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Abbreviated Title: Fully-Human Anti-BCMA-CAR T Cells NIH Protocol \#: *18-C-0125* OSP\#: 1807-1780 IBC\#: RD-18-III-02 NCT\#: NCT03602612 Version Date: April 26, 2021 Title: A Phase I Clinical Trial of T Cells Expressing a Novel Fully-Human Anti-BCMA CAR for Treating Multiple Myeloma NCI Principal Investigator: James N. Kochenderfer, M.D.  
Surgery Branch Center for Cancer Research National Cancer Institute, National Institutes of Health 10 Center Drive, Rm. 6B17, MSC 1203 Telephone: 240-760-6062 Email: kochendj@mail.nih.gov  
| Drug Name:    | Anti-BCMA-CARtransduced autologous  peripheral blood  lymphocytes (PBL)                                            | Cyclophosphamide   | Fludarabine                |
|---------------|--------------------------------------------|--------------------|----------------------------|
| IND Number:   | 18246                                      | 18246              | 18246                      |
| Sponsor:      | Center for Cancer                          | Center for Cancer  | Center for Cancer Research |
| Research      | Research                                   |                    |                            |
| Manufacturer: | Department of  Transfusion Medicine  (DTM) | Generic            | Generic                    |
| Supplier:     | Department of  Transfusion Medicine  (DTM) | CC Pharmacy        | CC Pharmacy                |
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precis background

Multiple myeloma (MM) is a malignancy of plasma cells.  
MM is nearly always incurable. T cells can be genetically modified to express chimeric antigen receptors (CARs) that specifically target malignancy-associated antigens.  
Autologous T cells genetically modified to express CARs targeting the B-cell antigen CD19 have caused complete remissions in a small number of patients with leukemia or lymphoma. These results demonstrate that CAR-expressing T cells have anti-malignancy activity in humans.  
B-cell maturation antigen (BCMA) is a protein expressed by normal plasma cells and the malignant plasma cells of multiple myeloma.  
BCMA is not expressed by normal cells except for plasma cells and some mature B cells.
We have constructed a novel anti-BCMA CAR that can specifically recognize BCMAexpressing target cells in vitro and eradicate BCMA-expressing tumors in mice.  
This CAR has an antigen-recognition domain made up of a single fully-human heavy chain variable region.  
We hypothesize that anti-BCMA-CAR-expressing T cells will specifically eliminate BCMAexpressing MM cells in patients Possible toxicities include cytokine-associated toxicities such as fever, hypotension, and neurological toxicities. Elimination of normal plasma cells and unknown toxicities are also possible.
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objectives primary

Determine the safety and feasibility of administering T cells expressing an anti-BCMA CAR
to patients with MM.
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eligibility

Greater than or equal to 18 years of age and less than or equal to age 73.  
Patients must have measurable MM defined as a serum M-protein ≥1.0 g/dL or a urine Mprotein ≥200 mg/24 hours or an involved serum free light chain (FLC) level ≥10 mg/dL
(provided FLC ratio is abnormal) or a biopsy-proven plasmacytoma of 2.0 cm or more in largest dimension, or greater than or equal to 30% bone marrow plasma cells.  
Patients must have previously received at least 3 different treatment regimens for MM. Patients must have prior exposure to an IMiD such as lenalidomide, and a proteasome inhibitor
Patients must have a creatinine level of < 1.5 mg/dL  
Patients must have a cardiac ejection fraction > 50%. An ECOG performance status of 0-2 is required.
Patients on any anticoagulant medications except aspirin are not eligible.  
No active infections are allowed.  
Absolute neutrophil count ≥ 1000/L, platelet count ≥ 55,000/L, hemoglobin ≥ 8g/dL ALT and AST ≤ 2.5-fold higher than the upper limit of normal.  
At least 14 days must elapse between the time of any prior systemic treatment (including corticosteroids) and the required leukapheresis.  
At least 14 days must elapse between the time of any prior systemic treatment (including corticosteroids) and initiation of protocol treatment.  
The patient's MM will need to be assessed for BCMA expression by flow cytometry or immunohistochemistry performed at the NIH. The myeloma must express BCMA. If unstained, paraffin-embedded bone marrow or plasmacytoma sections are available from prior biopsies, these can be used to determine BCMA expression by immunohistochemistry; otherwise patients will need to come to the NIH for a bone marrow biopsy or other biopsy of a plasmacytoma to determine BCMA expression. The sample for BCMA expression can come from a biopsy obtained at any time before enrollment.
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design

This is a phase I dose-escalation trial Patients will undergo leukapheresis  
T-cells obtained by leukapheresis will be genetically modified to express an anti-BCMA
CAR
Patients will receive a lymphocyte-depleting chemotherapy conditioning regimen with the intent of enhancing the activity of the infused anti-BCMA-CAR-expressing T cells.  
The chemotherapy conditioning regimen is cyclophosphamide 300 mg/m2 daily for 3 days and fludarabine 30 mg/m2 daily for 3 days. Fludarabine will be given on the same days as the cyclophosphamide.  
After the chemotherapy ends, the patients will have two days with no treatments and then receive an infusion of anti-BCMA-CAR-expressing T cells.  
The initial dose level will be 0.75x106 CAR+ T cells/kg of recipient bodyweight.
The cell dose administered will be escalated until a maximum tolerated dose is determined.  
Following the T-cell infusion, there will be a mandatory 9-day minimum inpatient hospitalization to monitor for toxicity.  
Outpatient follow-up is planned for 2 weeks, and 1, 2, 3, 4, 6, 9, and 12 months after the CAR T-cell infusion. Afterwards, follow-up will be every six months up to at least 5 years.  
| TABLE OF CONTENTS                                                                                                                                         |                                                                                                                                                        |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS                                                                                                                                                    |                                                                                                                                                        |
| TABLE OF CONTENTS ......................................................................................................................................4 |                                                                                                                                                        |
| STATEMENT OF COMPLIANCE                                                                                                                                   |                                                                                                                                                        |
| l                                                                                                                                                         | INTRODUCTION .......................................................................................................................................10 |
| 1.1                                                                                                                                                       | Study Objectives ...................................................................................................................................10 |
| 1.1.1                                                                                                                                                     | Primary Objective                                                                                                                                      |
| Exploratory Objectives ...........................................................................................................10                      |                                                                                                                                                        |
| 1.1.2                                                                                                                                                     |                                                                                                                                                        |
| 1.2                                                                                                                                                       | Background and Rationale ............................................................................................................10                |
| 1.2.1                                                                                                                                                     | Introduction                                                                                                                                           |
| 1.2.2                                                                                                                                                     | Multiple myeloma epidemiology and standard treatment .............................................11                                                   |
| 1.2.3                                                                                                                                                     | Allogeneic transplantation for MM ......................................................................................12                             |
| 1.2.4                                                                                                                                                     | T-cell gene therapy ...........................................................................................................................12      |
| 1.2.5                                                                                                                                                     | Chimeric antigen receptors                                                                                                                             |
| BCMA                                                                                                                                                      |                                                                                                                                                        |
| 1.2.6                                                                                                                                                     |                                                                                                                                                        |
| 1.2.7                                                                                                                                                     | Previous clinical experience with anti-BCMA CAR T cells ........................................18                                                     |
| 1.2.8                                                                                                                                                     | Anti-BCMA CAR development and preclinical testing .................................................20                                                  |
| 1.2.9                                                                                                                                                     | Rationale for immunosuppressive chemotherapy and selection of lymphocyte-                                                                              |
| depleting chemotherapy regimen                                                                                                                            |                                                                                                                                                        |
| Rationale for dose-escalation and starting dose..............................................................27                                           |                                                                                                                                                        |
| 1.2.10                                                                                                                                                    | Summary of risks and potential benefits ...............................................................................28                              |
| 1.2.11                                                                                                                                                    |                                                                                                                                                        |
| 2                                                                                                                                                         | ELIGIBILITY ASSESSMENT AND ENROLLMENT ..................................................................29                                             |
| 2.1                                                                                                                                                       | Eligibility Criteria .                                                                                                                                 |
| Inclusion Criteria ............................................................................................................................29         |                                                                                                                                                        |
| 2.1.1                                                                                                                                                     |                                                                                                                                                        |
| 2.1.2                                                                                                                                                     | Exclusion criteria ..................................................................................................................30                |
| 2.1.3                                                                                                                                                     | Recruitment Strategies ...............................................................................................................32               |
| 2.2                                                                                                                                                       | Screening Evaluation ..........................................................................................................................32      |
| 2.2.1                                                                                                                                                     | Screening activities performed after a consent for screening has been signed.......32                                                                  |
| 2.3                                                                                                                                                       | Participant Registration and Status Update Procedures                                                                                                  |
| 2.4                                                                                                                                                       | Treatment Assignment Procedures for the Registartion Purposes Only:.........................33                                                         |
| 3                                                                                                                                                         | STUDY IMPLEMENTATION .................................................................................................................34               |
| 3.1                                                                                                                                                       | Study Design                                                                                                                                           |  
4
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abbreviated title fully-human anti-bcma-car t

| Version Date: April 26, 2021                                                                                                                             |                                                                                                                                                   |       |
|----------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------|-------|
| General study plan ...................................................................................................................34                 |                                                                                                                                                   |       |
| 3.1.1                                                                                                                                                    |                                                                                                                                                   |       |
| 3.1.2                                                                                                                                                    | Protocol schema                                                                                                                                   |       |
| 3.1.3                                                                                                                                                    | Dose Limiting Toxicity-the dose limiting toxicity assessment period is 28 days. . 37                                                              |       |
| Dose Escalation ....................................................................................................................37                   |                                                                                                                                                   |       |
| 3.1.4                                                                                                                                                    |                                                                                                                                                   |       |
| 3.2                                                                                                                                                      | Dose Modifications/Delay ................................................................................................................39       |       |
| 3.3                                                                                                                                                      | D Drug Administration                                                                                                                             |       |
| 3.3.1                                                                                                                                                    | Leukapheresis ........................................................................................................................39          |       |
| Anti-BCMA-CAR-expressing T-cell preparation ......................................................40                                                     |                                                                                                                                                   |       |
| 3.3.2                                                                                                                                                    |                                                                                                                                                   |       |
| 3.3.3                                                                                                                                                    | Conditioning chemotherapy and anti-BCMA CAR T-cell administration-                                                                                |       |
| chemotherapy administration can be either inpatient or outpatient .........................................40                                            |                                                                                                                                                   |       |
| Potential repeat treatment ...................................................................................................42                         |                                                                                                                                                   |       |
| 3.3.4                                                                                                                                                    |                                                                                                                                                   |       |
| 3.4                                                                                                                                                      | Protocol Evaluation                                                                                                                               |       |
| Baseline evaluations and interventions                                                                                                                   |                                                                                                                                                   |       |
| 3.4.1                                                                                                                                                    |                                                                                                                                                   |       |
| 3.4.2                                                                                                                                                    | Studies to be performed on Day 0 and during the mandatory 9-day inpatient                                                                         |       |
| admission after cell infusion ........................................................................................................................44 |                                                                                                                                                   |       |
| Post-infusion outpatient evaluation ................................................................................45                                   |                                                                                                                                                   |       |
| 3.4.3                                                                                                                                                    |                                                                                                                                                   |       |
| 3.5                                                                                                                                                      | Study Calendar...................................................................................................................48               |       |
| 3.6                                                                                                                                                      | Gene-therapy-specific follow-up                                                                                                                   |       |
| Cost and Compensation                                                                                                                                    |                                                                                                                                                   |       |
| 3.7                                                                                                                                                      | 3.7.1                                                                                                                                             | Costs |
| 3.7.2                                                                                                                                                    | COMPENSATION .                                                                                                                                    |       |
| 3.7.3                                                                                                                                                    | Reimbursement                                                                                                                                     |       |
| Criteria for Removal from Protocol Therapy and Off Study Criteria                                                                                        |                                                                                                                                                   |       |
| 3.8                                                                                                                                                      |                                                                                                                                                   |       |
| 3.8.1                                                                                                                                                    | Criteria for removal from protocol therapy ...........................................................................54                          |       |
| 3.8.2                                                                                                                                                    | OFF-Study Criteria                                                                                                                                |       |
| 3.9                                                                                                                                                      | S STOPPING CRITERIA                                                                                                                               |       |
| 4                                                                                                                                                        | CONCOMITANT MEDICATIONS/MEASURES                                                                                                                  |       |
| 4.1                                                                                                                                                      | Antibiotic prophylaxis                                                                                                                            |       |
| 4.2                                                                                                                                                      | Blood product support                                                                                                                             |       |
| Anti-emetics                                                                                                                                             |                                                                                                                                                   |       |
| 4.3                                                                                                                                                      | Granulocyte colony-stimulating factor .............................................................................................56             |       |
| 4.4                                                                                                                                                      |                                                                                                                                                   |       |
| 4.5                                                                                                                                                      | Avoidance of corticosteroids ..................................................................................................................56 |       |
| 4.6                                                                                                                                                      | Toxicity management guidlines                                                                                                                     |       |  
S
5 BIOSPECIMEN COLLECTION...........................................................................................56  
| 5.1                                                                                                                                                                                                        | CORRELATIVE STUDIES FOR RESEARCH 56                                          |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------|
| 5.1.1                                                                                                                                                                                                      | Biospecimen collection before the start of the conditioning chemotherapy: 56 |
| 5.1.2                                                                                                                                                                                                      | Biospecimen collection Day 0 and after anti-BCMA-CAR T-cell infusion during  |
| the required hospitalization 57 5.1.3 Biospecimen collection during outpatient follow-up 57 5.1.4 Immunological Testing 58 5.1.5 Additional biopsies and additional blood draws 59 5.1.6 Future studies 59 |                                                                              |
| 5.2                                                                                                                                                                                                        | Sample Storage, Tracking and Disposition 59                                  |
| 5.2.1                                                                                                                                                                                                      | Samples Sent to Figg Lab 59                                                  |
| 5.2.2                                                                                                                                                                                                      | Sample Storage, Tracking and Disposition 60                                  |
| 5.2.3                                                                                                                                                                                                      | Protocol Completion/Sample Destruction 61                                    |
| 5.2.4                                                                                                                                                                                                      | Samples for Genetic/Genomic Analysis 61                                      |
| 6                                                                                                                                                                                                          | DATA COLLECTION AND EVALUATION 62                                            |
| 6.1                                                                                                                                                                                                        | Data Collection 62                                                           |
| 6.1.1                                                                                                                                                                                                      | Adverse event recording 63                                                   |
| 6.2                                                                                                                                                                                                        | DATA SHARING PLANS 63                                                        |
| 6.2.1                                                                                                                                                                                                      | Human Data Sharing Plan 63                                                   |
| 6.3                                                                                                                                                                                                        | Genomic Data Sharing Plan 64                                                 |
| 6.4                                                                                                                                                                                                        | Response Criteria 64                                                         |
| 6.4.1                                                                                                                                                                                                      | Important Considerations on response criteria 64                             |
| 6.4.2                                                                                                                                                                                                      | International Myeloma Working Group uniform response criteria: 65            |  
6.4.3 Minimal residual disease (MRD) criteria (requires complete response as defined above) 67 6.5 Toxicity Criteria..............................................................................................................68  
| 7     | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN 68   |
|-------|-----------------------------------------------------------------|
| 7.1   | Definitions 68                                                  |
| 7.2   | OHSRP Office of Compliance and Training / IRB Reporting 68      |
| 7.2.1 | Expedited Reporting 68                                          |
| 7.2.2 | IRB Requirements for PI Reporting at Continuing Review 68       |
| 7.3   | NCI Clinical Director Reporting 68                              |
| 7.4   | Institutional Biosafety Committee (IBC) Reporting Criteria 68   |  
6 Abbreviated Title: Fully-human Anti-BCMA-CAR T cells  
| Version Date: April 26, 2021                                                                                                               |                                                                                                                                                   |
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| Serious Adverse Event Reports to IBC ............................................................................68                        |                                                                                                                                                   |
| 7.4.1                                                                                                                                      |                                                                                                                                                   |
| 7.4.2                                                                                                                                      | Annual Reports to IBC .........................................................................................................69                 |
| 7.5                                                                                                                                        | NIH Required Data and Safety Monitoring Plan ........................................................................70                           |
| Principal Investigator/Research Team                                                                                                       |                                                                                                                                                   |
| 7.5.1                                                                                                                                      |                                                                                                                                                   |
| 7.5.2                                                                                                                                      | Safety Monitoring Committee (SMC) .....................................................................................70                         |
| 8                                                                                                                                          | Sponsor Protocol/ Safety Reporting ....................................................................................................70         |
| 8.1                                                                                                                                        | Definitions                                                                                                                                       |
| A Adverse Event                                                                                                                            |                                                                                                                                                   |
| 8.1.1                                                                                                                                      |                                                                                                                                                   |
| 8.1.2                                                                                                                                      | Serious Adverse Event (SAE) ...............................................................................................70                     |
| 8.1.3                                                                                                                                      | Life-threatening ..............................................................................................................................71 |
| SEVERITY                                                                                                                                   |                                                                                                                                                   |
| 8.1.4                                                                                                                                      |                                                                                                                                                   |
| 8.1.5                                                                                                                                      | Relationship to Study Product .......................................................................................................71           |
| 8.2                                                                                                                                        | Assessment of Safety Events                                                                                                                       |
| 8.3                                                                                                                                        | Reporting of Serious Adverse Events ..........................................................................................72                  |
| Reporting Pregnancy                                                                                                                        |                                                                                                                                                   |
| 8.4                                                                                                                                        |                                                                                                                                                   |
| 8.4.1                                                                                                                                      | MATERNAL EXPOSURE                                                                                                                                 |
| 8.4.2                                                                                                                                      | Paternal exposure..........................................................................................................................72     |
| Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND.....................73                                                  |                                                                                                                                                   |
| 8.5                                                                                                                                        | CLINICAL MONITORING                                                                                                                               |
| 9                                                                                                                                          |                                                                                                                                                   |
| 10                                                                                                                                         | STATISTICAL CONSIDERATIONS .......................................................................................................73              |
| 10.1                                                                                                                                       | Statistical Hypothesis ........................................................................................................................73 |
| Sample Size Determination ..............................................................................................................74 |                                                                                                                                                   |
| 10.2                                                                                                                                       |                                                                                                                                                   |
| 10.3                                                                                                                                       | Populations For Analyses ..................................................................................................................75     |
| 10.4                                                                                                                                       | Statistical Analyses                                                                                                                              |
| General Approach                                                                                                                           |                                                                                                                                                   |
| 10.4.1                                                                                                                                     |                                                                                                                                                   |
| 10.4.2                                                                                                                                     | Analysis of the Primary Endpoints .......................................................................................75                       |
| 10.4.3                                                                                                                                     | Analysis of the Secondary Endpoint(s) ...............................................................................75                           |
| 10.4.4                                                                                                                                     | Se                                                                                                                                                |
| 10.4.5                                                                                                                                     | Baseline Descriptive Statistics                                                                                                                   |
| 10.4.6                                                                                                                                     | Planned Interim Analyses ............................................................................................................75           |
| 10.4.7                                                                                                                                     | Sub-Group Analyses                                                                                                                                |
| 10.4.8                                                                                                                                     | Tabulation of individual Participant Data                                                                                                         |  
7
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abbreviated title fully-human anti-bcma-car t

| Version Date: April 26, 2021                                                                                                                              |                                                                                                                                                       |                                                                                                                     |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------|
| Exploratory Analyses                                                                                                                                      |                                                                                                                                                       |                                                                                                                     |
| 10.4.9                                                                                                                                                    |                                                                                                                                                       |                                                                                                                     |
| 11                                                                                                                                                        | HUMAN SUBJECTS PROTECTIONS .................................................................................................76                        |                                                                                                                     |
| 1.1                                                                                                                                                       | Rationale For Subject Selection .....................................................................................................76               |                                                                                                                     |
| Participation of Children ..............................................................................................................77                |                                                                                                                                                       |                                                                                                                     |
| 1.2                                                                                                                                                       |                                                                                                                                                       |                                                                                                                     |
| 11.3                                                                                                                                                      | Participation of Subjects Unable to Give Consent ................................................................77                                   |                                                                                                                     |
| 1.4                                                                                                                                                       | Evaluation of Benefits and Risks/Discomforts ..................................................................77                                     |                                                                                                                     |
| 11.4.1                                                                                                                                                    | Risk to family or relatives........................................................................................................78                 |                                                                                                                     |
| Risks of exposure to Ionizing Radiation                                                                                                                   |                                                                                                                                                       |                                                                                                                     |
| 11.4.2                                                                                                                                                    |                                                                                                                                                       |                                                                                                                     |
| 11.4.3                                                                                                                                                    | Risks of Scans and Contrast ......................................................................................................78                  |                                                                                                                     |
| 11.4.4                                                                                                                                                    | Risks of blood Sampling..............................................................................................................78               |                                                                                                                     |
| Risks of Bone marrow aspiration and biopsy .....................................................................78                                        |                                                                                                                                                       |                                                                                                                     |
| 11.4.5                                                                                                                                                    |                                                                                                                                                       |                                                                                                                     |
| 11.4.6                                                                                                                                                    | Risks of Intravenous Catheter                                                                                                                         |                                                                                                                     |
| 11.4.7                                                                                                                                                    | Risks of Apheresis ...............................................................................................................................79  |                                                                                                                     |
| Consent Process and Documentation ......................................................................................79                                |                                                                                                                                                       |                                                                                                                     |
| 11.5                                                                                                                                                      |                                                                                                                                                       |                                                                                                                     |
| 1.5.1                                                                                                                                                     | Consent Process for Adults Who Lack Capacity to Consent to Research                                                                                   |                                                                                                                     |
| Participation                                                                                                                                             |                                                                                                                                                       |                                                                                                                     |
| REGULATORY AND OPERATIONAL CONSIDERATIONS                                                                                                                 |                                                                                                                                                       |                                                                                                                     |
| 12                                                                                                                                                        | STUDY DISCONTINUATION AND CLOSURE ...............................................................................79                                   |                                                                                                                     |
| 12.1                                                                                                                                                      |                                                                                                                                                       |                                                                                                                     |
| 12.2                                                                                                                                                      | QUALITY ASSURANCE AND QUALITY CONTROL                                                                                                                 |                                                                                                                     |
| 12.3                                                                                                                                                      | CONFLICT OF INTEREST POLICY .................................................................................................80                       |                                                                                                                     |
| CONFIDENTIALITY AND P RIVACY .............................................................................................80                              |                                                                                                                                                       |                                                                                                                     |
| 12.4                                                                                                                                                      |                                                                                                                                                       |                                                                                                                     |
| 13                                                                                                                                                        | PHARMACEUTICAL INFORMATION .................................................................................................81                        |                                                                                                                     |
| 13.1                                                                                                                                                      | Retroviral Vector Containing the anti-BCMA CAR Gene ...............................................81                                                 |                                                                                                                     |
| Cells manufacturing                                                                                                                                       |                                                                                                                                                       |                                                                                                                     |
| 13.1.1                                                                                                                                                    |                                                                                                                                                       |                                                                                                                     |
| 13.1.2                                                                                                                                                    | TOXICITIES ........................................................................................................................................82 |                                                                                                                     |
| 13.1.3                                                                                                                                                    | Administration procedures: .....................................................................................................82                    |                                                                                                                     |
| 13.2                                                                                                                                                      | Commercial Agents:                                                                                                                                    |                                                                                                                     |
| Cyclophosphamide .......................................................................................................................82                |                                                                                                                                                       |                                                                                                                     |
| 13.2.1                                                                                                                                                    |                                                                                                                                                       |                                                                                                                     |
| 13.2.2                                                                                                                                                    | FLUDARABINE                                                                                                                                           |                                                                                                                     |
| 14                                                                                                                                                        | REFERENCES .                                                                                                                                          |                                                                                                                     |
| APPENDICES ............................................................................................................................................92 |                                                                                                                                                       |                                                                                                                     |
| 15                                                                                                                                                        | 15.1                                                                                                                                                  | Appendix A:-Performance Status Criteria .........................................................................92 |  
8  
Version Date: April 26, 2021  
| 15.2                                                           | Appendix B:Data Collection Elements Required By Protocol 93                     |
|----------------------------------------------------------------|---------------------------------------------------------------------------------|
| 15.3                                                           | Appendix C: Guidelines for management of common toxicities that occur after CAR |
| T-cell infusions 95 15.4 Appendix D: Infusion Instructions 100 |                                                                                 |  
9
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statement of compliance

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

Determine the safety and feasibility of administering T cells expressing a novel fully-human anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) with a heavy-chainonly antigen-recognition domain to patients with multiple myeloma (MM) and to determine a recommended phase 2 dose of these anti-BCMA CAR T cells 1.1.2 Exploratory Objectives  
Evaluate the *in vivo* persistence and phenotype of T cells expressing the anti-BCMA
CAR.  
Assess for associations between toxicity and immunologic parameters including blood CAR T-cell and cytokine levels.  
Assess gene expression of infused CAR T cells and associations of CAR T-cell gene expression with CAR T-cell *in vivo* persistence and anti-myeloma activity.  
Assess anti-BCMA-CAR-expressing T cells for anti-MM activity post infusion.
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1.2 background and rationale 1.2.1

Multiple myeloma (MM) is a malignancy of plasma cells that is almost always incurable. New therapies are needed for MM. Regressions of MM occurring in the setting of allogeneic stem cell transplantation (alloHSCT), and particularly after allogeneic donor lymphocyte infusions
(DLIs), provide evidence that cellular immune responses can have a clinically significant antimyeloma effect, but alloHSCT is associated with a significant transplant-related mortality and by chronic graft-versus-host disease.1,2 Many patients have obtained lengthy complete remissions of lymphoma or chronic lymphocytic leukemia after infusions of autologous T cells that were genetically modified to express chimeric antigen receptors (CARs) targeting the B-cell antigen CD19.3,4 Anti-CD19 CARs are now FDA-approved treatments. The lasting complete remissions of MM that sometimes occur in the setting of alloHSCT and the encouraging results obtained treating patients with anti-CD19-CAR-expressing T cells are rationales for attempting to develop CAR-T-cell therapies for MM. B-cell maturation antigen (BCMA) is a protein that is expressed by MM cells.5 BCMA is also expressed by normal plasma cells and by some normal B cells, but BCMA is not expressed by other normal cells.5 The very limited expression of BCMA in normal tissues makes BCMA a very promising target for CAR-T-cell therapies. We previously developed the first anti-BCMA CAR, and we have demonstrated that T cells expressing this CAR have BCMA-specific activity *in vitro* and *in vivo*.  
5 Anti-BCMA-CAR-expressing T cells can eradicate MM tumors in mice.5 We then conducted a phase I clinical trial of T cells expressing this first anti-BCMA-CAR. This is a protocol to evaluate a new CAR that differs from our previously-tested anti-BCMA CAR by having an antigen-recognition domain made up only of a fully-human heavy-chain variable domain. The new CAR also differs from the old CAR by having a 4-1BB rather than a CD28 costimulatory domain. The new anti-BCMA CAR trial will enroll patients with advanced MM persisting or progressing despite at least 3 prior therapies. Patients enrolled on the trial will receive a single cycle of chemotherapy that is designed to decrease endogenous lymphocyte counts because extensive evidence exists that depleting endogenous lymphocytes, and possibly other cells, with chemotherapy or total body irradiation dramatically increased the anti-tumor activity of adoptively transferred T cells.6-8 After the lymphocyte-depleting chemotherapy, patients will receive an infusion of autologous anti-BCMA-CAR T cells. The amount of T cellswill escalate with sequential dose levels until a maximum tolerated dose (MTD) is determined. If at least 3 patients are treated on each dose level without an MTD being established, up to 12 additional patients can be treated at the maximum feasible dose. The maximum feasible dose is defined as the dose of CAR T cells that can be feasibly produced.
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1.2.2 multiple myeloma epidemiology and

MM is a neoplasm of monoclonal plasma cells.9,10 For MM to be diagnosed, a patient must have a proliferation of monoclonal plasma cells plus other abnormalities such as anemia, renal insufficiency, hypercalcemia, and lytic bone lesions.10,11 MM is the second most frequently occurring hematologic cancer in the United States (U.S.).10 An estimated 22,350 new cases of MM were diagnosed in the U.S. in 2013, and an estimated 10,710 patients died from MM in the U.S. in 2013.12 Recent improvements in the therapy of MM have occurred.9,10,13 These improved treatments have increased the median survival of patients with newly-diagnosed MM from 3 years to slightly over 5 years, although the prognosis of newly-diagnosed MM patients varies widely.10,13 Current standard non-transplant therapies for MM include various combinations of dexamethasone, bortezomib and its analogs, lenalidomide and its analogs, prednisone, melphalan, and cyclophosphamide.9,10,13,14 Myeloablative doses of chemotherapy followed by autologous stem cell transplantation is a standard therapy for MM patients with good performance status, and adequate bone marrow stem cells.10,13 Compared to standard doses of chemotherapy for first treatment of MM, administration of myeloablative doses of chemotherapy followed by autologous stem cell transplantation improved progression-free survival in most trials and overall survival in some trials.10,13 Myeloablative chemotherapy followed by autologous transplantation can also be used as a treatment for relapsed MM in some cases.10,13,15 Use of the International Uniform Response Criteria for Multiple Myeloma is the most common approach for assessing clinical outcomes in MM.16 Despite the recent improvements in treatment, MM remains an almost always incurable disease.13,17,18 Patients obtaining remissions of MM almost always relapse.13 The median overall survival of patients with relapsed MM is 3 years or less.17 Survival is shorter for patients treated with lenalidomide plus dexamethasone who have received at least 2 prior lines of therapy compared to patients who received only one prior line of therapy.19 In patients with MM that was refractory to bortezomib, the median overall survival was only 9 months when the patient was also either ineligible for thalidomide or lenalidomide, or the patient had MM that was relapsed or refractory to thalidomide or lenalidomide.18 Utilizing the immune system to treat MM is one possible way to improve therapy of MM. So far, the clearest evidence of activity of the immune system against MM
comes from allogeneic transplantation studies.
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1.2.3 allogeneic transplantation for mm

Allogeneic stem cell transplantation (alloHSCT) can cure a fraction of patients with MM.20,21 Myeloablative alloHSCT can induce long-term complete remissions of MM, but also has a transplant-related mortality rate (TRM) of 20% to 50%.20,21 The high TRM of myeloablative alloHSCT led investigators to test nonmyeloablative alloHSCT for MM.20-22 A commonly used transplantation strategy is to administer myeloablative chemotherapy plus an autologous stem cell transplant and then to conduct a nonmyeloablative alloHSCT a short time later.1,20,21 In some studies, this strategy has been shown to yield higher rates of progression-free and overall survival when compared to the strategy of 2 sequential autologous stem cell transplants.1,22 One recent trial showed an 8-year progression-free survival rate of 22% for patients receiving an autologous transplant followed by a nonablative alloHSCT compared to an 8-year progressionfree survival of 12% for patients receiving sequential autologous transplants.1 These results showed a statistically significant advantage for the autologous followed by alloHSCT strategy, but also point out that the vast majority of patients are not cured by either approach. Because many nonmyeloablative transplant regimens that have been used to treat MM include very low doses of radiation or chemotherapy, nonmyeloablative alloHSCTs depend on an immunologic graft-versus-myeloma effect, and remissions of MM that occur after nonmyeloablative alloHSCT provide evidence that immune responses can be effective at eliminating MM.1,20,21 Direct evidence that lymphocytes can eliminate MM comes from donor lymphocyte infusions (DLIs).2,21 Twenty-two to twenty-eight percent of patients receiving DLIs to treat persisting MM after alloHSCT have achieved complete remissions (CRs).2,20,21 Of note, some of these patients obtaining CRs after DLIs did not receive any other therapies around the time of their DLI.2 These results demonstrate the ability of the immune system to eradicate MM.  
Unfortunately, both alloHSCT and DLIs utilize allogeneic lymphocytes; therefore, they are associated with the sometimes fatal complication of graft-versus-host disease2,20,21; in addition most patients receiving DLIs for MM do not obtain CRs, so developing of an effective autologous cellular immune therapy for MM would be a major advance.
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1.2.4 t-cell gene therapy

T cells can be prepared for adoptive transfer by genetically modifying the T cells to express receptors that specifically recognize tumor-associated antigens.23-30 Genetic modification of T
cells is a quick and reliable process, and clinical trials of genetically modified T cells targeting a variety of malignancies have been carried out.3,4,27,31,32 Genetically modified antigen-specific T
cells can be generated from peripheral blood mononuclear cells (PBMC) in sufficient numbers for clinical treatment within <10 days.3 Genetically modifying T cells with gammaretroviruses consistently causes high and sustained levels of expression of introduced genes without in vitro selection4,31,33,34, and genetic modification of mature T cells with gammaretroviruses has a long history of safety in humans.35-37 There are two general approaches for generating antigenspecific T cells by genetic modification: introducing genes encoding natural  T cell receptors
(TCRs) or introducing genes encoding CARs.24,26,27,29 CARs are fusion proteins incorporating antigen recognition moieties and T cell activation domains.28,38-40 The antigen-binding domains of most CARs currently undergoing clinical and preclinical development are antibody variable regions.24,28,38,40 TCRs recognize peptides presented by human leukocyte antigen (HLA)
molecules; therefore, TCRs are HLA-restricted, and a particular TCR will only be useful in patients expressing certain HLA molecules24,26,27,38, which limits the number of patients who could be treated with T cells genetically modified to express a TCR. In contrast, CARs recognize intact cell-surface proteins and glycolipids, so CARs are not HLA-restricted, and CARs can be used to treat patients regardless of their HLA types.24,27,41-43
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1.2.5 chimeric antigen receptors

Preclinical experiments evaluating CAR-expressing T cells as cancer therapy were initiated in 1993.44,45 These experiments led to a clinical trial of CAR-transduced T cells targeting the -
folate receptor on ovarian cancer cells; no tumor regressions were observed during this clinical trial.46 CARs that were capable of recognizing a variety of tumor-associated antigens have been evaluated in many centers.24,38 Preclinical studies have assessed a wide variety of factors that could affect in vivo function of CAR-expressing T cells. Multiple approaches for inserting CAR
genes into T cells by using gammaretroviruses4,31,33-35,47-49, lentiviruses3,50-53, or transposon systems54,55 have been assessed. Because all methods of T-cell genetic modification require a period of in vitro culture, various T-cell culture techniques have been evaluated.3,47,56 Different portions of CARs including antigen-recognition moieties, extracellular structural components, costimulatory domains such as the cytoplasmic portion of the CD28 protein, and T-cellactivation moieties such as the signaling domains of the CD3 protein can all be important to the in vivo function of CAR-expressing T cells, and all of these portions of CARs remain the subject of intensive investigation.38,47,51,57-59 Much of the preclinical work evaluating CARs has been performed with CARs targeting the B-cell antigen CD19.7,47-49,54,60,61 Data suggesting that T-cell costimulation played an important role in the activity of CAR-expressing T cells in vivo led investigators to add signaling moieties from the costimulatory molecule CD28 to CARs.49,58 These studies showed that adding CD28 moieties to CARs enhanced antigen-specific cytokine production and proliferation by anti-CD19 CAR T cells.58,62,63 T cells expressing CARs with CD28 signaling moieties and CD3 signaling domains were more effective than T cells expressing CARs without CD28 moieties at eradicating human leukemia cells from mice.62,63 Subsequently, CARs incorporating other signaling domains from costimulatory molecules such as 4-1BB (CD137) were developed.50 Anti-CD19 CARs containing the signaling domains of both 4-1BB and CD3 were superior to CARs containing the signaling domains of CD3 without any costimulatory domains at eradicating human malignant cells from mice.51,57 Similar to CD28, including 4-1BB signaling moieties in CARs led to increased CD19-specific proliferation and enhanced *in vivo* persistence.51 In contrast to T cells expressing a CAR with a CD28 moiety, the increased in vitro proliferation and prolonged in vivo persistence of T cells expressing a 41BB-containing CAR occurred whether or not the T cells were exposed to the antigen that the CAR recognized.51,57 Results from several clinical trials of anti-CD19 CAR T cells have been reported to date in peerreviewed papers.3,4,31,33,34,64-67 The first evidence of antigen-specific activity of anti-CD19 CAR
T cells in humans was generated during a clinical trial at the National Cancer Institute (NCI) in a patient who experienced a dramatic regression of advanced follicular lymphoma.33 This clinical trial utilized a gammaretroviral vector to introduce an anti-CD19 CAR containing the signaling domains of the CD28 and CD3 molecules.33 The first patient treated on this protocol had a large disease burden of follicular lymphoma. This first patient experienced no acute toxicities except for a low grade fever that lasted for 2 days, and he obtained a partial remission (PR) that lasted for 32 weeks.33 Bone marrow biopsies revealed a complete elimination of extensive bone marrow lymphoma that was present before treatment; in addition, normal B-lineage cells were completely eradicated from the bone marrow.33 The bone marrow B-cell eradication was confirmed by flow cytometry, and it persisted for over 36 weeks.33 B cells were also completely absent from the blood during this time, while T cells and other blood cells recovered rapidly.33 Seven months after the anti-CD19 CAR T cell infusion, progressive lymphoma was detected in the patient's cervical lymph nodes. The lymphoma remained CD19+, so the patient was treated a second time with anti-CD19 CAR T cells. The first and second treatment regimens were the same except the patient received a higher dose of cells with the second treatment. After the second treatment, the patient obtained a second ongoing partial remission.4 Seven more patients were subsequently treated with the same regimen of chemotherapy, antiCD19 CAR T cells, and high-dose IL-2.4 In 4 of 7 evaluable patients on the trial, administration of anti-CD19 CAR T cells was associated with a profound and prolonged B-cell depletion.4,33 In all 4 patients with B-cell depletion, the depletion lasted for over 36 weeks. The B-cell depletion could not be attributed to the chemotherapy that was administered because blood B-cells recovered to normal levels in 8 to 19 weeks in patients receiving the same chemotherapy plus infusions of T cells targeting NY-ESO or gp100, which are antigens that are not expressed by B
cells.33 Because normal B cells express CD19, prolonged normal B-cell depletion after antiCD19 CAR T-cell infusions demonstrated that CAR-expressing T cells had a powerful ability to eradicate CD19+ cells in humans. All of the patients with long-term B-cell depletion obtained either complete or partial remissions of their malignancies, and the 4 patients with long-term B cell depletion also developed hypogammaglobulinemia. Hypogammaglobulinemia in these patients was routinely treated with infusions of intravenous immunoglobulins. Of the eight patients treated, seven patients were evaluable for malignancy response; the one patient who was not evaluable died with pneumonia caused by influenza A.4 Six of the seven evaluable patients had remissions of their malignancies. Two of the remissions were complete remissions (CRs) of CLL.4 Both of these CRs were confirmed by multicolor flow cytometry of bone marrow cells.4 One of these CRs lasted 24 months, and the other is ongoing at 30 months.4 Most patients treated with this regimen of chemotherapy, anti-CD19 CAR T cells, and IL-2 experienced significant acute toxicities including fever, hypotension, and neurological toxicities such as delirium and obtundation.4 All of these toxicities peaked within 10 days after the cell infusion and resolved less than 3 weeks after the cell infusion.4 These acute toxicities correlated with serum levels of the inflammatory cytokines tumor necrosis factor and interferon-, and T cells producing these inflammatory cytokines in a CD19-specific manner were detected in the blood of patients after the anti-CD19 CAR T cell infusions.4 Subsequent studies with this same CAR
demonstrated the effectiveness of anti-CD19 CAR T cells against diffuse large B-cell lymphoma and led to multicenter phase II trials and an application for FDA approval.66
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1.2.6 bcma

One candidate antigen for immunotherapies for MM is BCMA (CD269) 68,69. BCMA RNA was detected universally in MM cells, and BCMA protein was detected on the surface of plasma cells from MM patients by several investigators 70-73. BCMA is a member of the tumor necrosis factor receptor (TNF) superfamily 74,75. BCMA binds B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) 75-77. Among nonmalignant cells, BCMA has been reported to be expressed mostly by plasma cells and a small subset of mature B cells 68,69,76,78,79.  
Mice deficient in BCMA were healthy and had a normal physical appearance 80,81. BCMAdeficient mice had normal numbers of B cells, but survival of long-lived plasma cells was impaired 78,80.  
A critical factor for any antigen being considered as a target for immunotherapies is the antigen's expression pattern in normal tissues. BCMA has been reported to be expressed in plasma cells and in some B cells but to otherwise have limited expression 68-70,79. To more completely assess the expression pattern of BCMA, we performed quantitative polymerase chain reaction (qPCR)
on a panel of cDNA samples from a wide range of normal tissues (**Figure 1**).5 As a positive control, we performed qPCR on cDNA from cells of a plasmacytoma that was resected from a patient with advanced MM.5 Ninety-three percent of the cells from the plasmacytoma sample were plasma cells as determined by flow cytometry. The BCMA expression of the plasmacytoma sample was dramatically higher than the BCMA expression of any other tissue (**Figure 1**). Not surprisingly, BCMA cDNA copies were detected in several hematologic tissues such as blood leukocytes, bone marrow, spleen, lymph node, and tonsil. Low levels of BCMA cDNA copies were detected in the samples of testis and trachea. In addition, low levels of BCMA cDNA copies were detected in most gastrointestinal organs such as duodenum, rectum, and stomach (**Figure 1**). One possible explanation for BCMA expression in gastrointestinal organs and the trachea was the known presence of plasma cells and B cells in tissues such as lamina propria and Peyer's Patches 82,83.  
In **Figure 1**, actin cDNA copies and BCMA cDNA copies were measured by qPCR in all of the samples, and the results were expressed as the number of BCMA cDNA copies per 105 actin cDNA copies.5 Figure 1  
Figure 1  
  
Table 1 Organs stained for BCMA by immunohistochemistry and found to lack of BCMA expression except on plasma **cells**  
| adrenal bladder bone breast cerebellum cerebral cortex duodenum eye fallopian tube esophagus stomach small intestine colon rectum heart kidney   |
|--------------------------------------------------------------------------------------------------------------------------------------------------|  
For a protein to be an appropriate target for CAR-expressing T cells aimed at MM, the protein  
 must be expressed on the surface of MM cells. By flow cytometry of bone marrow samples from patients with MM, we found that 6/10 tested samples uniformly expressed high levels of BCMA by flow cytometry. An example of this flow cytometry staining is shown in **Figure 2**.  
| lung ovary pancreas parathyroid pituitary placenta prostate skin spinal cord spleen skeletal muscle testis thymus thyroid trachea cervix   |
|--------------------------------------------------------------------------------------------------------------------------------------------|  
  
Figure 2  

As shown in **Figure 2** flow cytometry for BCMA (solid line) and isotype-matched control staining (dashed line) revealed BCMA expression on the surface of MM cells from a plasmacytoma of a patient with MM.5 The plot is gated on plasma cells, which made up 93% of the total plasmacytoma cells. In addition to the flow cytometry studies, we stained tissue sections from plasmacytomas of 3 patients with MM. In all 3 of the samples, the neoplastic plasma cells expressed cell-surface BCMA.5 1.2.7 Previous clinical experience with anti-BCMA CAR T cells This pre-clinical work led to the first in-humans clinical trial of CAR-Ts targeting BCMA at the NCI.84 An anti-BCMA CAR containing a murine scFv, a CD8 hinge and transmembrane region, a CD28 costimulatory domain, and a CD3 T-cell activation domain was created by synthesizing the appropriate DNA and ligating it into a gamma-retroviral backbone.84 Patients who had MM with uniform BCMA expression by either IHC or flow cytometry were enrolled.84 All participants received conditioning chemotherapy prior to infusion of CAR T cells with goals of depleting endogenous leukocytes, including depletion of regulatory T cells, and increasing serum cytokine levels of IL-15 and IL-7 to increase CAR-T activity.84-87 The chemotherapy regimen used was cyclophosphamide 300 mg/m2 and fludarabine 30 mg/m2 given daily on days -5 to -3 followed by an infusion of CAR-BCMA T cells on day 0.84 Twenty-six patients have received CAR-BCMA T cells treated with either 0.3x106, 1x106, 3x106, or 9x106 CAR+ T cells/kg of bodyweight; 16 patients were treated on the highest dose level. Five patients had a history of poor-prognosis chromosome 17p-deletion prior to protocol enrollment; results of 10 of these patients have been published.84 Toxicities were mild in patients receiving lower doses of CARTs (0.3-3.0x106 CAR-T/kg).84 As the dose of CAR-T escalated, patients began to have more symptoms of cytokine release syndrome.84 Patients treated at the highest dose level (9x106 CAR+T-cells/kg) experienced cytokine-release syndrome and a variety of grade 3 and 4 toxicities including neutropenia, thrombocytopenia, hypotension, and acute kidney injury that were managed without long-lasting complications.84 While CAR-BCMA T-cell toxicity was severe in some patients treated at the highest dose level, toxicities were mainly limited to the first two weeks after CAR-BCMA T-cell infusion. Because of Grade 3 and 4 CRS experienced by some patients with high bone marrow myeloma burdens, the clinical protocol was modified to only allow enrollment of patients with lower myeloma burdens defined as MM making up 30% or less of bone marrow cells. Two patients experienced delayed neutropenia and thrombocytopenia that started approximately one month after CAR T-cell infusion when blood counts had recovered from the chemotherapy administered before CAR-BCMA T-cell infusions. These patients were treated with filgrastim, eltrombopag, and prednisone based on the hypothesis that the cytopenias were caused by CAR T cells in the patient's bone marrow. At the time of delayed cytopenias, 15 percent of the first patient's bone marrow T cells were CAR+, and 16.5 percent of the second patient's bone marrow T cells were CAR+. In both cases, cytopenias resolved after approximately 1 month. A third patient had milder delayed thrombocytopenia and neutropenia that were treated with eltrombopag and G-CSF without corticosteroids. Of the 10 patients treated with lower doses of anti-BCMA CAR-Ts, one patient experienced a VGPR that lasted eight weeks, eight patients had SD that lasted from two to 12 weeks and one patient had a transient PR.84 Partial loss of BCMA expression on MM cells was demonstrated in one patient.84 13 of 16 evaluable patients treated at the highest dose level obtained objective anti-myeloma responses with 2 stringent complete responses, 8 very good partial responses, and 3 partial responses; the duration of responses varied. The longest response to date is 75 weeks. Eight of 10 evaluable patients treated with the highest dose of CAR-BCMA T cells obtained minimal residual disease negative status by bone marrow flow cytometry. Consistent with BCMA-specific T-cell activity, plasma cells were reduced on bone marrow core biopsies in all 8 evaluated patients and absent in 4 of these patients 2-3 months after CAR-BCMA T-cell infusion. Of the five patients who had a history of the poor-prognosis chromosome 17p-deletion prior to protocol enrollment, four obtained a PR or better. These results demonstrate that CARBCMA T cells can induce responses in poor-prognosis MM.  
A common concern raised with CAR-T therapy is whether soluble antigen may hinder efficacy. In the NCI trial, serum BCMA protein did not eliminate CAR-T cell activity against MM; moreover, serum BCMA levels were observed to drop significantly in patients who obtained responses to anti-BCMA CAR T cells.84 In agreement with pre-clinical work, the phase I clinical trial with anti-BCMA CART cells did not demonstrate undue damage to nonhematopoietic organs after infusions of T-cells.84,88 Cohen *et. al* at the University of Pennsylvania have published their initial findings from a phase I trial of anti-BCMA CAR-T without conditioning chemotherapy in patients with relapsed or refractory MM.89 One patient was treated with 1.8x108 CAR T cells, two with 2x108 CAR T cells and three patients at the highest dose level of 5x108 CAR T cells.89 Toxicities were similar to those published by Ali *et. al* from the NCI experience, except for an episode of grade 4 posterior reversible encephalopathy syndrome (PRES) that manifested as severe delirium, seizures, obtundation and cerebral edema.89 This was treated with steroids, anti-epileptics, and cyclophosphamide; there was no long-term neurological dysfunction.89 Reported results show one patient has an ongoing stringent CR at seven months with an MRD-negative bone marrow by flow cytometry.89 One patient who had pleural and dural MM involvement was found to have CAR-Ts cells in pleural fluid and CSF and achieved VGPR with resolution of extramedullary disease; this patient's VGPR lasted 5 months, and progression was associated with loss of BCMA expression on MM cells.89 Four patients had modest to minimal in vivo CAR T-cell proliferation and minimal to no anti-myeloma response.89 Of note, the trial reported by Cohen et al. utilized an anti-BCMA CAR with fully-human antibody variable regions. Full trial methods and results have yet to be published. A third anti-BCMA CAR-T clinical trial is a multicenter trial being conducted by bluebird bio Inc.90 Twenty-one patients with relapsed/refractory MM have been infused with bb2121, an antiBCMA CAR-T that expresses a CAR with the same scFv as the anti-BCMA CAR used at the NCI.84 In contrast to the NCI anti-BCMA CAR, bb2121 has a 4-1BB costimulatory motif, and it is encoded by a lentivirus.90 Bb2121 is administered after cyclophosphamide plus fludarabine lymphocyte-depleting chemotherapy.90 To date, the authors report that the safety profile of bb2121 has been favorable through doses as high as 800x106 CAR-Ts with no DLT observed.90 Seventy-one percent of patients have had CRS that was generally mild.90 Efficacy of bb2121 in this study has been promising. Of 3 patients receiving a CAR T-cell dose of 50x106 CAR T
cells, one patient obtained a PR, but no patients obtained responses of VGPR or CR.90 Among 18 patients receiving CAR T-cell doses greater than 50 x106 CAR-Ts, 15 patients have reached at least 60 days of follow-up, and 11 of 15 patients with at least 60 days of follow-up have obtained best responses of VGPR or CR.90 All patients with at least 60 days of follow-up receiving a cell dose of greater than 50 x106 CAR-Ts have obtained at least a PR.90 Fan *et al.* have reported early results from an on-going phase I anti-BCMA CAR trial that is enrolling patients with relapsed or refractory MM.91 The CAR used in this study is called LCARB38M. Among 19 patients reported, there was a 100% overall response rate.91 Eighteen patients obtained a stringent CR or a VGPR after receiving anti-BCMA CAR-T.91 CRS occurred in 74% of patients but was noted to be mild in most patients.91
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1.2.8 anti-bcma car development and

The anti-BCMA CAR to be assessed in this protocol contains an antigen recognition domain made up only of a single fully-human variable heavy chain (FHVH) domain rather than the traditional CAR antigen-recognition domain of a single-chain variable fragment (scFv). The FHVH33 heavy-chain variable region sequence was obtained as part of a research agreement with TeneoBio Inc. TeneoBio derived this heavy chain variable region by immunizing a proprietory rat that is transgenic for human heavy chain variable region domains. TeneoBio then selected heavy chain variable domains that bound with high affinity to human BCMA. Investigators in the laboratory of James Kochenderfer then generated 9 different CARs that each incorporated one of 4 different heavy-chain-only anti-BCMA CARs with different costimulatory domains. These CARs were assessed for specificity of antigen recognition and a wide variety of in vitro and in vivo functions. From these experiments the FHVH33 heavy-chain domain was found to be the one with the most sensitive and specific functional recognition of BCMA. After selecting FHVH33, the optimal overall CAR design was determined for evaluation in a clinical trial. The selected anti-BCMA CAR contains the hinge and transmembrane regions of the human CD8 molecule, the signaling moiety of the 4-1BB costimulatory molecule, and the signaling domains of the CD3 molecule. The CAR is designated FHVH33-CD8BBZ (**Figure**
3). Some of the experiments demonstrating the function of FHVH33-CD8BBZ are described in this section of this protocol. The clinical gene therapy vector encoding FHVH33-CD8BBZ is MSGV, which has been used in many prior clinical trials at the NCI.66,92 Figure 3  
  

Figure 4 shows anti-BCMA CAR expression on T cells from a multiple myeloma patient 7 days after transduction with gammaretroviruses encoding the anti-BCMA CAR. Transductions were carried out 2 days after the cultures were started, so the T cells had been in culture for a total of 7 days at the time of this analysis. The plots are gated on live, CD3+ lymphocytes.  
We also performed a series of in vitro assays to assess the function of anti-BCMA-CARexpressing T cells, and we found that FHVH33-CD8BBZ-CAR-expressing T cells exhibited BCMA-specific activities including CD107a upregulation and cytokine production in vitro.  
These experiments showed that anti-BCMA-CAR-expressing T cells are activated in a BCMAspecific manner.  
  
Figure 5 shows upregulation of CD107a, which indicates degranulation and correlates with cytotoxicity93 when anti-BCMA CAR-expressing T cells were cultured with the BCMAexpressing cell line BCMA-K562. CD107a was not upregulated when anti-BCMA-CARexpressing T cells were cultured for 4 hours with the BCMA-negative control cell line NGFRK562. Untransduced T cells did not upregulate CD107a when cultured with either BCMA-K562 or NGFR-K562 (not shown). The plots are gated on live CD3+ lymphocytes. T cells transduced with the FHVH33-CD828Z anti-BCMA CAR produced large amounts of IFN when they were cultured overnight with the BCMA-expressing cell lines BCMA-K562 and RPMI8226 (**Figure**
6). In contrast, the anti-BCMA CARs produced only background levels of IFN when they were cultured with the BCMA-negative target cell lines NGFR-K562, CCRF-CEM, and 293-GP. The anti-BCMA-CAR-transduced T cells also made minimal IFN when cultured without any target cell. The experiment depicted in **Figure 6** consisted of culturing the anti-BCMA-CARtransduced T cells with the indicated target cell lines overnight and then performing a standard IFN enzyme-linked immunosorbant assay (ELISA) to detect IFN in the culture supernatant.  
Figure 6  
  
A second ELISA experiment performed in a similar manner utilizing FHVH33-CD8BBZ antiBCMA CAR T cells from a different patient is shown in **Table 2**. In the experimental results shown in **Table 2**, three different types of effector T cells from the same multiple myeloma patient were evaluated, untransduced T cells, T cells expressing the FHVH33-CD828Z CAR, and T cells expressing the FHVH33-CD8BBZ CAR. FHVH33-CD828Z is identical to FHVH33-CD8BBZ except that FHVH33-CD828Z has a CD28 costimulatory domain while FHVH33-CD8BBZ has a 4-1BB costimulatory domain. The untransduced T cells expressed only background levels of IFN against all target cells. T cells expressing either FHVH33-
CD828Z or FHVH33-CD8BBZ specifically recognized BCMA as shown by the high levels of IFNg released when the CAR-expressing T cells were cultured with the BCMA+ target cells BCMA-K562 an RPMI8226; in contrast, the CAR-expressing T cells produced much lower levels of IFNg when cultured with the BCMA-negative target cell lines Panc10.05, U251, 293GP, and the primary cells normal human bronchial epitheial cells (NHBE), human microvascular endotherlial cells (HMVEC), and primary human intestinal epithelial cells
(InEpC). T cells alone made low levels of IFN for all T cell types evaluated. Compared to T
cells expressing FHVH33-CD828Z, T cells expressing FHH33-CD8BBZ produced higher levels of IFN when cultured with the RPMI8226 cell line. The RPMI8226 cell line expresses low levels of BCMA. In many IFN ELISA experiments and other functional assays, T cells expressing FHVH33-CD8BBZ have shown a greater ability than T cells expressing FHVH33- CD828Z to be activated by RPMI8226 cells. These results suggest that compared to FHVH33- CD828Z, T cells expressing FHVH33-CD8BBZ have a greater ability to be activated by target cells expressing low levels of BCMA. Another point made by the data in **Table 2** is that CARs containing a 4-1BB moiety produce low levels of IFN when cultured alone or with BCMAnegative target cells. This production of IFNg with CAR T cells cultured in the absence of the CAR target antigen is a very consistent finding with 4-1BB CARs that target a variety of antigens including anti-CD19 CARs in clinical use and other anti-BCMA CARs.51 Table 2 BCMA+ **BCMA-negative**  
| BCMA- K562        | RPMI- 8226   | Panc10. 05   | U251   | 293GP   | NHBE   | HMVEC   | InEpC   | T-cells   |    |
|-------------------|--------------|--------------|--------|---------|--------|---------|---------|-----------|----|
| Effector T cell   | Only         |              |        |         |        |         |         |           |    |
| Untransduced      | 179          | 406          | 76     | 41      | 92     | 54      | 54      | 33        | 25 |
| FHVH33-828Z 38548 | 13855        | 37           | 24     | 40      | 43     | 25      | 18      | 14        |    |
| FHVH33-8BBZ 70216 | 33465        | 306          | 360    | 238     | 423    | 610     | 500     | 367       |    |
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all values are pg/ml of

Table 3 shows ELISA results from an experiment in which effector T cells were cultured in the presence of BCMA protein at concentrations ranging from 150 ng/mL to 0 ng/mL. Effector T cells used in this experient were T cells from the same patient transduced with the SP6-CD828Z negative-control CAR, FHVH33-CD828Z, or FHVH33-CD8BBZ. Target cells used in this experiment were either BCMA-K562 cells or RPMI8226 cells or the BCMA-negative NGFRK562 cells. The T cells and target cells were cultured together overnight, and IFN was measured in the culture supernatant by a standard ELISA assay. The presence of soluble BCMA protein did not block recognition of the BCMA+ target cells, and BCMA protein also did not cause nonspecific activation of the FHVH33-CD8BBZ T cells. All numbers are pg/mL IFN.  
Table 3  
  
  
FHVH33-CD8BBZ
cells or BCMA-negative NGFR-K562 cells. CFSE was diluted to a greater degree, indicating more proliferation, when the T cells were cultured with BCMA-K562 target cells (open histogram in **Figure 8**) compared to when anti-BCMA-CAR T cells were cultured with BCMAnegative NGFR-K562 target cells (solid histogram in **Figure 8**). Note that the solid histogram representing culture of FHVH33-CD8BBZ T cells with NGFR-K562 cells is smaller than the open histogram representing culture of FHVH33-CD8BBZ T cells with BCMA-K562 cells because fewer T cells that were cultured with NGFR-K562 were left alive at the end of the assay.  
The assay was conducted as described previously.5 Figure 8 also includes a graph of CAR T-cell number at the beginning and at the end of the CFSE proliferation assay. The number of T cells transduced with FHVH33-CD8BBZ was compared to the number of T cells expressing FHVH33-CD828Z. In multiple experiments, we consistently found superior in vitro proliferation of T cells expressing FHVH33-CD8BBZ compared with T cells expressing FHVH33-CD828Z.  
Figure 8  
  
Figure 9  
  
MSGV-FHVH33-CD8BBZ
In the experiments depicted in **Figure 9** and **Figure 10**, anti-BCMA-CAR T cells were infused on Day 0, and no other treatments were administered. In the experiments reported in **Figure 9** and **Figure 10**, there were 5 mice in all groups.  
  
1.2.9 Rationale for immunosuppressive chemotherapy and selection of lymphocyte-depleting chemotherapy regimen We plan to administer a conditioning chemotherapy regimen of cyclophosphamide and fludarabine before infusions of anti-BCMA-expressing T cells because substantial evidence demonstrates an enhancement of the anti-malignancy activity of adoptively-transferred T cells when chemotherapy or radiotherapy are administered before the T cell infusions.6,8,94 In mice, administering chemotherapy or radiotherapy prior to infusions of tumor-antigen-specific T cells dramatically enhanced the anti-tumor efficacy of the transferred T cells.6,8,26,27,94,95 Administering chemotherapy or radiotherapy enhances adoptive T-cell therapy by multiple mechanisms including depletion of regulatory T cells and elevation of T-cell stimulating serum cytokines including interleukin-15 (IL-15) and interleukin-7 (IL-7), and possibly depletion of myeloid suppressor cells and other mechanisms.6,8,95,96 Removal of endogenous "cytokine sinks" by depleting endogenous T cells and natural killer cells caused serum levels of important T-cell stimulating cytokines such as IL-15 and IL-7 to increase, and increases in T-cell function and anti-tumor activity were dependent on IL-15 and IL-7.6 Experiments in a murine xenograft model showed that regulatory T cells could impair the anti-tumor efficacy of anti-CD19 CAR T
cells.97 Myeloid suppressor cells have been shown to inhibit anti-tumor responses.96 Experiments with a syngeneic murine model showed that lymphocyte-depleting total body irradiation (TBI) administered prior to infusions of anti-CD19-CAR-transduced T cells was required for the T cells to cure lymphoma.7 In these experiments, some mice received TBI, and other mice did not receive TBI. All mice were then challenged with lymphoma and treated with syngeneic anti-CD19-CAR T cells. Mice receiving TBI had a 100% cure rate and mice not receiving TBI had a 0% cure rate.7 Strong suggestive evidence of enhancement of the activity of adoptively-transferred T cells has been generated in humans.31,98,99 Very few clinical responses have occurred and very little evidence of in vivo activity has been generated in clinical trials of anti-CD19-CAR T cells administered without lymphocyte-depleting chemotherapy.31,34 In contrast, many regressions and evidence of long-term B-cell depletion have occurred in clinical trials in which patients received anti-CD19-CAR T cells after lymphocyte-depleting chemotherapy.3,4,33,52 The chemotherapy regimen that best increases the anti-malignancy efficacy of CAR-expressing T cells is not known, but the most commonly used chemotherapy regimens that have been used in clinical trials and that convincingly demonstrate persistence and in vivo activity of adoptively transferred T cells have included cyclophosphamide and fludarabine.3,4,32,33,98,99 Both cyclophosphamide and fludarabine are highly effective at depleting lymphocytes.98,99 One wellcharacterized and commonly used regimen is the combination of 300 mg/m2 of cyclophosphamide administered daily for 3 days and fludarabine 30 mg/m2 administered daily for three days on the same days as the cyclophosphamide.100 Multiple cycles of this regimen can be tolerated by heavily pretreated leukemia patients.66,100
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1.2.10 rationale for dose-escalation and

The clinical trial described in this protocol is planned as a dose escalation in which the number of anti-BCMA-CAR T cells administered to patients will be increased with sequential dose levels. The rationale for conducting a dose-escalation trial of a cellular therapy is based on two main sources of evidence. First, the anti-tumor efficacy of adoptively-transferred T cell treatments increases as the dose of T cells administered to mice increases.101-103 Second, in the setting of allogeneic transplantation, relapsed malignancy is often treated with infusions of allogeneic donor lymphocytes (DLIs).104,105 The incidence of graft-versus-host disease, which is caused by T cells attacking allogeneic antigens on host tissues, increases as the dose of T cells administered in DLIs increases'.104,105 The starting CAR T-cell dose of 0.75x106 CAR+ T cells/kg is based on experience in past CAR
clinical trials. The 0.75x106 CAR+ T cells/kg dose is considered a dose lower than that at which substantial toxicity has been observed. We believe starting at a lower dose is prudent given that this is the first trial of a heavy-chain-only CAR. The following clinical trials were used to guide the starting dose of this trial. Note that it was our goal to use a dose lower than the MTD of prior trials. NCI protocol 09-C-0082 (Kochenderfer et al Journal of Clinical Oncology, 2017), a trial of anti-CD19 CAR T cells had an MTD of 2x106 CAR+ T cells/kg.66 NCI protocol 14-C-0168, a trial of anti-BCMA CAR T cells had an MTD of 9x106 CAR+ T cells/kg.106 A multicenter trial of anti-BCMA CAR T cells conducted by bluebird bio, Inc. determined that a flat dose of 450 x106 CAR+ T cells was the optimal dose for the expansion phase of the study (unpublished results). The 450 x 106 flat dose of CAR T cells would be 4.5x106 CAR+ T cells/kg for a 100 kg patient. Because the CAR to be tested in the current trial and the CAR tested in the bluebird bio trial both contain a 4-1BB domain, we predict the activity of the CAR to be tested in the current trial, FHVH33-CD8BBZ will be most like the CAR in the bluebird trial.
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1.2.11 summary of risks and

This clinical trial is being performed to evaluate a genetically-modified T-cell therapy for multiple myeloma, which is an almost always incurable disease.13 Only patients with multiple myeloma who have persisting or relapsed myeloma despite at least 3 prior lines of therapy will be enrolled. The risks of the study fall into 4 general categories. First, chemotherapy that could cause cytopenias is part of the protocol. As with any chemotherapy that causes neutropenia and thrombocytopenia, this chemotherapy could cause toxicities such as infections and bleeding. The second category of toxicity is cytokine-release-type toxicities such as high fevers, hypotension and neurological toxicities such as delirium, obtundation, myoclonus, seizures, headache, and transient focal neurological toxicities including aphasia and focal paresis. These cytokinerelease-type toxicities have been detected in other clinical trials of CAR T cells during the first 2 weeks after anti-CD19 CAR T cells or anti-BCMA T cells were infused.4,106,107 In most cases, these toxicities have been transient with toxicities generally resolving within 2 days to 2 weeks. The third main category of toxicity is direct damage to normal tissues by the CAR T cells. This could happen because of unexpected cross-reactivity of the anti-BCMA CAR with proteins other than BCMA in vivo. We have not observed organ damage in our completed study of anti-BCMA
CAR T cells.106 A potential toxicity caused by anti-BCMA CAR T cells damaging normal cells is hypogammaglobulinemia due to depletion of plasma cells and a subset of mature B cells.  
Hypogammaglobulinemia has been a complication for many patients on clinical trials of antiCD19 CAR-expressing T cells and anti-BCMA T cells.4,33 . Hypogammaglobulinemia in these patients was routinely treated with infusions of intravenous immunoglobulins.4 Finally, genotoxicity is a theoretical risk of any type of integrating gene therapy. To our knowledge, genotoxicity, such as occurrence of a replication-competent retroviruses or transformation of T
cells caused by insertional mutagenesis has never occurred in a clinical trials of T-cell gene therapies.35,36 The specific gene therapy vector backbone, MSGV1, proposed for use in this clinical trial has been used in hundreds of patients over the past 13+ years by our group and others.4,25,108,109 The potential benefits to subjects enrolling on this trial include the possibility that the antiBCMA-CAR T cells can cause a significant anti-myeloma effect. Many patients enrolled on early trials of anti-CD19 CAR T cells and anti-BCMA CAR T cells obtained prolonged complete remissions of advanced malignancies3,4,106,110, so there is a chance that recipients of anti-BCMA
CAR T cells on this clinical trial of fully-human heavy-chain-only CARs could also derive a direct benefit from participation in this trial. Patients might also derive a benefit from knowing that they are contributing to the development of new cellular therapies for cancer. Aiding in the development of new therapies might help future patients.
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2 eligibility assessment and enrollment

Note: if a patient meets an eligibility requirement as outlined below, and is enrolled on the protocol but then is found to no longer meet the eligibility requirement after enrollment but before the start of protocol treatment, the treatment will be aborted or delayed until eligibility criteria are met. 2.1.1 Inclusion Criteria 2.1.1.1 Multiple Myeloma criteria BCMA expression must be detected on malignant plasma cells from either bone marrow or a plasmacytoma by flow cytometry or immunohistochemistry. If patient has plasmacytomas, one plasmacytoma must be biopsied to demonstrate BCMA expression. A specific quantitative level of BCMA expression for eligibility is not specified, but patients with multiple myeloma cells that are negative for BCMA by flow cytometry and immunohistochemistry on either bone marrow biopsy or plasmacytoma biopsy will not be enrolled. These assays must be performed at the National Institutes of Health (NIH). It is not required that the specimen used for BCMA determination comes from a sample that was obtained after the patient's most recent treatment. If paraffin embedded unstained samples of bone marrow involved with MM or a plasmacytoma are available, these can be shipped to the NIH for BCMA staining, otherwise new biopsies will need to be performed for determination of BCMA expression.  
BCMA expression will need to be documented on the majority of malignant plasma cells by flow cytometry at the NIH at some time after the original anti-BCMA CAR T-cell infusion in all patients undergoing a second anti-BCMA CAR T-cell infusion.  
Patients must have received at least 3 different prior treatment regimens for multiple myeloma Must have prior exposure to an "IMiD" such as lenolidamide and a proteasome inhibitor Patients must have measurable MM as defined by at least one of the criteria below.  
One or more of these abnormalities defines measurable multiple myeloma: Serum M-protein greater or equal to 1.0 g/dL. Urine M-protein greater or equal to 200 mg/24 h. Serum free light chain (FLC) assay: involved FLC level greater or equal to 10 mg/dL (100 mg/L) provided serum FLC ratio is abnormal.  
A biopsy-proven plasmacytoma at least 2.0 cm in largest dimension Bone marrow core biopsy with 30% or more plasma cells 2.1.1.2 Other inclusion criteria
Greater than or equal to 18 years of age and less than or equal to age 73.  
Able to understand and sign the Informed Consent Document.
Clinical performance status of ECOG 0-2  
Patients of both genders must be willing to practice birth control from the time of enrollment on this study and for four months after last day of receiving protocol treatment.  
Seronegative for HIV antibody. (The experimental treatment being evaluated in this protocol depends on an intact immune system. Patients who are HIV seropositive can have decreased immune-competence and thus are less responsive to the experimental treatment and more susceptible to its toxicities.)
A patient with a negative blood PCR test for hepatitis B DNA test can be enrolled. If hepatitis B DNA (PCR) testing is not available, patients with a negative hepatitis B surface antigen and negative hepatitis B core antibody can be enrolled.  
Patients must be tested for the presence of Hepatitis C antigen by PCR and be HCV RNA
negative in order to be eligible. Only if Hepatitis C PCR testing is not available in a timely manner, patients who are Hepatitis C antibody-negative can be enrolled.  
Absolute neutrophil count greater than or equal to 1000/mm3 without the support of filgrastim or other growth factors within the previous 10 days.  
Platelet count greater than or equal to 55,000/mm3 without transfusion support within the past 10 days.
Hemoglobin greater than 8.0 g/dL.  
Less than 5% plasma cells in the peripheral blood leukocytes Serum ALT and AST less or equal to 2.5 times the upper limit of the institutional normal. Serum creatinine less than or equal to 1.5 mg/dL. Total bilirubin less than or equal to 2.0 mg/dL, except in patients with Gilbert's Syndrome who must have a total bilirubin less than 3.0 mg/dL.  
At least 14 days must have elapsed since any prior systemic therapy at the time the patient starts the cyclophosphamide and fludarabine conditioning regimen, and patients' toxicities must have recovered to a grade 1 or less (except for toxicities such as alopecia or vitiligo).  
Because this protocol requires collection of autologous blood cells by leukapheresis in order to prepare anti-BCMA-CAR T cells, systemic anti-myeloma therapy including systemic corticosteroid steroid therapy of greater than 5 mg/day of prednisone or equivalent dose of another corticosteroid are not allowed within 2 weeks prior to the required leukapheresis.  
Normal cardiac ejection fraction (greater than or equal to 50% by echocardiography) and no evidence of hemodynamically significant pericardial effusion as determined by an echocardiogram.
For patients with past participation in gene-therapy, cryopreserved PBMC that have not been genetically-engineered must be available.  
2.1.2 Exclusion criteria Patients on any anticoagulants except aspirin. Patients that require urgent therapy due to tumor mass effects or spinal cord compression. Patients that have active hemolytic anemia. Patients with second malignancies in addition to multiple myeloma are not eligible if the second malignancy has required treatment within the past 3 years or is not in complete remission. There are two exceptions to this criterion: successfully treated non-metastatic basal cell or squamous cell skin carcinoma.  
Women of child-bearing potential who are pregnant or breastfeeding because of the potentially dangerous effects of the preparative chemotherapy on the fetus or infant. Women of child bearing potential cannot have a positive pregnancy test. Women of child-bearing potential are defined as all women except women who are post-menopausal or who have had a hysterectomy. Postmenopausal will be defined as women over the age of 55 who have not had a menstrual period in at least 1 year.  
Active systemic infections (defined as infections causing fevers or requiring antimicrobial treatment), active coagulation disorders or other major uncontrolled medical illnesses of the cardiovascular, respiratory, endocrine, renal, gastrointestinal, genitourinary, neurologic, or immune system, history of myocardial infarction, active cardiac arrhythmias including active atrial fibrillation, history of any arrhythmias other than sinus tachycardia or atrial fibrillation, currently taking any anti-arrythmic or congestive heart failure medications, active obstructive or restrictive pulmonary disease.  
Any form of primary immunodeficiency (such as Severe Combined Immunodeficiency Disease).
Systemic corticosteroid steroid therapy of greater than 5 mg/day of prednisone or equivalent dose of another corticosteroid (prednisone, dexamethasone, etc) is not allowed within 2 weeks prior to either the required leukapheresis or within 2 weeks prior to CAR T-cell infusion (and at any time after the CAR T cell infusion).  
History of severe immediate hypersensitivity reaction to any of the agents used in this study.  
Patient unwilling to undergo intensive care unit treatment including mechanical ventilation, cardiopulmonary resuscitation, vasoactive drugs, and hemodialysis.  
History of allogeneic stem cell transplantation Patients with current spinal cord compression (without intradural myeloma involvement). Patients who have a history (or current evidence) of cerebrospinal fluid multiple myeloma, or intra-dural central nervous system masses.  
Patients with active autoimmune skin diseases such as psoriasis or other active autoimmune diseases such as rheumatoid arthritis.  
Patients must not have required supplemental oxygen within the past month unless it was for a resolved infection.  
Patient must not have received genetically modified cells except on prior NCI gene therapy protocols.  
2.1.3 Recruitment Strategies This protocol may be abstracted into a plain language announcement posted on the NIH websites and on NIH social media platforms. Participants will be recruited from the current patient population at NIH, and local community physicians.
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2.2 screening evaluation

2.2.1 Screening activities performed after a consent for screening has been signed Note: Screening evaluation testing/procedures are conducted under the separate screening protocol, 01C0129 (Eligibility Screening and Tissue Procurement for the NIH Intramural Research Program Clinical Protocols). The following assessments must be completed within **30 days** prior to starting the chemotherapy conditioning regimen unless otherwise noted (if not, then the evaluation must be repeated):
Complete history and physical examination, including: weight, vital signs, ECOG Confirmation of diagnosis of MM by the NCI Laboratory of Pathology and confirmation of BCMA expression on malignant plasma cells from either bone marrow or a plasmacytoma by flow cytometry or immunohistochemistry. The sample used for this BCMA expression analysis can come from any time prior to enrollment on the protocol.  
Bone marrow aspirate and biopsy specifically ask for BCMA immunohistochemistry staining of the bone marrow biopsy. Order cytogenetics with interphase FISH (pretreatment aspirate only) and flow cytometry on the bone marrow aspirate. Specifically request 2 separate bone holes for bone marrow aspiration. The aspirate for hemepath (0.5 mL) and flow (2-2.5 mL) should be done from 1 bone hole and the aspirate for research (2-2.5 mL) should be done from a second bone hole followed by an aspirate for cytogenetics (1-2 mL) from the second bone hole. Note: Only bone marrow aspirates done before CAR T-cell treatment and 2 weeks after CAR T-cell infusion require aspirates through 2 bone holes. All other aspirates only need to be done through 1 bone hole.
The bone marrow biopsy must take place at some time after the patient's most recent myeloma treatment. If the patient agrees to future use of specimens when screening bone marrow is obtained, reserve one tube of bone marrow aspirate to be sent to the Surgery Branch Cell Production Facility SB-CPF. Bldg. 10 3W-3808. Phone: 240-858-3755. Bone marrow aspirate cells and supernatant will be cryopreserved. As many vials as possible with 10 million cells or less will be cryopreserved, and 2 vials of supernatant with 0.5 to 1 mL each will be cryopreserved. A bone marrow biopsy must be performed within 24 days of the start of the protocol-required conditioning chemotherapy. If this screening bone marrow aspirate and biopsy is performed within 24 days of the start of the conditioning chemotherapy, a second baseline bone marrow biopsy does not need to be performed.  
All patients must have flow cytometry staining of bone marrow or plasmacytoma cells for BCMA by Dr. Stetler-Stevenson's lab (NCI Laboratory of Pathology).  
EKG MRI of spine and pelvis (only if clinically indicated in patients with back pain or pelvic pain or a history of plasmacytomas of spine or pelvic)  
MRI of the brain
If necessary to assess disease, obtain a skeletal X-ray survey PET scan of torso and/or extremities (only if needed to evaluate disease)  
CT scans of areas with possible lesions/plasmacytomas This may include areas such as the head, neck, chest, abdomen and pelvis. (only if needed to evaluate disease)
Donor venous assessment DNA (PCR) for Hepatitis B and C; antibody testing for HIV, HTLV-I/II, T. cruzi
(Chagas agent), West Nile, and syphilis (RPR), only if DNA testing is not available, order Hepatitis C antibody, Hepatitis B core antibody, and hepatitis B surface antigen.  
Echocardiogram
(Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Phosphorus, Alkaline Phosphatase, ALT, AST, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid)  
CBC with differential and platelet count
Serum immunofixation electrophoresis Serum immunoglobulin free light chains Glucose-6-phosphate dehydrogenase (G6PD) deficiency screening  
TSH (T4 and T3)
Serum Cortisol β-HCG pregnancy test (serum or urine) on all women of child-bearing potential
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2.3 participant registration and status

Registration and status updates (e.g., when a participant is taken off protocol therapy and when a participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant Registration & Status Updates found here. 2.4 TREATMENT ASSIGNMENT PROCEDURES FOR THE REGISTARTION PURPOSES ONLY:  
| Number   | Name     | Description                                                                   |
|----------|----------|-------------------------------------------------------------------------------|
| 1        | Cohort 1 | Subjects enrolled to determine the MTD or  maximum feasible dose              |
| 2        | Cohort 2 | Subjects enrolled after the MTD or maximum  feasible dose has been identified |  
| Arms: Number   | Name                                                         | Description                                                                                                                                                                                                                                                                                                       |
|----------------|--------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1              | Conditioning  chemotherapy plus CAR  T-cells dose escalation | Patients will receive escalating doses (up to 5 planned) of  CAR+ T cells infused on day 0 + Cyclophosphamide: 300  mg/m2  IV infusion over 30 minutes on days -5, -4 and -3 + Fludarabine: 30 mg/m2  IV infusion over 30 minutes  administered immediately following the cyclophosphamide  on days -5, -4,and -3 |
| 2              | Conditioning  chemotherapy plus CAR  T-cells expansion phase | 6.0x106  cells/kg dose (maximum feasible dose) of CAR T  Cells + Cyclophosphamide: 300 mg/m2  IV infusion over  30 minutes on days -5, -4 and -3 + Fludarabine: 30 mg/m2 IV infusion over 30 minutes administered immediately  following the cyclophosphamide on days -5, -4,and -3                               |
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arm assignment

Patients in cohort 1 will be directly assigned to arm 1 Patients in cohort 2 will be directly assigned to arm 2
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3 study implementation 3.1 study

This protocol is a phase I dose-escalation study of autologous T cells that are genetically modified to express an anti-BCMA CAR. The protocol will enroll patients with multiple myeloma previously treated with at least 3 prior lines of therapy. Patients will be evaluated for general health, and multiple myeloma staging will be carried out. An assessment of BCMA expression will be an important part of the eligibility screening. Patients enrolled on the study will undergo leukapheresis, and anti-BCMA-CARexpressing T cells will be generated by transducing the patient's T cells with a gammaretrovirus encoding the anti-BCMA CAR. Patients will receive a conditioning chemotherapy regimen of cyclophosphamide 300 mg/m2 daily for 3 days and fludarabine 30 mg/m2 IV daily for 3 days on the same days. This is an extensively-used chemotherapy regimen that can be easily administered on an outpatient basis. Two days after the end of the conditioning chemotherapy, patients will receive a single infusion of anti-BCMA-CAR-expressing T cells. A minimum 9day hospitalization will be required after the cell infusion to monitor closely for acute toxicities. It is expected that some patients will need to be hospitalized for up to 30 days to allow all toxicities to resolve to the point that discharge from the hospital is prudent. Patients are required to stay within 60 minutes driving time from the Clinical Center until day 14 after the CAR T-cell infusion. Patients will then be evaluated for toxicity and multiple myeloma will be staged 2 weeks and 1, 2, 3, 4, 6, 9, and 12 months after the infusion. They will be followed every 6 months afterwards for at least 5 years. A small number of subjects may be eligible for re-enrollment if a patient is removed from the protocol before completing protocol treatment; or the patient is going to receive a second treatment. These patients would be required to meet all eligibility criteria at the time of reenrollment. Patients will be assigned a new sequential study number for the reenrollment study period. Any cryopreserved anti-BCMA CAR T cells produced from a patient who was removed from the study can be used to treat that patient after re-enrollment. We do not anticipate changes in the risk profile for the initial versus re-enrollment 3.1.2 Protocol schema  
  
  
  
  
  
  
  
  
  
  
  
Potential repeat treatments for patients with  
  
persistent myeloma 3.1.3 Dose Limiting Toxicity-the dose limiting toxicity assessment period is 28 days. DLTs are defined as follows (Note that DLTs will not occur in patients who do not receive CAR T-cell infusions but do receive the conditioning chemotheapy):  
Grade 3 toxicities possibly or probably or definitely related to the anti-BCMA CAR T
cells and lasting more than 9 days Grade 4 toxicities possibly or probably or definitely related to the anti-BCMA CAR T
cells.  
The following specific toxicities will not be DLTs:
Neutropenia (ANC<500/L) lasting 10 days or less is not a DLT
Neutropenia with an ANC>500/mL is not a DLT Anemia (Hgb<8 g/dL) lasting 10 days or less is not a DLT
Anemia with Hgb greater than or equal to 8 g/dL is not a DLT
Transfusion-dependent thrombocytopenia lasting 28 days or less is not a DLT Thrombocytopenia that is not transfusion-dependent is not a DLT Hypotension requiring continuous treatment with vasopressors for 72 hours or less is not a DLT.  
Fever is not a DLT. All cytopenias except neutropenia, anemia, and thrombocytopenia are not DLTs Asymtomatic electrolyte disturbances regardless of grade are not DLTs Prolonged QT interval as long as ventricular arrhythmias do not occur is not a DLT Grade 1, 2, or 3 creatine kinase elevation is not a DLT
Infections controlled by antibiotics are not DLTs 3.1.4 Dose Escalation
The trial will be a dose-escalation with 5 dose levels based on the patient's **actual** bodyweight.  
| Dose Escalation Schedule Dose Level Dose of IND Agent  Level -1 0.4x106  CAR+ T cells per kg of recipient bodyweight Level 1 0.75x106  CAR+ T cells per kg of recipient bodyweight Level 2 1.5x106  CAR+ T cells per kg of recipient bodyweight  Level 3 3.0x106  CAR+ T cells per kg of recipient bodyweight   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Dose Escalation Schedule Dose Level Dose of IND Agent  Level 4 6.0x106  CAR+ T cells per kg of recipient bodyweight Level 5 12.0x106  CAR+ T cells per kg of recipient bodyweight   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
A minimum of 3 patients will be enrolled at each dose level. There will be a minimum of 9 days between the CAR T-cell infusion of a patient and the start of the conditioning chemotherapy regimen for the next patient. This will cause a 14 day delay between sequential CAR T-cell infusions. If sufficient cells cannot be grown to meet the criteria for the assigned dose level but sufficient cells can be grown to meet the requirements of one dose level lower than the assigned dose level, the patient will be enrolled in the appropriate dose level for the number of cells infused. If a DLT occurs in an additional patient entered at a lower dose due to cell growth limitations, accrual will continue at this level as described in the dose-escalation scheme below. Accrual will be halted at the higher level until the dose level at the lower level is complete as described below. If sufficient cells cannot be produced to infuse the number of cells called for by one dose level lower than the called for dose level, the treatment will be aborted. A second attempt will be made to prepare cells for the patient if the patient agrees and if the patient still meets all eligibility criteria. Should none of the first 3 patients treated on a dose level experience a DLT, the first patient can be infused on the next higher dose level after a 28-day delay following CAR T-cell infusion of the third patient. Should 1 of 3 patients experience a dose limiting toxicity at a particular dose level, three more patients would be treated at that dose level. If 1/6 patients have a DLT at a particular dose level, the first patient can be infused on the next higher dose level after a 28-day delay following CAR T-cell infusion of the 6th patient. If a level with 2 or more DLTs in 3-6 patients has been identified, 3 additional patients will be accrued at the next-lowest dose for a total of 6, in order to further characterize the safety of the MTD. If 2 of 3 patients on Dose level 1 experience DLTs, accrual will proceed on dose level -1. The MTD is the dose at which a maximum of 1 of 6 patients has a DLT. After a MTD is defined, additional patients can be treated at the MTD. Up to 12 total additional recipients can be treated after an MTD is established to more completely characterize toxicity of the MTD. If cell growth limitations preclude administration of the MTD, the patient will receive as many cells as possible up to the MTD. If it proves to be technically impossible or impractical to achieve the higher dose levels due to cell production constraints and a MTD has not been reached, the highest achievable dose level will be declared the maximum feasible dose, and up to 12 additional recipients will be treated with the maximum feasible cell dose.  
The maximum feasible dose has been determined to be 6x106 CAR+ cells/kg based on technical difficulty preparing larger numbers of CAR+ T cells and lack of establishment of a MTD after treating at least 3 patients on all dose levels. Dose escalation will follow the rules outlined in the Table below:  
| Number of Patients with  DLTat a Given Dose Level     | Escalation Decision Rule                                                                                                                                                                                                                                                                                                                                                                                         |
|-------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 0 out of 3                                            | Enter up to 3 patients at the next dose level                                                                                                                                                                                                                                                                                                                                                                    |
| > 2                                                   | Dose escalation will be stopped. This dose level will be  declared the maximally administered dose (highest dose  administered). Up to three (3) additional patients will be  entered at the next lowest dose level if only 3 patients  were treated previously at that dose.                                                                                                                                    |
| 1 out of 3                                            | Enter up to 3 more patients at this dose level.  If 0 of these 3 patients experience DLT, proceed  to the next dose level.  If 1 or more of this group suffer DLT, then dose  escalation is stopped, and this dose is declared the  maximally administered dose. Up to three (3)  additional patients will be entered at the next  lowest dose level if only 3 patients were treated  previously at that dose. |
| <1 out of 6 at the highest  dose or 1 level below the |                                                                                                                                                                                                                                                                                                                                                                                                                  |
| maximally administered dose                           | This is the MTD and is generally the recommended phase  2 dose. At least 6 patients must be entered at the  recommended phase 2 dose.                                                                                                                                                                                                                                                                            |
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3.2 dose modifications/delay other toxicity

Patients may be removed from further treatment if they have active infections defined as infections causing fevers or infections requiring anti-microbial therapy that arise while patients are on-study but before the CAR T-cell infusion; however such patients are eligible for treatment if they meet all eligibility criteria after the infection resolves.  
If a patient experiences a grade 3 or greater toxicity (with the exception of cytopenias including neutropenia, lymphopenia, anemia, or thrombocytopenia) while on-study before the CAR T-cell infusion, the CAR T-cell infusion must be delayed until the toxicity improves to a grade 2 or less. Exceptions to this would be if the grade 3 toxicity was present at baseline or related to progressing disease.
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3.3 drug administration 3.3.1 leukapheresis

The patient will undergo a 10-20 liter leukapheresis (generally, 15 liters will be processed to target a yield of 6-10 x109 lymphocytes) in the Department of Transfusion Medicine (DTM)
Dowling Apheresis Clinic according to DTM standard operating procedures. The procedure requires dual venous access, and takes approximately 3-4 hours to complete. A central line will be placed if peripheral venous access is not sufficient.
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3.3.2 anti-bcma-car-expressing t-cell preparation

After cells are obtained by apheresis, further cell processing to generate anti-BCMA CARexpressing T cells will occur in the DTM according to standard operating procedures (SOPs).  
Either freshly-collected cells or cryopreserved cells can be used to initiate the cell-preparation process. In addition as stated in eligibility criteria, cryopreserved cells stored continuously in the Center for Cellular Engineering can be used to prepare autologous clinical cell products on other NCI cell therapy protocols, if the future cell therapy is conducted under a different NCI protocol. Peripheral blood mononuclear cells will be isolated. Sufficient cells for 2 complete cell productions will be retained in the Department of Transfusion Medicine; the excess cells will be sent to the Surgery Branch Cell Production Facility SB-CPF. Bldg. 10 3W-3808. Phone: 240858-3755for cryopreservation at 100 to 200 million PBMC per vial. These cells will be used in research. The anti-CD3 monoclonal antibody OKT3 will be used to stimulate T-cell proliferation. Two days after the start of the T-cell cultures, the cells will be transduced by exposing them to a supernatant containing replication-incompetent gammaretroviruses encoding the anti-BCMA CAR. The cells will continue to proliferate in culture. The total cell production process will take 7 to 9 days before cells are cryopreserved. Ten vials of the infused cells will be cryopreserved and stored in the Surgery Branch Cell Production Facility SB-CPF. Each vial will contain 10-20 million cells. Before infusion, the percentage of T cells expressing the anti-BCMA CAR will be determined by flow cytometry, and this percentage of BCMA+ T cells will be used in calculating the total number of cells to be infused to meet the dose requirements of the dose-escalation plan described in **3.1.4** In addition to flow cytometry, further testing of the cells will take place prior to infusion to evaluate for microbial contamination, replication-competent retroviruses, and viability. Details of this testing can be found in the appropriate DTM SOPs. When a patient is no longer eligible for retreatment on this protocol due to meeting any of the off-study criteria listed in section **3.8.2**, any remaining cryopreserved pretreatment PBMC collected on this protocol will be transferred from the Department of Transfusion Medicine to the Principal Investigator of this protocol for storage in the Surgery Branch Cell Production Facility SB-CPF. Bldg. 10 3W-3808. Phone: 240-858-3755 and possible use in research. Note: All cells sent to the Surgery Branch Cell Production facility will be transferred to NCI Frederick Biorepository. 3.3.3 Conditioning chemotherapy and anti-BCMA CAR T-cell administration-chemotherapy administration can be either inpatient or outpatient  
| Drug             | Dose                                                                           | Days                                |
|------------------|--------------------------------------------------------------------------------|-------------------------------------|
| Cyclophosphamide | 300 mg/m2  IV infusion over                                                    | Daily x 3 doses on days -5,         |
| 30 minutes       | -4, -3                                                                         |                                     |
| Fludarabine      | 30 mg/m2  IV infusion over  30 minutes administered  immediately following the | Daily x 3 doses on days -5,  -4, -3 |  
3.3.3.1 Overall summary of the treatment plan  
| Drug                                | Dose      | Days                   |
|-------------------------------------|-----------|------------------------|
| cyclophosphamide on day - 5, -4, -3 |           |                        |
| Anti-BCMA CAR T cells               | Variable. | One time dose on day 0 |  
Day -5, -4, and -3: Patients will receive pre-hydration with 1000 mL 0.9% sodium chloride I.V. over 1 to 3 hours. Patients will receive anti-emetics following NIH Clinical Center guidelines, but dexamethasone will not be administered. One suggested regimen is ondansetron 16 to 24 mg orally on days -5, -4, and -3 before chemotherapy (I.V. ondansetron can be substituted). Patients should be provided with anti-emetics such as lorazepam and prochlorperazine to use at home.  
On days -5, -4, and -3, cyclophosphamide at a dose of 300 mg/m2 I.V. will be diluted in 100 mL
5% dextrose solution and infused over 30 minutes. After the cyclophosphamide on days -5, -4, and -3, patients will receive 30 mg/m2 I.V. fludarabine in 100 mL 0.9% sodium chloride over 30 minutes. **Note: in patients with an estimated creatinine clearance of 30 - 70**
mL/minute/1.73m2**, the daily dose of fludarabine will be reduced by 20%.** Creatinine clearance will be estimated by the Chronic Kidney Disease Epidemiology Collaboration (CKDEPI) formula.  
Following the fludarabine infusion, patients will receive 1000 mL 0.9% sodium chloride I.V. over 1-2 hours. Furosemide will be given if needed.  
Days -2 and -1: No interventions except as needed for general supportive care such as antiemetics. To minimize bladder toxicity, patients should increase normal oral fluid intake to at least 2 L/day. Day 0: Anti-BCMA CAR T cells will be administered. Premedication for the cell infusion will be given approximately 30 minutes prior to the infusion. The premedications are acetaminophen 650 mg orally and diphenhydramine 12.5 mg IV. Cells are delivered to the patient care unit from the Department of Transfusion Medicine. Prior to infusion, the cell product identity label is double-checked by two authorized staff (MD or RN), and identification of the product and documentation of administration are entered in the patient's chart as is done for blood banking protocols. The cells are to be infused intravenously over 20 to 30 minutes via a central line with non-filtered tubing through an **INFUSION PUMP**, gently agitating the bag during infusion to prevent cell clumping. Cells may arrive on the unit in a syringe instead of a bag. In this case, the cells can be administered to the patient by pushing the cells through a free flowing normal saline line per nursing SOP, but still must be infused over 20-30 minutes. Details of the infusion procedure are included in Appendix D (Note: The cell infusion may be delayed up to a day per the PI's discretion) Days 0 to 9: Mandatory hospitalization for observation and treatment as necessary. Note: hospitalization for CAR T-cell patients is routinely extended; extension of hospitalization beyond the required 9 days is anticipated for CAR T cell toxicity management. In addition, patients are required to stay within 60 minutes driving time from the Clinical Center until day 14 after the CAR T-cell infusion. Guidelines for dealing with toxicities that often occur after CAR T cell infusions including hypotension, fever and tachycardia are given in **15.3**. A CBC with differential will be obtained daily. If the absolute neutrophil count becomes less than 500/microliter, filgrastim will be initiated at a dose of 300 micrograms daily subcutaneously for patients under 70 kg in weight and at a dose of 480 micrograms daily for patients over 70 kg in weight. Filgrastim will be given daily and then discontinued as soon as the absolute neutrophil count recovers to 2000/microliter.
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3.3.4 potential repeat treatment

Patients obtaining any response except progressive disease are potentially eligible for a repeat treatment consisting of conditioning chemotherapy followed by an infusion of anti-BCMA CAR
T cells. Retreatment will be at least 2 months after the original treatment. The dose of antiBCMA CAR T cells administered during repeat treatments will be the dose level currently enrolling patients receiving initial CAR T-cell infusions. The repeat treatment will include the same conditioning chemotherapy as the initial treatment. To be eligible for a repeat treatment, patients must have not experienced a DLT with their first treatment. Patients must also meet the same eligibility requirements listed in Section 2.1. BCMA expression must be documented after the first anti-BCMA CAR infusion and before the second BCMA CAR T-cell infusion. The patients must undergo screening evaluation as listed in Section 2.2 except infectious disease serology screening is not required to be repeated unless clinically indicated. Follow-up testing for retreatment will be the same as for the first treatment. The patient may be re-enrolled on the the study as a new patient to allow this re-treatment and these patients will be considered in the total sample size for this study. All patients receiving a second treatment will be identified as patients receiving repeat treatments in any reports of results of this trial. A maximum of 2 total treatments can be administered to any one patient, and at least 2 months must elapse between the first treatment and the second treatment. Second treatments including DLTs will not affect the dose escalation plan described in section 3.1.4. DLTs that occur in patients receiving a repeat treatment will not affect the dose escalation of patients receiving an initial treatment. However, excessive DLTs among re-treatments will result in a discontinuation of this practice. Specifically, if 2 of the first 3 patients re-treated, 3 or more of the first 6 re-treated patients, 4 or more out of the first 9 re-treated patients, or greater than 1/3 of the total patients receiving a repeat treatment experiences a DLT during repeat treatment, then repeat treatments will be discontinued altogether.
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3.4 protocol evaluation 3.4.1 baseline

Bone marrow aspirate and biopsy must be performed within 24 days of the start of the conditioning chemotherapy. If the screening bone marrow aspirate and biopsy were done within 24 days of the start of conditioning chemotherapy, the baseline bone marrow aspirate and biopsy should not be done unless clinically indicated. Specifically ask for BCMA, CD3, CD8, and CD4 immunohistochemistry staining of the bone marrow biopsy. Order cytogenetics with interphase FISH (pretreatment only) and flow cytometry on the bone marrow aspirate. Specifically request 2 separate bone holes for bone marrow aspiration. The aspirate for hemepath (0.5 mL) and flow (2-2.5 mL) should be done from 1 bone hole and the aspirate for research (2-2.5 mL) should be done from a second bone hole followed by an aspirate for cytogenetics (1-2 mL) from the second bone hole. Note: Only bone marrow aspirates done before CAR T-cell treatment and 2 weeks after infusion require aspirates throught 2 bone holes. All other bone marrow aspirates only need to be done through 1 bone hole. For all bone marrow aspirates, bone marrow supernatant must be collected and frozen in the same manner as serum is collected and frozen. BCMA immunohistochemical staining was performed using Ventana Ultra automated staining system (Ventana, Tucson, AZ) according to manufacturer's directions. Incubation in mouseantiBCMA primary antibody (Santa Cruz Biotechnology clone D6) was followed by anti-mouse secondary antibody incubation. Flow cytometry on bone marrow aspirates was performed by using standard methods and the Santa Cruz Biotechnology clone D6 antibody that is specific for BCMA.
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the following tests must be

Patients must have a central venous access before the time of cell infusion. This might require placement of a non-valved P.I.C.C line or other device.  
Physical exam with vital signs and oxygen saturation PET-CT scan in all patients CT scans of areas with possible lesions/plasmacytomas. This may include areas such as the head, neck, chest, abdomen and pelvis. (If needed to stage MM) Spine MRI if needed to stage MM
Skeletal survey. Additional scans and x-rays may be performed if clinically indicated based on patients' signs and symptoms.
Serum immunofixation electrophoresis 250 microgram cosyntropin stimulation test if suspicious for adrenal insufficiency based on low serum sodium or high serum potassium or hypotension or a history of adrenal insufficiency or low serum cortisol or other clinical indications (This may be performed after start of chemotherapy as long it is performed before the cell infusion)  
24-hour urine collection with immunofixation electrophoresis timed urine 24-hour urine collection for urine protein
Serum immunoglobulin free light chains Anti CMV antibody titer, HSV serology, and EBV panel, T cruzi serology, toxoplasmosis serology (Note: patients who are known to be positive for any of the above do not need to be retested; may be performed within 3 months of chemotherapy start date)  
Blood will be collected for research purposes. Draw 12 CPT tubes (8 mL each of blood will be collected prior to initiation of the conditioning chemotherapy regimen. This is a total of 96 mL of blood. Some of this blood will be used for immunology assays and some will be used for RCR assays. This blood can be collected on different days as long
as a total of 12 CPT tubes are collected prior to cell initiation of the chemotherapy. Send to the Surgery Branch Cell Production Facility SB-CPF. Bldg. 10 3W-3808. Phone: 240858-3755.  
In addition to the CPT tubes, draw 16 mL of blood to obtain serum for research purposes
(2 SST tubes, 8 mL per tube) within 3 days prior to the start of the chemotherapy. Send to the Figg lab; For sample pick-up, page 102-11964.
The following tests must be completed within 7 days of the start of the conditioning chemotherapy regimen:
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tbnk

Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg),
Phosphorus, Alkaline Phosphatase, ALT, AST, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK (creatine kinase), Uric Acid (to be repeated on the first day of the chemotherapy at the discretion of the PI)
2-microglobulin ABO typing
Ionized calcium CBC with differential and platelet count  
PT/PTT Fibrinogen Urinalysis; if results are abnormal, send for urine culture β-HCG pregnancy test (serum or urine) on all women of child-bearing potential C-reactive protein (CRP)
3.4.2 Studies to be performed on Day 0 and during the mandatory 9-day inpatient admission after cell infusion Vital signs including pulse oximetry will be monitored q1h x 4 hours (+/- 15 minutes)
after completion of the CAR T cell infusion and approximately every 4 hours (+/-15 minutes) otherwise unless otherwise clinically indicated.  
Daily physical exam CBC twice a day from day 0 until day 9 with differential daily. After day 9 do a CBC
with differential daily until discharge. (In the case of a later day infusion or early discharge, this may be only once a day.)
TBNK on the day of CAR T-cell infusion (day 0), day 7 and day 14 after infusion Chemistries twice a day starting from day 0 to day 9. After day 9 do chemistries once a day until discharge: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total,
Magnesium total (Mg), Phosphorus, Alkaline Phosphatase, ALT, AST, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Uric Acid, creatine kinase. (In the case of a later day infusion or early discharge, this may be only once a day.)
PT/PTT and fibrinogen daily while hospitalized
**Day 0 Research Blood:** 1 SST tube will be drawn on the morning of CAR cell infusion prior to infusion of CAR T cells. Send to the Figg lab; for sample pick-up, page 10211964.  
**Post-infusion Research blood:** Every Monday, Wednesday, and Friday during hospitalization, starting on the first Monday, Wednesday, or Friday after the day of CAR T-cell infusion and lasting up to 20 days after infusion of anti-BCMA-CAR-transduced T
cells, 56 mL of patient peripheral blood will be obtained (6 CPT tubes 8 mL each and 1 SST tube 8 mL). Send CPT tubes to the Surgery Branch Cell Production Facility SBCPF. Bldg. 10 3W-3808. Phone: 240-858-3755. Send SST tubes to the Figg lab; for sample pick-up, page 102-11964.  
**Additional Post-infusion research blood:** 1 SST tube will be drawn on the first Sunday after CAR T-cell infusion. This tube will be stored refrigerated on the nursing unit and processed first thing Monday morning at the latest. Send to the Figg lab; For sample pickup, page 102-11964.  
Note regarding research blood collection: If any of the above time points fall on federal holiday then, we may collect research blood on the next day. 3.4.3 Post-infusion outpatient evaluation After completion of therapy the patient will be followed for potential complications related to anti-BCMA–CAR T-cell infusion. The patient will be seen at the NIH in follow-up to evaluate disease status and late problems related to anti-BCMA –CAR T-cell infusion at days +14 (+/- 1 day), +30 (+/- 5 days), +60(+/- 7 days), +90(+/- 7 days), and +120(+/- 7 days); and at 180(+/- 14 days), 270 (+/- 14 days), and 365 (+/- 30 days) months after anti-BCMA –CAR T-cell infusion. After 12 months, the patient will be seen every 6 months (+/- 30 days) up to five years. All of these time points have allowable ranges (see Table in section 3.5). At these times patients will have the following tests performed to determine clinical response:
6 CPT tubes of Research Blood (48 mL) will be collected to obtain blood for immunological testing. Send to the Surgery Branch Cell Production Facility SB-CPF. Bldg. 10 3W-3808. Phone: 240-858-3755.  
1 SST tube (8 mL) of Research Blood will be obtained for serum collection.Send to the Figg lab; For sample pick-up, page 102-11964.
Note: after the first year of follow-up, research blood will be reduced to 4 CPT tubes
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(32 ml total) during required

PET scans will be performed if needed to stage disease as described in section **3.5.**
CT scans of areas with lesions/plasmacytomas starting 1 month after infusion. This may include areas such as the head, neck, chest, abdomen and pelvis. (Only if a plasmacytoma/lesion was detected before treatment)
MRI of spine (Only if a plasmacytoma/lesion was detected before treatment)  
Physical exam with vital signs and oxygen saturation (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Phosphorus, Alkaline Phosphatase, ALT, AST, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid PT/PTT, fibrinogen
TBNK Ionized calcium CBC with differential  
C-reactive protein (CRP)
Blood for serum 2-microglobulin Blood for immunoglobulin free light chains Blood for serum immunofixation electrophoresis 24-hour urine collection with immunofixation electrophoresis timed urine (can be omitted if not needed for staging after day 30).  
24-hour urine collection with protein timed urine (can be omitted if not needed for staging after day 30).  
Urinalysis: if results are abnormal, send for urine culture At the 2 week follow-up, bone marrow aspirate and biopsy must be performed. Aspirate must be sent for flow cytometry to the lab of Dr. Maryalice Stetler-Stevenson. BCMA staining must be requested for the flow cytometry. BCMA immunohistochemistry should also be requested on the bone marrow biopsy.Specifically ask for BCMA, CD3, CD8, and CD4 immunohistochemistry staining of the bone marrow biopsy. Specifically request 2 separate bone holes for bone marrow aspiration. The aspirate for hemepath (0.5 mL) and flow (2-2.5 mL) should be done from 1 bone hole and the aspirate for research (2-2.5 mL) should be done from a second bone hole.  
At 2 week follow-up, a peripheral blood sample will be collected. Peripheral blood must be sent for flow cytometry analysis to the lab of Dr. Maryalice Stetler-Stevenson to evaluate for malignant plasma cells, the BCMA expression on any malignant plasma cells present and CAR positive T cells.  
At the 2 month follow-up, a bone marrow aspirate and biopsy will be collected. Aspirate must be sent for flow cytometry to the lab of Dr. Maryalice Stetler-Stevenson. BCMA staining must be requested for the flow cytometry. BCMA immunohistochemistry should also be requested on the bone marrow biopsy. This may also be performed at day +30 if clinically indicated. Specifically ask for BCMA, CD3, CD8, and CD4 immunohistochemistry staining of the bone marrow biopsy. The aspirate for hemepath (0.5 mL), flow (2-2.5 mL) and research (2-2.5 mL) can be done from 1 bone hole.
At the 6-month, 18-month, 30 -month, and 42-month follow-up appointments, a bone marrow aspirate and biopsy will be performed. Aspirate must be sent for flow cytometry to the lab of Dr. Maryalice Stetler-Stevenson. BCMA staining must be requested for the flow cytometry. BCMA immunohistochemistry should also be requested on the bone marrow biopsy. Specifically ask for BCMA, CD3, CD8, and CD4 immunohistochemistry staining of the bone marrow biopsy. The aspirate for hemepath (0.5 mL), flow (2-2.5 mL) and research (2-2.5 mL) can be done from 1 bone hole.  
For each bone marrow aspirate performed, send one tube of bone marrow aspirate to Surgery Branch Cell Production Facility SB-CPF. Bldg. 10 3W-3808. Phone: 240-8583755. Bone marrow cells should be cryopreserved and the liquid bone marrow supernatant should also be saved at -80 degrees. As many vials as possible with 10 million cells or less will be cryopreserved, and 2 vials of supernatant with 0.5 to 1 mL
each will be cryopreserved.  
Gene-therapy-specific follow-up must be carried out as described in section 3.6 48  
Version Date: April 26, 2021
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3.5 study calendar

| Follow up                                                                                                                                                                                                 |                                                |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------|-------------------------|--------------------------|---------------------------|---------------------------|---------------------------|------------------------------------------------------------|----|----|----|----|
| Pre-cell infusion/  Day 0 Day  +7b                                                                                                                                                                        | Day+14  (+- 1  day) and  Day+30  (+/- 5  days) |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| Screening/ Baseline                                                                                                                                                                                       | Day  +60  (+/- 7  days)                        | Day  +90  (+/- 7  days) | Day  +120  (+/- 7  days) | Day  +180  (+/- 14  days) | Day  +270  (+/- 14  days) | Day  +365  (+/- 30  days) | Every 6 months  after day 365(+/-  30 days) up to 5  years |    |    |    |    |
| Procedures a                                                                                                                                                                                              |                                                |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| Clinical Assessments History and PE                                                                                                                                                                       | X                                              | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  | X  | X  |
| Vital signs, O2 saturationo                                                                                                                                                                               | X                                              | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  | X  | X  |
| Height, weight                                                                                                                                                                                            | Xs                                             | Xs                      |                          |                           |                           |                           |                                                            |    |    |    |    |
| Performance Score                                                                                                                                                                                         | X                                              | X                       |                          |                           |                           |                           |                                                            |    |    |    |    |
| Laboratory Assessments CBC with differential                                                                                                                                                              | X                                              | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  | X  | X  |
| Sodium, Potassium  Chloride, CO2, Creatinine,  Glucose, BUN, Albumin,  Calcium, Magnesium,  Phosphorus, Alkaline  Phosphatase, ALT, AST, T.  Bilirubin, D. Bilirubin,  LDH, Total Protein, CK,  Uric Acid | X                                              | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  | X  | X  |
| Serum C-reactive peptide                                                                                                                                                                                  | Xh                                             | Xm                      | Xm                       | X                         | X                         | X                         | X                                                          | X  | X  | X  | X  |
| PT/PTT, Fibrinogen                                                                                                                                                                                        | Xh                                             | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  | X  |    |
| Ionized calcium                                                                                                                                                                                           | Xh                                             | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  |    |    |
| Thyroid Stimulating  Hormone, T3                                                                                                                                                                          | XK                                             |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| 2-HCG (serum or urine                                                                                                                                                                                    | X                                              |                         |                          |                           |                           |                           |                                                            |    |    |    |    |  
49  
Version Date: April 26, 2021  
| Follow up                                                                                 |                                                |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
|-------------------------------------------------------------------------------------------|------------------------------------------------|-------------------------|--------------------------|---------------------------|---------------------------|---------------------------|------------------------------------------------------------|----|----|----|----|
| Pre-cell infusion/  Day 0 Day  +7b                                                        | Day+14  (+- 1  day) and  Day+30  (+/- 5  days) |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| Screening/ Baseline                                                                       | Day  +60  (+/- 7  days)                        | Day  +90  (+/- 7  days) | Day  +120  (+/- 7  days) | Day  +180  (+/- 14  days) | Day  +270  (+/- 14  days) | Day  +365  (+/- 30  days) | Every 6 months  after day 365(+/-  30 days) up to 5  years |    |    |    |    |
| Procedures a                                                                              |                                                |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| in women of childbearing  potential) 2-microglobulin                                     | Xh                                             | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  |    |    |
| Anti-CMV, HSV, EBV,  T.cruzi,toxoplasma                                                   | Xh,j                                           |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| Antibody screen for  Hepatitis B and C; HIV,  HTLV-I/II, West Nile, and  syphilis (RPR)   | X                                              |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| TBNK                                                                                      | Xh                                             | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  | X  | X  |
| G6PD                                                                                      | XK                                             |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| Urinalysis (culture prn)                                                                  | Xh                                             | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  |    |    |
| 24 hr urine with  immunofixation  electrophoresis timed  urine and urine 24 hour  protein | Xh                                             | X                       | Xf                       | Xf                        | Xf                        | Xf                        | Xf                                                         | Xf | Xf |    |    |
| ABO typing                                                                                | Xh                                             |                         |                          |                           |                           |                           |                                                            |    |    |    |    |
| Serum immunoglobulin  free light chains                                                   | X                                              | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  |    |    |
| Serum immunofixation electrophoresis                                                      | X                                              | X                       | X                        | X                         | X                         | X                         | X                                                          | X  | X  |    |    |
| Peripheral blood flow                                                                     | Xt                                             |                         |                          |                           |                           |                           |                                                            |    |    |    |    |  
50  
Version Date: April 26, 2021  
| Follow up                                             |                                                |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
|-------------------------------------------------------|------------------------------------------------|-------------------------|--------------------------|---------------------------|---------------------------|---------------------------|------------------------------------------------------------|-----|-----|----|----|
| Pre-cell infusion/  Day 0 Day  +7b                    | Day+14  (+- 1  day) and  Day+30  (+/- 5  days) |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| Screening/ Baseline                                   | Day  +60  (+/- 7  days)                        | Day  +90  (+/- 7  days) | Day  +120  (+/- 7  days) | Day  +180  (+/- 14  days) | Day  +270  (+/- 14  days) | Day  +365  (+/- 30  days) | Every 6 months  after day 365(+/-  30 days) up to 5  years |     |     |    |    |
| Procedures a                                          |                                                |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| cytometry Radiological Assessments Skeletal Survey    | X                                              |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| CT scan of disease  affected areas                    | Xu                                             | X i,e                   | Xe                       | Xe                        | Xe                        | Xe                        | Xe                                                         | Xe  | Xe  |    |    |
| PET                                                   | Xh                                             | Xf                      | Xf                       | Xf                        | Xf                        | Xf                        |                                                            |     |     |    |    |
| Brain MRI                                             | X                                              |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| Spine MRI e                                           | X                                              | X e                     | X e                      | X e                       | X e                       | X e                       | X e                                                        | X e | X e |    |    |
| Other Specific Assessments Serum Cortisol             | Xk                                             |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| 250 microgram  cosyntropin test c                     | Xh                                             |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| EKG, echocardiogram                                   | X                                              |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| RCR                                                   | X                                              | X                       | X                        | X                         | Xg                        |                           |                                                            |     |     |    |    |
| Central Venous catheter  placement                    | X                                              |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| Venous Assessment                                     | X                                              |                         |                          |                           |                           |                           |                                                            |     |     |    |    |
| Response Evaluation Bone marrow  aspirate/biopsy d, q | X                                              | Xl                      | X                        | X                         | Xv                        |                           |                                                            |     |     |    |    |
| Research Blood n. p. r                                | X h                                            | Xn                      | X                        | X                         | X                         | X                         | X                                                          | X   | X   | X  | X  |
| Adverse Events                                        |                                                | X                       | X                        | X                         | X                         | X                         | X                                                          | X   | X   | X  | X  |
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51 version date april 26

| Follow up                          |                                                |                         |                          |                           |                           |                           |                                                            |    |    |    |    |    |
|------------------------------------|------------------------------------------------|-------------------------|--------------------------|---------------------------|---------------------------|---------------------------|------------------------------------------------------------|----|----|----|----|----|
| Pre-cell infusion/  Day 0 Day  +7b | Day+14  (+- 1  day) and  Day+30  (+/- 5  days) |                         |                          |                           |                           |                           |                                                            |    |    |    |    |    |
| Screening/ Baseline                | Day  +60  (+/- 7  days)                        | Day  +90  (+/- 7  days) | Day  +120  (+/- 7  days) | Day  +180  (+/- 14  days) | Day  +270  (+/- 14  days) | Day  +365  (+/- 30  days) | Every 6 months  after day 365(+/-  30 days) up to 5  years |    |    |    |    |    |
| Procedures a                       |                                                |                         |                          |                           |                           |                           |                                                            |    |    |    |    |    |
| Concomitant Medications            | X                                              |                         | X                        | X                         | X                         | X                         | X                                                          | X  | X  | X  | X  | X  |  
asee section **2.2** and section **3.3.1** for details b see section **3.4.2** for details of testing during hospitalization c only if cortisol<18 mg/dL or clinical suspicion of adrenal insufficiency d bone marrow biopsy will include flow cytometry, BCMA expression, cytogenetics (pretreatment bone marrow aspirate only), amyloid staining
(pretreatment bone marrow biopsy only), and molecular studies of each sample.  
e Only if a plasmacytoma/lesion was detected before treatment f if useful for response assessment g RCR blood collection will only continue after the 1 year time-point if a previous RCR test has been positive, see section 3.6 for details h required at baseline, only needed at screening if needed to evaluate disease..  
i only D+30, no CTs or PETs indicated at D+14 j Patients who are known to be positive for any of these tests do not need to be retested; may be performed within 3 months of chemotherapy start date k only at Screening l Always do a bone marrow aspirate and biopsy on day 14; only do a bone marrow aspirate and biopsy on Day 30 if clinically indicated.  
Additional bone marrow biopsies and aspirates might be needed if clinically indicated due to low blood counts or for myeloma response assessment.  
m Done Daily during hositalization nIn addition to baseline research blood, 1 SST tube must be drawn on the morning of CAR T-cell infusion prior to CAR T-cell infusion o +/- 15 minutes pDraw 1 SST tube on the first Sunday after CAR T-cell infusion. This SST tube should be stored refrigerated on the nursing unit until Monday morning when it should be processed first thing Monday morning at the latest.  
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Version Date: April 26, 2021 qA bone marrow aspirate and biopsy with flow cytometry will also be performed to confirm suspected complete remission. Note that 2 separate bone holes will be required for the pretreatment and 2-week follow-up bone marrow aspirates. All other bone marrow aspirates will only require 1 bone hole.  
r In addition to the day 0 and 1st Sunday research blood mentioned above, research blood is drawn every Mon-Wed-Fri while the patient is inpatient.  
sWeight at admission is adequate even if not D0. Height is only required to be performed sometime before starting chemotherapy.  
t only D+14 u only if needed to stage MM
v Bone marrow aspirate and biopsy with flow cytometry will be perfomed at the 6-month, 18-month, 30-month, and 42- month timepoints
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3.6 gene-therapy-specific follow-up

Long-term follow up of patients receiving gene transfer is required by the FDA and must continue even after the patient comes off the treatment study. Physical examinations will be performed and documented annually for 5 years following cell infusion to evaluate long-term safety. After 5 years, health status data will be obtained from surviving patients via telephone contact or mailed questionnaires for 10 additional years for a total of 15 years after cell infusion. Blood will need to be collected at least annually for longterm follow-up of gene therapy. After 5 years, patients who are still on this study will be transferred to the long-term gene therapy protocol NCI protocol 15-C-0141 to complete long-term gene-therapy follow-up.  
Persistence of CAR transduced cells will be assessed by quantitative PCR and/or flow cytometry at 1, 2, 3, 4, 6 and 12 months after cell infusion, or until CAR-expressing cells are no longer detectable. If any patient shows a high level of persistence of CAR gene transduced cells or an increasing population of CAR gene transduced T cells at month 6 or later (by FACS staining or qPCR), the previously archived samples will be subjected to techniques that would allow the identification of predominant clonal populations of transduced cells.  
Patients' blood samples will be obtained and undergo analysis for detection of replication competent retroviruses (RCR) by PCR prior to cell infusion and at 3, 6, and 12 months post cell administration. If all of these samples are negative for RCR, blood collection for RCR will be discontinued after the 12 month time-point..  
S+L- culture-based replication-competent retrovirus testing will be conducted on the infusion CAR T cells of all patients
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3.7 cost and compensation 3.7.1

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

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

Prior to documenting removal from study, effort must be made to have all subjects complete a safety visit approximately 30 days after the last dose of study therapy. 3.8.1 Criteria for removal from protocol therapy Note that the treatment consists of a conditioning chemotherapy regimen followed by a T-cell infusion. Patients will be taken off treatment for the following:
Any DLT makes patients ineligible for repeat treatments.  
The patient no longer meets the eligibility criteria for the protocol after enrolling but before start of the chemotherapy conditioning regimen. If the reason that the patient is not eligible can be rapidly resolved within 2 weeks, the patient can proceed on treatment for up to two weeks, otherwise the patient must come off treatment and then off study as appropriate. An exception to this is that a platelet count of 50,000 or more is considered adequate to start chemotherapy.  
The patient started chemotherapy but cannot complete the entire treatment (ending with completed cell infusion) for any reason specified in the protocol or PI discretion. If the reason that the patient is no longer eligible can be rapidly resolved, the patient can proceed on treatment, otherwise the patient must come off treatment and then off study as appropriate.  
The patient receives any other treatment for multiple myeloma except bisphosphonates and repeat treatment on this protocol. Bisphosphonates are allowed when administered to prevent osteopenia more than 2 months after the anti-BCMA-CAR T-cell infusion.  
General or specific changes in the patient's condition render the patient unacceptable for further treatment on this study in the judgment of the investigator.  
Participant requests to be withdrawn from active therapy Investigator discretion Positive pregnancy test
3.8.2 Off-Study Criteria Patients will be taken off study for the following: (all patients coming off study will enroll on the long-term gene therapy follow-up protocol 15-c-0141)
The patient completes the study upon reaching 5 years after CAR T-cell infusion. The patient completes off treatment follow-up The patient voluntarily withdraws There is significant patient noncompliance PI discretion Death PI decision to end this study  
The patient no longer meets the eligibility criteria for the protocol after enrolling but before start of the chemotherapy conditioning regimen.  
Development of progressive or relapsed multiple myeloma after anti-BCMA CAR T cell infusion in patients not desiring or not eligible for re-treatment on this protocol.  
The patient receives any anti-myeloma therapy except bisphosphonates.  
Bisphosphonates are allowed 2 months or more after the CAR T-cell infusion.
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3.9 stopping criteria

If no responses of PR or CR occur after 2 patients are treated on the highest dose level, the protocol will be stopped.  
Instructions for how to proceed when toxicity occurs will be as instructed by the dose escalation section of the protocol.  
A death on study not attributible to progressive malignancy within 30 days of a cell infusion for the initial 5 subjects will be a cause for a pause to accrual pending discussion with the FDA and IRB.  
If 3 or more of the first 9 treated patients experience grade 4 toxicity possibly or probably attributable to CAR T cells within 30 days of cell infusion, this will be a cause for a pause to accrual to reassess the safety of the product pending amendment of the protocol approved/reviewed by the FDA and IRB.
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4 concomitant medications/measures 4.1 antibiotic

Patients with a CD4 T-cell count less than 200 will be maintained on pneumocystis prophylaxis with atovaquone or inhaled pentamidine. Patients with a CD4 T-cell count less than 200 will also be maintained on acyclovir or valacyclovir.
Patients with serum IgG level less than 600 mg/dL will receive intravenous immunoglobulin replacement as needed to maintain an IgG level above 600 mg/dL after at least 1 month post-CAR T-cell infusion. An example of an intravenous immunoglobulin infusion to be used for this purpose would be Gammunex 500 mg/kg given as a single dose. Intravenous immunoglobulin infusions should be preceded by premedication with diphenhydramine and acetaminophen, and rate of infusion should be started at low rates and escalated in a step-wise manner  
Neutropenic patients will start on broad spectrum antibiotics with a first fever of 38.30C
or greater or two fevers of 38.0 separated by at least 1 hour and concomitant ANC < 500/mL.  
Aminoglycosides will be avoided unless clear evidence of sepsis.
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4.2 blood product support

Leukocyte filters will be utilized for all blood and platelet transfusions with the exception of the CAR-transduced T cell infusions to decrease sensitization to transfused WBC and decrease the risk of CMV infection.  
Patients who are seronegative for CMV should receive CMV-negative blood products whenever possible.  
Using daily CBC's as a guide, the patient will receive platelets and packed red blood cells
(PRBC's) as needed. Attempts will be made to keep Hgb >8.0 gm/dL, and platelets >10,000/mm3. All blood products with the exception of the CAR-transduced T cells will be irradiated. Leukocyte filters will be utilized for all PRBC and platelet transfusions to decrease sensitization to transfused WBC's and decrease the risk of CMV infection.
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4.3 anti-emetics

Anti-emetics will follow NIH Clinical Center Guidelines (except that corticosteroids will be avoided).
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4.4 granulocyte colony-stimulating factor

A CBC will be obtained daily while the patient is inpatient. If the absolute neutrophil count becomes less than 500/microliter, Filgrastim will be initiated at a dose of 300 micrograms daily for patients under 70 kg in weight and a dose of 480 micrograms daily for patients over 70 kg in weight only in patients with absolute neutrophil counts less than 500/microliter. Filgrastim will be discontinued as soon as the absolute neutrophil count recovers to 2000/microliter.
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4.5 avoidance of corticosteroids

Patients should not take corticosteroids including prednisone, dexamethasone or any other corticosteroid at a dose equivalent to 5 mg/day or more of prednisone for any purpose without approval of the Principal Investigator.
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4.6 toxicity management guidlines

Guidelines for management of common CAR toxicities including cytokine release syndrome (CRS) and neurologic toxicity are in section **15.3**.
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5 biospecimen collection

Biospecimen collection on this protocol will consist of blood draws and acquisition of bone marrow aspirates and possible biopsies of plasmacytomas for research purposes. Blood and bone marrow collection is described above in Sections **2.2, 3.4**, and 3.5.