nct# 04407442
untitled h2

Phase II Study of Daratumumab in Combination with Azacitidine and Dexamethasone in Relapsed/Refractory Multiple Myeloma Patients Previously Treated with Daratumumab Protocol Number: CC \#20251 Investigational Product(s): Daratumumab and Azacitidine Version Number: 6.1 Version Date: 06/08/2022 IND Number: EXEMPT
Principal Investigator (Sponsor-Investigator)  
  
  
  
  
| Revision History             |            |
|------------------------------|------------|
| Protocol Version             | Date       |
| Version 1                    | 07/22/2019 |
| Version 2                    | 10/03/2019 |
| Version 3                    | 11/08/2019 |
| Version 4                    | 01/16/2020 |
| Version 5                    | 04/06/2020 |
| Version 6                    | 11/06/2020 |
| Version 6.1                  | 06/08/2022 |
| Proprietary and Confidential |            |  
The information in this document is considered privileged and confidential, and may not be disclosed to others except to the extent necessary to obtain Institutional Review Board approval and informed consent, or as required by federal and state laws.  
Persons to whom this information is disclosed should be informed that this information is privileged and confidential and that it should not be further disclosed.
nct# 04407442
protocol signature page

Protocol No.: 20251 1. I agree to follow this protocol version as approved by the UCSF Protocol Review Committee (PRC), Institutional Review Board (IRB), and Data and Safety Monitoring Committee (DSMC).  
2. I will conduct the study in accordance with Good Clinical Practices (ICH-GCP) and the applicable IRB, ethical, federal, state, and local regulatory requirements.  
3. I certify that I, and the study staff, have received the required training to conduct this research protocol. 4. I agree to maintain adequate and accurate records in accordance with IRB policies, federal, state and local laws and regulations.  
UCSF Principal Investigator Swetha Kambhampati, Alfred Chung Printed Name Signature Date
nct# 04407442
abstract

| Abstract Title                                                                                      | A                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Phase                                                                                                                                                                                                          | II   | Study   | of   | Daratumumab   | in   | Combination   | with   | Azacitidine   | and   |
|-----------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------|---------|------|---------------|------|---------------|--------|---------------|-------|
| Dexamethasone in Relapsed/Refractory Multiple Myeloma Patients Previously Treated  with Daratumumab |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                |      |         |      |               |      |               |        |               |       |
| Study Description                                                                                   | This is a single-arm, 2-stage, phase II study of the safety and efficacy of daratumumab  in combination with azacitidine and dexamethasone in relapsed/refractory multiple  myeloma (RRMM) patients who have previously been exposed to daratumumab. Based  on pre-clinical data we hypothesize that azacitidine, by upregulating the expression of  CD38, can potentially increase the antibody-dependent cell-mediated cytotoxicity and  efficacy of daratumumab on multiple myeloma cells and help reverse daratumumab  resistance. This study aims to assess the efficacy and safety of daratumumab and  azacitidine in relapsed refractory multiple myeloma. |                                                                                                                                                                                                                |      |         |      |               |      |               |        |               |       |
| Phase of Study                                                                                      | Phase 2 Daratumumab and Azacitidine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                |      |         |      |               |      |               |        |               |       |
| Investigational  Products Study population                                                          | Patients ≥ 18 years of age with relapsed or refractory multiple myeloma (RRMM) who  have progressed on ≥ 2 lines of prior therapy, including an immunomodulatory drugs  (IMiD) and proteasome inhibitor, and have previously been treated with daratumumab,  with most recent daratumumab treatment being at least 6 months prior to enrollment to  allow for CD38 normalization.                                                                                                                                                                                                                                                                                 |                                                                                                                                                                                                                |      |         |      |               |      |               |        |               |       |
| Primary Objective                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | To evaluate the efficacy, as determined by the overall response rate (ORR) of  daratumumab in combination with azacitidine and dexamethasone in RRMM  patients who have previously been exposed to daratumumab |      |         |      |               |      |               |        |               |       |
| Secondary Objectives                                                                                |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | To evaluate duration of response per IMWG response criteria.                                                                                                                                                   |      |         |      |               |      |               |        |               |       |
|                                                                                                    | To assess the safety and toxicity of azacitidine in combination with  daratumumab and dexamethasone                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                |      |         |      |               |      |               |        |               |       |
|                                                                                                    | To assess the 1-year OS and PFS of daratumumab in combination with  azacitidine and dexamethasone                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                                                                                |      |         |      |               |      |               |        |               |       |
|                                                                                                    | To evaluate the change in CD38 expression on plasma cells induced by  azacitidine in patients with RRMM and correlate this change with depth and  duration of response                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                |      |         |      |               |      |               |        |               |       |  
| Sample Size                                        | Simon's minimax two-stage design will be used.  The overall study (including the safety lead-in) plans to enroll 23 patients.  Safety Lead In: We expect to enroll 6 patients overall in the safety lead-in.  Stage 1: We expect to enroll a total of 13 patients during Stage 1 (including the 6  patients enrolled in the safety lead-in). Depending on the clinical response in Stage 1,  Stage 2 will commence. Stage 2: If there is ≥ 2 responses in 13 patients the study will enroll an additional 10  patients.  If there is ≤ 1 response in 13 patients the study will be stopped. Participants will continue study treatment until they are unable to tolerate treatment due  to toxicities or demonstrate progression. We anticipate the duration of study treatment  to be around 8 months.   |
|----------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Duration of Study  Treatment Duration of Follow up | Patients will be followed until 1 year after the last patient has received the last  administration of daratumumab. This is the first study to evaluate the safety and efficacy of azacitidine and daratumumab  in combination with dexamethasone in patients with relapsed and refractory myeloma.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| Unique Aspects of this  Study                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
nct# 04407442
list of abbreviations

| List of Abbreviations ADCC antibody-dependent cellular toxicity AE adverse event ALP alkaline phosphatase ALT alanine aminotransferase ANC absolute neutrophil count AST aspartate aminotransferase Aza Azacitidine BM Bone marrow BUN blood urea nitrogen CBC complete blood cell (count) CDC complement dependent cytotoxicity CNS central nervous system CR complete response CRC clinical research coordinator CRF case report form CT computerized tomography CTCAE Common Terminology Criteria for Adverse Events CTMS Clinical Trial Management System Dara Daratumumab Dexamethasone Dexamethasone DFS disease-free survival DLT dose limiting toxicity DMC Data Monitoring Committee DOR duration of response DSMC Data and Safety Monitoring Committee DSMP Data and Safety Monitoring Plan ECG/EKG electrocardiogram ECOG Eastern Cooperative Oncology Group FDA Food and Drug Administration FDG Fluorodeoxyglucose FLC free light chain GCP Good Clinical PracticePhase II - Daratumumab and Azacitidine   | Page 5 of 95   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|  
| List of Abbreviations GGT gamma-glutamyl transferase HBeAg hepatitis B "e" antigen HBV hepatitis B virus HCV hepatitis C virus HDFCCC Helen Diller Family Comprehensive Cancer Center HIPAA Health Insurance Portability and Accountability Act HIV human immunodeficiency virus ICF informed consent form ICH International Conference on Harmonization IND investigational new drug application IP investigational product IRB Institutional Review Board IV intravenous LDH lactate dehydrogenase Mab/MoAb Monoclonal Antibody MFI Median fluorescent intensity MM Multiple myeloma MR Minor response MRI Magnetic resonance imaging MRD minimal residual disease MTD maximum tolerated dose NCI National Cancer Institute ORR overall response rate OS overall survival PD disease progression Pd pharmacodynamics PFS progression free survival PI principal investigator PK pharmacokinetics PO Per os (by mouth, orally) PR partial response PRC Protocol Review Committee (UCSF) RRMM Relapsed refractory multiple myelomaPhase II - Daratumumab and Azacitidine   | Page 6 of 95   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|  
| SGPT VGPR WBC   |
|-----------------|  
SD stable disease SAE serious adverse event  
| SGOT   | serum glutamic oxaloacetic transaminase serum glutamic pyruvic transaminase very good partial response white blood cell count   |
|--------|---------------------------------------------------------------------------------------------------------------------------------|  
Protocol CC\#:20251  
| Table of Contents                                                                                                                         |                                                                                                                                                                    |                 |
|-------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------|
| Protocol Signature Page.                                                                                                                  |                                                                                                                                                                    |                 |
| Abstract .                                                                                                                                |                                                                                                                                                                    |                 |
| List of Abbreviations                                                                                                                     |                                                                                                                                                                    |                 |
| 1                                                                                                                                         | Introduction                                                                                                                                                       |                 |
| 1.1                                                                                                                                       | Background on Indication .                                                                                                                                         |                 |
| 1.2                                                                                                                                       | Background on the Investigational Product(s) and Associated Known Toxicities ...................................12                                                 |                 |
| D DARATUMUMAB                                                                                                                             |                                                                                                                                                                    |                 |
| Azacitidine                                                                                                                               |                                                                                                                                                                    |                 |
| 1.3                                                                                                                                       | Rationale for the Proposed Study .                                                                                                                                 |                 |
| 1.4                                                                                                                                       | Rationale for the Dose Selection/Regimen ...........................................................................................................26             |                 |
| 1.5                                                                                                                                       | Correlative Studies                                                                                                                                                |                 |
| 2                                                                                                                                         | Study Objectives .                                                                                                                                                 |                 |
| 2.1                                                                                                                                       | Hypothesis .                                                                                                                                                       |                 |
| 2.2                                                                                                                                       | Primary, Secondary, and Exploratory Objective(s) .......................................................................................30                         |                 |
| Primary Objectives:                                                                                                                       |                                                                                                                                                                    |                 |
| Secondary Objectives:                                                                                                                     |                                                                                                                                                                    |                 |
| 2.3                                                                                                                                       | Primary, Secondary, and Exploratory Endpoint(s) ..............................................................................................31                   |                 |
| Primary endpoints: .                                                                                                                      |                                                                                                                                                                    |                 |
| Secondary Endpoints: .                                                                                                                    |                                                                                                                                                                    |                 |
| Study Design                                                                                                                              |                                                                                                                                                                    |                 |
| 3                                                                                                                                         | 3.1                                                                                                                                                                | Characteristics |
| 3.2                                                                                                                                       | Eligibility Criteria .                                                                                                                                             |                 |
| Inclusion Criteria                                                                                                                        |                                                                                                                                                                    |                 |
| Exclusion Criteria .                                                                                                                      |                                                                                                                                                                    |                 |
| 3.3                                                                                                                                       | Inclusion and Recruitment of Women and Minorities .....................................................................................37                          |                 |
| 3.4                                                                                                                                       | D Duration of Treatment                                                                                                                                            |                 |
| 3.5                                                                                                                                       | D Duration of Follow Up                                                                                                                                            |                 |
| Primary Completion .                                                                                                                      |                                                                                                                                                                    |                 |
| 3.6                                                                                                                                       |                                                                                                                                                                    |                 |
| 3.7                                                                                                                                       | Study Completion                                                                                                                                                   |                 |
| 4                                                                                                                                         | Investigational Products .......................................................................................................................................38 |                 |
| Description, Supply and Storage of Investigational Products ...........................................................................38 |                                                                                                                                                                    |                 |
| 4.1                                                                                                                                       |                                                                                                                                                                    |                 |
| D DARATUMUMAB                                                                                                                             |                                                                                                                                                                    |                 |
| A A CONTROL IN THE CONTROLLING AND I                                                                                                      |                                                                                                                                                                    |                 |
| DEXAMETHASONE                                                                                                                             |                                                                                                                                                                    |                 |
| Accountability Records for Investigational Product(s)                                                                                     |                                                                                                                                                                    |                 |
| 4.2                                                                                                                                       |                                                                                                                                                                    |                 |
| Phase II - Daratumumab and Azacitidine                                                                                                    | Page 8 of 95                                                                                                                                                       |                 |  
| Version date: 06/08/2022                                                                                                                                                    | Protocol CC#:20251                                                                                                                                                   |                  |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|
| 4.3                                                                                                                                                                         | Ordering Investigational Product(s) ...........................................................................................................40                    |                  |
| 5                                                                                                                                                                           | Treatment Plan                                                                                                                                                       |                  |
| 5.1                                                                                                                                                                         | Dosage and Administration ........................................................................................................................40                 |                  |
| D DARATUMUMAB                                                                                                                                                               |                                                                                                                                                                      |                  |
| AA                                                                                                                                                                          |                                                                                                                                                                      |                  |
| DEXAMETHASONE                                                                                                                                                               |                                                                                                                                                                      |                  |
| 5.2                                                                                                                                                                         | Treatment Compliance ................................................................................................................................45              |                  |
| 5.3                                                                                                                                                                         | Other Investigational Procedures/Modalities ............................................................................................46                           |                  |
| 5.4                                                                                                                                                                         | Dose Limiting Toxicity                                                                                                                                               |                  |
| Dose Modifications and Dosing Delays ..........................................................................................................47                           |                                                                                                                                                                      |                  |
| 5.5                                                                                                                                                                         |                                                                                                                                                                      |                  |
| D DARATUMUMAB                                                                                                                                                               |                                                                                                                                                                      |                  |
| AAACITINE                                                                                                                                                                   |                                                                                                                                                                      |                  |
| DEXAMETHASONE                                                                                                                                                               |                                                                                                                                                                      |                  |
| 5.6                                                                                                                                                                         | Criteria for Permanent Discontinuation of Study Drug(s) ...............................................................................52                            |                  |
| 5.7                                                                                                                                                                         | Use of Concurrent/Concomitant Medications.........................................................................................................53                 |                  |
| Recommended Therapies                                                                                                                                                       |                                                                                                                                                                      |                  |
| 5.8                                                                                                                                                                         | D Dietary Restrictions .                                                                                                                                             |                  |
| 5.9                                                                                                                                                                         | Prohibited Medications .                                                                                                                                             |                  |
| Study Procedures and Schedule of Events .............................................................................................................54                     |                                                                                                                                                                      |                  |
| 6                                                                                                                                                                           | 6.1                                                                                                                                                                  | Study Calendar . |
| 6.2                                                                                                                                                                         | Participant Registration                                                                                                                                             |                  |
| 6.3                                                                                                                                                                         | Schedule of Procedures and Observations                                                                                                                              |                  |
| Pretreatment Period                                                                                                                                                         |                                                                                                                                                                      |                  |
| T Treatment Period                                                                                                                                                          |                                                                                                                                                                      |                  |
| Post-treatment/ Follow-Up                                                                                                                                                   |                                                                                                                                                                      |                  |
| 6.4                                                                                                                                                                         | Correlative Studies ..............................................................................................................................................64 |                  |
| CD38 Expression Changes                                                                                                                                                     |                                                                                                                                                                      |                  |
| Methylation Pattern Changes                                                                                                                                                 |                                                                                                                                                                      |                  |
| Tumor Microenvironment Changes .                                                                                                                                            |                                                                                                                                                                      |                  |
| 7                                                                                                                                                                           | Reporting and Documentation of Results ..............................................................................................67                              |                  |
| Evaluation of Efficacy: Antitumor Effect - Hematologic Tumors...............................................................67                                              |                                                                                                                                                                      |                  |
| 7.1                                                                                                                                                                         |                                                                                                                                                                      |                  |
| Response Categories                                                                                                                                                         |                                                                                                                                                                      |                  |
| Measurement of Endpoints for Response                                                                                                                                       |                                                                                                                                                                      |                  |
| Assessment of Disease Progression or Response ...........................................................................................................................69 |                                                                                                                                                                      |                  |
| Methods of Response Evaluation .                                                                                                                                            |                                                                                                                                                                      |                  |
| 7.2                                                                                                                                                                         | Evaluation of Safety                                                                                                                                                 |                  |
| 7.3                                                                                                                                                                         | Definitions of Adverse Events                                                                                                                                        |                  |
| Adverse Event (AE) .                                                                                                                                                        |                                                                                                                                                                      |                  |
| Adverse Events of Special Interest .                                                                                                                                        |                                                                                                                                                                      |                  |
| Phase II - Daratumumab and Azacitidine                                                                                                                                      | Page 9 of 95                                                                                                                                                         |                  |  
| UCSF Helen Diller Family Comprehensive Cancer Center                                                                                                              |                                                                                                                                                                           |                    |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------|
| Version date: 06/08/2022                                                                                                                                          | time the content of the                                                                                                                                                   | Protocol CC#:20251 |
| Serious Adverse Events (SAE) .                                                                                                                                    |                                                                                                                                                                           |                    |
| Unlisted (Unexpected) Adverse Event/Reference Safety Information ..........................................................................................73     |                                                                                                                                                                           |                    |
| 7.4                                                                                                                                                               | Recording of Adverse Events                                                                                                                                               |                    |
| 7.5                                                                                                                                                               | Follow-up of Adverse Events ...........................................................................................................................................73 |                    |
| Adverse Events Monitoring                                                                                                                                         |                                                                                                                                                                           |                    |
| 7.6                                                                                                                                                               |                                                                                                                                                                           |                    |
| 7.7                                                                                                                                                               | Expedited Reporting                                                                                                                                                       |                    |
| Reporting to the Data and Safety Monitoring Committee                                                                                                             |                                                                                                                                                                           |                    |
| Reporting to Institutional Review Board                                                                                                                           |                                                                                                                                                                           |                    |
| Expedited Reporting to the FDA .                                                                                                                                  |                                                                                                                                                                           |                    |
| Interventional IIS Janssen Scientific Affairs Requirements for Safety Data                                                                                        | Collection and Reporting ........................74                                                                                                                       |                    |
| 8                                                                                                                                                                 | Statistical Considerations and Evaluation of Results .............................................................................78                                      |                    |
| Sample Size Determination                                                                                                                                         |                                                                                                                                                                           |                    |
| 8.1                                                                                                                                                               | Analyses .                                                                                                                                                                |                    |
| 8.2                                                                                                                                                               |                                                                                                                                                                           |                    |
| Analyses Population                                                                                                                                               |                                                                                                                                                                           |                    |
| Se                                                                                                                                                                |                                                                                                                                                                           |                    |
| EFFicacy Analysis                                                                                                                                                 |                                                                                                                                                                           |                    |
| Correlative Studies Analysis                                                                                                                                      |                                                                                                                                                                           |                    |
| 8.3                                                                                                                                                               | Interim Analyses and Study Stopping Rules .........................................................................................................82                     |                    |
| 8.4                                                                                                                                                               | Accrual Estimates ................................................................................................................................................83      |                    |
| 9                                                                                                                                                                 | Study Management ...............................................................................................................................................83        |                    |
| Pre-study Documentation...................................................................................................................................83      |                                                                                                                                                                           |                    |
| 9.1                                                                                                                                                               |                                                                                                                                                                           |                    |
| 9.2                                                                                                                                                               | Institutional Review Board Approval ...................................................................................................................83                 |                    |
| 9.3                                                                                                                                                               | Informed Consent.................................................................................................................................................83       |                    |
| 9.4                                                                                                                                                               | Changes in the Protocol                                                                                                                                                   |                    |
| Handling and Documentation of Clinical Supplies ................................................................................................84                |                                                                                                                                                                           |                    |
| 9.5                                                                                                                                                               | Case Report Forms (CRFs) ..............................................................................................................................................84 |                    |
| 9.6                                                                                                                                                               | Oversight and Monitoring Plan                                                                                                                                             |                    |
| 9.7                                                                                                                                                               |                                                                                                                                                                           |                    |
| 9.8                                                                                                                                                               | Record Keeping and Record Retention                                                                                                                                       |                    |
| 10    Protection of Human Subjects .......................................................................................................................85      |                                                                                                                                                                           |                    |
| 10.1                                                                                                                                                              | Protection from Unnecessary Harm .......................................................................................................................85                |                    |
| 10.2                                                                                                                                                              | Protection of Privacy ...............................................................................................................................................85   |                    |
| Appendix 1                                                                                                                                                        | Performance Status Criteria .....................................................................................................86                                       |                    |
| Appendix 2                                                                                                                                                        | Data and Safety Monitoring Plan  ...................................................................................................87                                    |                    |
| Appendix 3                                                                                                                                                        | Prohibited Medications List..................................................................................................90                                           |                    |
| References ....................................................................................................................................................91 |                                                                                                                                                                           |                    |
| Phase II - Daratumumab and Azacitidine                                                                                                                            | Page 10 of 95                                                                                                                                                             |                    |
nct# 04407442
1 introduction 1.1 background on

In the last decade, the introduction of proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) has significantly prolonged survival of patients with multiple myeloma (MM).1,2 However, despite numerous advances in treatment, multiple myeloma is still considered an incurable disease with an overall 5-year survival rate of 51%.3 Daratumumab , the first-in-class CD38 monoclonal antibody, has significantly improved the treatment response of MM, due to plasma cell overexpression of protein CD38.4,5 Daratumumab binds CD38 with high affinity and induces tumor cell death through a variety of immune-mediated mechanisms of including complement-dependent cytotoxicity
(CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).6,7 Daratumumab monotherapy was approved for heavily pretreated relapsed refractory multiple myeloma (RRMM) patients8 based on the open-label, phase I/II
GEN501 study and phase II SIRIUS study which showed overall response rates of 36% and 29%.9,10 Daratumumab, in the randomized open-label trials POLLUX and CASTOR, was also shown to have high response rates in RRMM when combined with Lenalidomide and Bortezomib, respectively, with a 63% and 61% risk reduction of disease progression or death.11,12 More recently, trials such as ALCYONE13 and MAIA14 have lead to approval of front-line daratumumab combination therapies for untreated transplant-ineligible myeloma patients. ALCYONE showed that daratumumab, bortezomib, melphalan, and prednisone combination in this patient population had an overall response rate of 90.9% in the daratumumab group as opposed to 73.9% in the control group13, while MAIA study showed that daratumumab, lenalidomide, and dexamethasone had a complete response of 47.6% in the daratumumab group as opposed to 24.9% in the control group14. Given the favorable results from the CASSOPEIA trial in Europe showing that daratumumab in combination with bortezomib, thalidomide, and dexamethasone (VTd), the frontline therapy standard of care in Europe in transplant eligible myeloma patients, resulted in 29% stringent complete response rate as opposed to 20% in the control non-daratumumab group15. There is an ongoing phase II GRIFFIN trial assessing the safety and efficacy of daratumumab in combination with revlimid, velcade, and dexamethasone (RVd) in frontline transplant eligible patients (NCT02874742). Preliminary data has indicated that this study has met its primary endpoint, demonstrating a higher percentage of stringent complete response (sCR) in patients in the daratumumab group compared to the control RVd group. While we are still awaiting the final results from this trial, if these preliminary results are confirmed in the final analysis, we anticipate daratumumab to be approved in the future in combination with the current standard of care RVd in frontline transplant-eligible myeloma patients. Now daratumumab is also approved in the subcutaneous form, based on the results of the COLUMBA study. The subcutaneous form provides several additional benefits for both patients and healthcare providers including shorter administration time, lower rate of infusion related reactions, additional flexibility, and reduction in treatment burden to the patient as well as lower risk of volume overload in patients with cardiac or renal insufficiency.  
While daratumumab is well tolerated and has high efficacy, not all patients respond, and many patients eventually develop progressive disease and resistance to daratumumab. A retrospective study showed that these patients have poor prognosis with a median overall survival of 5.3 months and a postdaratumumab regimen overall response rate of 34% with limited treatment options.16 Another retrospective study analyzing outcomes of 275 multiple myeloma patients at 14 academic centers with disease refractory to CD38 monoclonal antibodies (MoABs), either daratumumab or isatuximab, found that this patient population had a median overall survival (OS) of 8.6 months, ranging from 11.2 months for patients not simultaneously refractory to an immunomodulatory (IMiD) agent and a proteasome inhibitor to 5.6 months for penta-refractory patients who are Phase II - Daratumumab and Azacitidine Page 11 of 95 refractory to CD38 MoABs, 2 proteasome inhibitors, and 2 IMiDs.17 These studies highlight that heavily pre-treated MM patients who are resistant to daratumumab have overall poor prognosis. Thus, research has focused on understanding markers and predictors of daratumumab response and mechanisms of daratumumab resistance, which will prove to be even more important as daratumumab is being moved to frontline therapy.
nct# 04407442
1.2 background on the investigational

Daratumumab, the first-in-class CD38 monoclonal antibody, has significantly improved the treatment response of  
  
MM, due to plasma cell overexpression of protein CD38. CD38 is highly expressed on myeloma cells but is expressed at relatively low levels on normal lymphoid and myeloid cells and in some tissues of non-hematopoietic origin, making it a relevant target for the treatment of multiple myeloma. Daratumumab is an immunoglobulin G1kappa (IgG1k) human mAb that binds to a unique CD38 epitope on CD38-expressing cells with high affinity and was developed by the immunization of human immunoglobulin transgenic mice with recombinant CD38 protein.7,18 Daratumumab binds CD38 with high affinity and induces tumor cell death through a variety of immune-mediated mechanisms including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis, direct apoptosis after cross-linking, and can modulate CD38 enzymatic activity.7,19 Preclinical studies have showed that daratumumab's ADCC is highly specific to CD38 given daratumumab has not shown to induce ADCC in CD38 negative cells. Preclinical studies have also shown that daratumumab is effective at inducing both CDC and ADCC against MM cells in the presence of bone marrow stromal cells suggestive of daratumumab's activity in the bone marrow microenvironment.7,18 Daratumumab has also shown to induce apoptosis with cross-linking. In addition, studies have shown that daratumumab has additional antimyeloma effects through immunomodulation and by eliminating a population of highly immunosuppressive CD38+ Tregs, T-cell MDSCs, and Bregs and thus stimulating T-cell effector functions.19 The immunomodulatory effects of daratumumab not only include reduction of CD38+
immunosuppressive cellular populations, but also include concomitant induction of helper and cytotoxic T-cell expansion, production of IFN-gamma in response to viral peptides, and increased TCR clonality and T-cell functional response suggestive of an improved adaptive immune response.19 In-vitro studies, using bone marrow mononuclear cells from patients with multiple myeloma (MM), demonstrated increased killing of tumor cells when daratumumab was combined with lenalidomide or bortezomib as well as with both lenalidomide and bortezomib. Additionally, the upregulation of CD38 by pomalidomide or lenalidomide can enhance the activity of anti-CD38 antibodies including daratumumab. These observations suggest a strong potential for the treatment of CD38-expressing malignancies using daratumumab as monotherapy and in combinations. The potential toxicity of daratumumab was evaluated in a repeat dose study in chimpanzees. The primary toxicities identified in chimpanzees were infusion-related reactions during the first, but not subsequent, daratumumab infusions and thrombocytopenia. The binding affinity of daratumumab is ≥15-fold higher for chimpanzee platelets than for human platelets, suggesting that thrombocytopenia may be less pronounced in humans. Depletion of specific lymphocyte phenotypic cell populations, as expected, based on the intended pharmacological effect of daratumumab, was observed in chimpanzees. No genotoxicity, chronic toxicity, carcinogenicity, or reproductive toxicity testing has been conducted.  
Phase II - Daratumumab and Azacitidine Page 12 of 95
nct# 04407442
clinical studies and efficacy

The promising preclinical data demonstrating the immunomodulatory effects of daratumumab has lead to extensive clinical trials testing the safety and efficacy of daratumumab in initially RRMM and now more recently newly diagnosed MM both transplant eligible and transplant ineligible. Daratumumab was initially approved in 2015 as monotherapy for patients with MM who have received at least 3 lines of therapy, including a proteasome inhibitor, IMiD, or who are double-refractory to a proteasome inhibitor and IMiD.9 Pooled analysis of GEN501 and SIRIUS trials evaluating daratumumab monotherapy show an ORR of 31%, median DOR of 7.6 months, median PFS and OS of 4 months and 20.1 months respectively in heavily pretreated RRMM
patients.10,20 The DRd and DVd regimens are approved by the FDA for use in patients with MM who have received at least one prior therapy based on the POLLUX and CASTOR trials respectively.11,21 POLLUX trial showed a higher ORR in the daratumumab arm, 93% vs 76%, and superior PFS of 83% vs 60%.11 CASTOR trial showed higher ORR
of 84% vs 63% in the daratumumab arm and improved PFS of 16.7 vs 7.1 months.21 DPd is approved for use in patients with MM who have received at least two prior therapies including lenalidomide and a proteasome inhibitor with an ORR of 60% based on the EQUULEUS trial.22 Daratumumab is also approved in combination with bortezomib, melphalan, and prednisone (VMP) for the treatment of newly diagnosed MM patients who are ineligible for autologous stem cell transplant (ASCT) based on the ALCYONE study which showed a 50% reduction in the risk of progression or death compared with VMP alone.13 Daratumumab in combination with lenalidomide and dexamethasone was recently approved in newly diagnosed MM patients who are ineligible for ASCT based on the MAIA trial showing a superior ORR in the daratumumab arm of 92.9% vs 81.3% and showing reduced risk of disease progression or death by 44% in the daratumumab arm.14 The CASSIOPEIA trial in Europe found that daratumumab in combination with VTd compared to VTd alone demonstrated superior sCR of 29% vs 20%, superior CR of 39% vs 26%, and superior achievement of minimal residual disease (MRD) negativity of 64% vs 44%.15 This lead to the recent FDA approval of D-VTD for the treatment of newly diagnosed patients with MM who are eligible for ASCT. Given this was the first study showing the clinical benefit of daratumumab plus standard of care in frontline treatment of transplant eligible patients with newly diagnosed MM, this has lead to the ongoing GRIFFIN trial studying daratumumab plus standard of care in United States VRd in frontline treatment of transplant eligible patients (NCT02874742). Daratumumab by intravenous (IV) infusion is currently approved in over 90 countries worldwide for the treatment of relapsed or refractory, and frontline MM. Daratumumab subcutaneous (SC) was developed to provide several potential benefits for both patients and healthcare providers and was approved based on the phase 3 non-inferiority, randomized COLUMBA study. Both co-primary endpoints, overall response rate and Ctrough at Cycle 3 Day 1, were non-inferior for SC daratumumab, compared to IV daratumumab. Advantages of the subcutaneous form include shorter administration time of 3 to 5 minutes (compared with 4 to 7 hours for IV infusion) and the lower rate of infusion related reactions (IRRs) give additional flexibility and reduction in treatment burden to the patient, as well as reducing health care professional time spent on administration. The smaller administration volume for daratumumab SC reduces the risk of volume overload in patients with cardiac or renal insufficiency. There is very little data at this time regarding the re-treatment of daratumumab in patients with RRMM who have previously been treated with daratumumab. There was a retrospective analysis recently published regarding the safety and efficacy of daratumumab in combination with pomalidomide and dexamethasone in patients who were daratumumab and/or pomalidomide-refractory. Cohort 1 (12 patients) was daratumumab- and POM-naive, and cohort 2 (22 patients) was daratumumab- and/or POM-refractory. A subgroup of 12 patients in cohort 2 (cohort 3) was daratumumab- and pomalidomide-refractory. The overall response rates (ORRs) were 91.7%, 40.9%, and 33.3% in cohorts 1, 2, and 3, respectively.23 There is no published data regarding retreatment with daratumumab monotherapy.  
There is an ongoing trial, Son of SAR study (NCT02514668), evaluating the efficacy of isatuximab monotherapy, another CD38 monoclonal antibody with similar efficacy to daratumumab monotherapy24, in patients previously treated with daratumumab. Preliminary unpublished results of this ongoing trial show a response rate of < 10%. Overall, an estimated 5,528 subjects have received daratumumab in the clinical development program from 37 studies, ie, 8 completed, 19 studies with completed primary analysis (of these, 3 studies have ongoing cohorts), and 10 full ongoing monotherapy and combination therapy clinical studies in newly diagnosed MM, smoldering MM, relapsed/refractory MM, and also non-myeloma diseases, including myelodysplastic syndrome (MDS), amyloidosis, lung cancer, acute lymphoblastic leukemia, natural killer T-cell lymphoma (NKTCL), and non-Hodgkin's lymphoma (NHL), as well as an early-access study and healthy volunteers. Of these, 1,756 subjects were exposed to daratumumab from monotherapy studies and 3,772 subjects were exposed to daratumumab from combination therapy (IB; version 16, dated 20 December 2019).
nct# 04407442
pharmacokinetics

Daratumumab is given intravenously (IV) at 16 mg/kg dose and when given as monotherapy for MM, is given once weekly for total of 8 doses (weeks 1-8), every 2 weeks for 8 doses (weeks 9-24), and every 4 weeks thereafter (starting week 25 and onwards). A study that reported the pharmacokinetic data from GEN501 and SIRIUS studies found that in both studies, daratumumab exhibited nonlinear pharmacokinetic characteristics. Decreasing daratumumab clearance with increasing dose suggests saturation of target-mediated clearance at higher dose levels, whereas decreasing clearance over time with repeated dosing may be due to tumor burden reductions as CD38-positive cells are eliminated. These and other pharmacokinetic data analyses support the use of the recommended dose regimen of daratumumab (16 mg/kg weekly for 8 weeks, every 2 weeks for 16 weeks, and every 4 weeks thereafter) to rapidly saturate target-mediated clearance during weekly dosing and maintain saturation when dosing every 2 or 4 weeks.25 Analysis of daratumumab pharmacokinetics have showed that daratumumab exhibits typical pharmacokinetics for an IgG1 monoclonal antibody. Mean serum concentrations of daratumumab peaked at the end of the first infusion and then declined in an apparent bi-exponential manner. V Z estimates indicate that daratumumab is confined primarily in the vascular system, with limited extravascular tissue distribution. Following the first full infusion, C max increased in approximate proportion to the increasing dose of daratumumab 1–24 mg/kg, and increased in a greater than dose-proportional manner after multiple doses. AUC increased in a greater than doseproportional manner after the first and multiple doses. Following the 16 mg/kg recommended dosing regimen
(weekly for 8 weeks, every 2 weeks for 16 weeks, and every 4 weeks thereafter), accumulation of daratumumab continued throughout the weekly dosing period and decreased slightly as patients entered the less intense dosing periods. Following the end of treatment, daratumumab remained detectable in most patients in the SIRIUS 16 mg/kg group at 8 weeks post-treatment. No subjects in the GEN501 or SIRIUS studies developed antibodies to daratumumab during treatment suggesting a low risk for immunogenicity with this therapeutic agent.25 Over the dose range from 1 to 24 mg/kg as monotherapy or 1 to 16 mg/kg in combination with other treatments, increases in area under the curve (AUC) were more than dose-proportional. Clearance was rapid at low doses and slower at higher doses; clearance also decreased with multiple doses. This PK profile was consistent with targetmediated disposition indicating target saturation at higher doses.26 Decreasing clearance over time with repeated dosing may be due to a reduction of tumor burden as CD38+ cells are eliminated.25 The PK of daratumumab was similar following monotherapy and combination therapies in multiple myeloma. The mean ± SD estimated terminal half-life of daratumumab associated with linear clearance was 18 ± 9 days when administered as monotherapy and 23 ± 12 days when administered as combination therapy.  
Exposure-response analyses described elsewhere (Xu et al, submitted)26 indicates that most patients (approximately 80%) following the recommended 16 mg/kg dose regimen were expected to achieve daratumumab serum concentrations correlated with 99% target (CD38) saturation and 90% of the maximal effect on ORR.25 The clinical pharmacology assessment of daratumumab SC monotherapy data are available from daratumumab SC-dosed subjects in a Phase 1/1b study MMY1004 [Part 2]) and Phase 3 study (MMY3012), Phase 2 combination therapy study MMY2040 and population pharmacokinetics and exposure-response analyses. In MMY1004, the 1800 mg dose achieved maximum Ctrough (Cycle 3 Day 1 predose) values that were similar or greater than the maximum Ctrough observed for the approved 16 mg/kg IV dose following the same dose schedule. The PK data from Part 2 supported the daratumumab SC 1800 mg dose selection for the Phase 3 study. The PK data from MMY3012 study demonstrated that daratumumab SC 1800 mg is non-inferior to daratumumab IV 16 mg/kg in terms of maximum Ctrough (Cycle 3 Day 1 predose), with the lower bound of the 90% CI for the geometric means ratio for daratumumab SC versus daratumumab IV for maximum Ctrough (Cycle 3 Day 1 predose) exceeding 80%, thereby meeting the predefined non-inferiority criterion. Additionally, daratumumab SC 1800 mg monotherapy consistently produced lower peak-to-trough fluctuations, similar or slightly higher trough levels over time, and lower peak concentrations compared with daratumumab IV 16 mg/kg monotherapy. Overall, consistent daratumumab concentrations were observed across the body weight ranges. As expected, slightly higher concentrations were observed for subjects with lower body weights. There was no apparent relationship between exposure and safety endpoints (SAEs, Grade 3 or higher TEAEs and neutropenia). The simulated trough concentrations following 6 weekly doses of daratumumab SC 1800 mg for combination therapy (daratumumab SC, bortezomib, melphalan, and prednisone [D-VMP], daratumumab SC, lenalidomide, and dexamethasone [D-Rd], daratumumab SC, bortezomib, lenalidomide, and dexamethasone [D-VRd]) were similar to monotherapy. Overall, daratumumab SC was well-tolerated with manageable side effects and a significantly reduced incidence of IRRs relative to daratumumab IV. The safety profile of daratumumab administered subcutaneously at a flat dose of 1800 mg continued to be generally comparable to that of the 16 mg/kg IV formulation.
nct# 04407442
safety

The most frequently reported adverse reactions (reported at a rate of  20%) in the POLLUX trial were infusion reactions, diarrhea, nausea, fatigue, pyrexia, upper respiratory traction infections, muscle spasm, cough, and dyspnea.11 The most frequently reported adverse events (reported at a rate of  20%) in the CASTOR trial were infusion reactions, diarrhea, peripheral edema, upper respiratory tract infection, and peripheral sensory neuropathy. Neutropenia and thrombocytopenia are also side effects of daratumumab.21 The safety results from all ongoing studies of daratumumab are summarized (through June 2017) below:
nct# 04407442
table 1-1 available safety findings

| Study Number (N  exposed to  Daratumumab)                                          | Safety Results Daratumumab Monotherapy Trials                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                          |
|------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------|
| MMY2002, GEN501, and  MMY1002 (Relapsed/Refractory MM,  156) Integrated analysis  | Most common TEAEs: fatigue (40%), nausea and anemia (28% each), back  pain (26%), cough (24%), neutropenia (23%), pyrexia (22%), upper respiratory  tract infection (22%), and thrombocytopenia (21%).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                          |
|                                                                                   | Grade 3/4 TEAEs in 57% of subjects; most common were anemia (17%),  thrombocytopenia (14%), neutropenia (12%), lymphopenia and pneumonia  (6%), leukopenia and hypertension (5% each), hypercalcemia (3%).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                          |
|                                                                                   | TEAEs leading to DC of study treatment in 4% of subjects; none considered  drug related.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |                                                                                                          |
|                                                                                   | SAEs in 34% of subjects; most common were pneumonia (6%) and pyrexia,  general physical health deterioration, and hypercalcemia (3% each).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                          |
|                                                                                   | Deaths due to progressive disease (7%), and 3 (2%) due to TEAEs  (cardiorespiratory arrest in the setting of H1N1 influenza, general physical  health deterioration secondary to aspiration pneumonia, and pneumonia).  None of the fatal TEAEs were considered related to daratumumab.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                          |
| SMM2001 (smoldering                                                                |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | SAEs were reported for 15 subjects (24% in the long intense, 2% in                                       |
| MM, 122)                                                                           | intermediate, and 10% in short intense treatment groups). No IRRs or deaths.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                          |
| MMY1004 - Relapsed/Refractory MM  (subcutaneous  administration, 53)               |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | A total of 8 subjects received daratumumab-MD 1200 mg and 45 subjects  received daratumumab- MD 1800 mg. |
|                                                                                   | Most common TEAEs (≥20% of all subjects): upper respiratory tract infection  (1200 mg: 38%; 1800 mg: 22%), insomnia (1200 mg: 38%; 1800 mg: 11%),  decreased appetite (1200 mg: 38%; 1800 mg: 7%), thrombocytopenia (1200 mg:  38%; 1800 mg: 18%), viral upper respiratory tract infection (1200 mg: 25%; 1800  mg: 13%), vomiting (1200 mg: 25%; 1800 mg: 13%), hyperuricaemia (1200 mg:  25%; 1800 mg: 2%), hypokalaemia (1200 mg: 25%; 1800 mg: 4%), blood  creatinine increased (1200 mg: 25%; 1800 mg: 4%), anemia (1200 mg: 25%;  1800 mg: 33%), fatigue (1200 mg: 25%; 1800 mg: 20%), pyrexia (1200 mg: 25%;  1800 mg: 27%), diarrhea (1200 mg: 25%; 1800 mg: 22%), headache (1200 mg:  25%; 1800 mg: 18%), cough (1200 mg: 25%; 1800 mg: 13%), epistaxis (1200  mg: 25%; 1800 mg: 4%), hypertension (1200 mg: 25%; 1800 mg: 7%), rhinitis  allergic (1200 mg: 25%; 1800 mg: 2%), nasal congestion (1200 mg: 25%; 1800  mg: 2%), rectal hemorrhage (1200 mg: 25%; 1800 mg: 0%), and asthenia (1200  mg: 13%; 1800 mg: 20%), |                                                                                                          |
|                                                                                   | Grade 3/4 TEAEs: in 63% and 49% of subjects in the 1200 mg and 1800 mg  cohorts, respectively. The most frequently reported Grade 3 or 4 TEAEs were in  the Blood and Lymphatic System Disorders System Organ Class (SOC), reported  in 38% and 31% of subjects in the 1200 mg and 1800 mg cohorts, respectively.  Two of 8 (25%) subjects in the 1200 mg cohort and 4% in the 1800 mg cohort  had Grade 3 or 4 hypertension.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                          |
|                                                                                   | SAEs: SAEs were reported in 26% of all subjects: 50% and 31% of subjects in  the 1200 mg and 1800 mg cohorts, respectively. The most frequently reported  serious TEAEs were in the Infections and Infestations SOC (1200 mg: 38%,  1800 mg: 16%), the Nervous System Disorders SOC (1200 mg: 13%, 1800 mg:  4%), and the Respiratory, thoracic and mediastinal disorders (1200 mg: 13%,  1800 mg: 2%).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                          |
|                                                                                   | No TEAEs leading to DC of study treatment. One subject in the  daratumumab-MD 1800 SC cohort died within 30 days of the last dose of  study drug from progressive disease.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                          |  
| MMY3010 (Relapsed/Refractory                                                                             | MM,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                     |
|----------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 687)                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | SAEs in 38% of subjects; most common were pneumonia (4%), hypercalcemia  (3%), pyrexia (3%), dyspnea (2%), thrombocytopenia (2%), acute kidney injury  (2%), and back pain (2%).                                                                                                                                    |
|                                                                                                         | Deaths: Forty-eight subjects (7%) reported TEAEs with an outcome of death.                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                                                                                                                                                     |
| LYM2001 (non-Hodgkin's                                                                                  | All subjects had at least 1 or more TEAE. Most common TEAEs were reported                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                     |
| lymphoma, 36)                                                                                            | from SOC of respiratory, thoracic and mediastinal disorders; cough (DLBCL  cohort: 47%; FL cohort: 38%; MCL cohort: 80%); SOC of gastrointestinal  disorders: abdominal pain (DLBCL cohort: 13%; FL cohort: 31%; MCL cohort:  20%), and nausea (DLBCL cohort: 33%; FL cohort: 6%; MCL cohort: 40%), and  SOC of general disorders and administration site conditions; fatigue (DLBCL  cohort: 27%; FL cohort: 19%; MCL cohort: 20%), and pyrexia (DLBCL cohort:  13%; FL cohort: 25%; MCL cohort: 40%) |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | SAEs in 15 subjects. All SAEs were reported in 1 subject each, except for  febrile neutropenia, pneumonia, general physical health deterioration, and  pyrexia (2 subjects each).                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | Deaths in 4 subjects: 2 due to progressive disease and 2 due to an adverse event  (chronic kidney disease and pneumonia).                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | IRRs - none. Daratumumab Combination Trials                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                     |
| GEN503 and MMY3003 (integrated;  daratumumab in  combination with  lenalidomide and  dexamethasone (318) |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Most common TEAEs (DRd treatment group): neutropenia (64%), diarrhea  (53%), upper respiratory tract infection (37%), fatigue (37%), anemia (36%),  cough (34%), muscle spasms (31%), constipation (31%), thrombocytopenia  (29%), viral upper respiratory tract infection (29%), nausea (28%), and pyrexia  (25%). |
|                                                                                                         | Grade 3/4 TEAEs (DRd treatment group): 89%; most common, neutropenia  (58%), anemia (16%), and thrombocytopenia (14%).                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | TEAEs leading to DC of treatment in 13% of subjects in the DRd treatment  group; most common were pneumonia (4 subjects, 1.3%), general physical  health deterioration (3 subjects, 0.9%), and septic shock (2 subjects, 0.6%).                                                                                                                                                                                                                                                                        |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | SAEs (DRd treatment group) in 63% of subjects; most common were  pneumonia (13%), influenza, febrile neutropenia (4%) and pyrexia (4% each),  and bronchitis, pulmonary embolism, lower respiratory tract infection, diarrhea  (3% each).                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | Deaths (DRd treatment group):7% of subjects died within 30 days of the last  dose of study drug: 21 due to TEAEs and 1 due to disease progression.                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                     |
| MMY1001 (daratumumab                                                                                     | in                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                     |
| combination                                                                                              | with                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                     |
| pomalidomide                                                                                             | and                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                     |
| dexamethasone, 240)                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Most common TEAEs: neutropenia (81%), anemia (55%), fatigue (52%),  diarrhea (50%), thrombocytopenia (43%), cough (41%), leukopenia (39%),  constipation (36%), nausea (34%), dyspnea (33%), pyrexia (33%), upper  respiratory tract infection (32%), muscle spasms (29%), vomiting (28%),  arthralgia (26%).       |
|                                                                                                         | Grade 3 or 4 TEAEs in 99% of subjects; most common were neutropenia  (79%), anemia (28%), leukopenia (24%), thrombocytopenia (19%),  lymphopenia (14%), and pneumonia and fatigue (13% each).                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | TEAEs leading to DC of treatment in 16% of subjects (no single AE reported  in >1 subject).                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | SAEs in 57% of subjects; most common were pneumonia (12%), sepsis (7%),  febrile neutropenia (5%), fall (4%), anemia, dyspnea, small intestinal  obstruction, and urinary tract infection (3% each).                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                     |
|                                                                                                         | Deaths: 9% of subjects died during study treatment or within 30 days after the  last dose of study drug; 7 due to AEs and 2 deaths due to disease progression.                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                                                     |  
| MMY1001  (daratumumab                                                                                       | plus                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|-------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| bortezomib                                                                                                  | and                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| dexamethasone, 30)                                                                                          |                                                                                                                                                                                                                                                                                                                         | SAEs were reported for 3 subjects in the DVd arm (Hyperglycemia [1 subject],  soft tissue infection, pneumonia, prerenal failure, diarrhea, respiratory failure  [1 subject], laboratory test interference [1 subject]. In DVMP arm, SAEs were  reported in 1 subject (cardiac failure). No subjects in the D-VTd arm were  reported to have SAEs.                                                                                                           |
|                                                                                                            | No deaths reported.                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| MMY1001 (daratumumab  in combination with  carfilzomib/dexamethasone +/- lenalidomide DKd =  20, DKRd = 20) |                                                                                                                                                                                                                                                                                                                         | AEs of Interest - Cardiac Events: In the DKd arm, cardiac disorder TEAEs of  interest were reported in 15% of subjects; 4 (5%) subjects had a Grade 3 or  Grade 4 event. No cardiac disorder TEAEs of Grade 5 was reported. In the  DKRd arm, cardiac disorder TEAEs of interest were reported in 41% of  subjects, with 1 subject (5%) having a Grade 3 or Grade 4 event (congestive  heart failure) that was also an SAE. No Grade 5 events were reported. |
|                                                                                                            | SAEs reported in 32% of subjects in the DKd arm (n=85); most common was  pneumonia (7%). SAEs were reported in 46% of subjects in the DKRd arm  (n=22). The most frequently reported SAEs were pulmonary embolism (n=3;  14%) and influenza and pyrexia (n=2; 9% each). All other SAEs were reported  in 1 subject each. |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | One death in the DKd arm due to progressive disease.                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| MMY1005  (daratumumab                                                                                       | in                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| combination with bortezomib                                                                                 | and                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| dexamethasone; Japan, 8)                                                                                    |                                                                                                                                                                                                                                                                                                                         | SAEs in 3 subject each (herpes zoster, prostate cancer, nasopharyngitis).                                                                                                                                                                                                                                                                                                                                                                                    |
|                                                                                                            | No IRRs.                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | No deaths.                                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| MMY3004  (daratumumab                                                                                       | in                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| combination                                                                                                 | with                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| bortezomib                                                                                                  | and                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| dexamethasone, 243)                                                                                         |                                                                                                                                                                                                                                                                                                                         | Most common TEAEs were thrombocytopenia (60%), peripheral sensory  neuropathy (50%), diarrhea (35%), upper respiratory tract infection (33%),  anemia (28%), and cough (28%).                                                                                                                                                                                                                                                                                |
|                                                                                                            | Grade 3/4 AEs were reported in 81% of subjects; most common were  thrombocytopenia (46%), anemia (15%), and neutropenia (14%).                                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | TEAEs leading to DC of treatment were reported for 10% of subjects; most  common were pneumonia (4 subjects; 2%), and cardiac failure congestive (2 subjects; 0.8%).                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | SAEs in 50% of subjects; most common were pneumonia (10%), and anemia,  bronchitis, thrombocytopenia, atrial fibrillation, upper respiratory tract infection,  and pyrexia (3% each).                                                                                                                                    |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | Deaths:15 subjects died within 30 days of the last dose of study drug; 13 deaths  were due to TEAEs and 2 were due to disease progression.                                                                                                                                                                               |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| MMY3007 (daratumumab  in combination with  bortezomib, melphalan and  prednisone, 700)                      |                                                                                                                                                                                                                                                                                                                         | Most common TEAEs were thrombocytopenia (51%), neutropenia (51%),  anemia (33%), peripheral sensory neuropathy (31%), diarrhea (24%), pyrexia  (22%), nausea (21%), upper respiratory tract infection (20%) and constipation  (18%).                                                                                                                                                                                                                         |
|                                                                                                            | Grade 3/4 TEAEs were reported in 74% of subjects, with neutropenia (39%)  and thrombocytopenia (36%) being the most frequently reported.                                                                                                                                                                                 |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | SAEs were reported in 37% of subjects, with pneumonia being the most  frequent (7%).                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | Deaths: Thirty-eight (5%) subjects had a Grade 5 TEAE.                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| MMY3008  (daratumumab                                                                                       | in                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| combination                                                                                                 | with                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| lenalidomide                                                                                                | and                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| dexamethasone, 729)                                                                                         |                                                                                                                                                                                                                                                                                                                         | Most common TEAEs were neutropenia (39%), diarrhea (34%), constipation  (33%), fatigue (28%), anemia (25%), nausea and edema peripheral (24% each),  insomnia (22%), muscle spasms (21%), and asthenia (20%).                                                                                                                                                                                                                                                |
|                                                                                                            | Grade 3/4 TEAEs were reported in 70% of subjects, with neutropenia being the  most frequently reported (32%).                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | SAEs were reported in 50% of subjects; most common were pneumonia (6%),  pyrexia and pulmonary embolism (3% each).                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|                                                                                                            | Deaths Thirty-four subjects (5%) had a Grade 5 TEAE.                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| Phase II - Daratumumab and Azacitidine                                                                      | Page 18 of 95                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
nct# 04407442
monitoring response on daratumumab

Daratumumab has been found to interfere with serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) assays due to co-migration of daratumumab with patients' M protein which impedes accurate quantification of endogenous M proteins.18,27 Given this can pose a problem when SPEP and IFE assays are needed to measure disease response, a clinical assay known as the daratumumab IFE reflex assay (DIRA) has been developed to mitigate daratumumab interference with IFE.27 DIRA uses a mouse anti-daratumumab antibody that binds to daratumumab and shifts the migration of daratumumab away from the M protein.27 The DIRA assay has been utilized in clinical trials of daratumumab to confirm disease response, and the assay's specificity, reproducibility, and ability to distinguish daratumumab mAb from endogenous M protein has been validated.9 In summary, daratumumab has been well tolerated and has demonstrated encouraging response rates in clinical trials both as a single agent and in combination regimens in the relapsed/refractory setting and now more recently in the frontline setting. Moreover, daratumumab in combination regimens has maintained a favorable safety profile without significant increase in toxicities.
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azacitidine

Azacitidine is FDA approved for the treatment of all subtypes of myelodysplastic syndrome (MDS) and was recently approved in 2018 in combination with venetoclax for newly diagnosed AML in patients who were  75 years of age or had comorbidities that precluded the use of intensive induction chemotherapy. Azacitidine is thought to have two main mechanisms of antineoplastic action—cytotoxicity, resulting from incorporation into RNA and DNA, and DNA hypomethylation, restoring normal growth control and differentiation in hematopoietic cells. Induction of DNA hypomethylation appears to require lower azacitidine doses than does cytotoxicity, as the concentration of azacitidine required for maximum inhibition of DNA methylation in vitro does not suppress DNA synthesis. Upon uptake by cells, azacitidine is phosphorylated to 5-azacytidine monophosphate by uridinecytidine kinase and then to diphosphate and triphosphate by pyrimidine monophosphate and diphosphate kinases, respectively. 5-azacitidine triphosphate is incorporated into RNA, disrupting nuclear and cytoplasmic RNA metabolism and inhibiting protein synthesis. 5-azacitidine diphosphate is reduced by ribonucleotide reductase to 5azacitidine-deoxycytidine diphosphate, which is then phosphorylated by nucleoside diphosphate kinases to 5-azadeoxycitidine triphosphate, which is incorporated into DNA. As a result, DNA synthesis is inhibited. Azacitidine is most toxic during the S-phase of the cell cycle, but the predominant mechanism of cytotoxicity has not been established. Azacitidine inhibits methylation of replicating DNA by stoichiometric binding with DNA methyltransferase 1, resulting in DNA hypomethylation. DNA hypermethylation at the CpG islands has been described in myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), and other malignancies.  
Molecular structure of Aza
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clinical studies

Effectiveness of azacitidine in high risk MDS was demonstrated in azacitidine-001, a Phase III international, multicenter, randomized controlled trial comparing azacitidine administered subcutaneously (SubQ) at 75 mg/m2 for 7 consecutive days every 28 days to conventional care regimen (CCR) which included either best supportive care, low dose cytarabine, or intensive chemotherapy as selected by investigators before randomization for 358 patients with high risk MDS.28 After a median follow-up of 21.1 months, the median overall survival was 24.5 months for the Phase II - Daratumumab and Azacitidine Page 19 of 95 azacitidine group vs 15 months for the conventional care group. Azacitidine resulted in higher rates of hematologic responses assessed by International Working Group (IWG) 2000 criteria and longer durations of response with median of 13.6 months in the azacitidine group compared to 5.2 months in the conventional care group. At 2 years, 50.8% of patients in the azacitidine group were alive compared with 26.2% in the conventional group.28 Azacitidine prolonged OS compared with CCR regardless of IPSS cytogenetic risk group. Subset analysis of the data has shown that the drug can be used safely in even the oldest patients with MDS and is superior to treatment with other established regimens, such as low-dose cytarabine.29 Azacitidine-001 showed that achievement of hematologic response was associated with improved OS with azacitidine treatment, and patients who achieved a hematologic response to azacitidine had significantly prolonged OS and reduced risk of death versus patients who achieved a response to CCR. In addition, stable disease or achievement of complete response (CR), marrow CR (mCR), partial response (PR) or hematologic response with alternative dosing schedules of azacitidine has been shown to significantly reduce the risk of death versus disease progression. In the azacitidine-001 study, the median time to first response with azacitidine was 2 cycles (range: 1–16), with 91% of responding patients achieving first response within 6 cycles and all but 1 achieving first response by cycle 12. Continued treatment improved response quality in 48% of patients, with a median of 3 cycles from first to best response. By cycle 12, 92% of responding patients achieved their best response.28 Based on this experience, some clinicians have advised administering at least six cycles of azacitidine, and the National Comprehensive Cancer Network guidelines for MDS recommend at least four to six cycles of azacitidine before assessing for treatment failure. Studies performed by Cancer and Leukemia Group B (CALGB) demonstrated that azacitidine had activity in MDS
and AML when given at 75 mg/m2 by intravenous infusion (CALGB 8421) or subcutaneous injection (CALGB 8921 and 9221) daily for 7 days in a 28-day cycle.30,31 The crossover phase III CALGB 9221 trial which was a multicenter randomized, open-label trial designed to compare the safety and efficacy of subcutaneous azacitidine with supportive care (observation group) in patients with any of the five subtypes of MDS, showed a significant effect of azacitidine on response rates (P< 0.0001), with an overall response rate of 60% in patients receiving azacitidine compared with 5% in those receiving only supportive care. Median time to leukemic transformation or death was 21 months for azacitidine arm versus 13 months for supportive care arm. Transformation to acute myelogenous leukemia occurred in 15% of patients on the azacitidine arm and in 38% of patients in the supportive care arm. Median OS was 18 months for azacitidine and 11 months for supportive care arm.30,31 Response rates in these CALGB trials were similar in subjects with all MDS subtypes and with AML. In a sub-analysis of azacitidine-001 of low-blast- count AML (20%–30% blasts) the 2-year OS rate for patients treated with azacitidine was 50%, and the median OS was 24.5 months compared with 16.0 months for patients treated with CCR and 16.4 months for patients not preselected to receive intensive chemotherapy (best supportive care or low-dose cytarabine). Survival benefits with azacitidine may not require CR because CR rates were similar for azacitidine versus CCR (18% vs. 16%), whereas rates of red blood cell transfusion independence were significantly higher with azacitidine (41% vs. 18%).32 Recently, the global phase III randomized open-label azacitidine- AML-001 study of azacitidine (75 mg/m2 per day on days 1–7 in 28-day cycles) versus CCR extended these findings in patients aged 65 years with newly diagnosed AML with >30% blasts. Azacitidine treatment demonstrated a clinically meaningful improvement in median OS of 10.4 months versus 6.5 months with CCR. Azacitidine significantly improved 1-year survival: 46.5% versus 34.2% with CCR, a 12.3% difference. In patients who did not achieve CR, median OS was prolonged from 6.9 months with azacitidine versus 4.2 months with CCR.33 Azacitidine in combination with venetoclax was approved in newly diagnosed AML in elderly patients  75 years of age or patients who had comoribidities that precluded the use of intensive induction chemotherapy based on M14-358 (NCT02203773), a large multicenter phase 1b dose-escalation and expansion study showing that the novel combination of venetoclax with decitabine or azacitidine was effective and well tolerated in elderly patients with AML. With a median time on study of 8.9 months, 67% of patients (all doses) achieved complete remission (CR) + CR with incomplete count recovery (CRi), with a CR + CRi rate of 73% in the venetoclax 400 mg + HMA cohort. Patients with poor-risk cytogenetics and those at least 75 years old had CR + CRi rates of 60% and 65%, respectively. The median duration of CR + CRi (all patients) was 11.3 months, and median overall survival (mOS) was 17.5 months; mOS has not been reached for the 400-mg venetoclax cohort.  
34
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azacitidine in multiple myeloma

While azacitidine is currently not approved in MM, there have been pre-clinical studies studying the role of azacitidine and DNMTi in MM. Studies have found that the DNA methylation score can predict for the sensitivity of human myeloma cell lines and primary MM cells of patients to DNMTi.35 Azacitidine has also been shown to exert apoptosisinducing and growth-inhibiting effects on MM cell lines and its mechanism may be related to the decrease of BCL2/BAX ratio, caspase-3 activation, and the arrest of the cell cycle.36 Another study found that azacitidine induces apoptosis of MM cells by downregulating two crucial cell survival pathways in MM by inhibiting the elaboration of IL-6 receptor alpha and IL-6 resulting in reduced expression of phosphor-STAT3 and Bcl-xl and by inhibiting both nuclear factor-kappaB nuclear translocation and DNA binding.37 Clinically DNMTi's such as azacitidine are currently being investigated in MM as monotherapy and in combination with standard therapies such as lenalidomide and dexamethasone (NCT01155583, NCT01050790). A recent phase 1b single center 3 x 3 dose escalation study looking at oral azacitidine (CC-486) in combination with lenalidomide and dexamethasone in patients with RRMM who had previously failed lenalidomide found the ORR was 37.5% at the MTD and clinical benefit rate was 50%. Median OS was 10.3 months and median PFS was 2.6 months. This study found that oral azacitidine, lenalidomide, and dexamethasone produced meaningful clinical responses in heavily treated Len refractory MM patients.38 However to this data there has been no trial assessing the safety and efficacy of daratumumab in combination with azacitidine and dexamethasone.
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standard dosing

The standard dose of azacitidine, based on the trials leading to its approval in AML and MDS, is 75 mg/m2 intravenously or subcutaneously for 7 days every 28 days.28,30,31,34 Because of the difficulty of continued administration for 7 days, in a randomized trial, MDS patients were given azacitidine subcutaneously in one of three schedules every 4 weeks for six cycles: azacitidine 5-2-2 (75 mg/m2 daily for 5 days, followed by 2 days of no treatment, and then 75 mg/m2 daily for 2 days for a total dose of 525 mg/m2 per cycle), azacitidine 5-2-5 (50 mg/m2 daily for 5 days, followed by 2 days of no treatment, and then 50 mg/m2 daily for 5 days for a total dose of 500 mg/m2 per cycle), or azacitidine 5 (75 mg/m2 for 5 days for a total dose of 375 mg/m2).39 All three alternative dosing regimens produced hematological improvements, red blood cell transfusion independence, and safety responses consistent with the approved azacitidine regimen. However, results suggest that the azacitidine 5 dosing regimen may be better tolerated with a more convenient dosing schedule than the two alternative dosing regimens.39
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safety

With respect to safety of azacitidine, in the CALGB 9921 trial, >95% patients in both groups reported treatmentassociated adverse events. azacitidine-treated subjects reported more frequently than observation arm subjects gastrointestinal events (nausea, vomiting, diarrhea, constipation, and anorexia), neutropenia, febrile neutropenia, thrombocytopenia, injection site events, arthralgia, dizziness, dyspnea, cough, and myalgia. Serious adverse events occurred more frequently in azacitidine-treated patients (60%) than in observation patients (36%). Serious adverse events most commonly resulting in hospitalization were thrombocytopenia, febrile neutropenia, fever, and pneumonia. There were no deaths attributed to azacitidine treatment. During the study periods, half of the deaths in the three trials were probably related to MDS and the other half were unrelated to MDS.31 Phase II - Daratumumab and Azacitidine Page 21 of 95 The most common reasons for azacitidine discontinuation, dose reduction, or therapy interruption were leukopenia, neutropenia, and thrombocytopenia. Patients who developed CR or PR first had further decreases in hematologic parameters. Most patients in both treatment arms received packed RBC and/or platelet transfusions and medications to treat adverse events. The frequency of adverse events decreased after the first two cycles of azacitidine therapy.31 The data described below reflect exposure to azacitidine for injection in 443 MDS patients from 4 clinical studies. Study 1 was a supportive-care controlled trial (subcutaneous administration), Studies 2 and 3 were single arm studies (one with subcutaneous administration and one with intravenous administration), and Study 4 was an international randomized trial (subcutaneous administration).  
Table below presents adverse reactions occurring in at least 5% of patients treated with azacitidine for Injection in Study 4. Similar to Studies 1 and 2 described above, duration of exposure to treatment with azacitidine for Injection was longer (mean 12.2 months) compared with best supportive care (mean 7.5 months).  
Table 1-2 Adverse Reactions Occurring In At Least 5% of Patients Treated With Azacitidine for  
Injection  
Number (%) of Patients  
Any Grade Grade 3/4  
| Injection                                                     | Number (%) of Patients                        |         |          |         |
|---------------------------------------------------------------|-----------------------------------------------|---------|----------|---------|
| Any Grade                                                     | Grade 3/4 Best  Supportive Care Only  (N=102) |         |          |         |
| Blood and lymphatic system disorders Anemia                   | 90 (51)                                       | 45 (44) | 24 (14)  | 9 (9)   |
| Febrile neutropenia                                           | 24 (14)                                       | 10 (10) | 22 (13)  | 7 (7)   |
| Leukopenia                                                    | 32 (18)                                       | 2 (2)   | 26 (15)  | 1 (1)   |
| Neutropenia                                                   | 115 (66)                                      | 29 (28) | 107 (61) | 22 (22) |
| Thrombocytopenia                                              | 122 (70)                                      | 35 (34) | 102 (58) | 29 (28) |
| Gastrointestinal disorders Abdominal pain                     | 22 (13)                                       | 7 (7)   | 7 (4)    | 0       |
| Constipation                                                  | 88 (50)                                       | 8 (8)   | 2 (1)    | 0       |
| Dyspepsia                                                     | 10 (6)                                        | 2 (2)   | 0        | 0       |
| Nausea                                                        | 84 (48)                                       | 12 (12) | 3 (2)    | 0       |
| Vomiting                                                      | 47 (27)                                       | 7 (7)   | 0        | 0       |
| General disorders and administration  site conditions Fatigue | 42 (24)                                       | 12 (12) | 6 (3)    | 2 (2)   |
| Injection site bruising                                       | 9 (5)                                         | 0       | 0        | 0       |
| Injection site erythema                                       | 75 (43)                                       | 0       | 0        | 0       |
| Injection site hematoma                                       | 11 (6)                                        | 0       | 0        | 0       |
| Injection site induration                                     | 9 (5)                                         | 0       | 0        | 0       |
| Injection site pain                                           | 33 (19)                                       | 0       | 0        | 0       |
| Injection site rash                                           | 10 (6)                                        | 0       | 0        | 0       |
| Injection site reaction                                       | 51 (29)                                       | 0       | 1 (1)    | 0       |
| Pyrexia                                                       | 53 (30)                                       | 18 (18) | 8 (5)    | 1 (1)   |
| Infections and infestations Rhinitis                          | 10 (6)                                        | 1 (1)   | 0        | 0       |
| Upper respiratory tract infection                             | 16 (9)                                        | 4 (4)   | 3 (2)    | 0       |
| Urinary tract infection                                       | 15 (9)                                        | 3 (3)   | 3 (2)    | 0       |
| Investigations Weight decreased                               | 14 (8)                                        | 0       | 1 (1)    | 0       |
| Best  Supportive  Care Only (N=102)                           |                                               |         |          |         |
| Azacitidine  for Injection (N=175)                            | Azacitidine  for Injection (N=175)            |         |          |         |
| Body                                                          | System                                        |         |          |         |
| Adverse Reactionsa                                            |                                               |         |          |         |
| Phase II - Daratumumab and Azacitidine                        | Page 22 of 95                                 |         |          |         |  
| Metabolism and nutrition disorders Hypokalemia   | 11 (6)   | 3 (3)   | 3 (2)       | 3 (3)   |
|--------------------------------------------------|----------|---------|-------------|---------|
| Nervous system disorders Lethargy                | 13 (7)   | 2 (2)   | 0           | 1 (1)   |
| Psychiatric disorders Anxiety                    | 9 (5)    | 1 (1)   | 0           | 0       |
| Insomnia                                         | 15 (9)   | 3 (3)   | 0           | 0       |
| Renal and urinary disorders Hematuria            | 11 (6)   | 2 (2)   | 4 (2)       | 1 (1)   |
| Respiratory,                                     | thoracic | and     | mediastinal |         |
| disorders Dyspnea                                | 26 (15)  | 5 (5)   | 6 (3)       | 2 (2)   |
| Dyspnea exertional                               | 9 (5)    | 1 (1)   | 0           | 0       |
| Pharyngolaryngeal pain                           | 11 (6)   | 3 (3)   | 0           | 0       |
| Skin and subcutaneous tissue disorders Erythema  | 13 (7)   | 3 (3)   | 0           | 0       |
| Petechiae                                        | 20 (11)  | 4 (4)   | 2 (1)       | 0       |
| Pruritus                                         | 21 (12)  | 2 (2)   | 0           | 0       |
| Rash                                             | 18 (10)  | 1 (1)   | 0           | 0       |
| Vascular disorders Hypertension                  | 15 (9)   | 4 (4)   | 2 (1)       | 2 (2)   |  
In Studies 1, 2 and 4 with subcutaneous administration of azacitidine for injection, adverse reactions of neutropenia, thrombocytopenia, anemia, nausea, vomiting, diarrhea, constipation, and injection site erythema/reaction tended to increase in incidence with higher doses of azacitidine for injection. Adverse reactions that tended to be more pronounced during the first 1 to 2 cycles of subcutaneous treatment compared with later cycles included thrombocytopenia, neutropenia, anemia, nausea, vomiting, injection site erythema/pain/bruising/reaction, constipation, petechiae, dizziness, anxiety, hypokalemia, and insomnia. There did not appear to be any adverse reactions that increased in frequency over the course of treatment. Overall, adverse reactions were qualitatively similar between the intravenous and subcutaneous studies. Adverse reactions that appeared to be specifically associated with the intravenous route of administration included infusion site reactions (e.g. erythema or pain) and catheter site reactions (e.g. infection, erythema, or hemorrhage). Most Commonly Occurring Adverse Reactions (Subcutaneous or Intravenous Route): nausea, anemia, thrombocytopenia, vomiting, pyrexia, leukopenia, diarrhea, injection site erythema, constipation, neutropenia, ecchymosis. The most common adverse reactions by intravenous route also included petechiae, rigors, weakness and hypokalemia.
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pharmacokinetics

In terms of pharmacology of azacitidine, it is rapidly absorbed following subcutaneous administration. Maximum plasma concentration is attained in about 30 minutes. Azacitidine is widely distributed, and is rapidly eliminated, with a mean plasma half-life of about 41 minutes.40 Metabolism of azacitidine involves deamination by cytidine deaminase followed by opening of the ring structure.41 Azacitidine and its metabolites are primarily excreted by the kidneys. The effects of intrinsic factors such as age, gender, race, renal impairment, or hepatic impairment on the pharmacokinetics of azacitidine have not been studied. In vivo drug-drug interaction studies have not been conducted.40 *In vitro* studies in cultured human hepatocytes indicate that azacitidine at a clinically relevant concentrations is not an inducer of CYP 1A2, 2C19, or 3A4/5.40
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1.3 rationale for the proposed

While daratumumab is well tolerated and has high efficacy, not all patients respond and many patients eventually develop progressive disease and resistance to daratumumab. These patients often have poor prognosis and limited treatment options. A retrospective study presented at ASH 2018 looking at MM patients who have progressed on daratumumab found that these patients have a median OS of 5.3 months and a post-daratumumab regimen ORR of 34% with limited treatment options. Another study analyzing outcomes of 275 MM patients at 14 academic centers with disease refractory to CD38 MoABs (daratumumab and isatuximab) found a median OS for the entire cohort of 8.6 months ranging from 11.2 months for patients not simultaneously refractory to an IMiD agent and a proteasome inhibitor to 5.6 months for penta-refractory patients (refractory to CD38 MoAb, 2 PIs, and 2 IMiDs). Overall response rate to first regimen after relapse post CD38 MoAB was 31% with median PFS and OS of 3.5 and 9.3 months. These studies demonstrate that MM patients refractory to CD38 MoAb such as daratumumab have poor prognosis and limited treatment options. Thus developing ways to improve the efficacy of daratumumab and overcome the resistance to daratumumab are very much in need, especially as trials have progressively lead to daratumumab's approval not only in the relapsed refractory setting but also in the frontline setting for MM patients. Pre-clinical data shows that CD38 expression on MM cells is associated with response to daratumumab therapy and a significant association has been seen between CD38 expression and daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC) as well as complement-dependent cytotoxicity (CDC) in ex vivo patients' samples with MM.42,43 Pre-clinical data also shows that there is rapid but reversible loss of cell surface CD38 expression on MM
cells during daratumumab treatment which may subsequently lead to immune escape and resistance to daratumumab and thus disease progression.19,43 There are several possible explanations for the reduction of CD38 levels during daratumumab treatment: 1) daratumumab may select for tumor cells with lower CD38 expression while preferentially eliminating MM cells with high CD38 levels 2) downregulation of CD38 may be an active process to evade daratumumab-mediated killing 3) in vitro studies suggest that binding of daratumumab to CD38 may cause redistribution of CD38 molecules 4) trogocytosis of CD38-daratumumab complexes and direct internalization may also result in a loss of CD38. This pre-clinical data showing that CD38 is a predictor of response to daratumumab and that loss of CD38 occurs during daratumumab treatment, suggests that methods to upregulate CD38 expression can increase the efficacy of daratumumab and decrease the resistance and immune escape to daratumumab. Thus efforts are underway to study the regulation of CD38 in MM. Recent studies have shown that DNA methylation patterns change during MM progression, and clinically aggressive subtypes such as plasma cell leukemias and high risk (4,14) MM have hypermethylation.44-46 DNA hypermethylation has been associated with heterochromatin formation and inactivation of tumor suppressor genes.47 DNA
methyltransferases (DNMTs) methylate cytosine residues in CpG islands and remodel chromatin. Currently two DNMT inhibitors (DNMTis) have been approved for treatment of acute myeloid leukemia and myelodysplastic syndrome: azacitidine and decitabine (DEC). These cytidine analogs function by incorporating into genome of proliferating cells during DNA synthesis, covalently binding DNMTs, targeting them for proteasomal degradation and causing passive loss of cytosine methylation in daughter cells.47 Based on analysis of publicly-available ENCODE data, Dr. Arun  
 Wiita's lab at UCSF found a CpG island in the first exon of CD38 and hypothesized that DNA methylation represses CD38 expression. CD38 was previously identified as a differentially methylated region in myeloma patient samples with a negative CpG island, DNA methylation and expression of CD38 gene  
Phase II - Daratumumab and Azacitidine Page 24 of 95
correlation between gene expression and DNA methylation.45 Dr. Wiita's lab performed additional analysis of DNA
methylation and gene expression data in three independent MM datasets and found an inverse correlation between CD38 promoter methylation status and gene expression between normal plasma cells and malignant plasma cells in MM. These findings supported our hypothesis of methylation-level regulation of CD38 transcription, and suggested that DNMTi treatment with azacitidine may be able to increase CD38 transcription in MM cells and that this increase can be exploited to augment the efficacy of daratumumab-mediated lysis of MM cells.  
Aza treatment increases CD38 cell surface expression To test the role of DNA methylation in regulation of CD38 expression, a panel of MM cell lines were treated with increasing doses of DNMTi azacitidine, and CD38 cell surface expression was measured by flow cytometry. Cells were treated for either 3 days or 5 days with flow cytometry analysis at 7 days, which allowed for DNMTi incorporation into newly synthesized DNA in replicating cells leading to DNMT degradation and loss of DNA methylation. This has induced a 1.2-2.4 increase in CD38 MFI in a dose dependent manner across all four cell lines.  
Importantly, at doses of > 1M azacitidine appears to upregulate CD38 equally if not more than previously investigated doses of the known inducers ATRA and Panobinostat.42,48 Next we determined whether upregulated CD38 MFIs increased functional daratumumab efficacy. Using an immortalized transgenic NK cell line to mediate lysis, we observed a significant increase in antibody dependent cellular cytotoxicity (ADCC) against DNMTi treated vs control. Importantly, this increase in ADCC was consistent with CD38 MFI upregulation. For example, 2 M azacitidine increased CD38 MFI 1.37  0.03 fold and increased ADCC lysis to 50.23  6.84 %. In addition, the additive effect of DNMTi and ATRA on CD38 upregulation was also seen in ADCC assays.  
Pre-clinical data from Dr. Wiita's lab shows that by derepressing the transcription of CD38 and upregulating the expression of CD38 on MM plasma cells, azacitidine can potentially increase the ADCC and efficacy of daratumumab in MM patients and help reverse daratumumab resistance that develops over time caused by loss of CD38 expression. However, there is no prior trial that has assessed the safety and efficacy of daratumumab combined with azacitidine for treatment of MM. This is the first study designed to assess the safety and efficacy of azacitidine combined with daratumumab and dexamethasone to see if the synergy of azacitidine and daratumumab, through upregulation of CD38 expression, can improve the efficacy of daratumumab alone and overcome the resistance to daratumumab that develops over time.  
Treatment with Aza significantly increases ADCC of Dara and the effect is additive with ATRA
The main risk of this trial and combining azacitidine and daratumumab is toxicity, particularly cytopenias and infections, though we anticipate that the combination will be safe and there are clear dose modifications and safety stopping rules in place for DLTs. The safety lead-in to the trial will allow for formal safety analysis in the beginning of the trial and mitigate the risks of toxicity and ensure that the patients enrolled in this phase II study are treated with a dose combination of azacitidine and daratumumab with an acceptable safety profile. Daratumumab has already been approved in MM and demonstrated adequate safety in combination with other agents such as revlimid, pomalidomide, and velcade. Azacitidine is approved in MDS and leukemia and demonstrated an acceptable safety profile in other non-myeloma hematologic malignancies in combination with other agents that also cause cytopenias such as venetoclax.34 It is anticipated that the combination of daratumumab and azacitidine, which have acceptable safety Phase II - Daratumumab and Azacitidine Page 25 of 95 profiles for currently approved indications, will have a manageable safety profile in combination. We believe that the potential benefits of improving response rates, CR rates, PFS and OS of relapsed refractory MM patients with poor prognosis by combining daratumumab and azacitidine outweighs the risks of possible increased toxicity with this combination.
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1.4 rationale for the dose

Daratumumab will be administered subcutaneously at the currently approved dose of 1800 mg, once weekly for 8 doses, then every two weeks for 8 doses, and then every 4 weeks thereafter until disease progression or unacceptable toxicity. First dose will be administered on Day 1 of Cycle 1. The rationale behind this dosing regimen stems from the GEN501 and SIRIUS trials that evaluated the safety and efficacy of daratumumab monotherapy.10,20 An analysis to understand and justify the recommended dose and dosing schedule for daratumumab in MM patients from a quantitative pharmacologic perspective was done. This analysis found that there was a maximum effect relationship between daratumumab exposure (i.e. maximal trough concentration) and overall response rate (ORR) and between daratumumab and target saturation.  
The daratumumab subcutaneous dose of 1800 mg was chosen based on the COLUMBA study49 which showed that subcutaneous daratumumab was non-inferior to intravenous daratumumab in terms of efficacy and pharmacokinetics and had a consistent safety profile to that of intravenous daratumumab in patients with relapsed refractory multiple myeloma An overall response was seen in 108 (41%) of 263 patients in the subcutaneous group and 96 (37%) of 259 in the intravenous group (relative risk 1·11, 95% CI 0·89–1·37). The geometric means ratio for Ctrough was 107·93% (90% CI 95·74–121·67), and the maximum Ctrough was 593 μg/mL (SD 306) in the subcutaneous group and 522 μg/mL (226) in the intravenous group.  
Azacitidine will be given subcutaneously or IV at standard dose 75 mg/m2 for 5 days consecutively every 4 weeks starting day -7 to day -3 of Cycle 1 (before first dose of daratumumab). The starting dose regimen of 75 mg/m2 is based on prior trials leading to azacitidine approval in MDS and AML, including the azacitidine-00128 and CALGB
8421, 8921, and 9221 trials.30,31 The dosing of azacitidine for 5 days consecutively (instead of 7) will be used for both patient convenience and to minimize toxicity. This is based on a study that showed that 5 days of consecutive azacitidine had similar efficacy to 7 days.39 Azacitidine prescribing information created by Celgene suggests dose adjustments for azacitidine based on hematologic toxicity and our dose de-escalation follows these recommendations.50 Dexamethasone 40 mg will be given orally once weekly on days 1, 8, 15, and 22 of each fourweek cycle starting day 1 of cycle 1 for at least the first two cycles. Dose modifications for azacitidine and dexamethasone based on toxicities are outlined in Section 5.5 There will be no dose modification for daratumumab. Treatment will be continued until progression or patient unable to tolerate treatment due to toxicities.  
  
Figure: Azacitidine upregulates *CD38* transcript expression. (A) Bar graph showing fold change in CD38 RNA (red) and cell surface protein (blue) in RPMI-8226 cells upon drug treatments. Data from 3d+4 and 5d+2 azacytidine treatments are grouped together and compared to ATRA and panobinostat treatment. Data is presented as mean ± SD from a representative experiment from two independent experiments. (B) CD38 expression on primary MM cells upon azacytidine treatment. Grey lines separate different patient samples (IDs on x-axis). Colors in the legend indicate the drug and concentration used. The percentage of CD138+ cells in each sample are given in brackets underneath the IDs. Data are presented as mean ± SD from 3 replicate wells.
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1.5 correlative studies

The correlative studies for this trial stem from preclinical work done in Dr. Arun Wiita's lab at UCSF. Dr. Priya Choudhry and Dr. Arun Wiita's work was recently published in *Leukemia* and is described below.  
1) To determine the *in vivo* change in CD38 expression on plasma cells in MM patients exposed to azacitidine
(primary objective)
Hypothesis: Exposure to azacitidine will result in an increased surface expression of CD38, as determined by flow cytometry. Pre-clinical work done in Dr. Arun Wiita's lab where a panel of MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12- PE) were treated with increasing doses of azacitidine and CD38 cell surface expression was analyzed by flow cytometry, showed that azacitidine consistently increased an increase in D38 median fluorescent intensity (MFI) in all cell lines. Wiita's lab treated cells for either 3 days or 5 days with flow cytometry analysis at 7 days (3d+4 or 5d+ 2, respectively). This extra time allowed for DNMTi incorporation into newly synthesized DNA in replicating cells over two doublings, leading to DNMT degradation and loss of DNA methylation. 3d+4 and 5d+2 treatments induced a 1.22.4 fold increase in CD38 MFI in a dose dependent manner for all four cell lines. 3 uM azacitidine, which is clinically equivalent to 75 mg/m2, consistently induced at least a 2 fold increase in CD38 MFI in all cell lines, in both the 3d+4 and 5d+2 treatments.  
  
Figure: Azacitidine treatment increases CD38 cell surface expression. (A) Bar graph of CD38 expression and cell survival upon treatment with azacytidine in the indicated cell lines. For each cell line the 3d, 3d+4 and 5d+2 treatments are compared side-by-side. Height of the bars indicates the fold change in CD38 MFI (left y-axis) and bold black line shows % cell viability (right y-axis). Both are normalized to DMSO treatment. Colors indicate the concentration of azacytidine used. Data are presented as mean ± SD from 2-3 independent experiments with triplicates in each experiment. * indicates a significant difference from DMSO treatment (p  0.05 using a paired student's t-test). (B) shows the histogram plots for CD38 expression for one representative experiment in XG1 cells. Histograms from 3d+4 azacytidine treatment are compared to the known CD38 upregulators ATRA and Panobinostat. The drugs used are indicated on the left, and fold change in CD38 MFI compared to DMSO control is given on the right. The grey dotted line indicates MFI in DMSO treated cells.  
The preclinical work done in Dr. Wiita's lab further demonstrates that azacitidine also increases CD38 transcription in treated RPMI-8226 cells using qRT-PCR thus leading to its increased expression. Intriguingly, treatment with azacitidine appeared to induce higher fold changes in CD38 expression at the RNA level compared to cell surface expression. This indicates that there may be additional mechanisms regulating CD38 expression posttranscriptionally. Though azacitidine is cleared very rapidly with respect to its pharmacokinetics, we hypothesize that the transcriptional effects of azacitidine will be prolonged and long-lasting due to incorporation into DNMT's and subsequent effects of drug on methylation after cellular division. The following correlative studies will be done contingent upon funding.  
2) To assess *in vivo* changes in whole genome methylation, including CD38 specific methylation, after treatment with azacitidine via performing whole genome reverse-phase bisulfite sequencing.  
Hypothesis: Exposure to azacitidine will result in decreased overall methylation of the plasma cell genome and thus indirectly lead to increased CD38 expression. Historically, determination of DNA methylation was largely confined to only a small fraction of the DNA methylome, usually to regulatory regions like gene promoters which are frequently associated with the CpG-rich CpG islands.51 Initially, based on analysis of publicly-available ENCODE data, Dr. Wiita's lab found a CpG island in the first exon of CD38 and hypothesized that DNA methylation represses CD38 expression. CD38 was previously identified as a differentially methylated region in myeloma patient samples with a negative correlation between gene expression and DNA methylation. They further performed additional analyses of DNA methylation and gene expression data in three independent MM datasets (GSE43860, GSE17306 and GSE12453) and found an inverse correlation between CD38 promoter methylation status and gene expression between normal plasma cells and malignant plasma cells in MM. These findings suggested that DNMTs may regulate CD38 transcription. However, surprisingly in Dr. Wiita's pre-clinical work, targeted bisulfite sequencing of the CD38 CpG island revealed almost complete hypomethylation at baseline in RPMI-8226 and KMS12-PE cells. Thus it was hypothesized that DNMTi treatment might instead upregulate CD38 indirectly. It was tested whether azacitidine upregulates CD38 via transcription factors PU.1 or ATF2 or through activation of interferon response, but this was not found to be true. It was tested whether DNMTi might function via TNF upregulation given TNF is regulated by DNA methylation and is known to upregulate CD38 expression in airway smooth muscle cells. Co-treatment with a TNF-neutralizing antibody completely abrogated azacitidine-induction of CD38 upregulation. Furthermore, azacitidine treatment indeed induced TNF secretion from RPMI-8226 cells. Finally, recombinant TNF increased surface CD38 in RPMI-8226 Phase II - Daratumumab and Azacitidine Page 28 of 95 cells. These results therefore confirm that indirect mechanisms can mediate DNMTi-induced CD38 upregulation and suggest the TNF pathway may play a leading role in this process. We anticipate that there may be other indirect mechanisms by which DNMTi treatment upregulates CD38 and by doing whole genome reverse-phase bisulfite sequencing in patients pre and post azacitidine treatment, we hope to assess for other genes that may be involved in CD38 upregulation that are regulated by methylation. Many approaches to analyze the DNA methylome include a bisulfite conversion step during which unmethylated cytosines are converted to uracil, while methylated cytosines remain as cytosines. Subsequent PCR manifests unmethylated cytosines as thymines, thus, allowing discrimination between the unmethylated and methylated cytosines. The gold standard to address the complete DNA methylome  
  
3) To assess the *in vivo* changes in multiple myeloma tumor microenvironment with treatment of azacitidine via CyTOF
Hypothesis: Azacitidine will lead to changes in the multiple myeloma bone marrow tumor microenvironment that may have immunoregulatory and anti-tumor effects Azacitidine may upregulate expression of CD38 indirectly through altering the bone marrow tumor microenvironment, specifically through upregulation of inflammatory cytokines such as TNF alpha. Given the limited clinical data with regards to the use of azacitidine in multiple myeloma patients, there is very little known about the in vivo impact of azacitidine on bone marrow stromal tumor microenvironment. In leukemia, pre-clinical data shows that azacitidine has a variety of immunomodulatory effects on leukemic cell lines including upregulation of CD86, downregulation of INDO, downregulation of PI-9, downregulation of MHC-class I molecules, upregulation of PD-1, and increase of CXCR4.52Another study looking at azacitidine's epigenetic effects found that azacitidine inhibits T-cell proliferation and activation, blocking cell cycle in the G0 to G1 phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor alpha and interferon gamma. The immunomodulatory effects of azacitidine have lead to an exploration of its role in the allogeneic transplantation setting in prevention of graft versus host disease. Pre-clinical studies have shown that azacitidine has a direct effect on immune response through affecting T-cell activation, proliferation, and secretion of proinflammatory cytokines. The effect of 5-azaC in T lymphocytes, which is caused by both an early effect on the expression of genes related to T-cell proliferation and activation and a delayed effect on the methylation pattern of genes such as *FOXP3*, lead to the hypothesis 5-azaC may play a role in the allogeneic transplantation setting as an immunomodulatory drug.53 A clinical study of 27 patients who had undergone a reduced intensity allogeneic transplantation for acute myeloid leukemia and were treated with monthly courses of azacitidine, found that administration of azacitidine increased the number of Tregs within the first 3 months after transplantation compared with a control population. azacitidine administration also induced a cytotoxic CD8(+) T-cell response to several tumor antigens and thus this study further examined the role of azacitidine after transplantation as a mechanism of augmenting a graft versus leukemia effect without a concomitant increase in graft versus host disease.54 Given that much of Dara's effects are mediated based on interactions with NK cells and macrophages (to clear the CD38-coated plasma cells) or modulating the microenvironment (potentially depleting specific NK subsets and Tregs), examining shifts in immune microenvironment after Azacitidine could play an important role in dissecting clinical effects of azacitidine and may help explain either increased or decreased efficacy of dara combination beyond that expected from CD38 upregulation alone. These findings could also inform possible combinations of azacitidine with other immunotherapies in the future. We propose to use CyTOF (Fluidigm) or mass cytometry to better understand the effects of azacitidine on the bone marrow tumor microenvironment. CyTOF, is a variation of flow cytometry in which antibodies are labeled with heavy metal ion tags rather than fluorochromes. Readout is by time-of-flight mass spectrometry. This allows for the combination of many more antibody specificities in a single samples, without significant spillover between channels.
nct# 04407442
2 study objectives 2.1 hypothesis

We hypothesize that in patients with relapsed and refractory multiple myeloma who have previously progressed on daratumumab, the combination of azacitidine, daratumumab, and dexamethasone will be effective and safe and lead to improved response rates through upregulation of CD38.
nct# 04407442
2.2 primary secondary and exploratory

The primary objective of this study is to evaluate the efficacy of adding azacitidine to daratumumab and dexamethasone, as measured by the overall response rate in patients with relapsed refractory multiple myeloma (RRMM) previously treated with daratumumab.
nct# 04407442
secondary objectives

To evaluate the duration of response per IMWG criteria.  
To evaluate the safety and tolerability of azacitidine in combination with daratumumab and dexamethasone.  
To evaluate progression free and overall survival in patients treated with azacitidine in combination with daratumumab and dexamethasone.  
To assess the changes in CD38 expression on plasma cells after treatment with azacitidine and correlate this change with the depth and duration of response of azacitidine in combination with daratumumab and dexamethasone.
nct# 04407442
2.3 primary secondary and exploratory

Rate of overall response (ORR) defined as sCR+CR+VGPR + PR as best response utilizing IMWG Uniform Response Criteria55 calculated in the time period from initiation of study treatment until discontinuation of treatment
nct# 04407442
secondary endpoints

Adverse events, as graded by National Cancer Institute Common Terminology Criteria for Adverse Events
(NCI CTCAE version 5.0), assessed from the initiation of study treatment until discontinuation of treatment Duration of response (DOR) defined as the duration from the date of initial documentation of a response (PR
or better) according to the IMWG criteria to the date of first documented evidence of progressive disease according to the IMWG criteria or death due to PD whichever occurred first.  
Overall survival which is defined from the date of first dose of study treatment to the date of death due to any cause or censored based on the date of last encounter if patient is alive or lost to follow-up.  
Progression-free survival which is defined as the duration from the date of first dose of study treatment to the date of first documented evidence of progressive disease or death, whichever occurs first.  
Change in CD38 surface expression of plasma cells after azacitidine treatment (the CD38 expression of plasma cells pre and post first cycle of azacitidine treatment in this trial will be compared)
Number of patients who achieve at least 1.5 fold increase in their CD38 expression by flow cytometry after azacitidine treatment Correlation of change in CD38 expression after azacitidine treatment to response including depth of response and duration of response