1 Baseline and C1D1 evaluations do not need to be repeated if performed at screening or baseline |                      |                    |    |  
1 Baseline and C1D1 evaluations do not need to be repeated if performed at screening or baseline in designated time frame. All evaluations will be done within 72 hours before treatment initiation on Day 1 of every cycle. If treatment does not start within 28 days after enrollment, screening evaluations will be repeated.  
2 480 mg of nivolumab via IV infusion on Day 1 of each cycle.  
3 10 mg of tadalafil PO on every day of every cycle.  
4 125 mg of vancomycin every 6 hours (500 mg daily dose) from Day 1 to day 21 of every cycle. 5 if clinically indicated. 6 If screening serology results indicate potential HBV or HCV infection, viral load will be tested before every nivolumab infusion 7 For vital signs see Section **3.2.** 8 Biochemical Profile: electrolytes, BUN, creatinine, AST, ALT, total and direct bilirubin, calcium, phosphorus, albumin, magnesium.  
9 CT scan of chest, abdomen and pelvis (or MRI of abdomen if clinically indicated) every 8 (+/-
1) weeks after start of study therapy.  
10 Mandatory tumor biopsies will be performed at baseline and any day during week 3 of cycle 2 while patient is taking oral vancomycin. If patient's disease progresses before C3D1, the posttreatment biopsy may be performed per PI discretion at the time of progression.  
11 +/- 1 week 12 Follow up visits are planned to be performed at 60 (+/- 14 days) and 90 (+/- 14 days) days after treatment discontinuation to evaluate patient's safety. After this visit, subjects will be followed every 6 months (± 1 month) for survival by phone call or e-mail.  
13 If subjects are not willing to come to NIH for FU visits, they will be contacted by phone call or e-mail for survival and adverse events.  
14 On Day 1 of cycles 1-4, then every 12 weeks until PD. 15 Before patient takes tadalafil on day 1 of every cycle.  
16 At baseline and C1D10, an IV line will be placed and a mixed meal test (Boost Plus Vanilla)
will be performed with collection of blood samples at 0 (+/- 10), 30 (+/- 10), 60 (+/- 15), 90 (+/- 20), 120 (+/- 20), 150 (+/- 20), and 180 (+/- 30) min for bile acid analysis. T=0 is the time patient ingests mixed meal. If patient is still on the protocol treatment after 6 months, patient will repeat the mixed meal test on day 10 of whatever cycle the patient is on.  
17 Before patient takes vancomycin on day 1 of every cycle. 18 On day 1 of every cycle. 19 At baseline and C1D10. If patient is still on the protocol treatment after 6 months, this will be performed on day 10 of whatever cycle patient is on.
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3.5 costs and compensation 3.5.1

NIH does not bill health insurance companies or participants for any research or related clinical care that participants receive at the NIH Clinical Center. If some tests and procedures performed outside the NIH Clinical Center, participants may have to pay for these costs if they are not covered by insurance company. Medicines that are not part of the study treatment will not be provided or paid for by the NIH Clinical Center.
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3.5.3 reimbursement

The NCI will cover the costs of some expenses associated with protocol participation. Some of these costs may be paid directly by the NIH and some may be reimbursed to the participant/guardian as appropriate. The amount and form of these payments are determined by the NCI Travel and Lodging Reimbursement Policy.
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3.6 criteria for removal from

Prior to removal from study, efforts must be made to have all subjects complete a safety visit approximately 90 days following the last dose of study therapy
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3.6.1 criteria for removal from

Participant requests to be withdrawn from active therapy Unacceptable Toxicity as defined in the Section **3.3** and **Appendix C**
Positive pregnancy test or intent to become pregnant Investigator discretion Initiation of therapy that prevents further administration of study treatment (See Section 4)
Progressive Disease. NOTE: While RECIST PD will be noted and recorded the immunerelated RECIST criteria (**Appendix E**) will be applied to determine discontinuation of study treatment.  
Intercurrent illness that prevents further administration of treatment
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3.6.2 off study criteria

Lost to follow-up Death Participant requests to be withdrawn from study  
Investigator discretion PI decision to end the study
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3.6.3 lost to follow-up

A participant will be considered lost to follow-up if he or she fails to return for the 60 or 90 day scheduled safety visits post-treatment or is unable to be contacted by the study site staff. The following actions must be taken if a participant fails to return to the clinic for a required study visit:
The site will attempt to contact the participant and reschedule the missed visit within 2 weeks and counsel the participant on the importance of maintaining the assigned visit schedule and ascertain if the participant wishes to and/or should continue in the study.  
Before a participant is deemed lost to follow-up, the investigator or designee will make every effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, an IRB approved certified letter to the participant's last known mailing address or local equivalent methods). These contact attempts should be documented in the participant's medical record or study file.  
Should the participant continue to be unreachable, he or she will be considered to have withdrawn from the study with a primary reason of lost to follow-up.
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4 concomitant medications/measures

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. Drugs with laxative properties and herbal or natural remedies for constipation should be used with caution through to 90 days after the last dose of immune checkpoint inhibitors. Drugs with significant ototoxicity should be used with caution
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4.1 excluded concomitant medications

Table 10: Prohibited concomitant medications  
| 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 noncancer-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])                                                                             |               |         |
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table 10 prohibited concomitant medications

| 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. |                                                                             |     |    |       |                |      |    |
| Viagra                                                                                                                                                                                                                                                                  | Should not be given concomitantly whilst the patient is  on study treatment |     |    |       |                |      |    |
| Microbiome changing drugs, including but                                                                                                                                                                                                                                | Should not be given concomitantly whilst the patient is                     |     |    |       |                |      |    |
| not limited to probiotics                                                                                                                                                                                                                                               | on study treatment                                                          |     |    |       |                |      |    |
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4.2 methods of contraception 4.2.1

Females of childbearing potential who are sexually active with a non-sterilized male partner must use effective method of contraception (**Table 11**) from the time of screening and must agree to continue using such precautions for 5 months after the last dose of nivolumab + oral vancomycin + tadalafil combination therapy. 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.
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4.2.2 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 7 months after the last dose of nivolumab + oral vancomycin + tadalafil combination therapy. Engaging 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 11**).
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4.2.3 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 11**. 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).  
| 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                      |            |                                                   |         |      |  
Table 11: Highly Effective methods of contraception (<1% failure rate)
a This is also considered a hormonal method
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4.3 blood donation

Subjects should not donate blood while participating in this study, and for 5 months (women) and 7 months (men) after the last dose of nivolumab + oral vancomycin + tadalafil combination therapy.