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Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM NIH Protocol \#: 17C0140 Version Date: 08/01/2022 NCT Number: NCT03233724 Title: Phase I/II Evaluation of Oral Decitabine/Tetrahydrouridine as Epigenetic Priming for Pembrolizumab Immune Checkpoint Blockade in Inoperable Locally Advanced or Metastatic Non-Small Cell Lung Cancers, Esophageal Carcinomas, or Pleural Mesotheliomas NCI Principal Investigator: David S. Schrump, M.D., M.B.A.  
Thoracic Surgery Branch (TSB), NCI, NIH 10 Center Drive, Room 4-3942 Bethesda, MD 20892-1502 Phone: 240-760-6239 Email: schrumpd@mail.nih.gov  
| Drug Names:   | Decitabine (DAC)                | Tetrahydrouridine (THU)   | Pembrolizumab   |
|---------------|---------------------------------|---------------------------|-----------------|
| Sponsor:      | Center for Cancer Research, NCI |                           |                 |
| IND Number:   | 126827                          |                           |                 |
| Manufacturer: | IriSys, LLC and                 | KP Pharmaceutical         | Generic         |
| DCTD, NCI     | Technology Inc.                 |                           |                 |
| Supplier:     | CC Pharmacy                     | CC Pharmacy               | CC Pharmacy     |  
Commercial Agents: None 1
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precis

Background:
Non-small cell lung cancers (NSCLC), esophageal carcinomas (EsC) and malignant pleural mesotheliomas (MPM) account for approximately 185,000 deaths annually in the United States, with over two thirds of patients presenting with advanced, incurable disease. 1st-line platinum-based chemotherapy for advanced NSCLC, EsC, or MPM produces transient responses at best, with most patients succumbing to disease within 12-16 months following diagnosis.  
Recent randomized clinical trials have demonstrated response rates approximating 20% in unselected patients with advanced NSCLC or EsC, and nearly 45% in patients with tumors exhibiting high level expression of programmed death ligand 1 (PD-L1) following administration of pembrolizumab, a humanized monoclonal anti-PD-1 antibody.  
Approximately 17% of unselected MPM patients have exhibited objective responses following administration of pembrolizumab or other PD-1 inhibitors.  
Preclinical studies have demonstrated that epigenetic drugs such as DNA demethylating agents and histone deacetylase (HDAC) inhibitors can "prime" cancer cells and tumor microenvironments thereby enhancing efficacy of immune checkpoint inhibitors.  
Although a potent DNA demethylating agent, Decitabine has poor bioavailability and inconsistent distribution in solid tumors due to rapid inactivation by cytidine deaminase (CDA) which is present in high levels in many organs.  
Recent studies in rodents and nonhuman primates, as well as a Phase 1 clinical trial
(NCT\#01685515) in patients with sickle cell disease have demonstrated that oral tetrahydrouridine (THU), an inhibitor of CDA, significantly enhances bioavailability/solidtissue-distribution of low dose oral DAC, thereby enhancing systemic DNA demethylation with acceptable toxicities.  
Preliminary results of a recent clinical trial suggest that oral DAC-THU can increase the frequency and magnitude of responses to immune checkpoint inhibitors in lung cancer patients with low or absent intra-tumoral PD-L1 expression.  
These data support further evaluation of oral DAC-THU in combination with immune checkpoint inhibitors for therapy of thoracic malignancies.  
Objectives:
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phase i

To define pharmacokinetics, toxicities and maximum tolerated dose of oral DAC-THU in combination with pembrolizumab in patients with inoperable, or unresectable, locally advanced or metastatic NSCLC, EsC, or MPM.  
Phase II
To determine clinical response by RECIST criteria to oral DAC-THU in combination with pembrolizumab in patients with inoperable, or unresectable, locally advanced or metastatic NSCLC, EsC, or MPM.
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eligibility inclusion criteria

Male or female, 18 years or older with histologically or cytologically-proven, inoperable, or unresectable locally advanced, or metastatic NSCLC, EsC, or MPM.  
Measurable disease. Patients with high PD-L1 expression (≥ 50%) and low PD-L1 expression (0-49%) in cancer cells by immunohistochemistry are eligible.  
NSCLC patients with no prior systemic treatment, or those with prior first line treatment including an immune checkpoint inhibitor are eligible for study.  
MPM patients who have received, refused, or are ineligible for first line chemotherapy are eligible.  
Patients with EsC including Seiwert-Stein Type I and Type II gastro-esophageal junction
(GEJ) carcinomas who have received or refused standard of care first line therapy and/or targeted therapy are eligible.  
Patients who received DNA demethylating agents or PD-1/PD-L1 inhibitors for another malignancy may be eligible for study if there were no dose-limiting immune related events, and there has been either no clinical evidence of disease or minimal residual disease that has been stable for at least three years.  
Willingness to undergo tumor biopsies if safely accessible per PI discretion before and after treatment.  
ECOG performance status 0 - 2. No evidence of unstable or decompensated myocardial disease; adequate pulmonary reserve.  
Adequate renal, hepatic and hematopoietic function.
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exclusion criteria design

The Phase I component will be a standard 3+3 design combining high and low PD-L1 expressers with incremental dose escalation of oral DAC-THU to define MTD.  
Simon 2-stage design for Phase II studies will be used to determine clinical response at the MTD.  
Patients with any targetable mutation for which there is approved first or second line therapy.  
Serious cardiovascular conditions.  
Active Hepatitis A, Hepatitis B or Hepatitis C. Human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS)-
related illness.  
Other active infection requiring systemic therapy.  
Pregnant or breastfeeding women.  
Patients who are receiving systemic corticosteroids.  
Patients receiving another investigational agent.  
Another malignancy.  
Patients will receive oral DAC-THU on T-W for two weeks out of every 3 for 9 weeks.  
Pembrolizumab will be administered on Wednesday, Thursday or Friday at a fixed intravenous dose of 200 mg every 3 weeks.  
One cycle is three weeks; one course is 9 weeks. Treatment evaluation using RECIST 1.1 every 10 +/- 1 weeks.  
Those patients exhibiting disease progression or unacceptable toxicities will be removed from study. Patients exhibiting stable disease or disease regression will be offered an additional course of therapy followed by treatment evaluation. Treatment will continue in this manner until off-study criteria have been met.  
Once the MTD for DAC/THU has been identified, the MTD dose level will be expanded by 4 patients to confirm its safety. Then, including these 10 patients at the MTD, a total of 10 NSCLC patients with high (50% or greater) intratumoral PD-L1 expression and 10 NSCLC patients with low (0-49%) intratumoral PL-L1 expression will be accrued to the first stage of each of two separate Phase II cohorts using individual Simon optimal designs. If 5 or more patients of the 10 first stage NSCLC patients in the high PD-L1 cohort respond to treatment, the cohort will be expanded to 23 patients. If 11 of 23 of these patients respond to treatment, the trial will be deemed positive for NSCLC with high PD-L1 expression. If 2 or more of the 10 first stage NSCLC patients in the low PD-L1 expression cohort respond to treatment, the cohort will be expanded to 29 patients. If 6 or more of these 29 patients experience a response, the trial will be deemed positive for NSCLC with low PD-L1 expression. Up to 10 EsC patients, including those considered to be part of the Phase I component after the MTD has been identified, will be enrolled into a separate cohort to examine responses to DAC-THU/pembrolizumab at the MTD. If 2 or more of these 10 EsC patients respond to treatment, these findings may warrant an amendment or a separate Phase II trial to determine response rates to DAC-THU/pembrolizumab in EsC patients. Similarly, if 2 or more of 10 MPM patients respond to treatment, these findings may warrant an amendment or a separate Phase II trial to determine response rates to DACTHU/pembrolizumab in MPM.  
Biopsies of index lesions will be obtained at baseline and at treatment evaluation following the first course of therapy for analysis of pharmacodynamic endpoints.  
Patients will be followed for toxicity for at least 30 days after treatment has been discontinued, start of new anti-cancer treatment or until death, whichever occurs first.  
4  
| TABLE OF CONTENTS PRÉCIS 2 TABLE OF CONTENTS 5 STATEMENT of COMPLIANCE 10 1 INTRODUCTION 10 1.1 Study Objectives 10 1.1.1 Primary Objectives 10 1.1.2 Secondary Objectives 10 1.2 Background and Rationale 10 1.2.1 Background and Significance: 10 1.2.2 Pharmacokinetics of DAC Alone Following Intravenous (IV), Subcutaneous (SC) and  Oral Administration in Non-Human Primates 18 1.2.3 Identification of Dose and Timing of Oral THU Required to Increase Oral  Bioavailability of DAC in Non-Human Primates 19 1.2.4 Effects of Oral THU on DAC Oral Bioavailability and Inter-Individual Variability in  Non-Human Primates 19 1.2.5 Effects of Repeat Administration of Oral THU-DAC on Blood Counts, DNA  Methylation and Fetal Hemoglobin Levels in Non-Human Primates 20 1.2.6 Clinical Pharmacokinetics: THU Alone 24 1.2.7 Clinical Pharmacokinetics: DAC Alone 24 1.2.8 Clinical Safety: THU Alone 25 1.2.9 Clinical Safety and Efficacy: Oral THU and DAC in Combination, Repeat Dose Phase  1/2 Study in SCD 26 1.2.10 Pembrolizumab 38 1.2.11 Preliminary Experience with Oral DAC-THU and Immune Checkpoint Inhibitors  in NSCLC 44 2 ELIGIBILITY ASSESSMENTAND ENROLLMENT 49 2.1 Eligibility Criteria 49 2.1.1 Inclusion Criteria 49 2.1.2 Exclusion Criteria 50 2.1.3 Recruitment Strategies 51 2.2 Screening Evaluation 52 2.3 Participant Registration and Status Update Procedures 52 2.3.1 Treatment Assignment Procedures 53 3 STUDY IMPLEMENTATION 53   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Version Date:  08/01/2022                                                                                                                            |                                                                                                                                                     |                                                                                                                      |
|------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|
| 3.1                                                                                                                                                  | Study Design .                                                                                                                                      |                                                                                                                      |
| Study Schema .                                                                                                                                       |                                                                                                                                                     |                                                                                                                      |
| 3.1.1                                                                                                                                                | Definition of Dose Limiting Toxicity (DLT) ....................................................................................55                   |                                                                                                                      |
| 3.1.2                                                                                                                                                | Dose Escalation                                                                                                                                     |                                                                                                                      |
| 3.1.3                                                                                                                                                |                                                                                                                                                     |                                                                                                                      |
| D Drug Administration                                                                                                                                |                                                                                                                                                     |                                                                                                                      |
| 3.2                                                                                                                                                  | 3.2.1                                                                                                                                               | DAC-THU                                                                                                              |
| Pembrolizumab                                                                                                                                        |                                                                                                                                                     |                                                                                                                      |
| 3.2.2                                                                                                                                                |                                                                                                                                                     |                                                                                                                      |
| 3.2.3                                                                                                                                                | Additional Modifications for Subjects with Grade 2 Infusion-Related Reactions ....60                                                                |                                                                                                                      |
| 3.2.4                                                                                                                                                | Severe Hypersensitivity Reactions and Flu-like Symptoms ..........................................60                                                |                                                                                                                      |
| 3.2.3                                                                                                                                                | CR Self –Administered Study Drugs Policy ......................................................................61                                   |                                                                                                                      |
| 3.3                                                                                                                                                  | Dosing Delays and Modifications .....................................................................................................61             |                                                                                                                      |
| 3.3.1                                                                                                                                                | General Guidance for Dose Modifications ............................................................................61                              |                                                                                                                      |
| 3.3.2                                                                                                                                                | Treatment Delays and Modifications for Medical Needs ................................................62                                             |                                                                                                                      |
| 3.3.3                                                                                                                                                | Dose Reductions .................................................................................................................................62 |                                                                                                                      |
| 3.3.4                                                                                                                                                | Dosage Modification and Management of Immune-Related Adverse Events ..........62                                                                    |                                                                                                                      |
| 3.3.5                                                                                                                                                | Dose Modification of DAC-THU                                                                                                                        |                                                                                                                      |
| 3.3.6                                                                                                                                                | Dose Delay of Pembrolizumab                                                                                                                         |                                                                                                                      |
| 3.4                                                                                                                                                  | On Study Protocol Evaluation (Appendix 3: Study Calendar) ................................................73                                        |                                                                                                                      |
| Baseline Assessments ...........................................................................................................................73   |                                                                                                                                                     |                                                                                                                      |
| 3.4.1                                                                                                                                                |                                                                                                                                                     |                                                                                                                      |
| 3.4.2                                                                                                                                                | T Treatment Evaluation                                                                                                                              |                                                                                                                      |
| 3.4.3                                                                                                                                                | Research Evaluations (Refer to Appendix 4, Unless Otherwise Indicated Below)...74                                                                   |                                                                                                                      |
| Follow-Up Evaluations                                                                                                                                |                                                                                                                                                     |                                                                                                                      |
| 3.4.4                                                                                                                                                |                                                                                                                                                     |                                                                                                                      |
| 3.5                                                                                                                                                  |                                                                                                                                                     |                                                                                                                      |
| Costs                                                                                                                                                |                                                                                                                                                     |                                                                                                                      |
| 3.5.1                                                                                                                                                |                                                                                                                                                     |                                                                                                                      |
| 3.5.2                                                                                                                                                | COMPENSATION .                                                                                                                                      |                                                                                                                      |
| 3.5.3                                                                                                                                                | Reimbursement                                                                                                                                       |                                                                                                                      |
| Criteria for Removal from Protocol Therapy and Off Study Criteria ........................................75                                         |                                                                                                                                                     |                                                                                                                      |
| 3.6                                                                                                                                                  | 3.6.1                                                                                                                                               | Criteria for Removal from Protocol Therapy .......................................................................75 |
| 3.6.2                                                                                                                                                | OFF-Study Criteria                                                                                                                                  |                                                                                                                      |
| 3.6.3                                                                                                                                                | Lost to Follow-Up...........................................................................................................................76      |                                                                                                                      |
| 4                                                                                                                                                    | CONCOMITANT MEDICATIONS/MEASURES                                                                                                                    |                                                                                                                      |
| 4.1                                                                                                                                                  | Hematologic and Blood Product Support                                                                                                               |                                                                                                                      |
| 4.2                                                                                                                                                  | Antidiarrheals .                                                                                                                                    |                                                                                                                      |
| 4.3                                                                                                                                                  | Electrolyte Replacement ........................................................................................................................76  |                                                                                                                      |
| Proton Pump Inhibitors ...........................................................................................................................76 |                                                                                                                                                     |                                                                                                                      |
| 4.4                                                                                                                                                  |                                                                                                                                                     |                                                                                                                      |
| S                                                                                                                                                    | CORRELATIVE STUDIES                                                                                                                                 |                                                                                                                      |
| 6                                                                                                                                                    |                                                                                                                                                     |                                                                                                                      |
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| Version Date:  08/01/2022                                                                                                                                  |                                                                                                                                                                 |                                                                                                |
|------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------|
| 5.1                                                                                                                                                        | Biospecimen Collection and Processing for Research ...................................................................77                                        |                                                                                                |
| Pharmacokinetic (PK) Sampling .................................................................................................77                          |                                                                                                                                                                 |                                                                                                |
| 5.1.1                                                                                                                                                      | Studies on Tumor Tissue and Peripheral Blood ..................................................................77                                               |                                                                                                |
| 5.1.2                                                                                                                                                      | Immune Subset Analyses                                                                                                                                          |                                                                                                |
| 5.1.3                                                                                                                                                      | Circulating Tumor Cells ......................................................................................................................77                |                                                                                                |
| 5.1.4                                                                                                                                                      |                                                                                                                                                                 |                                                                                                |
| Sample Storage, Tracking and Disposition .......................................................................................78                         |                                                                                                                                                                 |                                                                                                |
| 5.2                                                                                                                                                        | 5.2.1                                                                                                                                                           | Samples Managed by Dr. Figg's Blood Processing Core (BPC) ..................................78 |
| 5.2.2                                                                                                                                                      | Procedures for Storage of Tissue Specimens in the Laboratory of Pathology ..........79                                                                          |                                                                                                |
| 5.2.3                                                                                                                                                      | Thoracic Epigenetics Laboratory                                                                                                                                 |                                                                                                |
| Trepel Laboratory ................................................................................................................................79       |                                                                                                                                                                 |                                                                                                |
| 5.2.4                                                                                                                                                      |                                                                                                                                                                 |                                                                                                |
| 5.2.5                                                                                                                                                      | Sample Destruction, End of Protocol Procedures                                                                                                                  |                                                                                                |
| 6                                                                                                                                                          | DATA COLLECTION AND EVALUATION ...................................................................................80                                            |                                                                                                |
| 6.1                                                                                                                                                        | DATA COLLECTION                                                                                                                                                 |                                                                                                |
| 6.1.1                                                                                                                                                      | Routine Data Collection ............................................................................................................80                          |                                                                                                |
| 6.1.2                                                                                                                                                      | Exclusions to Routine Data Collection                                                                                                                           |                                                                                                |
| 6.2                                                                                                                                                        | DATA SHARING Plans .                                                                                                                                            |                                                                                                |
| 6.2.1                                                                                                                                                      | Human Data Sharing Plan ..........................................................................................................81                            |                                                                                                |
| 6.2.2                                                                                                                                                      | Genomic Data Sharing Plan........................................................................................................82                             |                                                                                                |
| 6.3                                                                                                                                                        | Response Criteria ...........................................................................................................................................82 |                                                                                                |
| Confirmatory Measurement/Duration of Response                                                                                                              |                                                                                                                                                                 |                                                                                                |
| 6.3.1                                                                                                                                                      |                                                                                                                                                                 |                                                                                                |
| 6.3.2                                                                                                                                                      | Definitions .......................................................................................................................................82           |                                                                                                |
| 6.3.3                                                                                                                                                      | Disease Parameters ........................................................................................................................83                   |                                                                                                |
| Methods for Evaluation of Measurable Disease ...................................................................84                                         |                                                                                                                                                                 |                                                                                                |
| 6.3.4                                                                                                                                                      |                                                                                                                                                                 |                                                                                                |
| 6.3.5                                                                                                                                                      | Response Criteria for Radiographic Studies                                                                                                                      |                                                                                                |
| Toxicity Criteria ......................................................................................................................................87 |                                                                                                                                                                 |                                                                                                |
| 6.4                                                                                                                                                        |                                                                                                                                                                 |                                                                                                |
| 7                                                                                                                                                          | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN ....87                                                                                               |                                                                                                |
| 7.1                                                                                                                                                        | DEFINITIONS                                                                                                                                                     |                                                                                                |
| 7.2                                                                                                                                                        | OHSRP Office of Compliance and Training/IRB Reporting ......................................................87                                                  |                                                                                                |
| 7.2.1                                                                                                                                                      | Expedited Reporting .....................................................................................................................87                     |                                                                                                |
| 7.2.2                                                                                                                                                      | IRB Requirements for PI Reporting Continuing Review .................................................87                                                         |                                                                                                |
| NCI Clinical Director Reporting .........................................................................................................87                |                                                                                                                                                                 |                                                                                                |
| 7.3                                                                                                                                                        |                                                                                                                                                                 |                                                                                                |
| 7.4                                                                                                                                                        | NIH Required Data and Safety Monitoring Plan .............................................................................8                                     |                                                                                                |
| 7.4.1                                                                                                                                                      | Principal Investigator/Research Team                                                                                                                            |                                                                                                |
| 8                                                                                                                                                          | SPONSOR SAFETY REPORTING                                                                                                                                        |                                                                                                |
| 8.1                                                                                                                                                        | DEFINITIONS                                                                                                                                                     |                                                                                                |
| 8.1.1                                                                                                                                                      | A Adverse Event                                                                                                                                                 |                                                                                                |
| 7                                                                                                                                                          |                                                                                                                                                                 |                                                                                                |
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| Version Date: 08/01/2022 8.1.2 Serious Adverse Event (SAE) 88 8.1.3 Life-threatening 89 8.1.4 Severity 89 8.1.5 Relationship to Study Product 89 8.2 Assessment of Safety Events 89 8.3 Reporting of Serious Adverse Events 90 8.4 Waiver of Expedited Reporting to CCR 90 8.5 Reporting Pregnancy 90 8.5.1 Maternal Exposure 90 8.5.2 Paternal Exposure 91 8.6 Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND 91 8.7 Sponsor Protocol Deviation Reporting 91 9 CLINICAL MONITORING 91 10 STATISTICAL CONSIDERATIONS 92 10.1 Methods of Analysis 94 11 HUMAN SUBJECTS PROTECTIONS 95 11.1 Rationale for Subject Selection 95 11.2 Participation of Children 95 11.3 Participation of Subjects Unable to Give Consent 95 11.4 Evaluation of Benefits and Risks/Discomforts 95 11.4.1 Risks 95 11.4.2 Benefits 96 11.5 Consent Process and Documentation 97 11.5.1 Consent Process for Adults Who Lack Capacity to Consent to Research  Participation 97 12 REGULATORY AND OPERATIONAL CONSIDERATIONS 97 12.1 Study Discontinuation And Closure 97 12.2 Quality Assurance And Quality Control 98 12.3 Conflict Of Interest Policy 98 12.4 Confidentiality and Privacy 99 13 PHARMACEUTICAL INFORMATION 99 13.1 Decitabine 99 13.1.1 Synonyms 99 13.1.2 Description 100 13.1.3 Chemical Names 100 13.1.4 Chemical Identification 100 13.1.5 Formulation 1008   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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abbreviated title dac-thu/pembro nsclc esc

| Version Date : 08/01/2022                                                                                                                                    |                                                                                                                                                   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------|
| Stability................................................................................................................................100                 |                                                                                                                                                   |
| 13.1.6                                                                                                                                                       | S Storage                                                                                                                                         |
| 13.1.7                                                                                                                                                       |                                                                                                                                                   |
| 13.1.8                                                                                                                                                       | Administration ...................................................................................................................101             |
| 13.1.9                                                                                                                                                       | TOXICITIES .                                                                                                                                      |
| 13.2 Tetrahydrouridine..................................................................................................................................102  |                                                                                                                                                   |
| 13.2.1                                                                                                                                                       | NAMES                                                                                                                                             |
| 13.2.2                                                                                                                                                       | Mode of Action                                                                                                                                    |
| How Supplied: ....................................................................................................................102                        |                                                                                                                                                   |
| 13.2.3                                                                                                                                                       | Storage: .                                                                                                                                        |
| 13.2.4                                                                                                                                                       |                                                                                                                                                   |
| 13.2.5                                                                                                                                                       | Stability:...............................................................................................................................102      |
| 13.2.6                                                                                                                                                       | Route of Administration: ...................................................................................................102                   |
| 13.2.7                                                                                                                                                       | Method of Administration: ..................................................................................................102                   |
| 13.2.8                                                                                                                                                       | Toxicities: ..................................................................................................................................103 |
| 13.3                                                                                                                                                         | Pembrolizumab                                                                                                                                     |
| 13.3.1                                                                                                                                                       | Description:                                                                                                                                      |
| 13.3.2                                                                                                                                                       | How Supplied: ....................................................................................................................103             |
| 13.3.3                                                                                                                                                       | Reconstitution/Dose Preparation: .................................................................................103                             |
| 13.3.4                                                                                                                                                       | Stability/Storage: ......................................................................................................................103      |
| 13.3.5                                                                                                                                                       | Administration: ...............................................................................................................103                |
| 13.3.6                                                                                                                                                       | Toxicities: ...............................................................................................................................104    |
| REFERENCES .                                                                                                                                                 |                                                                                                                                                   |
| 14                                                                                                                                                           | APPENDICES AND SUPPLEMENTARY TABLES.........................................................................113                                   |
| 15                                                                                                                                                           |                                                                                                                                                   |
| 15.1 Supplementary Tables                                                                                                                                    |                                                                                                                                                   |
| 15.1.1                                                                                                                                                       | Supplementary Table 1: Cancer/Testis Genes and Their Chromosomal Locations                                                                        |
| (Partial List)                                                                                                                                               |                                                                                                                                                   |
| 15.1.2                                                                                                                                                       | Supplementary Table 2: Frequency of CT Gene Expression in Various Human                                                                           |
| Cancers as Determined by Semi-Quantitative RT-PCR............................................................117                                             |                                                                                                                                                   |
| 15.2 Appendices .........................................................................................................................................120 |                                                                                                                                                   |
| 15.2.1                                                                                                                                                       | Appendix 1: Performance Status Criteria.................................................................120                                       |
| 15.2.2                                                                                                                                                       | Appendix 2: Sample Medication Self-Administration Record and Patient Diary                                                                        |
| 121                                                                                                                                                          |                                                                                                                                                   |
| Appendix 3: Study Calendar.........................................................................................123                                       |                                                                                                                                                   |
| 15.2.3                                                                                                                                                       | Appendix 4: Pharmacokinetic Samples and Research Bloods ..............................126                                                         |
| 15.2.4                                                                                                                                                       |                                                                                                                                                   |
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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 define pharmacokinetics, toxicities and maximum tolerated dose of oral DAC-THU in combination with pembrolizumab in patients with inoperable, or unresectable, locally advanced or metastatic NSCLC, EsC, or MPM. (Phase I Component)
To determine clinical response by RECIST criteria to oral DAC-THU in combination with pembrolizumab in patients with inoperable, or unresectable, locally advanced or metastatic NSCLC, EsC, or MPM. (Phase II Component)
1.1.2 Secondary Objectives To examine if oral DAC-THU modulates DNA methylation, as well as gene, micro-RNA,
and endogenous retroviral expression profiles of NSCLC, EsC, and MPM cells and alters the tumor micro-environment.  
To examine if oral DAC-THU modulates plasma tumor DNA methylation, circulating tumor cells and peripheral immune subsets.
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1.2 background and rationale 1.2.1

Lung cancer is the leading cause of cancer-related mortality worldwide and presently accounts for ~1.8 million annual deaths globally, of which nearly 160,000 occur in the United States (1, 2). The vast majority of these malignancies are directly attributable to cigarette smoking. Survival is stage dependent, and ranges from > 80% for patients with early stage tumors to less than 15% for patients with locally advanced or disseminated disease (3). The majority present with inoperable disease; as such median overall survival for lung cancer patients is 12-16 months. Approximately 80% of pulmonary carcinomas are non-small cell lung cancers (NSCLC) comprised primarily of adeno-, squamous cell and large cell undifferentiated carcinomas with distinct histologic and molecular features. The remaining 20% are highly lethal small cell lung cancers (SCLC) (4) which exhibit varying degrees of neuroendocrine differentiation, and are typically widely metastatic at presentation with a high propensity for recurrence despite initial, often dramatic responses to chemotherapy (5). Esophageal cancer (EsC) is the sixth leading cause of cancer-related mortality worldwide, accounting for ~509,000 deaths annually of which nearly 16,000 occur in the US (1, 6). Esophageal squamous cell cancers (ESCC) predominate in high incidence areas in Asia, South America, and the Middle East; these neoplasms typically arise in the upper and mid-esophagus, and are attributable to smoking, ethanol and cultural dietary practices. Esophageal adenocarcinomas (EAC) predominate in Western Europe and the US; these malignancies typically arise in the distal esophagus and gastro-esophageal junction and are attributable to obesity, gastro-esophageal reflux, Barretts esophagus, and to a lesser extent, cigarette smoking. Most patients (~70%) present with locally advanced or metastatic disease. Survival is stage dependent, ranging from ~40% for localized disease, to 20% for regional disease, and 5% for disseminated disease. With the exception of Her2 overexpression in a small percentage of cases, esophageal cancers typically have no actionable mutations (7, 8). Malignant pleural mesotheliomas (MPM) are rare, lethal neoplasms attributable primarily to environmental or occupational exposure to asbestos and related fibers, influenced in some cases by predisposing germline mutations (9-12). An estimated 40,000 individuals succumb to mesotheliomas worldwide each year despite asbestos being banned in many industrialized countries (13, 14). Most patients present with advanced, inoperable disease; median survival of these individuals is approximately 6 months with supportive care alone, and 12 months with systemic chemotherapy (15, 16). Whereas patients with limited disease burden may benefit from cytoreductive surgery- typically in the context of multimodality therapy, the role and extent of surgery in patients with MPM remains controversial (16-18). MPM typically have no actionable mutations. Immune checkpoint inhibitors are revolutionizing treatment of diverse malignancies including NSCLC, gastroesophageal carcinomas (2), and MPM. Humanized monoclonal antibodies such as pembrolizumab, which inhibit engagement of programmed death ligand -1 (PD-L1) on cancer cells and immune regulatory cells with programmed death-1 (PD-1) receptors on activated T cells (19), break tolerance and mediate objective responses in approximately 20% in patients with advanced NSCLC and gastroesophageal carcinomas when administered as second line therapy (20, 21). Response rates appear to be higher in smokers/former smokers due to increased mutational burden and neoantigen load (22-25), as well as patients whose tumors expressed high levels of PD-L1(20, 25). The favorable toxicity profiles, impressive, durable response rates and survival benefit of pembrolizumab have led to FDA approval of this immune checkpoint inhibitor for second and third line therapy of NSCLC and gastroesophageal carcinomas, respectively, irrespective of tumor PD-L1 expression status, as well as first line therapy for non-squamous NSCLC with 50% or greater expression of PD-L1 and no EGFR or ALK mutations (http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm526430.htm). Although presently not approved for the treatment of MPM, pembrolizumab is listed in NCCN guidelines for second-line treatment of these neoplasms, with far fewer toxicities compared to chemotherapy. Epigenetic Strategies for Cancer Therapy: Alterations in chromatin structure perturb gene expression during malignant transformation (26). DNA demethylation results in de-repression of endogenous retroviruses, loss of imprinting, and activation of cancer-testis genes, which normally exhibit stage-specific expression during germ cell development in testes or placenta, and either no expression or highly restricted expression in normal somatic tissues. In the context of genome wide DNA demethylation, numerous tumor suppressor genes are silenced by site specific DNA hypermethylation and/or polycomb repressive complexes. These alterations in chromatin structure, which recapitulate epigenomic states in normal germ cells provide the rationale for epigenetic therapy for cancer (27-29). Aberrant activation of cancer-testis (CT) genes (also referred to as cancer-germline genes) in somatic cells during malignant transformation results in expression of highly restricted tumor antigens that induce serologic as well as cell-mediated immune responses in cancer patients; as such, cancer-testis antigens (CTAs) have emerged as attractive targets for cancer immunotherapy (30). To date, over 200 CTAs have been identified, approximately half of which are encoded on the X chromosome (31) (**Figure 1**). CT-X chromosome (CT-X) genes are normally expressed in spermatogonia, and typically comprise extended families associated with inverted DNA repeats. The non-X CT genes tend to be expressed during later stages of germ cell differentiation (i.e. spermatocytes), and do not appear to be associated with extended families or inverted repetitive DNA sequences (32, 33). Relative to autosomal CT genes, CT-X genes are more frequently activated in cancer cells, and particular gene families appear to be coordinately de-repressed in a tumor-specific manner.  
  
In general, the frequency and magnitude of CT-X gene de-repression in human cancers coincides with advanced stage of disease. Typically, tumors with CT-X mRNA copy numbers > 10% of testes exhibit corresponding proteins detectable by immunohistochemistry techniques. In contrast, tumors with CT-X mRNA levels < 1% of testes do not have detectable antigen expression. With the exception of synovial sarcomas or Hodgkin Lymphomas that tend to exhibit homogeneous expression of NY-ESO-1 and CT-45, respectively, the majority of human cancers exhibit extremely focal CTA expression. These observations, together with recent reports demonstrating high level CT-X gene expression in cancer stem cells (35-37), raise the possibility that CT-X antigens are preferentially expressed in pluripotent tumor cells. As such, pharmacologic interventions that enhance expression and recognition of CT-X antigens may be novel strategies to eradicate cancer stem cells contributing to systemic metastases.  
Oral Decitabine and Tetrahydrouridine: Decitabine (5-AZA-2-deoxycytidine, DAC) was originally synthesized in 1964 by Pliml and Storm. This analogue of deoxycytidine has been extensively evaluated as an experimental anti-neoplastic and differentiation agent (38). Unlike cytidine analogues such as cytarabine, the sugar moiety of decitabine is unmodified, enabling incorporation of the analogue into DNA without termination of chain elongation or cytotoxicity at low doses. Through mechanisms that have not been fully delineated, the incorporated DAC induces rapid ubiquitination and proteosomal degradation of the "maintenance" DNA methyltransferase (DNMT) 1, but not "de-novo" DNMTs 3a or 3b (39, 40). In normal cells, DNMT1 functions primarily to restore DNA methylation following DNA replication; in cancer cells, DNMT1 is frequently over-expressed and contributes to de-novo DNA methylation and aberrant silencing of tumor suppressor genes; DNMT1 is indispensable for viability of cancer stem cells (41, 42). Depletion of DNMT1 in cancer cells by DAC results in passive DNA demethylation, and de-novo induction of a variety of genes including those encoding cancer-testis antigens, as well as epigenetically silenced tumor suppressors (43). At high concentrations DAC exhibits antimetabolite, DNA damaging, cytotoxic effects.  
In published studies we have demonstrated induction of NY-ESO-1 and MAGE-A3 as well as p16 in  
 NSCLC, EsC, and MPM cells but not normal respiratory epithelial cells or fibroblasts following 72 hour exposure to DAC (44, 45); up-regulation of NY-ESO-1 and MAGE-A3 persisted for approximately one month following DAC treatment, and facilitated recognition and lysis of tumor cells by cytotoxic T-lymphocytes (CTL) or PBL expressing T cell receptors for these CT as shown in Figure 2 and **Figure 3**) (45-48). We have also demonstrated eradication of pulmonary metastases in immunocompetent mice following systemic treatment with DAC and subsequent adoptive transfer of CTL specific for the murine CTA, P1A (**Figure 4**) (49).  
  
  
A Phase I dose escalation study was undertaken to examine the feasibility of using gene induction strategies to simultaneously augment immunogenicity and apoptosis in thoracic malignancies (50). Because DAC is an S-phase agent with a plasma t ½ alpha and t ½ beta of 7 and 35 minutes, respectively, following intravenous infusion (51), the study utilized a continuous 72-hour infusion to optimize the duration of DAC exposure in cells which are cycling at different rates within solid tumors, in an attempt to recapitulate treatment conditions in our preclinical experiments. The primary endpoints included identification of the MTD of DAC administered under these conditions, clinical response at the MTD, and analysis of NY-ESO-1, MAGE-A3 and p16 expression in tumor tissues before and after DAC treatment. The MTD of DAC administered by 72h continuous infusion was 60 mg/m2 for patients with three or more prior treatment regimens, and 75 mg/m2 for individuals with two or less prior treatments. Myelosuppression was the most common dose limiting toxicity. No objective responses were observed in 25 evaluable patients; however, stabilization of disease was observed in eight individuals. Two patients with advanced lung cancers refractory to prior therapy remained on study for approximately 12 months until being removed either because of inability to image residual disease in a previously radiated hilum, or slow disease progression. Pharmacokinetic analysis revealed average steady-state DAC plasma concentrations approximating 10 to 20 ng/ml (~50 nM), which corresponded with threshold concentrations for target gene induction in our preclinical tissue culture experiments.  
14 Immunohistochemistry analysis revealed induction of NY-ESO-1, MAGE-A3, and p16 in 5 of 13,  
 6 of 20, and 4 of 22 patients, respectively, whose tumor biopsies were sufficient for analysis.  
Virtually all of the non–small cell lung cancers that exhibited induction of NY-ESO-1, MAGEA3, and/or p16 had intense focal protein expression. In contrast, NY-ESO-1 or MAGE-A3 protein expression was markedly uniform in one small-cell lung cancer specimen following DAC exposure (representative results of NY-ESO-1, MAGE-A3, and p16 immunohistochemical analyses are depicted in **Figure 5**). Four patients exhibited simultaneous up-regulation of NY-ESO-1 and MAGE-3; two patients exhibited p16 with either NY-ESO-1 or MAGE-A3 induction, and one lung cancer patient exhibited simultaneous induction of NY-ESO-1, MAGE-A3, and p16 in tumor tissues following DAC infusion. Post-treatment antibodies to NY-ESO-1 were observed in three patients who exhibited NY-ESO-1 induction in biopsy specimens following DAC infusion. The lack of purified recombinant MAGE-A3 protein precluded analysis of immune recognition of this CTA. Of particular interest was a lung cancer patient with recurrent endobronchial tumor that exhibited prolonged stabilization of disease following DAC treatment. This patient underwent numerous bronchoscopic biopsies, which were of sufficient quality to enable reliable qRT-PCR analysis of target gene expression. NY-ESO-1 as well as MAGE-A3 mRNA copy numbers increased dramatically, whereas p16 expression remained relatively stable over the course of DAC treatment (Figure 6).  
  
Immunohistochemistry and serum ELISA (performed in a blinded manner), revealed NY-ESO-1 expression as well as NY-ESO-1 antibodies at the time of the fifth biopsy and thereafter. These data are consistent with a cumulative molecular treatment effect in this individual and suggest that chronic exposures to DNA demethylating agents are necessary to activate gene expression in solid tumors. As this trial was nearing completion, we attempted to perform more comprehensive evaluation of gene expression in lung cancer cells mediated by DAC exposure. In particular, we sought to ascertain the effects of DAC treatment relative to gene expression profiles detected in lasercaptured tumor cells and adjacent histologically normal bronchial epithelia from resected lung cancer specimens. Consistent with our observations regarding DAC treatment of lung cancer cells in vitro (data not shown), considerable interpatient heterogeneity was observed in baseline as well as post-treatment gene expression profiles of laser captured tumor cells. Data derived from eight resected specimens were sufficient for exploratory statistical analysis, allowing detection of induction/repression of gene expression with threshold of 1.2-fold and using P < 0.05 (two-tailed t test) to define potentially important modulation of gene expression. The limited number of arrays from DAC-treated patients precluded rigorous statistical analysis of gene modulation in these individuals. Seventy-five genes were induced, whereas 324 genes were repressed > 2-fold following DAC treatment. Interestingly, those genes that were induced or repressed > 2- fold by DAC seemed to be down-regulated or overexpressed, respectively, in resected primary lung cancers relative to adjacent, histologically normal bronchial epithelial cells (**Figure 7**). Sixty of the 75 genes induced, and 287 of 324 genes repressed, by DAC were mapped to the PANTHER database. Subsequent Gene Ontology analysis identified several potentially enriched functional groups, including signal transduction and protein modification. Although limited and exploratory in nature, these data suggested that prolonged DAC exposure can shift global gene expression profiles in lung cancer cells from a malignant signature to one more reflective of normal respiratory epithelial cells (50).  
  
Inhibition of gene-silencing chromatin modifying enzymes such as DNMT1 induces terminal differentiation of myeloid and other cancer cells *in vitro* (reviewed in ref (52)). These differentiation-mediated cell cycle exits, which are similar to those that occur during normal tissue differentiation, do not require p53, and are readily induced in p53/p16-null cancer cells (53). The same non-cytotoxic conditions also increase differentiation of normal progenitors, but in contrast, increase self-renewal of normal hematopoietic stem cells (52, 54). In cancer cells, DAC treatment facilitates differentiation-driving transcription factor-mediated up-regulation of maturation factors which antagonize MYC-function (e.g., CEBPE, p27/CDKN1B) and terminate proliferation independent of p53. In contrast, normal stem cells express master stem cell transcription factors (e.g., HLF, HOXB4), and activate stem cell genes and stem cell fate in response to DAC treatments (52). These observations suggest that dosing of DAC to avoid systemic toxicities may be a novel strategy to induce growth arrest, differentiation or apoptosis of pluripotent cancer cells (52, 54). When administered by infusion or subcutaneous injection, DAC has pharmacologic limitations that impede translation to chronic epigenetic therapy for solid tumors. For example, DNMT-1 depletion requires DAC levels to overlap with cellular S-phase entry, yet the plasma half-life of DAC is < 20 minutes, which severely curtails the probability of such overlap. This problem is not solved by increasing DAC dose, since off-target effects from higher Cmax cause cytotoxicity (i.e.  
myelosuppression) that paradoxically decreases efficacy by restricting the frequency of drug administration. Similarly, continuous infusion is likely not the answer, since toxic increases of DAC can occur in some tissues while inadequate exposure remains in others. This unbalanced distribution may be attributable to widely diverging tissue expression of cytidine deaminase (CDA), the enzyme that rapidly deaminates DAC and the related DNMT1-depleting cytidine analogue 5-azacytidine (5Aza) (55). Moreover, CDA expression is particularly high in the intestines and liver, suggesting that CDA could be a barrier to oral bioavailability of cytidine analogues (**Figure 8**).  
  
One potential strategy to overcome these limitations is to combine oral DAC with a CDA inhibitor (oral tetrahydrouridine, THU), which has been used extensively with no appreciable toxicities in cancer patients. THU is a competitive inhibitor of CDA with Ki values of 3-5 x 10-8M (56, 57).  
The very high affinity of THU for CDA may be due to its resemblance to a tetrahedral intermediate of cytidine formed by addition of water across the 3, 4 double bond (57). CDA binds THU approximately 4-orders of magnitude more tightly than uridine, the substrate for the reverse reaction catalyzed by CDA (57). Slow onset of inhibition suggests that structural reorganization precedes the formation of a stable enzyme-inhibitor complex (57). Preclinical studies in mice and nonhuman primates (described in references (58, 59) and extensively summarized in the Investigators Brochure) have established that oral THU administered prior to oral DAC increases bioavailability of DAC approximately 10-fold, prolongs plasma t½ from < 20 minutes to > 3 hours, and produces significant in vivo pharmacodynamic effects at a DAC oral dosage of 2.5-5 mg/m2
(59). Preclinical studies in non-human primates and results of a recent proof of concept Phase I clinical trial in sickle cell disease (SCD) are summarized below.
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1.2.2 pharmacokinetics of dac alone

The pharmacokinetics of DAC were assessed in female baboons following IV (n=2), SC (n=2),
and oral (n=2) administration. The average initial concentration (C0) following IV DAC 10 mg/m2
(0.5 mg/kg) in baboons PA7482 and PA7472 was > 3 µM (690 ng/mL), with mean alpha and beta half-lives 2.13 and 180.5 minutes respectively (**Figure 9**). SC DAC 10 mg/m2 in PA7258 and PA7254 produced peak drug levels < 0.5 µM (114 ng/mL) and a wider concentration-time profile than IV DAC at the same dose (**Figure 9**). Oral DAC 200 mg/m2 (10 mg/kg) in PA7470 and PA7484 produced peak levels of ~0.2 µM (46 ng/mL) and ~ 0.01 µM (2.3 ng/mL) respectively, and a wider concentration-time profile than SC administration (Figure 9).
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1.2.3 identification of dose and

In two female baboons, PA7470 and PA7484, selected for high and low bioavailability of oral DAC alone respectively, THU 400 mg/m2 (20 mg/kg), 60 minutes before DAC 100 mg/m2 (5 mg/kg), produced higher DAC concentrations than THU 40 mg/m2 (2 mg/kg) (**Table 1**) (59). In the same baboons after a wash-out period, THU 400 mg/m2 60 minutes before DAC produced higher DAC concentrations than THU 400 mg/m2 administered simultaneously or 30 minutes prior to DAC (**Table 1**) (59).
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1.2.4 effects of oral thu

Eight female baboons were treated with oral DAC 200 mg/m2 (10 mg/kg) (59). The median AUClast with oral DAC alone was 463 min*ng/ml, with a range between 190 to 6279 min*ng/mL (33fold variation) (**Table 1**). After a wash-out period of > 2 weeks, the same animals were treated with oral THU 400 mg/m2 60 minutes prior to DAC 100 mg/m2 (5 mg/kg). The median AUClast with THU-DAC (decitabine at half the dose) was 2284 min*ng/ml, with a range between 534 to 7515 min*ng/mL (14-fold variation) (**Table 1**). The increase in AUClast in the THU group was statistically significant (p=0.02, Wilcoxon test) (**Table 1**), even though AUClast was calculated over 25% more time in the DAC alone group (240 minutes versus 180 minutes for THU-DAC). Furthermore, in many animals in the THU-DAC group, the concentration-time trends suggested DAC levels may have continued to increase beyond the last measured time-point at 180 minutes.  
Hence, AUClast values for THU-DAC are underestimates. The coefficient of variation (as a measure of inter-individual variability) of AUClast was 141 in the DAC alone group and 97 in the THU-DAC group. The increase in AUClast and accompanied by decrease in coefficient of variation with THU-DAC was because the largest increases in DAC AUClast occurred in animals which had the poorest response to decitabine alone (**Table 1**): this converged the pharmacokinetic profiles in low oral bioavailability animals towards the animals with higher intrinsic oral bioavailability
(**Table 1**). The average decitabine Cmax was 0.05 µM (10.85 ng/mL) for DAC alone and 0.12 µM
(26.98 ng/mL) for THU-DAC (decitabine at half the dose).  
| AUClast (min*ng/mL)#             |             |               |                            |                      |
|----------------------------------|-------------|---------------|----------------------------|----------------------|
| Baboon Number                    | Weight      | Decitabine 10 | Decitabine 5               | THU 20 mg/kg 60 mins |
| (kg)                             | mg/kg alone | mg/kg alone   | before Decitabine 5  mg/kg |                      |
| PA7482                           | 12.3        | 49.98         | Not done                   | Not quantifiable     |
| PA7484                           | 11.5        | 190.35        | 252.73                     | 5621.05              |
| PA7256                           | 19.8        | 299.02        | Not done                   | 760.27               |
| PA7472                           | 10.7        | 327.25        | Not done                   | 444.825              |
| PA7254                           | 14.4        | 463           | Not done                   | 2587                 |
| PA7255                           | 19.9        | 807.8         | Not done                   | 533.47               |
| PA7258                           | 12.6        | 2863.6        | Not done                   | 2284.48              |
| PA7470                           | 9.6         | 6278.78       | 1219.98                    | 7515.32              |
| MeanSD                          | 1604  2262 | 2821  2749   |                            |                      |
| MedianIQR                       | 463  2565  | 2284  5088   |                            |                      |
| MedianIQR per                   | 160  226   | 457  1017    |                            |                      |
| mg of decitabine* Fold-variation | 30-fold    | 14-fold      |                            |                      |
| Coefficient of                   | 141         | 97            |                            |                      |
| Variation                        |             |               |                            |                      |  
Table 1: The largest increases in AUClast **produced by co-administration of oral THU occurred in baboons** which had the lowest AUClast with DAC alone
\#AUClast calculated over 240 minutes for decitabine alone, 180 minutes for THUdecitabine. *p=0.02. Wilcoxon test. SD=standard deviation. IQR = inter-quartile range
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1.2.5 effects of repeat administration

To evaluate pharmacodynamic effects with repeat dose administration, oral THU-DAC 2X/week for 8 weeks was administered to baboons PA7484 and PA7472 (59). DAC doses were 10 and 5 mg/m2 respectively, administered by oral gavage 60 minutes after oral THU 400mg/m2, dosages that were expected to produce Cmax within the range (0.005-0.5 µM) that was demonstrated to deplete DNMT1 in normal hematopoietic precursors without causing significant DNA damage or cytotoxicity (**see Investigators Brochure)**. In PA7484, platelet counts increased during the first 6 weeks of drug administration, with a decrease during the last 2 weeks (but not below the lower limit of normal) (**Figure 10A**). In the same animal, neutrophil counts decreased during the first 3 weeks of drug administration (but not below the lower limit of normal), and increased during the last 2 weeks (**Figure 10B**). In PA7472, platelet counts increased during drug administration (**Figure 10A**), while neutrophil counts decreased but not below the lower limit of normal (Figure 10B). These alterations in blood counts are characteristic of the shifts in hematopoietic differentiation produced by DNMT1 depletion by DAC (60, 61). In PA7472, a bone marrow aspirate 96 hours after THU-DAC administration provided sufficient cells for analysis of DNA
damage/repair by H2AX staining. There was a small increase in H2AX compared to negative control that was not suggestive of major DNA damage, although early DNA damage induction would have been missed (**Figure 10C**). Oral THU-DAC was also administered 3X/week with 50% of the above DAC doses, again with concurrent increases in platelet and decreases in neutrophil counts (**Figure 11**). One potential application of long-term DNMT1-depleting therapy is to increase fetal hemoglobin (HbF) in red blood cells as a means to treat hemoglobinopathies such as sickle cell disease (as envisaged in the proposed clinical trial). HbF levels increased progressively during treatment in both PA7484 and PA7472, although at late time-points in PA7484, there was a partial reverse of this trend (**Figure 10D**). In repeat dose experiments with 50% of the above DAC doses administered 3X/week, progressive HbF increases were again noted (**Figure 11**). One intended biological effect of therapy is to hypomethylate promoter CpG that regulate expression of target genes (e.g., -globin gene [HBG] promoter CpG). HBG promoter CpG that are hypomethylated at the developmental stages associated with HbF expression (and which therefore potentially play a role in regulating HBG expression) were identified by comparing methylation of -globin gene cluster CpG in DNA isolated from baboon fetal liver, fetal bone marrow, and adult bone marrow erythroid precursors (**Figure 10E**) (methylation levels of embryonic [HBE] and -globin gene
[HBB] CpG were similar in fetal and adult erythroid precursors; in both fetal and adult erythroid precursors, CpG in the locus control region, an enhancer for the -globin genes, were hypomethylated compared to the methylation level in white blood cells). The developmentally responsive HBG promoter CpG, but not other CpG such as HBE and HBB gene promoter CpG, were hypomethylated by up to 20% by the 2X/week oral THU-DAC administration to PA7484 and PA7472, and by up to 50% by 10 days of daily administration of SC decitabine 5 mg/m2 in PA7002 (**Figure 10E**). The SC treatment with DAC alone was used as a positive control and increased HbF levels up to 81.3% (data not shown).  
  
Abbreviated Title : DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
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1.2.6 clinical pharmacokinetics thu alone

As previously discussed, THU is a competitive inhibitor of CDA, the enzyme that is largely responsible for the metabolism of cytidine analogues (62). THU has no therapeutic utility as a separate entity, instead, it is frequently used in clinical studies to inhibit CDA and block the metabolism of cytidine analogues which are co-administered as the active therapeutic agent. The clinical pharmacokinetics of THU have been extensively characterized (63). In these studies, with the exception of time-to-peak THU levels, the pharmacokinetics of intravenous, SQ and oral THU at a dose of 200 mg/m2 were essentially identical (63). By each route of administration, the initial and secondary half-lives were ~8 and 80 minutes, respectively with a plasma half-life or approximately 7 hours. THU elimination was primarily in the urine and with intact drug but no metabolites identified in urine or plasma.  
These observations were extended through the use of [14C]-THU (64). In this study, 50 mg/kg
[
14C]-THU was rapidly cleared from the blood with a plasma half-life of about 1 hour following intravenous administration. Approximately 10% of the same dose was absorbed from the GI tract following oral administration. The main pathway of excretion was through the kidneys with approximately 50% of the administered drug excreted within 24h and 100% elimination within 48h.THU at 10, 25, and 50 mg/kg given 15 minutes before [3H]-cytosine arabinoside (ara-C) at a dose of 0.003 mg/kg produced an approximately two-fold increase in ara-C blood levels at all times measured from 5 minutes to 4 hours, with only slight increases in the half-life of ara-C (64). A dose-related effect of THU upon the deamination of ara-C was obvious only during the time from 15 minutes to 1 hour after the injection of 3H-ara-C. The inhibitory effect of THU upon CDA was also reflected in a considerably increased ratio of ara-C/uracil arabinoside in the urine.
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1.2.7 clinical pharmacokinetics dac alone

The clinical pharmacokinetics of three doses of DAC administered as 6-hour infusions are shown  
 in **Figure 12** (65). In this study, steady state concentrations are reached within 1-2h of the onset of the infusion, depending upon the dose administered, and are maintained throughout the infusion cycle. Upon cessation of the infusion, decitabine is rapidly removed from circulation (t ½α = 7 minutes) with a slower β-elimination phase (t ½β = 35 minutes).
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1.2.8 clinical safety thu alone

Administration of THU to humans by the IV, SQ (SQ), and oral (PO) routes, in combination with various cytidine analogues, in studies spanning over 30 years, suggest it has a benign toxicity profile (63, 64, 66-70) (summarized in **Table 2**). Reports of adverse events following treatment are infrequent. In a study of THU alone administered by IV, SQ and PO routes at a single dose of 200 mg/m2 to 5 patients with malignant melanoma, there was no mention of toxicities (63). Similarly, THU administered at a daily dose of approximately 900-1800 mg/m2 for 5 consecutive days together with cytosine arabinoside did not produce toxicities other than those expected with cytosine arabinoside alone (70). A similar experience is described when THU was administered at 700 mg/m2 daily for 4 consecutive days together with cytosine arabinoside (68). Currently, THU is being used in three NIH-sponsored clinical trials: at 350-500 mg/m2 administered daily for 3-5 consecutive days in combination with 5-fluoro-2'-deoxycytidine (e.g. NCT00077051), or daily for 7 or more days in combination with other oncotherapeutics (e.g. NCT00378807, NCT00521183).  
| Table 2: Clinical trials with tetrahydrouridine (published data). THU dose THU  Combination drug Number  Route of  patients   | Toxicity attributed to THU?   | Reference                                                                                       |                |               |       |         |    |
|-------------------------------------------------------------------------------------------------------------------------------|-------------------------------|-------------------------------------------------------------------------------------------------|----------------|---------------|-------|---------|----|
| 350                                                                                                                           | mg/m2                         | Q12                                                                                             | IV             | IV            | Ara-C | 100-200 |    |
| hours x 4 days                                                                                                                | mg/m2 Q12 hours x 4  days     | 32                                                                                              | None reported  | (68)          |       |         |    |
| 350 mg/m2  daily x 5                                                                                                          | IV                            | IV                                                                                              | 5-fluoro-2'-   |               |       |         |    |
| days                                                                                                                          | deoxycytidine                 | 5-80                                                                                            |                |               |       |         |    |
| mg/m2  daily x 5 days                                                                                                         | 11                            | None reported                                                                                   | (71)           |               |       |         |    |
| 2,100                                                                                                                         | -                             | 2,800                                                                                           | IV             | IV            | Ara-C | 1,200   | -  |
| mg/m2  total dose                                                                                                             | 1,600                         | mg/m2                                                                                           | and            |               |       |         |    |
| carboplatin                                                                                                                   | 900                           |                                                                                                 |                |               |       |         |    |
| mg/m2  total dose                                                                                                             | 8                             | Myelosuppression,  unacceptable hepatotoxicity,  diarrhea. No attribution to  direct THU effect | (72)           |               |       |         |    |
| 2 - 4 mg/kg (approx  75-150 mg/m2 ) for  3 consecutive days  every week                                                       | oral                          | Oral                                                                                            | 5-azacytidine  |               |       |         |    |
| 0.2mg/kg                                                                                                                      | for                           | 3                                                                                               |                |               |       |         |    |
| consecutive                                                                                                                   | days                          |                                                                                                 |                |               |       |         |    |
| every week                                                                                                                    | 2                             | None reported                                                                                   | (69)           |               |       |         |    |
| 25                                                                                                                            | -                             | 50                                                                                              | mg/kg          |               |       |         |    |
| (approx 900 - 1800  mg/m2 ) daily x 5  days                                                                                   | IV                            | IV Ara-C 0.1 - 0.2  mg/kg (approx 4 - 8  mg/m2 ) daily x 5 days                                 | None reported. | Toxicities    |       |         |    |
| were as expected from Ara-C  alone                                                                                            | (70)                          |                                                                                                 |                |               |       |         |    |
| 200 mg/m2  x 1                                                                                                                | IV, SQ  and  oral             | none                                                                                            | 5              | None reported | (63)  |         |    |
| 350 mg/m2 daily x 5  days every 21 days  OR for 5 days in  each of 2  consecutive weeks  every 28 days                        | IV                            | IV                                                                                              | 5-fluoro-2'-   |               |       |         |    |
| deoxycytidine                                                                                                                 | 2.5-                          |                                                                                                 |                |               |       |         |    |
| 180 mg/m2 /day x 5  days every 21 days  OR for 5 days in each  of 2 consecutive  weeks every 28 days                          | 58                            | None reported                                                                                   | (73)           |               |       |         |    |
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1.2.9 clinical safety and efficacy

A recent Phase I clinical trial was performed to evaluate the toxicities and potential efficacy of oral DAC in combination with oral THU in patients with SCD (Saunthararajah, et al, submitted for publication). The objectives of the study were to (i) extend DAC half-life/Tmax to hours instead of minutes, to increase S-phase dependent DNMT1-depletion (ii) without high DAC Cmax that causes off-target effects and cytotoxicity (intended Cmax > 5nM and < 200nM), (iii) decrease within and between patient variability in DAC distribution (that is, to produce stable distribution of DAC into tissues that otherwise provide sanctuary to myeloid cancer cells from DAC treatment effects), (iv) increase HbF to clinically meaningful levels in patients with SCD. The timings of administration between oral THU and oral DAC, and the dosages of THU and DAC in this clinical trial, were selected based on the above pharmacologic/pharmacodynamics objectives and studies in mice, and non-human primates (58, 59).  
Pharmacokinetic results: For the purposes of non-cytotoxic DNMT1-depletion, a wide concentration-time profile with low peak concentration or Cmax but extended exposure time or extended Tmax is desired (**Figure 13**) (59). To evaluate effects of different routes of administration on the DAC concentration-time profile, plasma DAC levels were compared after intravenous (IV), subcutaneous (SQ) and oral administration to non-human primates (baboons). IV DAC 0.5 mg/kg produced peak plasma drug concentrations (Cmax) > 600 nM and plasma t½ < 5 minutes (Figure 13). SQ DAC at the same dose produced Cmax ~300 nM and t½ ~40 minutes. Oral DAC at 20-fold greater dose (10 mg/kg) produced widely differing Cmax of ~220 and 10 nM in two baboons, with t½ > 100 minutes (**Figure 13A**). Thus, DAC oral absorption is very limited and very variable, although it does produce the wide concentration-time profile desired for non-cytotoxic DNMT1depletion. Pre-clinical studies in mice and baboons suggested that combining oral THU with oral DAC could address the very poor oral bioavailability and very wide inter-individual variability, creating the rationale for this first-in-man clinical trial of oral THU-DAC (**Figure 13**) (59). Samples for PK analyses were obtained at 0, 2, 4 and 24 hours in 12 of the 15 subjects who received study drug (dictated by venous access). DAC levels were measurable even in subjects receiving the lowest DAC dose of 0.01 mg/kg. A dose-dependent increase in plasma DAC was observed. The highest dose of oral Dec, 0.16 mg/kg, produced DAC plasma concentrations at 2 hours between 39-54 nM. The dose level below this, 0.08 mg/kg, produced concentrations at 2 hours of between 9-21 nM. These concentrations of DAC were in the range predicted to have non-cytotoxic epigenetic therapeutic effects (59), and accordingly, these dose levels were the only ones also associated with clinical pharmacodynamic effects, as described below. The model fit suggested Cmax was at approximately 1 hour, prior to the 2 hour time-point measured in this study. At all dose levels, by 4 hours, DAC plasma levels had decreased to < 50% of the level measured at 2 hours.  
Version Date : 08/01/2022  
  
Adverse events: Non-cytotoxic modification of hematopoietic differentiation by DAC is expected to simultaneously increase platelet counts and decrease absolute neutrophil counts (shown in vitro and in clinical studies with DAC alone ( 60 , 61 , 74 , 75 )). This expected pattern in blood counts was seen in subjects receiving oral DAC 0.08 and 0.16 mg/kg (dose levels 4 and 5) with the steepest blood count changes observed at the highest dose (**Figure 14**). Platelet and neutrophil counts on therapy did not reach the *a priori* defined triggers for dose modification, that is, neither a platelet count of > 1200 x 109/L, nor an absolute neutrophil count < 1.5 x 109/L, were reached (the highest platelet count on therapy was 1122 x 109/L in a subject with a pre-treatment count of 733 x 109/L,
and the lowest absolute neutrophil count on therapy was 1.6 x 109/L in a subject with a pretreatment count of 1.8 x 109/L) (**Figure 14**). Of particular relevance to this protocol, absolute lymphocyte counts remained stable during and subsequent to the eight-week THU-DAC therapy (**Figure 15**). The pattern of adverse events was similar between subjects receiving placebo or oral THU-DAC in all dose cohorts (**Table 3A**). There were no significant adverse events, including no significant gastro-intestinal adverse events, attributed to study drug (**Table 3B**). The most frequent adverse event in placebo and study drug treated subjects was pain from vaso-occlusive sickle cell crisis (**Table 3A**). There were no Grade 4 adverse events in placebo or study drug treated subjects (**Table 3**). No subjects discontinued placebo or study drug because of adverse events.  
  
  
Efficacy/Pharmacodynamic Effects: Hemoglobin F levels were measured by HPLC through the Clinical Pathology laboratory and also by flow cytometric quantification of HbF in individual red cells. Significant increases in HbF were observed by both these measurements at dose levels 4 and 5 (DAC 0.08 and 0.16 mg/kg respectively), with the greatest increases occurring at dose level 5 (**Figure 16**). These HbF increases were associated with trends for increasing total hemoglobin, decreasing LDH and total bilirubin (as biomarkers of hemolysis) and decreasing D-dimer levels (as a biomarker of coagulation pathway activation and vaso-occlusion) (**Figure 17**). Increases in HbF were not sustained once study drug was stopped in Week 8 (**Figure 16**). Hematologic side-effects and toxicity: There were no hematologic toxicities warranting treatment hold (please see above). Non-hematologic toxicity: No significant non-hematologic toxicities were observed (please see above). In summary, results from the aforementioned Phase 1 clinical trial of oral THU-DC demonstrate that the non-human primate data essentially mapped to subjects with high risk SCD: combining a mini-dosage of oral DAC (0.16 mg/kg; ~5 mg/m2) with THU 10 mg/kg produced major improvement in DAC tissue distribution and exposure, creating a wide DAC concentration-time profile that is desired for DNMT1-depletion without cytotoxicity in vivo. This DAC oral dosage compared very favorably to parenteral dosages FDA approved for administration to humans (2045 mg/m2/day for 3-5 days every 4-6 weeks). The THU-DAC was very well-tolerated without significant gastro-intestinal or other drug-related adverse events with repeat administration 2X/week for 8 weeks. Changes in hematologic parameters were those expected from non-cytotoxic DNMT1-depletion by DAC, with simultaneous increases in platelet counts and hemoglobin and decreases in absolute neutrophil counts. The platelet and absolute neutrophil count changes did not cross thresholds that would have triggered treatment holds (> 1,200 x 109/L for platelets, < 1.5 x 109/L for neutrophils). Increases in HbF that would be considered clinically significant if generated by standard of care HU therapy were observed at DAC dosage of 0.08 and 0.16 mg/kg. The HbF increases were associated with favorable trends in total hemoglobin and in peripheral blood markers of hemolysis and coagulopathy/vaso-occlusion - serum LDH, total bilirubin and D-dimer.  
| Version Date : 08/01/2022                                                                                 |                  |                                                                                           |          |                      |       |    |    |    |    |    |
|-----------------------------------------------------------------------------------------------------------|------------------|-------------------------------------------------------------------------------------------|----------|----------------------|-------|----|----|----|----|----|
| A                                                                                                         | Number of Events | PLACEBO (n=10) COHORT 1 (n=3) COHORT 2 (n=3) COHORT 3 (n=3) COHORT 4 (n=3) COHORT 5 (n=3) |          |                      |       |    |    |    |    |    |
| Grade                                                                                                     | Grade            | Grade                                                                                     | Grade    | Grade                | Grade |    |    |    |    |    |
| SYMPTOM                                                                                                   |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Pruritus                                                                                                  | 1                | 1                                                                                         | 3        |                      |       |    |    |    |    |    |
| Upper respiratory infection                                                                               |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Diarrhea                                                                                                  | 1                | 1                                                                                         | 1        |                      |       |    |    |    |    |    |
| 1                                                                                                         |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Vomiting                                                                                                  | 2                | -                                                                                         | 1        | 1                    | 1     |    |    |    |    |    |
| Urinary tract infection                                                                                   |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Pain                                                                                                      | 8                | 13                                                                                        | 4        | 3                    | 1     | 3  | m  | 3  | 2  | 2  |
| Acute Chest Syndrome                                                                                      |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Pneumonia                                                                                                 |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Headache                                                                                                  | 3                | 1                                                                                         | 1        | 1                    |       |    |    |    |    |    |
| Acute kidney injury                                                                                       |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Hematuria                                                                                                 | 1                | 1                                                                                         |          |                      |       |    |    |    |    |    |
| Nausea                                                                                                    | 1                | 1                                                                                         | 2        | 1                    |       |    |    |    |    |    |
| Constipation                                                                                              | 1                | 1                                                                                         |          |                      |       |    |    |    |    |    |
| Rash maculo-papular                                                                                       | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Skin hyperpigmentation                                                                                    | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Hypomagneses                                                                                              | 1                | 1                                                                                         |          |                      |       |    |    |    |    |    |
| Hypokalemia                                                                                               | 2                |                                                                                           |          |                      |       |    |    |    |    |    |
| 1                                                                                                         |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Leukocytosis                                                                                              | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Anemia                                                                                                    | 1                | 1                                                                                         |          |                      |       |    |    |    |    |    |
| Leukocytosis                                                                                              |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Hypomagnesemia                                                                                            |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Chest pain - cardiac                                                                                      | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Hyperkalemia                                                                                              |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Hypertension                                                                                              | 1                | 1                                                                                         |          |                      |       |    |    |    |    |    |
| Dysmenormea, Oligomenorrhea                                                                               |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Transaminitis                                                                                             | 2                |                                                                                           |          |                      |       |    |    |    |    |    |
| Light-headed                                                                                              | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Abdominal pain or cramps                                                                                  | 2                | 1                                                                                         | 4        | 1                    |       |    |    |    |    |    |
| Calve crambs                                                                                              | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Vaginal spotting                                                                                          |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Allergic rhinti                                                                                           |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Pinpoint puplis                                                                                           |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Enlarged tons it                                                                                          |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Tachycardia                                                                                               | 2                |                                                                                           |          |                      |       |    |    |    |    |    |
| Drowsiness                                                                                                | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Pulmonary embolus                                                                                         |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Otitis media                                                                                              | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Decreased range of motion                                                                                 |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Diachoresis                                                                                               | 1                |                                                                                           |          |                      |       |    |    |    |    |    |
| Vaginal yeast infection                                                                                   |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Lip ulcer                                                                                                 |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Lower extremity swelling                                                                                  | 1                | 1                                                                                         |          |                      |       |    |    |    |    |    |
| Skin ulceration                                                                                           |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Leukopenia                                                                                                |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Wheezing                                                                                                  |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Iron deficiency                                                                                           |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Toungue swelling                                                                                          | 3                |                                                                                           |          |                      |       |    |    |    |    |    |
| Cough                                                                                                     |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| B                                                                                                         | COHORT 1         | COHORT 2                                                                                  | COHORT 3 | COHORT 4    COHORT 5 |       |    |    |    |    |    |
| Grade                                                                                                     | Grade            | Grade                                                                                     | Grade    | Grade                |       |    |    |    |    |    |
| ADVERSE EVENT                                                                                             | 3 4              | 1 2 3 4                                                                                   | 3 4      | 1 2 3 4 1 2 3 4      |       |    |    |    |    |    |
| Pruritus                                                                                                  | Probable x1      |                                                                                           |          |                      |       |    |    |    |    |    |
| Diarrhea                                                                                                  | Unlikely x1      | Possible x1                                                                               |          |                      |       |    |    |    |    |    |
| Vomiting                                                                                                  | Possible x1      | Unlikely xl                                                                               |          |                      |       |    |    |    |    |    |
| Urinary tract infection                                                                                   | Unlikely x1      |                                                                                           |          |                      |       |    |    |    |    |    |
| Unlikely x1                                                                                               | Unlikely x1      |                                                                                           |          |                      |       |    |    |    |    |    |
| Nausea                                                                                                    |                  |                                                                                           |          |                      |       |    |    |    |    |    |
| Rash maculo-papular                                                                                       | Unlikely x1      |                                                                                           |          |                      |       |    |    |    |    |    |
| Abdominal pain or cramps                                                                                  | Probable x1      |                                                                                           |          |                      |       |    |    |    |    |    |
| Calve cramps                                                                                              | Probable x1      |                                                                                           |          |                      |       |    |    |    |    |    |
| Leukopenia                                                                                                | Possible x1      |                                                                                           |          |                      |       |    |    |    |    |    |
| Table 3: Adverse events in subjects treated with placebo and with oral THU-DAC at the various dose levels |                  |                                                                                           |          |                      |       |    |    |    |    |    |  
(Cohorts 1-5). A) All adverse events as per CTCAE v 4.0. There were no Grade 4 adverse events. B) Adverse events with attribution other than unrelated to study drug per the judgement of the treating clinical team.  
31  
  
Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date : 08/01/2022 Horizontal grey line shows 0.5 g/dL threshold associated with better survival. D) Proportion of RBCs expressing high levels of HbF (F-cells) in subjects receiving THU-DAC 0.08 or 0.16 mg/kg. E) Raw F-cell flow cytometry data in Cohort 4 and 5 subjects.  
  
Because FOXP3 is regulated by epigenetic mechanisms ( 76 ), it is possible that DAC-THU therapy will mediate expansion and enhanced immunosuppressive function of Tregs in cancer patients. Invitro treatment of human Tregs with DAC induced hypomethylation of the Treg specific DNA
demethylated region (TDMR), which is required for stable expression of FOXP3, and increased expression of Treg specific genes; however, the cells did not exhibit full suppressive function ( 77 ). On the other hand, 5-Aza which is converted to DAC before incorporation into DNA increases Tregs and significantly reduces airway inflammation in mice ( 78 ). Furthermore, adoptive transfer of Tregs treated in vitro with DAC mediates resolution of lung inflammation in a murine ARDS model ( 79 ).  Collectively these data suggest that DNMT depletion may enhance Treg function in humans; as such strategies to inhibit Treg activity may facilitate more effective antitumor effector responses in cancer patients receiving DAC-THU therapy.  
Rationale to Combine DAC-THU with Immune Checkpoint Inhibitors for Treatment of Thoracic Malignancies: Although substantial preclinical studies including those cited above as well as a recent report by Kim et al (80) support evaluation of epigenetic regimens in combination with immune checkpoint inhibitors, observations that 3 of 5 lung cancer patients previously treated with sequential azacytidine/etinostat without success exhibited objective responses to anti-PD-1 or anti-PD-L1 therapy (81-83) provided the clinical impetus to develop combinatorial epigenetic immunotherapies for cancer. Numerous trials have been initiated to evaluate azacytidine with or without etinostat or other HDAC inhibitors and either pembrolizumab or nivolumab in cancer patients who have failed first-line cytotoxic or TKI therapies (84). Several recent studies have further evaluated potential mechanisms by which DNA demethylating  
  
agents augment antitumor immunity **Figure 18**. Wrangle et al (85) examined gene expression profiles in several lung cancer lines following treatment with low dose 5-azacytidine (AZA) which is converted to DAC upon incorporation into DNA, and observed that AZA up-regulated cancertestis antigen expression as well as numerous pathways mediating innate and adaptive immunity.  
In addition, AZA up-regulated PD-L1, which could dampen cellular immune responses to cancer cells. The effects of AZA on immune pathways in lung cancer cells were very similar to those observed in HCT116 colon cancer cells following knock-out of DNMT1 and DNMT3b. Li et al (86) examined gene expression profiles in a large panel of breast, colorectal and ovarian cancer lines following treatment with low dose AZA, and observed up-regulation of cancer-testis antigens as well as significant enrichment for immunomodulatory pathways including antigen processing and presentation, cytokines/chemokines, and interferon signaling. Based on expression profiling, an AZA-IMune (AIM) gene set was defined, which appeared to be up-regulated in primary tumor tissues from cancer patients receiving AZA treatment. More recently, Chiappinelli et al (87) observed that low dose AZA de-represses endogenous retroviruses (ERV) which trigger the double stranded RNA (dsRNA) sensing machinery to induce a type I interferon response similar to a viral response. Analysis of melanoma, breast, ovarian, colon and lung cancers in TCGA revealed that cancers with high levels of viral defense genes were more likely to have better outcomes. Furthermore, high-level intratumoral expression of viral defense genes was associated with improved outcomes in melanoma patients receiving the immune checkpoint inhibitor, antiCTLA-4. Roulois et al (88) observed that low dose AZA targets colorectal cancer initiating cells
(CICs) by induction of "viral mimicry" (**Figure 8**) secondary to up-regulation of ERV, activation of the MDA5/MAVS RNA recognition pathway and up-regulation of IRF7. Knock-down ofMDA5/MAVS, or IRF7 significantly attenuated the inhibitory effects of AZA on proliferation and clonogenic potential of CICs.  
A series of experiments have been performed in the Thoracic Epigenetics Laboratory, TSB to examine endogenous retroviral (ERV) expression in lung cancer cells, and to evaluate the feasibility of using DNA hypomethylating agents to augment ERV expression as a potential adjunct to immune check point inhibitor therapy for lung cancer. We also sought to identify potential PD endpoints that could be incorporated into our epigenetic-immunotherapy protocols. Briefly, SYBR Green quantitative RT-PCR techniques were used to examine expression of nine ERV family sequences (MLTA10, MLT1B, MER21C, ERVL, MLT1C627, MER4D, MER57B1, MTE2B4) and cancer-germline genes in normal human bronchial epithelial (NHBE) cells, normal small airway epithelial cells (SAEC), 13 NSCLC lines, 20 SCLC lines, and 5 NSCLC lines and NHBE treated with 5-aza-2'-deoxycytidine (DAC; 0.1 uM x 72 hours). ERV expression patterns in lung cancer lines and normal respiratory epithelial cells were primer specific with no consistency in SCLC or NSCLC lines (**Figure 19**). Lung cancer lines generally showed higher ERV expression than normal cells. MLT1B, ERVL, and MLT1C627 were up-regulated more than two-fold in 82%, 73%, and 70% of lung cancer lines, respectively, compared to normal respiratory epithelial cells. Activation of ERV in lung cancer lines did not appear to coincide with de-repression of cancer-testis genes. DAC mediated relatively modest (two-fold average) up-regulation of ERV, with MLTA10 (2.7 fold), MER21C (3.6 fold) and ERVL (2.6 fold) showing highest induction.  
Collectively these data demonstrate that cultured lung cancer cells exhibit heterogeneous endogenous ERV expression profiles, as well as variable patterns of ERV induction following exposure to DNA demethylating agents.  
  
Abbreviated Title : DAC-THU/Pembro: NSCLC, EsC, PM
Version Date : 08/01/2022  
  
  
Recently a series of preclinical experiments have been performed by Drs. Vamsi Velchetti and Yogen Saunthararajah at the Cleveland Clinic to examine potential efficacy of DAC-THU and anti-PD-1 therapy for lung cancer. Briefly, immunocompetent C57/BL6 mice were inoculated via tail vein with Lewis lung cancer cells. Eighteen days later, mice were imaged to confirm lung colonization and then randomized to receive PBS SC, DAC 0.2mg/kg SC, or DAC (0.1mg/kg) SC/ THU (10mg/kg IP) q M-W-F. On Day 32, mice were bled for flow cytometry experiments and subsequently euthanized when ill-appearing. DAC-THU therapy significantly prolonged survival relative to DAC alone or PBS (**Figure 20**). Combination treatment markedly decreased DNMT1 and to a lesser extent, MYC protein levels while increasing p27 expression, consistent with the aforementioned p53- independent cell cycle exit effect of DNMT1 depletion. DAC-THU therapy also significantly up-regulated intratumoral MAGE-A1 and MAGE-A3 cancer-testis gene expression, as well as intratumoral interferon-γ levels. Furthermore, relative to PBS controls, mice treated with either DAC or DAC-THU exhibited significantly higher peripheral CD4 lymphocytes
(approaching those in untreated, nontumor bearing mice), as well as significantly decreased MDSC
in peripheral blood (**Figure 21**).  
  
In additional experiments, C57/BL6 mice with Lewis lung cancer metastases were randomized to receive PBS SC, DAC (0.1 mg/kg) SC/THU (10 mg/kg IP) q M-W-F, a murine anti-PD-1 antibody (100 ug) q 5 days, or a combined DAC-THU/ anti-PD-1 regimen. Both DAC-THU and anti-PD-1 alone prolonged survival; a combinatorial effect was observed, with 40% of mice receiving DACTHU-anti-PD-1 therapy appeared to be cured of their disease (**Figure 22**; left panel). Relative to tumor bearing mice treated with PBS alone, mice receiving epigenetic treatment, immunotherapy, or combination DAC-THU and anti-PD-1 therapy exhibited significantly higher peripheral CD4 and CD8 lymphocyte counts (**Figure 22**; right panel). Collectively these findings, together with recent observations that DNA demethylating agents and other epigenetic drugs enhance the efficacy of immune checkpoint inhibitors by blocking immunosuppressive activities of Tregs and MDSC in the tumor microenvironment provide strong rationale for evaluation of DAC-THU and pembrolizumab for cancer therapy.
untitled h1
1.2.10 pembrolizumab

The importance of immune surveillance in controlling outgrowth of neoplastic transformation was initially postulated by Ehrlich (89) nearly a century ago, and subsequently revised by Burnet (90). Recent accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in various malignancies (91). In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T-cells / FoxP3+ regulatory T-cells seems to correlate with improved prognosis and long-term survival in many solid tumors. The programmed death (PD)-1 receptor-ligand interaction is a major pathway utilized by tumors to establish equilibrium and ultimately escape immune surveillance (92). PD-1 (encoded by the gene Pdcd1) is an Ig superfamily member related to CD28 and CTLA-4 that is normally expressed on the surface of activated T-cells, and functions to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2). The mechanism by which PD-1 attenuates T-cell responses is similar to, but distinct from that of CTLA-4 as both molecules regulate an overlapping set of signaling proteins. PD-1 is expressed on activated lymphocytes including peripheral CD4+ and CD8+ T-cells, B-cells, T regs and natural killer cells. The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic tissues as well as in various tumors. Both ligands are type I
transmembrane receptors containing both IgV- and IgC-like domains in the extracellular region and contain short cytoplasmic regions with no known signaling motifs. Binding of either PD-1 ligand to PD-1 inhibits T-cell activation triggered through the T-cell receptor. PD-L1 is normally expressed at low levels on various non-hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only detectably expressed on antigen-presenting cells in lymphoid tissue or chronic inflammatory environments. PD-L2 is thought to control immune Tcell activation in lymphoid organs, whereas PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues. Although healthy organs express little (if any) PD-L1, a variety of malignancies including lung cancers express abundant levels of this T-cell inhibitor; PD-L1 expression is also elevated in tumor associated macrophages and fibroblasts. As a result, the PD1/PD-L1 pathway has emerged as an attractive target for therapeutic intervention in a wide variety of human malignancies (19, 84). Pembrolizumab is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/kappa isotype which directly inhibits interaction of PD-1 with its ligands, PD-L1 and PD-L2 (20). In 2015, pembrolizumab was approved in the US for second-line therapy of NSCLC (93), and in 2016, this immune checkpoint inhibitor was approved as first-line therapy for NSCLC with high level (≥ 50%) PD-L1 expression. In 2017, the FDA approved pembrolizumab for recurrent locally advanced, or metastatic gastroesophageal cancer with PD-L1 expression (CPS) greater than or equal to 1. In a Phase I trial (Keynote 001), 495 patients with advanced NSCLC were assigned to either training or validation groups, and received pembrolizumab at 2 mg/kg or 10 mg/kg every three weeks, or 10 mg/kg every two weeks (94). Fatigue, decreased appetite and pruritus were the most common side effects (single digit numbers); severe pneumonitis was observed in 1.8% of patients, including one death. The ORR, median PFS and median OS for treatment naïve patients were 24.8%, 6.0 months and 16.2 months, respectively, vs 18%, 3.0 months, and 9.3 months, respectively, for previously treated patients. Subsequent analysis demonstrated ORR and median PFS of 43.9% and 6.1 months vs 50.0% and 12.5 months, respectively, for treatment naïve and previously treated patients whose tumors had a proportion score (PS) of at least 50% cancer cells showing positive membrane staining for PD-L1 (94). In a Phase III trial (Keynote 024), Reck et al (95) randomly assigned 305 patients with untreated, advanced NSCLC with no sensitizing EGFR or ALK mutations and PD-L1 expression on 50% or more tumors cells to receive either pembrolizumab (200 mg Q3W) or platinum-based chemotherapy. Cross-over from the chemotherapy to pembrolizumab was allowed. The objective response rate was 44.8% in the pembrolizumab arm compared to 27.8% in the chemotherapy arm. Median progression free survival was 10.3 months (95% CI: 6.7 months to not reached) compared to 6.0 months (95% CI: 4.2 to 6.2 months) in the chemotherapy arm. Median duration of response was longer (not reached; range 1.9 to 14.5 months) in the pembrolizumab arm compared to 6.3 months (range: 2.1 to 12.6 months). Grade 3, 4, or 5 treatment related adverse events were significantly lower in the pembrolizumab arm compared to the chemotherapy arm (26.6% vs 53.3%).  
| Table 4: Systemic Toxicities of Pembrolizumab vs Chemotherapy in Treatment Naïve NSCLC Patients.   |                  |           |                  |           |
|----------------------------------------------------------------------------------------------------|------------------|-----------|------------------|-----------|
| Chemotherapy Group                                                                                 |                  |           |                  |           |
| Pembrolizumab Group                                                                                |                  |           |                  |           |
| Adverse Event                                                                                      | (N=154)          | (N=150)   |                  |           |
| Any Grade                                                                                          | Grade 3, 4, or 5 | Any Grade | Grade 3, 4, or 5 |           |
| number of patients (percent)                                                                       |                  |           |                  |           |
| Treatment-related f                                                                                | 13 (73.4)        | 41 (26.6) | 135 (90.0)       | 80 (53.3) |
| Any                                                                                                | 3 (21.4)         | 29 (18.8) | 31 (20.7)        | 29 (19.3) |
| Serious                                                                                            |                  |           |                  |           |
| Led to discontinuation                                                                             | 1 (7.1)          | 8 (5.2)   | 16 (10.7)        | 9 (6.0)   |
| Led to death                                                                                       | 1 (0.6)          | 1 (0.6)   | 3 (2.0)          | 3 (2.0)   |
| Occurred in ≥10% of patients in either                                                             |                  |           |                  |           |
| group?                                                                                             | 15 (9.7)         | 65 (43.3) | 3 (2.0)          |           |
| Nausea                                                                                             | 0                |           |                  |           |
| Anemia                                                                                             | 8 (5.2)          | 3 (1.9)   | 66 (44.0)        | 29 (19.3) |
| Fatigue                                                                                            | 16 (10.4)        | 2 (1.3)   | 43 (28.7)        | 5 (3.3)   |
| Decreased appetite                                                                                 | 14 (9.1)         | 0         | 39 (26.0)        | 4 (2.7)   |
| Diarrhea                                                                                           | 2 (14.3)         | 6 (3.9)   | 20 (13.3)        | 2 (1.3)   |
| Neutropenia                                                                                        | 1 (0.6)          | 0         | 34 (22.7)        | 20 (13.3) |
| Vomiting                                                                                           | 4 (2.6)          | 1 (0.6)   | 30 (20.0)        | 1 (0.7)   |
| Pyrexia                                                                                            | 16 (10.4)        | o         | 8 (5.3)          | o         |
| Constipation                                                                                       | 6 (3.9)          | 0         | 17 (11.3)        | 0         |
| Stomatitis                                                                                         | 4 (2.6)          | 0         | 18 (12.0)        | 2 (1.3)   |
| Decreased neutrophil count                                                                         | 0                | o         | 20 (13.3)        | 6 (4.0)   |
| Increased blood creatinine level                                                                   | 3 (1.9)          | o         | 15 (10.0)        | 1 (0.7)   |
| Decreased platelet count                                                                           | 0                | o         | 18 (12.0)        | 9 (6.0)   |
| Thrombocytopenia                                                                                   | 0                | 0         | 17 (11.3)        | 8 (5.3)   |
| Decreased white-cell count                                                                         | 1 (0.6)          | o         | 16 (10.7)        | 3 (2.0)   |
| Dysgeusia                                                                                          | 1 (0.6)          | o         | 15 (10.0)        | o         |
| Immune-mediated                                                                                    |                  |           |                  |           |
| Any                                                                                                | 45 (29.2)        | 15 (9.7)  | 7 (4.7)          | 1 (0.7)   |
| Hypothyroidism                                                                                     | 14 (9.1)         | 2 (1.3)   |                  |           |
| Hyperthyroidism                                                                                    | 12 (7.8)         | 0         | 2 (1.3)          | 0         |
| Pneumonitis                                                                                        | 9 (5.8)          | 4 (2.6)   | 1 (0.7)          | 1 (0.7)   |
| Infusion reaction                                                                                  | 7 (4.5)          | 0         | 2 (1.3)          | 0         |
| Severe skin reaction                                                                               | 6 (3.9)          | 6 (3.9)   | 0                | 0         |
| Thyroiditis                                                                                        | 4 (2.6)          | 0         | 0                | o         |
| Colitis                                                                                            | 3 (1.9)          | 2 (1.3)   | 0                | 0         |
| Myositis                                                                                           | 3 (1.9)          | 0         | 0                | 0         |
| Hypophysitis                                                                                       | 1 (0.6)          | 1 (0.6)   | 0                | 0         |
| Nephritis                                                                                          | 1 (0.6)          | 1 (0.6)   | 0                | 0         |
| Pancreatitis                                                                                       | 1 (0.6)          | 1 (0.6)   | 0                | 0         |
| Type 1 diabetes mellitus                                                                           | 1 (0.6)          | 1 (0.6)   | o                | o         |  
In a Phase 2, open-label, single-arm, multicohort study (KEYNOTE-059), 259 patients with recurrent gastric or gastroesophageal cancer received pembrolizumab, 200 mg intravenously every 3 weeks until disease progression, investigator or patient decision to withdraw, or unacceptable toxic effects ( 96 ). The primary end points were objective response rate and safety. Secondary end points included response duration. Expression of PD-L1 was assessed by immunohistochemistry.  
Tumors were considered positive if the combined positive score (CPS) of PD-L1 positive cells
(tumor are well as lymphoid cells) per total number of cells was ≥ 1%. The objective response rate was 11.6% (95% CI, 8.0%-16.1%; 30 of 259 patients), with complete response in 2.3% (95% CI,
0.9%-5.0%; 6 of 259 patients). Median (range) response duration was 8.4 (1.6+ to 17.3+) months
(+ indicates that patients had no progressive disease at their last assessment). Objective response rate and median (range) response duration were 15.5% (95% CI, 10.1%-22.4%; 23 of 148 patients) and 16.3 (1.6+ to 17.3+) months and 6.4% (95% CI, 2.6%-12.8%; 7 of 109 patients) and 6.9 (2.4 to 7.0+) months in patients with PD-L1-positive and PD-L1-negative tumors, respectively. Fortysix patients (17.8%) experienced 1 or more Grade 3 to 5 treatment-related adverse events. Two patients (0.8%) discontinued because of treatment-related adverse events, and 2 deaths were considered related to treatment. A subsequent analysis compared outcomes for three patient cohorts in this trial (97). Cohort 1 patients received pembrolizumab alone after ≥ 2 prior lines of therapy. Cohort 2 patients received pembrolizumab + cisplatin (80 mg/m2 Day 1) + 5-fluorouracil (800 mg/m2 Days 1-5 Q3W) or capecitabine (in Japan only, 1000 mg/m2 twice daily) as first-line. Cohort 3 patients received pembrolizumab alone as first-line. Patients were enrolled in Cohorts 1 and 2 regardless of tumor PD-L1 expression; only patients with PD-L1-positive tumors (combined positive score of ≥ 1%)
were enrolled in Cohort 3. In all cohorts, pembrolizumab was given at 200 mg Q3W for up to 2 years. Median (range) follow-up was 6 (1-25), 14 (2-24), and 18 (2-21) months for Cohorts 1 (259 patients), 2 (25 patients) and 3 (31 patients), respectively. Confirmed ORR (95% CI) were 16% (11-23) in PD-L1-positive, and 6% (3-13) in PD-L1-negative tumors in Cohort 1, 73% (45-92) in PD-L1-positive, and 38% (9-76) in PD-L1-negative tumors in Cohort 2, and 26% (12-45) in Cohort 3. Median PFS (95% CI) were 2 (2-2), 7 (6-11), and 3 (2-6) months in Cohorts 1, 2, and 3, respectively. Median OS (95% CI) were 6 (4-7) and 14 (9-not estimable) months for Cohorts 1 and 2 respectively, and not reached (9-21) in Cohort 3. In Cohorts 1, 2 and 3, Grade 3-5 treatmentrelated adverse events (TRAE) incidence occurred in 46 (18%), 19 (76%), and 7 (23%) patients in Cohorts 1, 2, and 3, respectively. In a recent randomized Phase III trial, 395 patients with previously treated advanced gastric or gastro-esophageal junction cancers with PD-L1 CPS ≥ 1% were randomized to receive pembrolizumab 200 mg q 3 weeks for up to 2 years or standard dose paclitaxel (98). Median overall survival was 9.1 months (95% CI 6.2–10.7) with pembrolizumab and 8.3 months (7.6– 9.0) with paclitaxel (hazard ratio [HR] 0.82, 95% CI 0.66–1.03; one-sided p=0.0421). Median progression-free survival was 1.5 months (95% CI 1.4–2.0) with pembrolizumab and 4.1 months (3.1–4.2) with paclitaxel (HR 1.27, 95% CI 1.03–1.57). Grade 3–5 treatment related adverse events occurred in 42 (14%) of the 294 patients treated with pembrolizumab and 96 (35%) of the 276 patients treated with paclitaxel. Whereas pembrolizumab did not significantly improve overall survival compared with paclitaxel as second-line therapy for advanced gastric or gastro-oesophageal junction cancer with PD-L1 CPS of 1 or higher, it is noteworthy that the survival curves for the treatment cohorts crossed at 8 months in favor of the pembrolizumab arm suggesting a delayed and sustained benefit from the immunotherapy (**Figure 23**). The authors stated that this violation of the proportional hazards assumption suggests that alternative statistical methods such as a weighted log-rank test should be used to evaluate potential efficacy of immunotherapy in clinical trials. Post-hoc analysis using a weighted log-rank test that placed more weight on the events that occurred around the middle of the follow-up duration (i.e., around the time at which the overall survival curves crossed) and less weight on events that occurred early or late resulted in a one-sided p-value of 0.0009. The survival curves and results of the weighted log-rank test, as well as the relative toxicity profiles of pembrolizumab and paclitaxel clearly suggest that pembrolizumab is preferable to chemotherapy for second-line treatment of gastric or gastroesophageal junction carcinomas.  
  
In a multi-institutional phase 1b trial, 25 previously treated MPM patients received pembrolizumab 10 mg/kg q2 weeks (99). Five (20%) patients experienced Grade 3 treatment-related adverse events. Three (12%) patients required dose interruption because of immune-related adverse events. No treatment-related deaths or discontinuations were observed. Five patients (20%) had a partial response; objective response: 20% (95% CI 6·8-40·7); 13 patients (52%) had stable disease. The responses were durable (median response duration 12·0 months [95% CI 3·7 to not reached]). These preliminary findings demonstrated safety and potential efficacy of pembrolizumab in MPM patients. In a subsequent study, Metaxas et al (100) reported results of 93 MPM patients who received pembrolizumab off-label in Switzerland and Australia. 48 patients (52%) received pembrolizumab as second-line treatment. 68 patients (73%) had epithelioid MPM, and 67 (72%) had an Eastern Cooperative Oncology Group performance status of 0 or 1. 66 of the 93 patients (71%) had PDL1 expression analysis: 68% were negative, 18% were intermediate, and 14% had high intratumoral PD-L1 expression. The overall response rate (ORR) was 18%, with a median progressionfree survival (mPFS) of 3.1 months (**Figure 24**); median overall survival was 7.2 months. ORR
was 37%, mPFS was 3.7 months, and the median overall survival was 10.2 months in patients with ECOG PS of 0 or 1 and only one previous systemic treatment (n = 35), Patients with nonepitheloid histological subtype showed an improved ORR (24% versus 16% [p = 0.54) and mPFS (5.6 versus 2.8 months [p = 0.02]). Compared with intermediate and negative PD-L1 expression, high PD-L1 expression was associated with an improved ORR (44% versus 42% versus 11% [p = 0.01]) and mPFS (6.2 versus 3.9 versus 2.7 months [p = 0.04]). Toxicities in MPM patients were similar to those observed in patients with other malignancies.  
  
In an abstract presented at ESMO 2019, Popat and colleagues (101) reported preliminary results of the Phase III European Thoracic Oncology Platform (ETOP 9-15) PROMISE-meso trial. 144 patients with relapsed MPM following platinum-based chemotherapy were randomized 1:1 to receive 200 mg of pembrolizumab q 3 weeks (n = 73) or physician's choice of gemcitabine or vinorelbine at standard doses (n = 71). Treatment was administered until disease progression by RECIST1.1 criteria up to 2 years, and patients were assessed every 9 weeks for 6 months, followed by every 12 weeks thereafter. Upon disease progression, those in the chemotherapy arm were permitted to cross over to the PD-1 inhibitor arm. The primary endpoint of the trial was PFS; secondary endpoints were ORR, time to treatment failure (TTF), overall survival (OS), investigator-assessed PFS, and adverse events (AEs). Outcomes by PD-L1 status served as a correlative endpoint. Baseline characteristics were similar between arms. The median age was 70 years, most patients had epithelioid histology (88.9%), and the majority had an ECOG performance status of 1 (75.1%). Prior therapies included carboplatin/pemetrexed, cisplatin/pemetrexed, platinum or without pemetrexed, and cisplatin/pemetrexed plus carboplatin/pemetrexed. The ORR was 22% with pembrolizumab compared with 6% in those who received chemotherapy (P = .004). The median DOR was 4.6 months for pembrolizumab and 11.2 months with chemotherapy (95% CI, 2.2-10.3; 95% CI, 6.2-15.3). Median PFS was 2.5 months for pembrolizumab compared with 3.4 months for chemotherapy (HR, 1.06; 95% CI, 0.73-1.53; P = .76). The 6-month PFS rates were 25.0% and 27.4% with pembrolizumab and chemotherapy, respectively. At a median follow-up of 11.8 months, the median OS was 10.7 months in the pembrolizumab arm versus 11.7 months in the chemotherapy arm (HR, 1.04; 95% CI, 0.661.67; P = .85). Even when censoring for crossover, there was no OS benefit observed with pembrolizumab (HR, 1.44; 95% CI, 0.77-2.67; P = .25), nor for inverse probability weighting (HR, 1.07; 95% CI, 0.67-1.71; P = .79). PFS was similar in both PD-L1–positive and –negative subsets. Toxicities of pembrolizumab therapy were similar to those previously reported. Collectively, the above results indicate that pembrolizumab has activity in MPM patients. Although pembrolizumab is not approved for MPM, it is listed in the current NCCN guidelines for palliative therapy of this disease. In light of the profound immunosuppressive microenvironment associated with MPM (102), there is clear rationale to utilize epigenetic priming regimens as a strategy to enhance the efficacy of immune checkpoint inhibitors for MPM therapy.
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1.2.11 preliminary experience with oral

A Phase II trial of oral DAC-THU and Nivolumab as second line therapy for NSCLC  
 (NCT02664181) was recently conducted at the Cleveland Clinic and the NCI (D. Schrump, PI). Patients were randomized 2:1 to receive oral DAC/THU on two consecutive days each week with Nivolumab every two weeks, or Nivolumab alone. Two of five (40%) evaluable patients on the experimental arm had objective responses, one of which was a CR (**Figure 25**). Interestingly, both of these patients were never smokers (1 ALK positive (PDL1 unknown) patient and 1 HER2 insertion site mutation (PDL1- 1-49%). 1 other patient who had PD-L1 0% had SD after 2 cycles. Typically, patients who are never smokers exhibit low mutational burden, and low or no detectable PD-L1 expression; response rates following immune checkpoint inhibitor therapy in patients with these lung cancers range from 0-5% (103, 104). These preliminary findings suggest that oral DAC-THU modulates the tumor and/or microenvironment to enhance efficacy of immune checkpoint blockade in NSCLC patients. In light of current controversies regarding the accuracy of determining PD-L1 expression in NSCLC based on single biopsies (105), and inconclusive data pertaining to the validity of PD-L1 expression as a biomarker of response to immune checkpoint inhibitors (106, 107), these findings support the use of this oral immune-modulatory regimen in conjunction with immune checkpoint inhibitors for the treatment of thoracic malignancies. Four patients were treated on this protocol before enrollment was voluntarily halted (without formal suspension) to allow for revision of the dosing regimen. Briefly, patient \#1 with medically inoperable, treatment naïve, synchronous primary NSCLC commenced oral DAC-THU (0.22 mg/kg) 3 days per week on 4/16/2018. During Week 4 she developed precipitous Grade 4 neutropenia after receiving a total DAC dose of 300 mg (2.7 mg/kg). She progressed to complete agranulocytosis several days later and was transferred to the intensive care unit for neutropenic fever and commenced broad-spectrum intravenous antibiotics. Due to ongoing agranulocytosis as well as mild anemia and thrombocytopenia, a bone marrow aspirate was performed which demonstrated hematopoietic stem cells with erythrocyte and platelet differentiation but no myeloid precursors. Although these findings were suggestive of anticipated reprogramming of hematopoietic stem cell differentiation (Section **1.2.9**), the magnitude of the granulocytopenia in the context of the investigational treatment regimen raised the possibility of autoimmune agranulocytosis. As such, the patient received IVIG, high-dose steroids, cyclosporine, and granulocyte infusions, ultimately recovering her blood counts approximately three weeks later.  
She commenced a gradual taper of her prednisone and cyclosporine, and was removed from treatment on 5/31/2018, and removed from study on 6/8/2018. Subsequently, she received stereotactic XRT and cryoablation for local control of her cancers. She remains alive with no clinical evidence of active disease as of June 2019. Patient \#2 with inoperable, treatment naïve NSCLC also received DAC-THU (0.22 mg/kg) 3 days per week, commencing on 5/7/2018. When patient \#1 developed severe neutropenia, DAC-THU was held for 1 week (Week 3; Cycle 1) for patient \#2 despite normal blood counts. The following week, patient \#2 received DAC-THU for 2 days instead of three without hemato-toxicity. She developed Grade 4 ANC during Week 5 after receiving a total DAC dose of 160 mg (1.80 mg/kg). Because her clinical picture was similar to that of patient \#1, she was hospitalized for observation; bone marrow aspirate revealed hematopoietic stem cells with very few myeloid precursors but preservation of erythrocyte and platelet lineages. Once again, given concerns regarding possible autoimmune agranulocytosis and ongoing neutropenia in patient \#1, patient \#2 received IVIG, high-dose steroids, and cyclosporine, without granulocyte infusions with resolution of her Grade 4 neutropenia within 1 week. She continued her steroid taper for approximately six weeks until she was removed from treatment and taken off protocol on 6/21/2018. Thereafter, she received XRT for new brain metastases and initiated standard of care palliative chemotherapy at an outside institution, ultimately succumbing to her disease on 2/9/2019.  
As a result of the aforementioned hematologic toxicities, no patients were accrued to the trial until we had a better understanding of their etiology, and subsequent patients received DAC-THU at dose level -1 per protocol. On 1/7/2019, patient \#3 with treatment naïve stage IV NSCLC commenced DAC-THU (0.2 mg/kg) twice-weekly. During Week 6 after receiving a total DAC dose of 150 mg (2.01 mg/kg), the patient developed Grade 4 neutropenia. Bone marrow aspirate demonstrated significantly decreased myeloid precursors with preservation of hematopoietic stem cells and erythroid and platelet lineages. IVIG, steroids, and cyclosporine were initiated. Four days later, the patient developed febrile neutropenia with pseudomonal bacteremia. He commenced a prolonged course of IV antibiotics due to the bacteremia and associated ecthyma gangrenosum involving the dorsum of the right hand. Within 8 days of initiating immunosuppression, the Grade 4 neutropenia had resolved to Grade 2. Steroids and cyclosporine were tapered over the course of two months. CT scans obtained as part of the evaluation for sepsis demonstrated a major response to therapy (**Figure 26**). Following consultation with the CCR-SD and immunotherapy experts at the NCI, Cleveland Clinic, and NYU Medical Center, the patient 45 resumed pembrolizumab without DAC-THU. He now continues pembrolizumab q3 weeks per  
 protocol with ongoing major response as of May 2019. Patient \#4 who had received chemotherapeutic pleurodesis but no systemic therapy for stage IV NSCLC commenced twice weekly DAC/THU on 1/28/2019. Because of her small size (59.7 kg) and lymphopenia requiring intermittent interruptions in drug administration, the patient's DAC dose (0.17 mg/kg) was less than the other patients, and she only received a total dose of 70 mg (1.17 mg/kg) during the 9-week course of therapy. She experienced only one Grade 3 ANC, and developed thrombocytosis requiring ASA prophylaxis suggestive of reprogramming of hematopoietic stem cells due to DNMT1 depletion. At treatment evaluation, staging studies demonstrated modest disease progression. Molecular profiling of tissue obtained from a posttreatment excisional biopsy suggested the presence of an atypical EGFR mutation not detected by NGS of her pretreatment biopsy tissue; Sanger sequencing confirmed the mutation. The patient was removed from treatment on 4/3/2019 and is now experiencing a major response to osimertinib evidenced by resolution of palpable chest wall masses and complete cessation of narcotics. Staging studies have not as yet been performed. These findings are provocative given observations that a patient with EGFR mutant NSCLC refractory to EGFR TKI had a near CR after resuming TKI
therapy following treatment with DAC-THU and nivolumab on protocol NCT02664181.  
In light of the severe neutropenias observed in the first three patients, and following notification of the CCR Director of Clinical Sciences, accrual to this trial was voluntarily (but not formally) suspended in order to revise the DAC-THU dosing as well as the window for assessing DLT. Data pertaining to the aforementioned patients are summarized in **Table 5**. The severe granulocytopenias observed in these patients have now been attributed to reprogramming of hematopoietic stem cells due to DNMT1 depletion rather than autoimmunity, suggesting that the PD effect has been occurring albeit too dramatically following DAC-THU therapy. Consistent with this experience all eight patients treated with DAC-THU and Nivolumab on NCT
NCT02664181 experienced Grade 3 or greater neutropenia; 5 patients experienced Grade 4 neutropenia, including one patient who was hospitalized for neutropenic fever and Grade 4 pneumonia/pneumonitis. Consequently, the dose regimen is now modified such that drug will be administered on two consecutive days (preferably Tuesday and Wednesday to facilitate bloodwork on Mondays and Thursdays) two out of every three weeks (instead of every week) for 9 weeks. Extrapolation of data from **Table 5**, demonstrates that the same doses of DAC (and THU) over 9 vs 4-6 weeks would result in > 30% decreases in intensity of DAC-THU dosing. Additionally, the revised DAC dosing regimen will be based on the total amount of DAC (mg/kg) administered per course. As such, the revised target of 2.5 mg/kg DAC over a 9-week course will be the new DL1, which corresponds to a 54% reduction from the dose initially attempted in this protocol and a 15% reduction from the exposures in SCD patients (108). The dosing per weight has been tightened (i.e.; q 10 kg rather than q 20 kg) to avoid relative under- or overdosing of patients at the extremes of each weight range. Lastly, the window for assessment of DLT is now revised to include Cycles 1 and 2, rather than only Cycle 1 of Course 1 to better monitor toxicities, and modify drug dosing, accordingly, while optimizing epigenetic priming for immune checkpoint blockade.  
| Number  of Caps  (5 mg  each  pill)   | DAC  per  Day  (mg)    | Total   |    |    |    |    |     |      |      |
|---------------------------------------|------------------------|---------|----|----|----|----|-----|------|------|
| Number  of DAC  Days                  | Total  DAC  Dose  (mg) |         |    |    |    |    |     |      |      |
| Days  per  Week                       | Total  DAC             |         |    |    |    |    |     |      |      |
| (mg/kg)                               | DAC                    |         |    |    |    |    |     |      |      |
| per BSA  (mg/M2)                      |                        |         |    |    |    |    |     |      |      |
| Patient                               | Weight                 | BSA     |    |    |    |    |     |      |      |
| (kg)                                  | (M2)                   |         |    |    |    |    |     |      |      |
| 1                                     | 112.8                  | 2.19    | 5  | 25 | 3  | 12 | 300 | 2.66 | 15.2 |
| 2                                     | 88.8                   | 1.99    | 4  | 20 | 3  | 8  | 160 | 1.8  | 17.3 |
| 3                                     | 74.3                   | 1.93    | 3  | 15 | 2  | 10 | 150 | 2.01 | 11.5 |
| 4                                     | 59.7                   | 1.59    | 2  | 10 | 2  | 7  | 70  | 1.17 | 20.9 |  
| Cumulative DAC Exposures over Nine-Week Treatment Dose/day (mg/kg) # Days Total Dose (mg/kg) % Change   |      |    |     |     |
|---------------------------------------------------------------------------------------------------------|------|----|-----|-----|
| DAC-THU/Pembro                                                                                          | 0.2  | 27 | 5.4 |     |
| DAC-THU/Nivo                                                                                            | 0.2  | 18 | 3.6 | -33 |
| DAC-THU (SCD)                                                                                           | 0.16 | 18 | 2.9 | -47 |
| DAC/THU/Pembro (rev)                                                                                    | 0.21 | 12 | 2.5 | -54 |  
Two additional patients have been treated on the protocol since the dosing was amended. The first patient with extensive tobacco and ethanol exposure and underlying CAD presented initially with potentially resectable stage III distal esophageal adenocarcinoma. Restaging after induction chemo-XRT revealed new cervical lymph node disease that was biopsy proven to be EsC. He commenced DAC-THU and Pembro at the modified dose and exhibited no treatment related toxicities. Restaging after the first course of treatment revealed regression of disease not reaching PR. He commenced his second course of therapy and prior to his first DAC infusion he experienced a non-STEMI, which was deemed to be unrelated to DAC-THU or pembrolizumab. He underwent uneventful multivessel stenting. Because of the MI, he was no longer eligible to continue therapy. The second patient was a 35 year old male with stage IV esophageal cancer who was referred for study after progressing on standard chemotherapy and Herceptin. He had disease within the esophagus extending from the thoracic inlet to the cardia with bulky nodal disease in the neck and mediastinum. Due to bulk of disease and stricture, a pediatric endoscope could barely pass through the esophagus. He also tolerated the first course of therapy well with no significant treatment related toxicities. Staging studies following Course 1 revealed stable disease. Endoscopy revealed a more pliable and distensible esophagus allowing easy passage of an adult scope. The patient was now able to eat solid foods. Because of incapaciting pain, he received a palliative course of XRT to the cervical nodes. Repeat imaging demonstrated complete resolution of the left cervical lymphadenopathy (**Figure 27**). Although the regression of the lymphadenopathy could have been attributable to priming of the microenvironment by the palliative radiation that enhanced the efficacy of the immune checkpoint blockade, the magnitude of the response (CR in the neck) exceeded that which one would have anticipated from sublethal XRT and pembrolizumab alone. Furthermore, consistent with the endoscopic findings, there was less FDG uptake in the esophagus. Additionally, there was metabolic response in the retroperitoneal lymph nodes.  
Pretreatment Post-Course1 and Palliative XRT to left cervical **region**  
  
While at home in West Virginia as the COVID-19 pandemic started to hit the US, he developed line sepsis. His port had been used for all blood draws at home as well as his pembro infusions. The port was removed, and he commenced IV antibiotics. Since he could not undergo TEE to rule out endocarditis, Infectious Disease consultants mandated six weeks of IV antibiotics. Because this would have significantly impacted his ability to receive investigational therapy and in light of the evolving restrictions regarding patient care at the NIH Clinical Center, he was removed from study so that he could commence cytotoxic chemotherapy while completing his antibiotic regimen.
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2 eligibility assessmentand enrollment 2.1

2.1.1.1 Histologically or cytologically confirmed, inoperable or unresectable locally advanced, or metastatic NSCLC, esophageal cancers including Seiwert-Stein Type I and Type II gastro-esophageal junction (GEJ) carcinomas, or MPM.  
2.1.1.2 NSCLC patients with no prior systemic treatment, or those with prior first line treatment including an immune checkpoint inhibitor, are eligible for study.  
2.1.1.3 Patients with esophageal and gastro-esophageal junction (GEJ) cancers are potentially eligible for study if they have received or refused first line standard of care cytotoxic therapy, and subsequent targeted therapy if appropriate.  
2.1.1.4 Patients with MPM are eligible for study if they have received, refused or are ineligible for first line chemotherapy.  
2.1.1.5 Patients who received DNA demethylating agents or PD-1/PD-L1 inhibitors for another malignancy may be eligible for study if there were no dose-limiting immune related events, and there has been either no clinical evidence of disease or minimal residual disease that has been stable for at least three years.  
2.1.1.6 Patients must have analysis of PD-L1 expression in cancer cells quantitated by immunohistochemistry analysis.  
2.1.1.6.1 Patients in Cohort 1 (Dose Escalation) may have any level of expression. 2.1.1.6.2 Patients in Cohort 2 (Dose Expansion: NSCLC with high PD-L1) must have ≥ 50%
expression in cancer cells.  
2.1.1.6.3 Patients in Cohort 3 (Dose Expansion: NSCLC with low PD-L1) must have 0-49%
expression. Note: Patients in this cohort must have been offered and refused standard of care platinum-based chemotherapy.  
2.1.1.6.4 Patients in Cohort 4 (Dose Expansion: EsC) may have any level of expression. 2.1.1.6.5 Patients in Cohort 5 (Dose Expansion: MPM) may have any level of expression. 2.1.1.7 Measurable disease, per RECIST 1.1. (see Section **6.3.3** for the evaluation of measurable disease).  
2.1.1.8 Willingness to undergo tumor biopsies if safely accessible per PI discretion before and after treatment.  
2.1.1.9 Age ≥18 years. Because no dosing or adverse event data are currently available on the use of Decitabine (DAC) and Tetrahydrouridine (THU) in combination with Pembrolizumab in patients < 18 years of age, children are excluded from this study, but will be eligible for future pediatric trials.  
2.1.1.10 ECOG performance status of ≤ 2 (see **Appendix 1**). 2.1.1.11 Patients must be without evidence of unstable or decompensated myocardial disease; and must have adequate pulmonary reserve evidenced by FEV1 and DLCO ≥ 35% predicted; oxygen saturation equal to or greater than 90% on room air by pulse oximetry or ABG (to be drawn if pulse oximetry < 90% on room air).  
2.1.1.12 No immunosuppressive medications except non-systemic corticosteroids. 2.1.1.13 Patients must have normal organ and marrow function as defined below:  
|  leukocytes                  | ≥ 3,000/mcL                                                                                                                                       |                                                    |
|-------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------|
|  absolute neutrophil count   | ≥ 1,500/mcL (without transfusion or cytokine  support)                                                                                            |                                                    |
|  absolute lymphocyte count   | > 800/mcL                                                                                                                                         |                                                    |
|  platelets                   | ≥ 100,000/mcL                                                                                                                                     |                                                    |
|  PT                          | no more than 2 seconds above the ULN                                                                                                              |                                                    |
|  total bilirubin             | < 1.5 X institutional upper limit of normal OR                                                                                                    |                                                    |
|  direct bilirubin            |                                                                                                                                                   | ≤ ULN for patients with total bilirubin > 1.5  ULN |
|  serum albumin               | ≥ 2.0 mg/dL                                                                                                                                       |                                                    |
|  AST(SGOT)/ALT(SGPT)         | ≤ 2.5 X institutional ULN                                                                                                                         |                                                    |
|  creatinine                  | ≤ 1.6 mg/ml OR                                                                                                                                    |                                                    |
|  creatinine clearance (eGFR) | ≥ 60 mL/min/1.73 m2  for patients with  creatinine levels above institutional normal  at the time DAC-THU and pembrolizumab  treatment commences. |                                                    |  
creatinine clearance (eGFR) ≥ 60 mL/min/1.73 m2 for patients with creatinine levels above institutional normal at the time DAC-THU and pembrolizumab treatment commences.  
2.1.1.14 Patients with history of brain metastases except those with meningeal carcinomatosis or leptomeningeal disease may be eligible for treatment a minimum of 1 week following completion of gamma knife or whole brain radiotherapy, or 4 weeks following surgical resection of brain metastasis provided post-treatment MR scan reveals no evidence of active disease, and no ongoing need for systemic steroids.  
2.1.1.15 Patients with laboratory evidence of autoimmune disease (e.g., positive ANA or lupus anticoagulant) without associated symptoms; vitiligo, or mild autoimmunity not impacting the function of organs, such as Hashimoto or psoriasis may be eligible for study.  
2.1.1.16 The effects of DAC-THU and pembrolizumab on the developing human fetus are unknown. For this reason and because antineoplastic agents as well as other therapeutic agents used in this trial are known to be teratogenic, women of child-bearing potential and men who engage in sexual activity must agree to use 2 forms of contraception at least 1 of which must be highly effective (intrauterine device [IUD], hormonal, tubal ligation; not highly effective includes barrier method) prior to study entry, for the duration of study participation and for 60 days after completion of the study treatment. Should a woman become pregnant, or suspect she is pregnant, while she or her partner is participating in this study the study participant should inform the study physician immediately.  
2.1.1.17 Ability of subject to understand and the willingness to sign a written informed consent document.
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2.1.2 exclusion criteria

2.1.2.1 Patients with cancers harboring targetable mutations for which there is approved first or second line therapy, unless standard of care therapy refused.  
2.1.2.2 Clinically significant cardiovascular / cerebrovascular disease as follows: cerebral vascular accident / stroke (< 6 months prior to enrollment), myocardial infarction (< 6
50  
months prior to enrollment), unstable angina, congestive heart failure (New York Heart Association Classification Class ≥ II), serious cardiac arrhythmia, clinically significant bleeding or clinically significant pulmonary embolism.  
2.1.2.3 Active Hepatitis A, Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV RNA
[qualitative] is detected).  
2.1.2.4 Human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome
(AIDS)-related illness due to unknown effects of DAC-THU on systemic immunity.  
2.1.2.5 Other active infection requiring systemic therapy. 2.1.2.6 Pregnant women are excluded from this study because DAC-THU may have the potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with DACTHU, breastfeeding should be discontinued if the mother is treated with DAC-THU.  
These potential risks may also apply to other agents used in this study.  
2.1.2.7 Other severe acute or chronic medical or psychiatric conditions or laboratory abnormality that may increase the risk associated with study participation or study drug administration, or may interfere with the interpretation of study results, and in the judgment of the investigator would make the patient inappropriate for entry into this study.  
2.1.2.8 Patients who are receiving systemic corticosteroids. 2.1.2.9 Patients with history of or active autoimmune disease including thyroiditis, colitis, nephritis, neuropathy or pneumonitis.  
2.1.2.10 Patients receiving another investigational agent. 2.1.2.11 An additional malignancy that is progressing or requires active treatment. Exceptions include basal cell carcinoma of the skin or squamous cell carcinoma of the skin that has undergone potentially curative therapy, or in-situ cervical or anal cancer, or ductal carcinoma in-situ.  
2.1.2.12 History or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the trial, interfere with the subject's participation for the full duration of the trial, or is not in the best interest of the subject to participate, in the opinion of the treating investigator.  
2.1.2.13 Psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the trial.  
2.1.2.14 Thrombocytosis defined as platelet count > 1,200,000/mcL.
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2.1.3 recruitment strategies

This study will be posted on NIH websites and on NIH social media forums. Participants may also be recruited through self-referrals, physician referrals, and referrals from the NIH Clinical Center (CC) Office of Patient Recruitment. Patients may also be referred to this study based on confirmation of histology (done by the Laboratory of Pathology) under protocol 06C0014.
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2.2 screening evaluation

Note: Screening evaluation testing/procedures are conducted under the separate screening protocol 06C0014 OR the consent for study 01C0129 (provided the procedure is permitted on that study) per PI discretion. Assessments and procedures to confirm study eligibility should be completed as follows: Within 4 Weeks Prior to Protocol Enrollment:
a. Medical history and physical exam including vital signs and performance status.  
b. Confirmation of histology (archival or fresh tissue if archival tissue is insufficient),
PD-L1 expression and mutation status (EGFR and ALK) by the Laboratory of Pathology (any time prior to enrollment). This confirmation will be done under protocol 06C0014 (patients will be co-enrolled).  
c. Blood tests:
i. CBC with differential ii. Acute Care Panel: Sodium, potassium, chloride, bicarbonate, creatinine, glucose, BUN, eGFR
iii. Hepatic Panel: Alkaline phosphatase, ALT, AST, total and direct bilirubin iv. PT/PTT
d. Viral Markers (anti-HIV, anti-HCV, HBsAg, anti-HAV IgM); HCV RNA if needed.  
e. 12-lead EKG. Patients with EKG evidence of cardiac ischemia (ST
depression/elevation greater than or equal to 2 mm), or arrhythmia will undergo cardiology evaluation to determine eligibility.  
f. Echocardiogram g. X-rays, contrast enhanced CT scans of chest, abdomen, pelvis, as well as brain MR,
and fused PET-CT scans to evaluate the status of disease (all imaging must be obtained within 4 weeks prior to starting therapy).  
h. Pulmonary function tests (PFTs) with and/or without bronchodilators. i. Arterial Blood Gas test (ABG) on room air (to be drawn if pulse oximetry < 90% on room air).  
j. Lupus Anticoagulant and ANA
Within 3 Days Prior to Protocol Enrollment:
a. Women of child-bearing potential will have a urine or serum hCG pregnancy test.
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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

| Number          | Name                                             | Description                                                |
|-----------------|--------------------------------------------------|------------------------------------------------------------|
| 1               | Dose-Escalation                                  | Subjects enrolled to dose escalation cohorts               |
| 2               | Dose Expansion: NSCLC                            | NSCLC subjects with high PD-L1 tumor expression            |
| with high PD-L1 | enrolled at the MTD after the MTD is established |                                                            |
| 3               | Dose Expansion: NSCLC                            | NSCLC subjects with low PD-L1 tumor expression             |
| with low PD-L1  | enrolled at the MTD after MTD is established     |                                                            |
| 4               | Dose Expansion: EsC                              | EsC subjects enrolled at the MTD after MTD is  established |
| 5               | Dose Expansion: MPM                              | MPM patients enrolled at the MTD after MTD is  established |
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arms

| Number   | Name            | Description                                               |
|----------|-----------------|-----------------------------------------------------------|
| 1        | Dose Escalation | DAC-THU + pembrolizumab at escalating doses               |
| 2        | Dose Expansion  | DAC-THU + pembrolizumab at the dose established in  Arm 1 |
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arm assignment

Subjects in Cohort 1 will be assigned to Arm 1. Subjects in Cohort 2, Cohort 3, Cohort 4 and Cohort 5 will be assigned to Arm 2.
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3 study implementation 3.1 study

This trial is unique in that it is intended to establish the rationale and conditions for the use of oral DAC-THU and pembrolizumab therapy for NSCLC, EsC, and MPM. The goal is to establish the basis for subsequent evaluation of this treatment regimen in combination with other agents targeting MDSC, or adoptive immunotherapy regimens in patients with thoracic malignancies. Patients potentially eligible for trial will be evaluated, and undergo subsequent eligibility assessment in the Thoracic Surgery Clinic/NCI. Patients meeting eligibility criteria will undergo baseline (if fresh tumor biopsy was not performed at screening) and post treatment tumor biopsies either by percutaneous image-guided FNA techniques in Interventional Radiology (Clinical Center/NIH) or endoscopic/thoracoscopic techniques in the CC operating room by Thoracic Surgery staff using standard of care practice guidelines. Tumor biopsies may be omitted at the discretion of the PI if risks outweigh potential research benefits. The Phase I component will be a standard 3+3 design starting with oral DAC-THU on two consecutive days (preferably Tuesday and Wednesday) for two weeks out of three weeks for 9 weeks with incremental dose escalation as shown in Section **3.1.3**. Patients with high PD-L1 tumor expression will be combined with patients with low PD-L1 tumor expression to minimize accrual numbers required for the Phase I component of the study. Pembrolizumab will be administered on Wednesday, Thursday or Friday at a fixed intravenous dose of 200 mg q 3 weeks. One cycle is three weeks; one course is 9 weeks. Treatment evaluation including repeat tumor biopsy will be performed after one course of therapy (Week 10 +/- one week). Those patients exhibiting disease progression or unacceptable toxicities will be removed from study. Patients exhibiting stable disease or disease regression will be offered an additional course of therapy followed by treatment evaluation (without mandatory biopsy). Treatment will continue in this manner until off-study criteria have been met. Once the MTD for DAC-THU has been identified, the MTD dose level will be expanded by 4 patients to confirm safety. Then, including these 10 patients at the MTD, a total of 10 NSCLC
patients with high intratumoral PD-L1 expression and 10 NSCLC patients with low intra-tumoral PD-L1 expression will be evaluated in the first stage of two separate Phase II cohorts each using a Simon optimal design (Phase II; Stage I). If 5 or more of 10 NSCLC patients with high PD-L1 expression respond to treatment, the cohort will be expanded to 23 patients (Phase II; Stage II) to determine ORR at the MTD. If 2 or more of 10 patients with low PD-L1 expression respond to treatment, the cohort will be expanded to 29 patients (Phase II; Stage II) to determine ORR at the MTD. Up to 10 EsC patients including any patients treated in the expansion cohort of the Phase I component of the trial will be accrued to a separate cohort at the MTD. If 2 or more of these 10 EsC patients respond to therapy, the protocol may be amended to enable further evaluation of the investigational treatment regimen in EsC patients. Similarly, up to 10 MPM patients including any patients treated in the expansion cohort of the Phase I component of the trial will be accrued to a separate cohort at the MTD. If 2 or more of these 10 MPM patients respond to therapy, the protocol may be amended to enable further evaluation of the investigational treatment regimen in these patients.  
  
Treatment evaluation including staging studies and biopsy will be performed on Days 64 and 65 +/- 7 days. DAC-THU will be given on two consecutive days as indicated, with Tuesday/Wednesday optimal but not absolute. Pembrolizumab may be administered on Wednesday, Thursday, or Friday for administrative convenience.
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3.1.2 definition of dose limiting

DLT is defined as: Any Grade 3 or greater toxicity that cannot be attributed to a cause other than study treatment during the first two cycles of Course 1 of DAC-THU and/or pembrolizumab therapy such as disease progression or intercurrent illness.  
Any toxicity that cannot be attributed to a cause other than study treatment (DAC-THU
and/or Pembrolizumab) that results in study treatment discontinuation.
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3.1.3 dose escalation

The revised dosing regimen selected for this trial is based on pharmacodynamic evidence of systemic hypomethylation without significant systemic toxicities mediated by oral DAC-THU in patients with sickle cell disease in a recent Phase 1 clinical trial (108), as well as experience with DAC-THU/Pembrolizumab in NSCLC (Section **1.2.11.2**). The frequency of DAC-THU dosing will increase using a conventional 3+3 Phase I study design attempting to maximize systemic DNMT1 depletion while avoiding Grade 3 or greater systemic toxicities (primarily neutropenia/thrombocytopenia). *Due to the uncertainty regarding cumulative toxicities, no intrapatient escalation will be allowed*.  
Dose escalation will proceed as indicated in **Table 7** using three patients per dose level
(irrespective of Tumor PD-L1 expression) until DLT within the first 6 weeks (two cycles) of DACTHU therapy is observed. If one of three patients at any given dose level experiences DLT, up to three additional patients will be treated at this dose level. If only one of six patients exhibit DLT,
subsequent patients will be enrolled into the next higher dose level. As soon as two patients at any given dose level develop DLT, no additional patients will be entered at that level. Subsequent patients will be accrued into the preceding dose level; if DLT is observed in less than two of six patients treated at this lower level, this dose will represent maximum tolerated dose (MTD).  
Table 7: DAC-THU Dosing Schema for High (50% or Greater) and Low (0-49%) PD-L1 Expression Groups  
Dose Level DAC DAC THU THU Frequency^  
mg/kg/course mg/kg/day* mg/kg/course mg/kg/day* wk **1,2,4,5,7,8**  
| Expression Groups                                |            |              |            |                |            |
|--------------------------------------------------|------------|--------------|------------|----------------|------------|
| Dose Level                                       | DAC        | DAC          | THU        | THU            | Frequency^ |
| mg/kg/course                                     | mg/kg/day* | mg/kg/course | mg/kg/day* | wk 1,2,4,5,7,8 |            |
| -1                                               | 2.0        | 0.17         | 120        | 10             | T;W        |
| 1                                                | 2.5        | 0.21         | 120        | 10             | T;W        |
| 2                                                | 3.0        | 0.25         | 120        | 10             | T;W        |
| *on days administered ^ T: Tuesday; W: Wednesday |            |              |            |                |            |
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3.2 drug administration 3.2.1 dac-thu

Timing between THU and decitabine: Oral THU capsules should be ingested within two hours prior to or two hours after a meal followed 60 minutes later by oral decitabine capsules. The capsules are to be ingested twice weekly on consecutive days as indicated in **Table 7** ('Smart Caps' that can remind patients about timing of administration should be considered for use).  
| 3.2.1.1   | THU Dosing   |
|-----------|--------------|
| Weight    | THU          |
| (kg)      | (caps/dose)  |
| 40-59     | 2            |
| 60-79     | 3            |
| 80-99     | 4            |
| 100-119   | 5            |
| 120-139   | 6            |