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1 Version Date:May 27, 2020 Abbreviated Title: Anti-CD19 and anti-CD20 CAR T cells CC **Protocol \#:** *20-C-0008A*
IBC \#:19-VI-07 Version Date: May 27, 2020 NCT Number: NCT04160195 Title: T Cells Expressing Fully-human Anti-CD19 and Anti-CD20 Chimeric Antigen Receptors for Treating B-cell malignancies and Hodgkin Lymphoma 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-CD19 and anti-CD20 CAR T cells   |
|---------------|---------------------------------------|
| IND Number:   | 19203                                 |
| Sponsor:      | Center for Cancer Research            |
| Manufacturer: | NIH DTM                               |  
Commercial Agents: Cyclophosphamide, Fludarabine, OKT3
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precis

Background:
- Improved treatments for a variety of treatment-resistant malignancies including B-cell lymphomas, andchronic lymphocytic leukemia (CLL) and Hodgkin lymphoma are needed.  
- A particular need is development of new treatments for chemotherapy-refractory B-cell malignancies.  
- 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 patients with leukemia or lymphoma. These results have established anti-CD19 CAR T cells as an important therapy for relapsed lymphoma, but only about 40% of patients receiving anti-CD19 CAR T cells have durable complete remissions.  
- Most B-cell malignancies express CD19 and CD20, but expression of CD19 and CD20 can be lost or diminished.  
- The malignant cells of Hodgkin lymphoma, Hodgkin Reed-Sternberg cells, originate from B cells, which is the rationale for treating Hodgkin lymphoma with T cells targeting CD19 and CD20.  
- CD19 and CD20 are not expressed by normal cells except for B cells and some plasma cells.  
- We have constructed a novel gene therapy construct that encodes a fully-human anti-CD19 CAR with a CD28 domain and a fully-human anti-CD20 CAR with a 4-1BB domain.  
- T cells expressing this CAR construct, called Hu1928-Hu20BB, can specifically recognize CD19 and CD20-expressing target cells in vitro and eradicate CD19 or CD20-expressing tumors in mice.  
- One CAR expressed in this CAR construct, Hu19-CD828Z has been tested in humans before. The other CAR in the total construct, Hu20-CD8BBZ, has not been tested in humans before.  
- Possible toxicities include cytokine-associated toxicities such as fever, hypotension, and neurological toxicities. Elimination of normal B cells is probable, and unknown toxicities are also possible.
Objectives:
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primary

- Determine the safety and feasibility of administering T cells expressing a novel fullyhuman anti-CD19 and anti-CD20 CAR construct to patients with advanced B-cell malignancies and Hodgkin lymphoma.
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exploratory

- Evaluate serum cytokine levels and associationswith anti-malignancy efficacy and toxicity
- Evaluate clinical predictors of anti-lymphoma responses and toxicity.  
- Evaluate phenotype of infused CAR T cells and CAR T cells from the blood of patients.
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eligibility

- Patients must have any B-cell lymphoma, or CLL/SLL, Gray-zone lymphoma, nodular lymphocyte-predominance Hodgkin lymphoma, or classical Hodgkin lymphoma with any CD19 or CD20 expression on Reed-Sternberg cells. Lower grade lymphomas transformed to DLBCL are potentially eligible as is primary mediastinal B-cell lymphoma and all other subtypes of DLBCL. Burkitt and mantle cell lymphoma are potentially eligible.  
- Patients must have malignancy that is measurable on a CT scan or by flow cytometry of bone marrow or blood.  
- Patients must have a creatinine of 1.5 mg/dL or less and a normal cardiac ejection fraction.  
- An ECOG performance status of 0-1 is required. - No active infections are allowed including hepatitis B or hepatitis C. - Absolute neutrophil count≥1000/µL, platelet count≥50,000/µL, hemoglobin≥8g/dL - Serum ALT and AST less or equal to 3 times the upper limit of the institutional normal unless liver involvement by malignancy is demonstrated.  
- At least 14 days must elapse between the time of any prior systemic treatment (including corticosteroids) and protocol-required leukapheresis or CAR T-cell infusion. Thirty days must elapse from therapy with antibodies targeting CD19 or CD20 and CAR T-cell infusion.
- The patient's malignancy will need to be assessed for CD19 and C20 expression by flow cytometry or immunohistochemistry performed at the NIH. If unstained, paraffinembedded bone marrow or lymphoma tissue sections are available from prior biopsies, these can be used to determine CD19 and CD20 expression by immunohistochemistry; otherwise, patients will need to come to the NIH for a biopsy to determine CD19 and CD20 expression. The sample for CD19 and CD20 expression must come from a biopsy obtained after any CD19 or CD20-targeted therapies such as monoclonal antibodies if such antibodies or CAR T-cell therapies have been received by the patient.  
- For classical Hodgkin lymphoma only, a biopsy from any time from any institution that shows any CD19 or CD20 expression on Reed-Sternberg cells is adequate for eligibility. CD19 or CD20 expression on the Reed-Sternberg cells that is weak or only present on some Reed-Sternberg cells by immunohistochemistry is compatible with protocol eligibility.
- For all lymphoma types except for classical Hodgkin lymphoma, either CD19 or CD20 expression must be "uniform". "Uniform" CD19 or CD20 expression is defined as no obvious lymphoma population lacking antigen expression can be present. Antigen expression can be assessed by either immunohistochemistry or flow cytometry.
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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 the Hu1928-
Hu20BB CAR construct.  
- Patients will receive a lymphocyte-depleting chemotherapy conditioning regimen with the intent of enhancing the activity of the infused CAR-expressing T cells.  
- The chemotherapy conditioning regimen is cyclophosphamide 500 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.  
- Two days after the chemotherapy ends, patients will receive an infusion of anti-CARexpressing T cells.  
- The initial dose level of this dose-escalation trial will be 0.66x106 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 is a mandatory 14-day inpatient hospitalization to monitor for toxicity.  
- Outpatient follow-up is planned for 2 weeks,16 days (neurologic checks), 21 days
(neurologic checks) and 1, 2, 3, 4, 6, 9, and 12 months after the CAR T-cell infusion; less frequent follow-up is required more than 1 year after infusion. Long-term genetherapy follow-up for a total of 15 years after infusion is required.
5 Version Date:May 27, 2020  
| Allo-HSCT   | Allogeneic hematopoietic stem cell transplant   |
|-------------|-------------------------------------------------|
| AML         | Acute myeloid leukemia                          |
| BSL         | Biosafety level                                 |
| CAR         | Chimeric antigen receptor                       |
| CFSE        | Carboxyfluorescein diacetate,succinimidyl ester |
| CLL         | Chronic lymphocytic leukemia                    |
| CR          | Complete remission                              |
| CRO         | Central Registration Office                     |
| CRP         | C-reactive protein                              |
| CTCAE       | Common Terminology Criteria for Adverse Events  |
| DLBCL       | Diffuse large B-cell lymphoma                   |
| DCI         | Donor Cell Infusion                             |
| DTM         | Department of Transfusion Medicine              |
| ELISA       | Enzyme-linked immunosorbent assay               |
| HLA         | Human leukocyte antigen                         |
| IBC         | Institutional Biosafety Committee               |
| OSP         | Office of Science Policy                        |
| PBMC        | Peripheral blood mononuclear cells              |
| PD          | Progressive disease                             |
| PET         | Positron emission tomography                    |
| PR          | Partial remission                               |
| RCR         | Replication competent retroviruses              |
| SD          | Stable disease                                  |
| SMC         | Safety Monitoring Committee                     |
| scFv        | Single chain variable fragment                  |
| TBNK        | T cell, B cell, and NK cell blood test          |
| TCR         | T cell receptor                                 |
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table of abbreviations

| TABLE OF CONTENTS                                                                                                                      |                                                                                                                         |
|----------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS .                                                                                                                               |                                                                                                                         |
| TABLE OF ABBREVIATIONS .                                                                                                               |                                                                                                                         |
| TABLE OF CONTENTS.                                                                                                                     |                                                                                                                         |
| STATEMENT OF COMPLIANCE                                                                                                                |                                                                                                                         |
| l                                                                                                                                      | INTRODUCTION .                                                                                                          |
| 1.1                                                                                                                                    | Study Objectives                                                                                                        |
| 1.1.1                                                                                                                                  | Primary Objective                                                                                                       |
| 1.1.2                                                                                                                                  | Secondary Objective(s)                                                                                                  |
| 1.1.3                                                                                                                                  | Exploratory Objective(s)                                                                                                |
| 1.2                                                                                                                                    | Background and Rationale .                                                                                              |
| 1.2.1                                                                                                                                  | Introduction                                                                                                            |
| 1.2.2                                                                                                                                  | B-cell malignancies:  epidemiology and standard treatment...................................... 14                      |
| 1.2.3                                                                                                                                  | Rationale for treating Hodgkin lymphoma with anti-CD19/anti-CD20 T cells .... 14                                        |
| T-cell gene therapy                                                                                                                    |                                                                                                                         |
| 1.2.4                                                                                                                                  |                                                                                                                         |
| 1.2.5                                                                                                                                  | Chimeric antigen receptors preclinical background                                                                       |
| 1.2.6                                                                                                                                  | CD19 .                                                                                                                  |
| 1.2.7                                                                                                                                  | CD20 .                                                                                                                  |
| 1.2.8                                                                                                                                  | CD20 expression on B-cell lymphomas and treatment of lymphoma with antibodies                                           |
| targeting CD20 .                                                                                                                       |                                                                                                                         |
| Clinical results with anti-CD19 CAR T cells                                                                                            |                                                                                                                         |
| 1.2.9                                                                                                                                  |                                                                                                                         |
| 1.2.10                                                                                                                                 | Anti-CD20 CARs .                                                                                                        |
| 1.2.11                                                                                                                                 | Development and preclinical testing of a CAR construct encoding both an anti-                                           |
| CD19 CAR and an anti-CD20 CAR.......................................................................................................23 |                                                                                                                         |
| 1.2.12                                                                                                                                 | Rationale for immunosuppressive chemotherapy and selection of lymphocyte-                                               |
| depleting                                                                                                                              |                                                                                                                         |
| 1.2.13                                                                                                                                 | Rationale for dose-escalation                                                                                           |
| 1.2.14                                                                                                                                 | Rationale for fully-human CARs                                                                                          |
| 1.2.15                                                                                                                                 | Summary of risks and potential benefits .............................................................................35 |
| 2                                                                                                                                      | ELIGIBILITY ASSESSMENT AND ENROLLMENT ........................................................36                        |
| 2.1                                                                                                                                    | Eligibility Criteria                                                                                                    |
| 2.1.1                                                                                                                                  | Inclusion Criteria                                                                                                      |
| 2.1.2                                                                                                                                  | Exclusion criteria 38                                                                                                   |
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| 2.1.3                                                                         | Recruitment Strategies 40                                                       |
|-------------------------------------------------------------------------------|---------------------------------------------------------------------------------|
| 2.2                                                                           | Screening Evaluation 40                                                         |
| 2.2.1                                                                         | Screening activities performed prior to obtaining informed consent 40           |
| 2.2.2                                                                         | Screening activities performed after a consent for screening has been signed 40 |
| 2.3                                                                           | Participant Registration and Status Update Procedures 41                        |
| 2.4                                                                           | Treatment Assignment Procedures (For registration purposes only): 41            |
| 3                                                                             | STUDY IMPLEMENTATION 42                                                         |
| 3.1                                                                           | Study Design 42                                                                 |
| 3.1.1                                                                         | General study plan 42                                                           |
| 3.1.2                                                                         | Protocol schema 44                                                              |
| 3.1.3                                                                         | Dose Limiting Toxicity 45                                                       |
| 3.1.4                                                                         | Dose Escalation 46                                                              |
| 3.2                                                                           | Pre-Treatment Phase 49                                                          |
| 3.2.1                                                                         | Leukapheresis 49                                                                |
| 3.2.2                                                                         | Hu1928-Hu20BB CAR-expressing T-cell preparation 49                              |
| 3.3                                                                           | Drug Administration 49                                                          |
| 3.3.1                                                                         | Overall summary of the treatment plan 50                                        |
| 3.4                                                                           | Dose Modifications/Delay 51                                                     |
| 3.5                                                                           | Potential repeat treatment 51                                                   |
| 3.6                                                                           | Protocol Stopping Rules 52                                                      |
| 3.7                                                                           | Protocol Evaluation 52                                                          |
| 3.7.1                                                                         | Baseline Evaluations 52                                                         |
| 3.7.2                                                                         | Studies to be performed on Day 0 and during the mandatory 14-day inpatient      |
| admission after cell infusion 54 3.7.3 Post-infusion outpatient evaluation 55 |                                                                                 |
| 3.8                                                                           | Study Calendar 57                                                               |
| 3.9                                                                           | Gene-therapy-specific follow-up 61                                              |
| 3.9.1                                                                         | Clinical Evaluation 61                                                          |
| 3.9.2                                                                         | Testing for persistence of CAR transduced cells 61                              |
| 3.9.3                                                                         | Replication competent retrovirus (RCR) testing 61                               |
| 3.10                                                                          | Cost and Compensation 61                                                        |
| 3.10.1                                                                        | Costs 62                                                                        |
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| 3.10.2   | Compensation 62                                                       |
|----------|-----------------------------------------------------------------------|
| 3.10.3   | Reimbursement 62                                                      |
| 3.11     | Criteria for Removal from Protocol Therapy and Off Study Criteria 62  |
| 3.11.1   | Criteria for removal from protocol therapy 62                         |
| 3.11.2   | Off-study Criteria 63                                                 |
| 4        | CONCOMITANT MEDICATIONS/MEASURES 63                                   |
| 4.1      | Antibiotic prophylaxis 64                                             |
| 4.2      | Blood product support 64                                              |
| 4.3      | Anti-emetics 64                                                       |
| 4.4      | Granulocyte colony-stimulating factor 64                              |
| 4.5      | Avoidance of corticosteroids 64                                       |
| 4.6      | Excluded medications 64                                               |
| 4.7      | Toxicity management guidelines 65                                     |
| 4.8      | Management of patients with Burkitt and other high-grade lymphomas 65 |
| 5        | BIOSPECIMEN COLLECTION 65                                             |
| 5.1      | CORRELATIVE STUDIES FOR RESEARCH 65                                   |
| 5.1.1    | Immunological Testing 67                                              |
| 5.1.2    | Additional biopsies and additional blood draws 69                     |
| 5.1.3    | Future studies 69                                                     |
| 5.1.4    | Samples Sent to Figg Lab 69                                           |
| 5.2      | Sample Storage, Tracking and Disposition 70                           |
| 5.2.1    | Samples Managed by Dr. Figg's Blood Processing Core (BPC) 70          |
| 5.2.2    | Sample Storage, Tracking, and Disposition for Surgery Branch 71       |
| 5.2.3    | Protocol Completion/Sample Destruction 71                             |
| 5.2.4    | Samples for Genetic/Genomic Analysis 72                               |
| 6        | DATA COLLECTION AND EVALUATION 73                                     |
| 6.1      | Data Collection 73                                                    |
| 6.1.1    | Adverse event recording: 74                                           |
| 6.2      | Data Sharing Plans 74                                                 |
| 6.2.1    | Human Data Sharing Plan 74                                            |
| 6.3      | Genomic Data Sharing Plan 74                                          |
| 6.4      | Response Criteria 74                                                  |
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abbreviated title anti-cd19 and anti-cd20

| Version Date: May 27, 2020                                                                                               |                                                                                                                                             |             |
|--------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------|-------------|
| Response criteria: .                                                                                                     |                                                                                                                                             |             |
| 6.4.1                                                                                                                    |                                                                                                                                             |             |
| 6.4.2                                                                                                                    | Response criteria for CLL: .                                                                                                                |             |
| 6.5                                                                                                                      | T Toxicity Criteria                                                                                                                         |             |
| NIH Reporting Requirements / DATA SAFETY MONITORING PLAN ................................ 78                             |                                                                                                                                             |             |
| 7                                                                                                                        | 7.1                                                                                                                                         | Definitions |
| 7.2                                                                                                                      | OHSRP Office of Compliance and Training Reporting ...........................................................78                             |             |
| 7.2.1                                                                                                                    | Expedited Reporting                                                                                                                         |             |
| 7.2.2                                                                                                                    | NIH Intramural IRB Requirements for PI Reporting at Continuing Review........ 78                                                            |             |
| 7.3                                                                                                                      | NCI Clinical Director Reporting                                                                                                             |             |
| 7.4                                                                                                                      | Institutional Biosafety Committee (IBC) Reporting Criteria ........................................... 78                                   |             |
| Serious Adverse Event Reports to IBC .................................................................................78 |                                                                                                                                             |             |
| 7.4.1                                                                                                                    |                                                                                                                                             |             |
| 7.4.2                                                                                                                    | Annual Reports to IBC.                                                                                                                      |             |
| 7.4.3                                                                                                                    | Clinical Trial Information .                                                                                                                |             |
| 7.4.4                                                                                                                    | Progress Report and Data Analysis                                                                                                           |             |
| NIH required Data and Safety Monitoring Plan                                                                             |                                                                                                                                             |             |
| 7.5                                                                                                                      |                                                                                                                                             |             |
| 7.5.1                                                                                                                    | Safety Monitoring Committee (SMC)                                                                                                           |             |
| 8                                                                                                                        | SPONSOR PROTOCOL/ SAFETY REPORTING ...................................................................................80                    |             |
| Definitions                                                                                                              |                                                                                                                                             |             |
| 8.1                                                                                                                      |                                                                                                                                             |             |
| 8.1.1                                                                                                                    | A Adverse Event                                                                                                                             |             |
| 8.1.2                                                                                                                    | Serious Adverse Event (SAE) .........................................................................................................80     |             |
| 8.1.3                                                                                                                    | Life-threatening                                                                                                                            |             |
| Severity                                                                                                                 |                                                                                                                                             |             |
| 8.1.4                                                                                                                    |                                                                                                                                             |             |
| 8.1.5                                                                                                                    | Relationship to Study Product                                                                                                               |             |
| 8.2                                                                                                                      | Assessment of Safety Events                                                                                                                 |             |
| 8.3                                                                                                                      | Reporting of Serious Adverse Events................................................................................................82       |             |
| Reporting Pregnancy                                                                                                      |                                                                                                                                             |             |
| 8.4                                                                                                                      |                                                                                                                                             |             |
| 8.4.1                                                                                                                    | MATERNAL EXPOSURE                                                                                                                           |             |
| 8.4.2                                                                                                                    | Paternal exposure                                                                                                                           |             |
| Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND................... 83                                 |                                                                                                                                             |             |
| 8.5                                                                                                                      |                                                                                                                                             |             |
| 9                                                                                                                        | CLINICAL MONITORING                                                                                                                         |             |
| 10                                                                                                                       | STATISTICAL CONSIDERATIONS ..............................................................................................................83 |             |
| 10.1                                                                                                                     | Statistical Hypothesis .                                                                                                                    |             |
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| Version Date:May 27, 2020 10.1.1 Primary objectives 83 10.1.2 Secondary objectives 83 10.2 Sample Size Determination 83 10.3 Populations For Analyses 84 10.4 Statistical Analyses 84 10.4.1 General Approach 84 10.4.2 Analysis of the Primary Endpoints 84 10.4.3 Analysis of the Secondary Endpoint(s) 84 10.4.4 Safety Analyses 85 10.4.5 Baseline Descriptive Statistics 85 10.4.6 Planned Interim Analyses 85 10.4.7 Sub-Group Analyses 85 10.4.8 Tabulation of individual Participant Data 85 10.4.9 Exploratory Analyses 85 11 COLLABORATIVE AGREEMENTS 85 12 HUMAN SUBJECTS PROTECTIONS 85 12.1 Rationale For Subject Selection 85 12.2 Participation/selection rationale 86 12.3 Participation of Children 86 12.4 Participation of Subjects Unable to Give Consent 86 12.5 Risk/Benefit Assessment 86 12.5.1 Known Potential Benefits 87 12.5.2 Known Potential Risks 87 12.5.3 Assessment of Potential Risks and Benefits for Recipients 88 12.6 Consent Process and Documentation 89 ... The initial consent process as well as re-consent, when required, may take place in person in a  private area (e.g., clinic consult room) or remotely (e.g., via telephone or other NIH approved  remote platforms) per discretion of the designated study investigator and with the agreement of  the participant/consent designee(s) 89 13 REGULATORY AND OPERATIONAL CONSIDERATIONS 89 13.1 Study Discontinuation and Closure 89 13.2 Quality Assurance and Quality Control 90 13.3 Conflict of Interest Policy 90   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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| 14                                                                                                                                                                                                                                                             | PHARMACEUTICAL INFORMATION 90                                                   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------|
| 14.1                                                                                                                                                                                                                                                           | Gamma-Retroviral Vector Containing the anti-CD19/anti-CD20 CAR Gene 91          |
| 14.1.1                                                                                                                                                                                                                                                         | Source/Acquisition and Accountability 91                                        |
| 14.1.2                                                                                                                                                                                                                                                         | Cells manufacturing 91                                                          |
| 14.1.3                                                                                                                                                                                                                                                         | Toxicities 91                                                                   |
| 14.1.4                                                                                                                                                                                                                                                         | Administration procedures 91                                                    |
| 14.2                                                                                                                                                                                                                                                           | Commercial Agents 91                                                            |
| 14.2.1                                                                                                                                                                                                                                                         | Cyclophosphamide 91                                                             |
| 14.2.2                                                                                                                                                                                                                                                         | FLUDARABINE 92                                                                  |
| 15                                                                                                                                                                                                                                                             | REFERENCES 93                                                                   |
| 16                                                                                                                                                                                                                                                             | APPENDICES 107                                                                  |
| 16.1                                                                                                                                                                                                                                                           | Appendix A: Performance Status Criteria 107                                     |
| 16.2                                                                                                                                                                                                                                                           | Appendix B: Data Collection Elements Required By Protocol 108                   |
| 16.3                                                                                                                                                                                                                                                           | Appendix C: Guidelines for management of common toxicities that occur after CAR |
| T-cell infusions 110 16.4 Appendix D: Infusion Instructions 115 16.5 Appendix E: CARTOX (Adapted from Neelapu et al, 2018): 118 16.6 Appendix F: Cytokine Release Syndrome Grading Assessment (Adapted from Lee et  al, 2019). ASTCT CRS Consensus Grading 119 |                                                                                 |
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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; a 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 antiCD19 and anti-CD20 CAR construct to patients with advanced B-cell malignancies and Hodgkin lymphoma. 1.1.2 Secondary Objective(s)
- Evaluate the in vivo persistence and peak blood levels of CAR T cells. - Assess for evidence of anti-malignancy activity by T cells expressing both an anti-CD19 CAR and an anti-CD20 CAR
- Assess the relationship of CD19 and CD20 expression on treatment outcomes.  
1.1.3 Exploratory Objective(s)
- Evaluate serum cytokine levels and associationswith anti-malignancy efficacy and toxicity
- Evaluate clinical predictors of anti-lymphoma responses and toxicity. - Evaluate phenotype of infused CAR T cells and CAR T cells from the blood of patients.  
We have developed anti-CD19 CAR T-cell therapies for B-cell malignancies, but we have found that some cases of B-cell lymphoma are CD19-negative before CAR T-cell treatment, and other cases lose CD19 expression after CAR T-cell treatment. To address the issue of CD19-negative B-cell lymphomas, we have designed a CAR construct that leads to simultaneous expression of 2 CARs on the same T cell, one CAR targeting CD19 and the other targeting CD20. Both CD19 and CD20 are B-cell antigens with very restricted tissue expression patterns. The anti-CD19 CAR expressed by this construct is Hu19-CD828Z that has been previously tested as a single CAR in a phase I clinical trial. In this trial, 55% of patients obtained complete remissions, and toxicity was manageable and limited to expected CAR-related toxicities. Four of 20 patients on this trial of Hu19-CD828Z lost CD19 expression, and this loss of CD19 expression is a major factor prompting us to develop CAR T-cell strategies targeting 2 antigens simultaneously. CD20 is a B-cell antigen that is expressed at high levels on most B-cell lymphomas, especially the most common B-cell lymphoma, diffuse large B-cell lymphoma. CD20 is the target of the commonly-used monoclonal antibody rituximab. Consistent with the limited expression pattern of CD20, Rituximab is remarkably free of toxicities. We chose CD20 as the second target, along with CD19, for the CAR construct to be tested in this clinical protocol because CD20 has high expression on many lymphomas and an expression pattern that is apparently limited to B cells. We designed an antiCD20 CAR called Hu20-CD8BBZ. This CAR has a fully-human anti-CD20 single-chain variable fragment (scFv) and also includes a 4-1BB costimulatory domain; therefore, one interesting aspect of this clinical trial is to assess the function of T cells expressing 2 CARswith one CAR containing a CD28 costimulatory domain and the other CAR containing a 4-1BB costimulatory domain. The CAR construct is designated Hu1928-Hu20BB; it is encoded by the MSGV1 gamma-retroviral vector. T cells expressing this anti-CD19 and anti-CD20 CAR construct can eradicate tumors in mice. We propose to conduct a phase I clinical trial of B cell lymphomas, Hodgkin lymphoma, and chronic lymphocytic leukemia.Chronic lymphocytic lymphoma patients will only be enrolled if they do not have leukemia cells in their blood at the time of protocol enrollment. Patients enrolled on the trial will receive a single cycle of chemotherapy that is designed to decrease endogenous lymphocyte counts because extensive evidence demonstrates that depleting endogenous lymphocytes, and possibly other cells, with chemotherapy or total body irradiation dramatically increases the anti-tumor activity of adoptively transferred T cells. After the lymphocyte-depleting chemotherapy, patients will receive an infusion of autologous CAR T cells. The T cell dose will escalate with sequential cohorts of patients until a maximum tolerated dose is determined.
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1.2.2 b-cell malignancies epidemiology and

Annually in the United States, approximately twenty-three thousand people die of B cell malignancies 1. B cell malignancies have quite heterogeneous clinical manifestations and prognoses. Chronic lymphocytic leukemia (CLL) is a common disease that is incurable by chemotherapy 2. Recently, the new tyrosine-kinase inhibitor drug ibrutinib has revolutionized the treatment of CLL, so most patients will be treated with ibrutinib or another signal transduction inhibitor before participating in a phase I clinical trial. The tyrosine-kinase inhibitors have very high response rates, and progression-free survivals of greater than 2 years have been reported3, but follow-up is short, and some patients receiving these agents do develop progressive CLL that in some cases has transformed to a higher grade3. A B-cell malignancy for which new therapies are urgently needed is mantle cell lymphoma. This disease is almost always incurable by chemotherapy and has an aggressive course that is characterized by short responses to chemotherapy 4. The tyrosine kinase inhibitor ibrutinib has been shown to have significant activity against mantle cell lymphoma, but in most cases it is not curative, so new therapies for mantle cell lymphoma are a great need5. Follicular lymphoma is a common lymphoma with an extremely variable course 6. Patients with follicular lymphoma have a median survival of about 4.5 years after first relapse 7,8. Follicular lymphoma is susceptible to a variety of therapies, but many of the therapies are toxic, and patients are seldom cured of follicular lymphoma, so improved therapies are needed for follicular lymphoma 9. Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy. DLBCL is often curable by standard chemotherapy, and autologous stem cell transplants are sometimes able to cure patients with relapsed large cell lymphoma 10.  
However, patients with DLBCL that is refractory to 1st salvage chemotherapy have response rates to 2nd salvage therapy of only 14 to 43% 11-15. Patients with DLBCL refractory to 2nd-line chemotherapy have very poor prognoses with a reported median survival of only 4 months and a 1-year survival rate of only 4% 15. Allogeneic stem cell transplantation is a potentially curative treatment option for patients with advanced B cell malignancies, but allogeneic stem cell transplantation generally has a treatment-related mortality rate of 10-30% and substantial chronic morbidity due to graft-versus-host disease 16,17. All in all, improved therapies for B cell malignancies are clearly needed.
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1.2.3 rationale for treating hodgkin

Classical Hodgkin lymphoma (cHL) is characterized pathologically by the presence of a multinucleated cell called the Reed Sternberg cell intermixed with smaller mononuclear Hodgkin cells in an extensive background of inflammatory cells. The malignant cells in cHL typically express CD30 and most often lack expression of many B cell markers including CD19 and CD20 18. The malignant Hodgkin Reed Sternberg (HRS) cells make up <1-2% of the total cellular infiltrate in Hodgkin lymphoma masses because the malignant cells are surrounded by an extensive inflammatory infiltrate 19,20. The multinucleated Reed Sternberg-like cells have been consistently shown to have reduced proliferative capacity as compared to the Hodgkin cells as demonstrated by their reduced potential to form colonies 21, incorporate bromodeoxyuridine22, divide on time-lapse imaging23, and inability to form tumors in nude mice 24. The cellular origin of the HRS cells was at first difficult to determine given their lack of expression of typical B cell markers and because the malignant cells are surrounded by an extensive nonmalignant inflammatory infiltrate leading to contamination. However, improvements in microdissection techniques combined with analysis of immunoglobulin rearrangements showed that the cellular origin of the HRS cells traces back to the germinal center B lymphocytes 25,26.  
These initiating germinal center cells express CD19. In fact, a more recent study demonstrated that there are circulating clonotypic CD19+ cells in the peripheral blood of Hodgkin lymphoma patients that may give rise to the HRS cells seen on pathologic examination27. Patients with classical Hodgkin lymphoma have an excellent response rate to front-line multi-agent chemotherapy. Unfortunately, the approximately 20% of patients who do not respond to front line treatment or relapse after salvage chemotherapy or autologous transplant have a less optimistic prognosis. The CD30 antibody-drug conjugate brentuximab vedotin (BV) has recently changed treatment algorithms for relapsed cHL. In the initial study, BV had an overall response rate of 75% and complete response rate of 34% 28. However, long-term follow-up of these same patients shows a PFS at 5 years of 22% and many patients require additional therapy 29. Therefore, while BV represents a large advance in the treatment of cHL, BV treatment alone is curative in only a small fraction of patients. Pembrolizumab represents another therapeutic strategy for patients with relapsed Hodgkin lymphoma and has FDA approval based on a phase II study showing an overall response rate of 69% and complete response rate of 22.4% 30 . Similarly, nivolumab has a 69% overall response rate and a 16% complete response rate31. Finally, anti-CD30 CAR T cells have also been investigated in patients with cHL in two published trials 32,33. In the Ramos et al paper, a 2/7 patients achieved prolonged complete responses lasting almost 2 years or more 33. In the Wang et al paper, 7/ 18 patients achieved a partial response but these were unfortunately shortlived with a median response duration ranging from 2-9 months with one ongoing response at 6.5 months 32. Collectively, these multiple new agents for classical Hodgkin lymphoma represent encouraging steps forward but do not provide efficacy in all patients and often do not lead to complete responses that are durable. We purpose that Hu1928-Hu20BB CAR T cells may represent an additional avenue for treatment of Hodgkin lymphoma. These cells may be efficacious in Hodgkin lymphoma by targeting any cases of CD20 positive disease, by depleting the CD19 positive clonotypic B lymphocytes that are the origin of the HRS cells, and by impacting non-malignant immune cells in the tumor microenvironment. First, Hu1928-Hu20BB CAR T cells may work to target any cases of CD20 positive lymphoma. Nodular lymphocyte predominant Hodgkin lymphoma expresses CD20 in 98% of cases 34. In contrast, only approximately 20% of cases of classical Hodgkin lymphoma express CD20 35. Importantly, within the CD20 expressing tumors, the median percentage of HRS cells expressing CD20 was 55% 35. Studies regarding whether CD20 expression is associated with a worse prognosis have demonstrated inconsistent results with some demonstrating an association with better prognosis 36, others with worse prognosis 37, and still others showing no change in prognosis 38. The expression of CD20 in nodular lymphocyte predominant Hodgkin lymphoma and in some cases of classical Hodgkin lymphoma made investigation into treatment with the antiCD20 monoclonal antibody rituximab a logical choice. In patients with nodular lymphocyte predominant Hodgkin lymphoma, overall response rate to rituximab is 100% 39. Studies of rituximab in classical Hodgkin lymphoma have been less successful. In the relapsed Hodgkin lymphoma setting, rituximab has single agent activity with an overall response rate of around 20% 40 and also may have activity when added to gemcitabine 41. Initial studies of adding rituximab in the upfront setting to ABVD were promising 42,43 but a follow-up randomized study was limited by poor accrual and did not show a benefit in either event free survival or overall survival 44. Similarly, studies of adding rituximab to escalated BEACOPP also failed to show a benefit 45,46. Rituximab may be insufficient for the treatment of Hodgkin lymphoma because it does not cause complete eradication of B cells. In particular, studies of rituximab in primates have shown it causes depletion of only 34-78% of B cells in lymph nodes 47. Similarly, in humans rituximab treatment leads to the rapid clearance of B cells from the peripheral blood 48 but with some persistence in the lymph nodes 49,50. We have observed that our anti-CD19 CAR T cells cause a more complete eradication of B cells than rituximab with many patients experiencing prolonged B cell depletion lasting over a year 51,52. This more complete eradication of B cells may lead to a better therapeutic response as compared to rituximab. Second, Hu1928-Hu20BB CAR T cells may work to target the clonotypic CD19 positive cells that may serve as HRS stem cells. There is a precedent for the concept of targeting CD19 positive progenitor cells with anti-CD19 CAR T cells in diseases where the mature malignant cells do not express CD19 but may arise from CD19 positive progenitors. These studies have been performed in both myeloma and Hodgkin lymphoma. An initial case report in myeloma demonstrated a 12month complete response in a heavily pre-treated patient who received anti-CD19 autologous CAR T cells following myeloablative chemotherapy 53. This response was despite the absence of CD19 expression as detected by flow cytometry on 99.95% of the patient's myeloma cells 53. A followup study using the same approach with this patient and 9 others showed that 2/10 patients undergoing anti-CD19 second autologous transplant patients experienced a longer duration of remission as compared to their first transplant. For example, 181 days with first transplant versus 479 days with second anti-CD19 autologous transplant 54. This is significant because the duration of response to second autologous transplant is generally much shorter than the response to first autologous transplant. There has also been a phase 1 study of CD19 CAR T cells in Hodgkin lymphoma55. This study was limited because it used electroporation of non-viral RNA to deliver the CAR construct and this results in very short-lived CAR T cells. Furthermore, the study only infused 4 patients which limits evaluation of response. However, even in this small patient population and using a very transiently expressed construct, responses were seen in 2/4 infused patients and one of these responses was a complete response. This suggests that the underlying concept of targeting Hodgkin lymphoma through CD19 modulation can be effective. In our previous viral CD19 CAR studies, we have observed significantly longer CAR T persistence 56 52 and therefore expect our Hu1928-Hu20BB CAR T cells to have greater efficacy and longer term response than that seen in this non-viral CD19 CAR T study. Finally, anti-CD19 anti-CD20 CAR T cells may cause responses by impacting the tumor microenvironment. The malignant HRS cells are surrounded by an extensive inflammatory infiltrate composed of fibrous tissue in addition to immune cells including T cells, B cells, macrophages, eosinophils, and others 18. This inflammatory cellular background appears to provide a tumor microenvironment that may support growth of the malignant cells57. In a previous study with anti-CD19 CAR T cell therapy comparing pre and post-treatment biopsies, the immune gene signature is markedly altered following treatment 58. These effects on local inflammatory cells could impact tumor bulk and response to Hu1928-Hu20BB CAR T cells.
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1.2.4 t-cell gene therapy

In an attempt to develop effective immunotherapies for cancer that are less toxic than allogeneic stem cell transplantation, many investigators have developed T-cell gene therapy approaches to specifically target T cells to tumor-associated antigens59. T cells can be prepared for adoptive transfer by genetically modifying the T cells to express receptors that specifically recognize tumorassociated antigens.59-66 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.59,67-70 Genetically modified antigen-specific T cells can be generated from peripheral blood mononuclear cells (PBMC) in sufficient numbers for clinical treatment within 10 days.68 Genetically modifying T cells with gamma-retroviruses consistently causes high and sustained levels of expression of introduced genes without in vitro selection67,70-72, and genetic modification of mature T cells with gamma-retroviruses has a long history of safety in humans.7375 There are two general approaches for generating antigen-specific T cells by genetic modification: introducing genes encoding natural αβT cell receptors (TCRs) or introducing genes encoding chimeric antigen receptors (CARs).59,61,63,65 CARs are fusion proteins incorporating antigen recognition moieties and T cell activation domains.64,76-78 The antigen-binding domains of most CARs currently undergoing clinical and preclinical development are antibody variable regions.61,64,76,78 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 molecules59,61,63,76, 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.59,61,79-81
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1.2.5 chimeric antigen receptors preclinical

Preclinical experiments evaluating CAR-expressing T cells as cancer therapy were initiated in 1993.82,83 These experiments led to a clinical trial of CAR-transduced T cells targeting the αfolate receptor on ovarian cancer cells; no tumor regressions were observed during this clinical trial.84 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 gamma retroviruses67,70-73,85-87, lentiviruses68,88-91, or transposon systems92,93 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.68,85,94 Different portions of CARs including antigen-recognition moieties, extracellular structural components, costimulatory domains such as the cytoplasmic portion of the CD28 protein, and T-cell-activation moieties such as the signaling domains of the CD3ζ 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.76,85,89,95-98 Much of the preclinical work evaluating CARs has been performed with CARs targeting the Bcell antigen CD19.85-87,92,99-101 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.87,96 These studies showed that adding CD28 moieties to CARs enhanced antigen-specific cytokine production and proliferation by anti-CD19 CAR T cells.96,102,103 T cells expressing CARs with CD28 signaling moieties and CD3ζ signaling domains were more effective than T cells expressing CARs without CD28 moieties at eradicating human leukemia cells from mice.102,103 Subsequently, CARs incorporating other signaling domains from costimulatory molecules such as 4-1BB (CD137) were developed.88 Anti-CD19 CARs containing the signaling domains of both 4-1BB and CD3ζ were superior to CARs containing the signaling domains of CD3ζ without any costimulatory domains at eradicating human malignant cells from mice.89,95 Similar to CD28, including 4-1BB signaling moieties in CARs led to increased CD19-specific proliferation and enhanced in vivo persistence.89 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 4-1BB-containing CAR occurred whether or not the T
cells were exposed to the antigen that the CAR recognized.89,95
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1.2.6 cd19

CD19 is commonly used as the target of CAR T cells because it is expressed on most malignant B cells 104,105, but the only normal cells that express CD19 are B cells and perhaps follicular dendritic cells 105,106. Importantly, CD19 is not expressed on pluripotent hematopoietic stem cells 107. While destruction of normal B cells is a drawback to targeting CD19, several factors indicate that destruction of normal B cells is tolerable. When patients receive the anti-CD20 monoclonal antibody rituximab, the number of normal B cells is severely depressed for several months 108, yet patients that receive chemotherapy plus rituximab do not have an increased rate of common infections when compared to patients who receive chemotherapy alone.109 Finally, patients can be treated with intravenous infusions of IgG if necessary to increase IgG levels.110
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1.2.7 cd20

B-lymphocyte antigen CD20 is an activated-glycosylated phosphoprotein expressed on the surface of all B-cells. CD20 is encoded by the *MS4A1* gene in humans. 79.110,111 CD20 is expressed on all stages of B cell development except the first and last, i.e., not on either early proB cells or plasma cells.112 CD20 expression on normal cells is limited to B cells except for possible expression on a small subset of T cells.113,114 115 CD20 is also found on B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, transformed mycosis fungoides, 116 and melanoma cancer stem cells.117 The expression of CD20 is regulated by the chemokine signaling through the CXCR4/SDF1 axis, and this can be impaired by drugs interfering with microenvironmental interactions.118 CD20 has no known natural ligand and its function is to enable optimal B-cell immune responses, specifically against T-independent antigens.119 CD20 may function as a calcium channelin the cell membrane. It has been shown that CD20 plays a role in the microenvironmental interactions of B cells,118 and B-cell receptor signaling. In chronic lymphocytic leukemia, when the anti-CD20 monoclonal antibody rituximab is used in clinical therapy, it seems to primarily eliminate the malignant B cells that express high levels of CD20 and have a high potential for activation of the B-cell receptor signaling pathway.120Rituximab combined with chemotherapies could down-regulate the CD20 expression, however, when CD20-negative cells were treated with 5-aza-2'-deoxycytidine in vitro, the expression of CD20 mRNA was stimulated within 3 days, resulting in the restoration of both cell surface expression of the CD20 protein and rituximab sensitivity. These findings suggest that some epigenetic mechanisms may be partly related to the down-regulation of CD20 expression after rituximab treatment.121 Because the cell surface density of CD20 is higher than that of CD19 in normal B cells, 122 CD20 became a popular target for the monoclonal antibodies.
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1.2.8 cd20 expression on b-cell

CD20 is an antigen that is expressed on mature B cells but not expressed on early B-cell progenitors or more mature plasma cells123. In addition, CD20 is broadly expressed in a range of B-cell malignancies124 . Previous studies showed that CD20 is expressed on more than 90% of cases of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and CLL 125. Moreover, the degree of expression of CD20 varies between the different B-cell malignancies with CLL, for example, expressing CD20 at a lower level than seen in DLBCL 125. The variable expression of CD20 on different malignancies is believed to account, at least partially, for the different responses of individual B-cell malignancies to CD20 targeted therapy 126. The expression of CD20 on most B-cell malignancies made CD20 an attractive therapeutic target. The first CD20 targeted monoclonal antibody, rituximab, resulted in a dramatic improvement in the prognosis of many B-cell malignancies. For example, patients with DLBCL treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) showed an improvement in 10 year overall survival from 27.6% in the CHOP group to 43.5% in the R-CHOP group127. Rituximab is also effective in a range of other B-cell malignancies. In particular, rituximab has marked efficacy in follicular lymphoma both in combination with chemotherapy 128-130and as a single agent 131. It is also effective in combination chemotherapy for CLL 132,133 and improves the rate of complete response in mantle cell lymphoma 134.  
There are ongoing efforts to improve upon rituximab to target CD20 more effectively. Resistance to rituximab can arise through down-regulation of CD20 on the malignant cell surface by multiple different mechanisms 126. The next generation CD20 antibodies have been developed to target CD20 more effectively even in the context of lower CD20 expression level as is seen in CLL. These antibodies include ofatumumab 135 and obinutuzumab 136, both of which are FDA approved for treatment of CLL. In follicular lymphoma, a head-to-head comparison of obinutuzumab to rituximab did show a slight improvement in progression free survival (3 year PFS 80% versus 73.3%) but at the cost of increased toxicity with obinutuzumab 137. This suggests that further antibody modifications or alternative methods to target CD20 are needed.
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1.2.9 clinical results with anti-cd19

Results from several clinical trials of anti-CD19 CAR T cells have been reported to date in peerreviewed papers.67,68,70-72,138-142 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 in a patient who experienced a dramatic regression of advanced follicular lymphoma.71 This clinical trial utilized a gamma retroviral vector to introduce an anti-CD19 CAR containing the signaling domains of the CD28 and CD3ζ molecules.71 The anti-CD19 CAR-transduced T cells were prepared by using a 24-day in vitro culture process. The clinical treatment regimen consisted of lymphocyte-depleting chemotherapy followed by an infusion of anti-CD19 CAR T cells and a course of high-dose interleukin-2 (IL-2). 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 after treatment.71 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.71 The bone marrow B-cell eradication was confirmed by flow cytometry, and it persisted for over 36 weeks.71 B cells were also completely absent from the blood during this time, while T cells and other blood cells recovered rapidly.71 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 partial remission that is ongoing over 9 years posttreatment.70 Seven more patients were subsequently treated with the same regimen of chemotherapy, antiCD19 CAR T cells, and high-dose IL-2.70 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.70,71 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.71 Because normal B cells express CD19, prolonged normal B-cell depletion after anti-CD19 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.70 Six of the seven evaluable patients had remissions of their malignancies. Two of the remissions were complete remissions (CRs) of CLL.70 Both of these CRs were confirmed by multicolor flow cytometry of bone marrow cells.70 One of these CRs lasted 24 months, and the other is ongoing at 48 months.70 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.70 All of these toxicities peaked within 10 days after the cell infusion and resolved less than 3 weeks after the cell infusion.70 These acute toxicities correlated with serum levels of the inflammatory cytokines tumor necrosis factor and interferon-γ, 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.70 We continued studies using the same CAR as in our previously reported anti-CD19 CAR T-cell reports. In these more recent studies, IL-2 was not administered to patients and the T-cell production process was shortened from 24 days to 10 days. The elimination of IL-2 administration was done in an attempt to lessen toxicity, and the shortening of the cell production process was done in an attempt to both simplify the cell production and to increase in vivo T-cell persistence and proliferation. We reported the results of this modified clinical protocol in a very recent paper143. In summary this paper reported treatment of 15 patients with advanced B-cell malignancies. Nine patients had diffused large B-cell lymphoma (DLBCL), 2 patients had indolent lymphomas, and 4 patients had chronic lymphocytic leukemia (CLL). Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19-CAR T cells. Of 15 patients, 8 obtained complete remissions (CRs), 4 patients obtained partial remissions, 1 patient had stable lymphoma, and 2 patients were not evaluable for response. CRs were obtained by 4 of 7 evaluable patients with chemotherapyrefractory DLBCL; 3 of these 4 CRs are ongoing with durations ranging from 9 to 22 months.  
Acute toxicities including fever, hypotension, delirium, and other neurological toxicities occurred in some patients after infusion of anti-CD19-CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly of an unknown cause 16 days after cell infusion.  
CAR T cells were detected in the blood of patients at peak levels ranging from 9 to 777 CAR+ T
cells/µL. Elimination of exogenous IL-2 from our protocol did eliminate the toxicity that is known to occur with administration of high-dose IL-2, but cytokine-release type toxicity attributable to the CAR T cells still remained143 In an attempt to further reduce the overall toxicity of our anti-CD19 CAR treatment protocol, we substantially reduced the dose of the chemotherapy regimen administered before CAR T-cell infusions. We treated 9 patients with B-cell lymphoma who received a single infusion of 1x106 anti-CD19-CAR-expressing T cells/kg bodyweight preceded by a low-dose chemotherapy regimen of 3 daily doses of cyclophosphamide 300 mg/m2 and fludarabine 30 mg/m2 administered on the same days.56 As expected, the severity of neutropenia and thrombocytopenia was reduced with the low dose chemotherapy compared to high-dose chemotherapy. We went on to treat a total of 22 patients with either a type of diffuse large B-cell lymphoma (19 patients), follicular lymphoma (2 patients), or mantle cell lymphoma (1 patient).56 The overall response rate was 73% with 55% complete remissions.56 Most importantly, of 12 patients obtaining complete remissions, 11 are still in ongoing complete remissions of up to 36 months duration.56 We also went on to show that long-term remissions of diffuse large B-cell lymphoma do not depend on persisting CAR T cells.144 This work led to several multicenter trials of the same anti-CD19 CAR used in the other trials described in the last 4 paragraphs, and ultimately to the first FDA-approved CAR Tcell therapy for lymphoma. The multicenter trials on diffuse large B-cell lymphoma confirmed results from the original NCI clinical trials. The effectiveness of anti-CD19 CAR T cells against advanced B-cell malignancies, particularly CLL and ALL has been reported by multiple groups90,140-142,145 Multiple groups have recently demonstrated the 70% to 90% complete remission rates when anti-CD19 CAR T cells are used to treat ALL140,142,145 These impressive response rates have been associated with significant toxicity that can be divided into 2 main categories. The first category is "cytokine-release syndrome" that consists mainly of fever, tachycardia, hypotension, fatigue, and in some cases myocardial dysfunction; these toxicities typically last for a few days to approximately 2 weeks before resolving140,142,145 . The second main category is neurological toxicity that sometimes occurs in patients not suffering from the typical cytokine-release syndrome toxicities. Commonly observed neurological toxicities include aphasia, tremor, seizures, and ataxia; similar to other toxicities, the neurological toxicities typically last from 1 or two days to 3 weeks before resolving142,145 . Neurologic toxicity was the most problematic aspect of treating patients with the FMC63-28Z CAR. Fifty-five percent of patients on our final clinical trial of this CAR for lymphoma at the NCI had Grade 3 or 4 neurologic toxicity. All of this early work on anti-CD19 CAR T-cell therapies has led to multicenter trials for most types of B-cell lymphoma and FDAapproval of the first anti-CD19 CAR product for treatment of DLBCL.146 To further reduce toxicity of CAR T cells and to reduce the potential immunogenicity of our original anti-CD19 CAR, we designed a new anti-CD19 CAR. This CAR had a fully-human scFv to reduce immunogenicity, and it had a CD8α hinge and transmembrane domain that we hypothesized would reduce T-cell cytokine production.98 The CD8α hinge and transmembrane domain was shown to deliver a weaker activation stimulus compared to the CD28 hinge and transmembrane domain in the FMC63-28Z CAR used in all of our prior clinical trials.98 This weaker activation stimulus consistently led to lower levels of cytokine production by T cells expressing CARs with CD8α hinge and transmembrane domains compared with CARs containing CD28 hinge and transmembrane domains.98 We tested the CAR that resulted from this preclinical work on CARs with human antibody variable domains and CD8α hinge and transmembrane domains, Hu19-CD828Z in a phase I clinical trial.147 Twenty patients were treated on this clinical trial of Hu19-CD828Z CAR T cells. The anti-lymphoma efficacy was very similar to that of FMC63-28Z CAR T cells. Fifty-five percent of patients receiving Hu19-CD828Z CAR T cells obtained complete remissions, and responses were durable with 40% of responses ongoing with durations of 5 to 29 months duration.147 The striking difference between results of the Hu19-CD828Z trial and our earlier work with FMC6328Z CAR T cells was the much lower rate of neurologic toxicity with Hu19-CD828Z CAR T cells.147 Only 5% of patients had Grade 3 or 4 neurologic toxicity with Hu19-CD828Z CAR T cells compared with 55% of patients having Grade 3 or 4 neurologic toxicity with FMC63-28Z CAR T cells.147 The anti-lymphoma response and event-free survival of patients on the trial of Hu19-CD828Z are in *Figure 1*. One issue that arose during the trial of Hu19-CD828Z was loss of CD19 expression on lymphoma cells. We documented CD19 loss on 4 of 20 treated patients on the Hu19-CD828Z trial; this finding led us to develop an approach that could target CD19 and another antigen simultaneously. Because of the proven anti-lymphoma activity and favorable toxicity profile of Hu19-CD828Z, we have chosen to include Hu19-CD828Z along with an antiCD20 CAR in a bicistronic construct called Hu19-CD828Z.  
Figure 1 Duration of response and event-free survival from Hu19-CD828Z clinical trial.
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1.2.10 anti-cd20 cars

Clinical experience with anti-CD20 CARs is much more limited than the clinical experience with anti-CD19 CARs. Preclinical work with anti-CD20 CARs has been published and includes studies of the interaction of CARs with serum anti-CD20 antibodies and a study of antigen expression levels on the activity of CAR T cells.148 Substantial work has been performed in developing bispecific CARs in which 1 CAR contains binding domains for both CD20 and CD19.149-151 These bispecific CARs differ from the Hu1928-Hu20BB CAR used in this protocol. The bispecific CARs encode 1 CAR with 2 binding domains; in contrast, our bicistronic approach has 2 different CARs expressed simultaneously by 1 T cell. Three papers have been published on CAR T-cell clinical trials, but the trials contained a very small number of patients. Two papers dealt with antiCD20 CAR trials that used electroporation for T-cell modification. One trial reported only 1 treated patient;139 the other reported 7 patient treated of which only one obtained an objective antilymphoma response, a partial response (PR).152 One possible reason for the unimpressive results is that electroporation led to only short persistence of CAR+ T cells of only 1 to 3 weeks.152 A
more recent trial used an anti-CD20 CAR with both CD28 and 4-1BB costimulatory domains.153 This CAR was encoded by a lentivirus vector; only 3 patients have been reported.153 Two patients maintained pre-existing complete responses (CRs), and one patient obtained a PR.153
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1.2.11 development and preclinical testing

In order to simultaneously target 2 lymphoma-associated antigens with 1 T cell, we designed CAR constructs that encoded 2 CARs. One CAR included in all constructs wasthe previously-described Hu19-CD828Z that has been shown to be save an effective in a prior phase I clinical trial. The second CAR targeted CD20 and included a 4-1BB moiety. We evaluated several different anti-
CD20 scFvs as the antigen-recognition domain of the anti-CD20 CAR before selecting a singlechain variable fragment (scFv) from the fully-human antibody 2.1.2.154 We designated the antiCD20 CAR containing the 2.1.2 scFv Hu20-CD8BBZ; Hu20-CD8BBZ also contained CD8α hinge and transmembrane domains, a 4-1BB domain, and the cytoplasmic part of CD3ζ.  
The bicistronic construct also incorporated the anti-CD19 CAR Hu19-CD828Z that contained a single chain variable fragment (scFv) from the fully-human anti-CD19 antibody 47G4, CD8α hinge and transmembrane domains, the CD28 cytoplasmic domain, and the cytoplasmic part of CD3ζ.155 Both the Hu19 and 2.1.2 (Hu20) antibodies were derived by immunizing mice transgenic for human immunoglobulin genes.154,155 The entire bicistronic construct was encoded by the MSGV1 gamma-retroviral vector backbone.156 The Hu19-CD828Z and Hu20-CD8BBZ CARs were separated by a ribosomal skip domain that caused both CARs to be expressed as separate proteins. The concept of a T cell simultaneously expressing 2 CARs is depicted in *Figure* 2, and diagrams of the bicistronic CAR
construct, Hu19-CD828Z, and Hu20-CD8BBZ are shown in *Figure* 3.  
Figure **2 A bicistronic CAR construct leads to expression of 2 CARs by the same T cell.**  
 Figure 3 :Diagrams of the CAR construct Hu1928-Hu20BB and individual CAR in the construct.  
  
A diagram depicting the cleavage at the ribosomal skip sequence and expression of 2 separate CAR proteins is shown in *Figure* 4. This diagram depicts the furin enzyme cleavage or the 2A
(F2A in this case) from the C-terminus of the Hu19-CD828Z CAR. In this diagram, SS stands for signal sequence, and HTM stans for hinge and transmembrane domain.  
  
Figure 5 shows expression of Hu19-CD828Z and Hu20-CD8BBZ on the surface of T cells five days after transduction with gamma-retroviruses encoding the Hu1928-Hu20BB CAR construct. For this experiment, whole peripheral blood mononuclear cells (PBMC) were started in culture on day 0 by stimulating with an anti-CD3 monoclonal antibody in interleukin (IL)-2-containing medium. Transductions were carried out 2 days after the cultures were started, and the T cells were assessed for CAR expression 6 days later, when cells had been in culture for a total of 8 days. The plots in Figure Figure 5 are gated CD4+ or CD8+ live, CD3+ lymphocytes.The CAR staining in Figure 5 was performed with the Kip-1 antibody, which binds to the Hu19 scFv that is in Hu19- CD828Z. Kip-1 does not bind to Hu20-CD8BBZ. Note that all T cells transduced in this work were transduced in the same manner as described above.  
26 Version Date:May 27, 2020 Figure 5: **CAR expression on T cells by Kip-1 antibody staining**  
  
Figure 6 shows T cells from the same cultures shown in *Figure* 5, but in *Figure* 6 staining with the Kip-4 antibody is shown. Kip-4 binds to the scFv of the Hu20-CD8BBZ CAR but does not bind to the Hu19-CD8BBZ CAR. Kip-4 is not as sensitive or specific as Kip-1.  
  
We also performed a series of in vitro assays to assess the function of anti-CD19-CAR-expressing T cells, and we found that CD19-CAR-expressing T cells exhibit CD19-specific activities including CD107a upregulation and cytokine production in vitro. These experiments showed that anti-CD19-CAR-expressing T cells are activated in an antigen-specific manner.  
*Figure* 7 shows upregulation of CD107a on CD8+ T cells, which indicates degranulation and correlates with cytotoxicity.157 when T cells were transduced with the Hu1928-Hu20BB, the T
cells could recognize CD19+ CD19-K562 target cells, CD20+ CD20-K562 target cells, and ST486 target cells that expressed low levels of CD19 and moderate levels of CD20. The negative-control target cells NGFR-K562 and CCRF-CEM were recognized at background levels. CD107a was not upregulated when anti-CD19-CAR-expressing T cells from Donor 1 were cultured for 4 hours with the negative control cell lines NGFR-K562 and CCRF which do not express CD19. Hu19- CD828Z-expressing T cells recognized CD19-K562 and ST486 but not CD20-K562. Hu20- CD8BBZ recognized CD20-K562 and CCRF-CEM but not CD19-K562. Untransduced T cells from Donor 1 did not upregulate CD107a when cultured with either CD19-K562 or NGFR-K562 except at background levels. The plots are gated on live CD3+ lymphocytes.  
  
Figure 8 shows a 4-hour cytotoxicity assay in which "Toledo" CD19+/CD20+ target cells were incubated with T cells transduced with Hu1928-Hu20BB, Hu19-CD828Z, or Hu20-CD8BBZ. All 3 CARs could kill the ST486 cells that express both CD19 and CD20.  
Figure 8 : Cytotoxicity of a lymphoma cell line by CAR-expressing T cells  
  
  
number of human proteins for reactivity of antibody-based reagents. This approach can be used with whole intact CAR T cells that are fluorescently labeled. We provided RetrogenixTM with untransduced human T cells and T cells from the same donor that expressed Hu20-CD8BBZ. The Hu20-CD8BBZ T cells were labeled and used to screen the 5647 human plasma membrane proteins. The only differences in binding between Hu20-CD8BBZ T cells and untransduced T cells was for CD20, which was expected for this anti-CD20 CAR, and CD27. CD27 binding was very weak and inconsistent, but nonetheless, we transduced 293T cells in my lab and assessed for reactivity against Hu20-CD8BBZ T cells in an IFNγ enzyme-linked immunosorbent assay
(ELISA). We found no release above background when Hu20-CD8BBZ T cells were exposed to CD27+ target cells; therefore, we concluded that the Hu20-CD8BBZ CAR does not functionally recognize CD27.  
We also performed our usual method of specificity staining in my lab of assessing the ability of CAR-expressing T cells to recognize primary human cells and human cell lines. We cultured CARexpressing T cells or untransduced T cells from the same patient with target cells overnight, and then performed a standard IFNγ ELISA to see if T cells are activated, as indicated by IFNγ release, when the T cells are cultured with target cells (Table 1-3). Ideally, the anti-CD19/anti-CD20 Hu1928-Hu20BB CAR T cells should only react with CD19+ and/or CD20+ target cells.  
We used the target cell lines and human primary cells as described in this paragraph. We previously transduced K562 cells to express CD19 (CD19-K562) or low-affinity nerve growth factor (NFGRK562).85 We also transduced K562 cells to express CD20. All of these genes were transferred to K562 cells by standard methods with the MSGV gamma-retroviral vector. The NGFR-K562 cells served as CD19-negative control cells. The following cells CD19+ and or CD20+ cell lines were used: NALM6 cells are CD19+ and CD20-negative by flow cytometry (acute lymphoid leukemia from DSMZ, Braunschweig, Germany). ST486, and Toledo were obtained from ATCC; both of these cells lines express both CD20 and CD19, but CD19 expression is very weak on ST486 cells. ST486 -/- cells had CD19 eliminated by CRISPR/Cas9 in our lab.  
The CD19-negative cell lines were used: melanoma cell line 624, the leukemia cell line NGFRK562, the T-cell leukemia cell line CCRF-CEM; A549 (a lung carcinoma cell line); MDA-MB231
(a breast cancer cell line), TC71 (a Ewings sarcoma cell line), COLO205 (a colon carcinoma cell line), U251 (a glioblastoma cell line). Panc10.05 (a pancreatic carcinoma cell line), and A431-H9 (an epidermoid (skin) carcinoma cell line that was transduced with the gene for mesothelin). Reactivity of CAR T cells with human primary cells was also assessed (Table 3). Fibroblasts were primary human skin fibroblasts; endothelial cells were primary human endothelial cells. The primary microvascular endothelial cells and primary hepatocytes were from Lonza. The primary human cardiac myocytes were from Dr. Yongshun Lin, NHLBI, NIH In each experiment, the result for effector T cell cultured alone was also given.  
T cells transduced with the anti-CD19 CAR produced large amounts of IFNγ when they were cultured overnight with the CD19-expressing cell lines but only small amounts of IFNγ when cultured with CD19-negative cell lines or CD19-negative primary human cells (Table 1). All cytokine values in Tables 1-3 are IFNγ levels in picograms/mL. The % CAR columns show the percentage of T cells in the ELISA assay that expressed the indicated CAR.  
Abbreviated Title: Anti-CD19 and anti-CD20 CAR T-cells Version Date: May 27, 2020 Table 1 : IFN7ELISA with Hu1928-Hu20BB and its constituent CARs shows antigen-specific recognition.  
| ST486-CD19-                     |           |                             |      |      |      |     |     |     |    |
|---------------------------------|-----------|-----------------------------|------|------|------|-----|-----|-----|----|
| CD19-K562 CD20-K562 Nalm6 ST486 | NGFR-K562 | CEM    T-cells Alone % CAR+ |      |      |      |     |     |     |    |
| -                               |           |                             |      |      |      |     |     |     |    |
| Untransduced                    | 41        | 368                         | 18   | 39   | 197  | 530 | 12  | 7   | o  |
| Hu1928-Hu20BB                   | 10103     | 21162                       | 6685 | 79   | 8317 | 781 | 88  | 52  | 44 |
| Hu19-CD828Z                     | 13280     | 36                          |      |      |      |     |     |     |    |
| 808                             | 6524      | 1433                        | 641  | 918  | 39   | 20  |     |     |    |
| Hu20-CD8BBZ                     | 171       | 4014                        | 1647 | 4620 | 4506 | 115 | 137 | 128 | 48 |  
Table 2: IFNr ELISA shows lack of reactivity of Hu1928-Hu20BB T cells with a panel of hmman tumor cell lines.  
| K562-  K562-  A431-   Panc   | Colo   | T-cells    |     |                                                   |     |     |     |    |     |     |     |     |
|------------------------------|--------|------------|-----|---------------------------------------------------|-----|-----|-----|----|-----|-----|-----|-----|
| T cells CD19   CD20          | H9     | 10.05 U251 | 205 | Hep G2  MDA231  A549   Tc71    624   Alone  %CAR+ |     |     |     |    |     |     |     |     |
| Untransduced <12             | <12    | <12        | <12 | 76                                                | <12 | <12 | 873 | 66 | 15  | <12 | <12 | 0.1 |
| Hu1928-Hu20BB 9343  6127     | 79     | 88         | 124 | 27                                                | 90  | 459 | 75  | 97 | 104 | 74  | 51  |     |  
All values are IFN γ in pg/mL except the last column, which is the percentage of each culture that expressed the Indicated CAR by flow cytometry.  
T cells were cultured with the indicated target cells overnight, and an IFN γ ELISA was performed.  
CD19-K562 expresses CD19 and CD20-K562 expresses CD20 All other targets listed lack both CD19 and CD20  
| CD19-         |       |
|---------------|-------|
| T cells       | K562  |
| Untransduced  | 56    |
| Hu1928-Hu20BB | 5536  |
| Hu19-CD828    | 13946 |
| Hu20-CD8BB    | 255   |  
Version Date: May 27, 2020 Table 3 : IFN r ELISA shows lack of reactivity of Hu1928-Hu20BB T cells with a panel of hman tumor cell lines.  
All values are IFN y in pg/mL except the last column, which is the percentage of each culture that expressed the Indicated CAR by flow cytometry.  
T cells were cultured with the indicated primary human target cells overnight, and an IFN r ELISA was performed. CD19-K562 expresses CD19 and CD20-K562 expresses CD20 All other targets listed lack both CD19 and CD20 One potential problem with anti-CD20 CARs is possible blocking by serum anti-CD20 antibodies that many patients enrolled on this trial will have received. Anti-CD20 monoclonal antibodies such as Rituximab might block binding of CAR T cells to lymphoma cells, or they could cause nonspecific activation of CAR T cells. Prior reports have assessed rituximab levels in the serum of patients and found that the median serum rituximab concentration to be 38.3 µ g/mL in patients who had received rituximab in the past 4 months. We assessed the impact that soluble rituximab  
| Proximal      | Renal   |                    |                    |         |        |                              |      |      |
|---------------|---------|--------------------|--------------------|---------|--------|------------------------------|------|------|
| CD20-   NGFR- | tubular | Skeletal   Hepatic | cortical   Mammary | T-cells |        |                              |      |      |
| K562          | K562    | cells              | muscle             | cells   | eptih. | epith. cells   Alone  % CAR+ |      |      |
| 31            | 40      | 54                 | 15                 | 36      | 108    | <12                          | <12  | 0.21 |
| 10929         | 109     | 128                | 79                 | 35      | 174    | 144                          | 106  | 84.7 |
| 118           | 85      | 38                 | 27                 | 20      | 107    | 19                           | 15   | 81.0 |
| 374           |         |                    |                    |         |        |                              |      |      |
| 10197         | 280     | 268                | 146                | 48      | 251    | 24                           | 90.9 |      |  
might have on anti-CD20 CAR T cells by doing ELISA assays in which CAR T cells, CD20+
target cells, and graded concentrations of rituximimab were added together in overnight cultures.  
After the cultures IFNg ELISAs were performed on the culture supernatant. We found that rituximab did decrease IFNg release in a dose-dependent manner, but it never eliminated the ability of the CAR T cells to recognize lymphoma. We also found that rituximab did not cause nonspecific CAR activation (Table 4). All cytokine values in Table 4 are pg//mL. rituximab concentrations are at the top of the table. Target cells use were ST486 -/- cells that express CD20 but not CD19. Human IgG was added to some wells as a control.  
Version Date: May 27, 2020 Table 4 : Impact of rituximab on Hu1928-Hu20BB CAR T cells.  
  
Next, we performed murine experiments to test Hu1928-Hu20BB and its constituent CARs. In the Figure 10  
  
In this model, Hu1928-Hu20BB and Hu20-CD8BBZ were much more effective than Hu19-
CD828Z. We hypothesize that Hu19-CD828Z was reacting against some residual CD19 that was expressed despite the attempt at CD19 abrogation. In this experiment, 4 million ST486 (-/-) cells were injected 6 days to establish palpable intradermal tumors prior to CAR T cell infusion. There were 5 mice/group. In all mouse experiments, mice received only 1 infusion of CAR T cells and no other interventions. Mice were sacrificed when tumors reached 1.5 cm in longest dimension. Note that mice did not exhibit any toxicity attributable to CAR T cells in any of our murine experiments.  
  
Chemotherapy regimen We plan to administer a conditioning chemotherapy regimen of cyclophosphamide and fludarabine before infusions of CAR-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.160-162 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.59,63,160-163 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.160,161,163,164 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-7.160 Experiments in a murine xenograft model showed that regulatory T cells could impair the anti-tumor efficacy of anti-CD19 CAR T cells.165 Myeloid suppressor cells have been shown to inhibit anti-tumor responses.164 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.101 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 antiCD19-CAR T cells. Mice receiving TBI had a 100% cure rate and mice not receiving TBI had a 0% cure rate.101 Strong suggestive evidence of enhancement of the activity of adoptively-transferred T cells has been generated in humans.67,166,167 Very few clinical responses have occurred and very little evidence of in vivo activity has been generated in clinical trials of autologous anti-CD19-CAR T
cells administered without lymphocyte-depleting chemotherapy.67,72 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.68,70,71,90 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/or fludarabine.68-71,166,167 Both cyclophosphamide and fludarabine are highly effective at depleting lymphocytes.166,167 One well-characterized 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.168 Multiple cycles of this regimen can be tolerated by heavily pretreated leukemia and lymphoma patients.168
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1.2.13 rationale for dose-escalation

The clinical trial described in this protocol is planned as a dose escalation in which the number of Hu1928-Hu20BB 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.169-171 Second, in the setting of allogeneic transplantation, relapsed malignancy is often treated with infusions of allogeneic donor lymphocytes (DCIs).172,173 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 DCIs increases'.172,173
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1.2.14 rationale for fully-human cars

Immune responses against genetically-modified T cells have previously been reported 139,174,175. Because treatment with Hu1928-Hu20BB CAR T cells is expected to eliminate recipient B cells, the most concerning type of immune response against is a cytotoxic T-cell response directed against foreign components of the CAR. Such cytotoxic T-cell responses against suicide-gene-modified T cells have previously been documented 174. The CARs used in all published CAR studies to date used antibody components that were derived from murine antibodies. It would be expected that in at least some patients these murine components would be immunogenic. Supporting this belief are data from a small number of patients that indicate that T-cell responses can develop against the FMC63 single chain variable fragment (scFv) that is a part of most anti-CD19 CARs that are currently being tested. Jensen and coworkers and Lee and coworkers have reported T-cell responses against the FMC63 murine scFv 139,175. Riddell and coworkers have reported cytotoxic T-cell responses against the FMC63 scFv (Riddell, Presentation at the American Society of Hematology Annual Meeting, 2013). Because this protocol intends to give patients multiple doses of CAR-modified T cells, the risk of developing anti-CAR immune responses is possibly even higher than in most previous clinical trials that only administered a single infusion of CAR T cells. All in all, use of a fully-human CAR will decrease the risk of antiCAR T-cell responses that we hypothesize could interfere with CAR T-cell efficacy especially when multiple infusions of T cells are given.  
The use of fully-human CARs might increase persistence of CAR T cells after a first dose of CAR T cells. In our prior trials of murine anti-CD19 CARs, persistence of CAR T cells in the blood has been limited (*Figure 10*)143, we hope that the fully-human anti-CD19 CAR will have longer persistence. The graphs shown in *Figure 10* are absolute numbers of CAR T cells in the blood of 5 patients as determined by a quantitative PCR assay that is specific for the CAR. These results show the usual pattern of T-cell persistence with the current anti-CD19 CAR used in ETIB CAR trials. The CAR is called FMC63-28Z, and it contains a murine scFv. After infusion, CAR T cells rapidly rise to a peak. The cells do not persist in the blood long-term. Cells were undetectable in the blood by approximately 2 months after infusion.
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1.2.15 summary of risks and

This clinical trial is being performed to evaluate a genetically-modified T-cell therapy for advanced B-cell malignancies, which are often incurable diseases 13,15,176. Only patients with B-cell malignancies persisting despite at least 2 prior lines of therapy will be enrolled. The risks of the study fall into 5 general categories. First, chemotherapy that is part of the protocol treatment could cause cytopenias. 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, tachycardia, transient abnormalities of liver function, and hypotension. These cytokine-release-type toxicities have been detected in other clinical trials of CAR T cells during the first 2 weeks after anti-CD19 CAR T cells were infused.70,141 A third category of potential toxicities are neurological toxicities such as delirium, obtundation, myoclonus, seizures, headache, and transient focal neurological toxicities including aphasia and focal paresis. In previous anti-CD19 CAR trials, cytokine-release toxicities and neurological toxicities have been limited in duration with toxicities generally resolving within 2 days to 3 weeks. The fourth possible category of toxicity is direct damage to normal tissues by the CAR T cells. This could happen because of unexpected crossreactivity of the anti-CD19 and anti-CD20 CAR with proteins other than CD19 and CD20 in vivo.  
This trial will be the first of a CAR containing the fully-human 47G4 variable regions, so crossreactivity with normal proteins is not inconceivable. We have performed extensive testingof 47G4 CARs by culturing 47G4-CD28Z CAR T cells with a variety of human cell lines, and we have not seen recognition of cell lines that did not express CD19. A fifth category of toxicity caused by anti-CD19 CAR T cells is hypogammaglobulinemia due to depletion of all B cells and some plasma cells. Hypogammaglobulinemia has been a complication for many patients on clinical trials of anti-CD19 CAR-expressing T cells.70,71 Hypogammaglobulinemia in these patients was routinely treated with infusions of intravenous immunoglobulins.70 The potential benefits to subjects enrolling on this trial include the possibility that the Hu1928- Hu20BBCAR T cells can cause a significant anti-malignancy effect. Many patients enrolled on earlier trials of anti-CD19 CAR T cells obtained prolonged complete remissions of advanced malignancies68,70,177, so there is a chance that recipients of the Hu1928-Hu20BBCAR T cells that are being evaluated in this protocol could derive a direct benefit from participation in this trial. In contrast, some patients did not obtain remissions on prior anti-CD19 CAR trials and in some patients the remissions were not lasting, so further research aimed at improving CAR T-cell therapies is needed.
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2 eligibility assessment and enrollment

Note: If a patient meets an eligibility requirement as outlined belowand 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. 2.1.1 Inclusion Criteria
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2.1.1.1 malignancy criteria

Note: As of approval of Amendment A, no patients with Hodgkin lymphoma can be enrolled until at least 6 patients with B-cell malignancies are treated without incidence of Guillain-Bare syndrome  
- Patients must have any B-cell lymphoma, or CLL/SLL, Gray-zone lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, or classical Hodgkin lymphoma with any CD19 or CD20 expression on Reed-Sternberg cells. Lower grade lymphomas or CLL transformed to DLBCL are potentially eligible as is primary mediastinal B-cell lymphoma and all other subtypes of DLBCL. Burkitt and mantle cell lymphoma are potentially eligible.  
- For classical Hodgkin lymphoma only, a biopsy from any time from any institution that shows any CD19 or CD20 expression on Reed-Sternberg cells is adequate for eligibility. CD19 or CD20 expression on the Reed-Sternberg cells that is weak or only present on some Reed-Sternberg cells by immunohistochemistry is compatible with protocol eligibility.  
- For all lymphoma types except for classical Hodgkin lymphoma, either CD19 or CD20 expression must be "uniform". "Uniform" CD19 or CD20 expression is defined as no obvious lymphoma population lacking antigen expression is present. Antigen expression can be assessed by either immunohistochemistry or flow cytometry.  
- Only when insufficient biopsy material is available to allow CD19 and CD20 expression assessment at the NIH, CD19 and/or CD20 staining performed at another institution can be used
- DLBCL patients must have received at least two prior chemotherapy-containing regimens at least one of which must have contained doxorubicin and a monoclonal antibody. Follicular lymphoma patients must have received at least 2 prior regimens including at least 1 regimen with chemotherapy. All other B-celllymphoma and leukemia patients must have had at least 1 prior chemotherapy-containing regimen. All patients with CLL or small lymphocytic lymphoma must have had prior treatment with ibrutinib or another signal transduction inhibitorand venetoclax.
- Hodgkin lymphoma patients must have:
o had at least 3 prior lines of therapy. o had at least 1 prior cytotoxic chemotherapy-containing regimen. o had prior exposure to brentuximab vedotin.  
o had undergone autologous stem cell transplant or been transplant ineligible or refused autologous transplantation  
- Eligibility will be expanded to include CD19 and CD20-negative classical Hodgkin lymphoma if any 2 patients with classical Hodgkin lymphoma and CD19/CD20 expression on RS cells have durations of response 6 months or greater (responses could be PRs or CRs) or a CR of 3 months or greater.
- All patients must have measurable malignancy as defined by at least one of the criteria below.  
Lymphoma or leukemia masses that are measurable (minimum 1.5 cm in largest diameter) by CT scan is required for all diagnoses except CLL. All masses must be less than or equal to 10.0 cm in the largest diameter.  
For a lymphoma mass to count as measurable malignancy, it must have abnormally increased metabolic activity when assessed by positron emission tomography (PET) scan. CLL masses do not need to have increased activity on PET scan.  
For CLL and lymphoma with only bone marrow involvement no mass is necessary, but if a mass is not present, bone marrow malignancy must be detectable by flow cytometry in lymphoma and CLL. Note that leukemia cells must make up 1% or less of peripheral blood lymphocytes in CLL patients for these patients to be eligible.
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2.1.1.2 other inclusion criteria

- Greater than or equal to 18 years of age.  
- Able to understand and sign the Informed Consent Document. - Clinical performance status of ECOG 0-1 - Room air oxygen saturation of 92% or greater - Patients of both sexes must be willing to practice birth control from the time of enrollment on this study and for four months after receiving the protocol treatment.  
- 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.
- Platelet count greater than or equal to 50,000/mm3 without transfusion support
- Hemoglobin greater than 8.0 g/dl. - For CLL only, less than or equal to 1% malignant cells in the peripheral blood lymphocytes must be documented by flow cytometry of blood within 2 weeks of protocol enrollment.  
- Serum ALT and AST less or equal to 3 times the upper limit of the institutional normal unless liver involvement by malignancy is demonstrated.
- Serum creatinine less than or equal to 1.5 mg/dl.  
- Total bilirubin less than or equal to 2.0 mg/dl.  
- 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 within 4 weeks of treatmentstart.  
- Patients must not take corticosteroids including prednisone, dexamethasone or any other corticosteroid for 14 days before apheresis and CAR T-cell infusion. Patients must also not take corticosteroids at doses higher than 5 mg/day of prednisone or equivalent at any time after the CAR T cell infusion.
- Patients must be able to understand and be willing to sign a written informed consent. - Patients who have either been previously treated on protocols of genetically-modified T
cells on a clinical trial at the NCI or received T cells modified with the MSGV or MSGV1 gamma-retroviral vectors at any institution are potentially eligible under these conditions:  
At least 3 months have elapsed since the last genetically-modified T-cell therapy that the patient received, and there is no evidence of replication-competent retroviruses (evidence must be provided from prior protocol Principal Investigator), and persisting genetically-modified T cells are either not detectable in the patient's blood or detectable at levels less than or equal to 0.2% of blood T cells as measured by flow cytometry using the Kip-1 antibody in the flow cytometry lab of the NCI Laboratory of Pathology (Maryalice Stetler-Stevenson's lab).
2.1.2 Exclusion criteria:
- Patients that require urgent therapy due to tumor mass effects or spinal cord compression.  
- Patients must not have received any anti-CD20 or anti-CD19 antibody products in the past 30 days prior to CAR T-cell infusion.  
- Any history of receiving PD-1 or PD-L1 inhibitors. - Patients that have active hemolytic anemia. - HIV-positive patients are excluded because HIV causes complicated immune deficiency and study treatment can pose more risks for these patients.  
- Patients with second malignancies in addition to their B-cell malignancy are not eligible if the second malignancy has required treatment (including maintenance therapy) 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.  
- Pregnant women are excluded from this study because study therapy can cause fetal harm.  
Because there is potential risk for adverse events in nursing infants secondary to treatment of the mother with study therapy, breastfeeding should be discontinued if the mother is treated with study drugs.  
- Active uncontrolled systemic infections (defined as infections causing fevers and infections requiring intravenous antibiotics when intravenous antibiotics have been administered for less than 72 hours), active coagulation disorders or other major uncontrolled medical illnesses of the cardiovascular, respiratory, endocrine, renal, gastrointestinal, genitourinary or immune system, history of myocardial infarction, history of ventricular tachycardia or ventricular fibrillation, active cardiac arrhythmias (active atrial fibrillation is not allowed, resolved atrial fibrillation not requiring current treatment is allowed (anticoagulants count as current treatment) ), active obstructive or restrictive pulmonary disease, active autoimmune diseases such as rheumatoid arthritis.  
- Hospitalization within the 7 days prior to enrollment. - Any form of primary immunodeficiency (such as Severe Combined Immunodeficiency Disease).  
- Prior allogeneic stem cell transplant - Systemic lymphoma treatment of any type and corticosteroid steroid therapy of any dose greater than 5 mg/day or more of prednisone or equivalent is not allowed within 14 days prior to the required leukapheresis, or the initiation of the conditioning chemotherapy regimen. Corticosteroid creams, ointments, and eye drops are allowed.  
- History of severe immediate hypersensitivity reaction to any of the agents used in this study.  
- Patients on systemic anticoagulant therapy except aspirin. - Active central nervous system metastases or cerebrospinal fluid malignancy. Patients with known brain metastases will be excluded from this clinical trial because of their poor prognosis and because they often develop progressive neurologic dysfunction that would confound the evaluation of neurologic and other adverse events.  
- Any current neurologic disorders except migraine headaches.
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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 prior to obtaining informed consent Minimal risk activities that may be performed before the subject has signed a consent include the following:
- Email, written, in person or telephone communications with prospective subjects  
- Review of existing medical records to include H&P, laboratory studies, etc.  
- Review of existing MRI, x-ray, or CT images
- Review of existing photographs or videos
- Review of existing pathology specimens/reports from a specimen obtained for diagnostic purposes.  
2.2.2 Screening activities performed after a consent for screening has been signed
The following activities will be performed only after the subject has signed the consent for study \#01-C-0129 on which screening activities will be performed. Assessments performed at outside facilities or on another NIH protocol within the timeframes below may also be used to determine eligibility once a patient has signed the consent.
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the following assessments must be

- Complete history and physical examination, including, weight, height, and vital signs. Note in detail the exact size and location of any lesions that exist.  
- Confirmation of diagnosis of a B-cell malignancy by the NCI Laboratory of Pathology and confirmation for B-cell malignancies of uniform CD19 and/or CD20 expression on the malignant cells from either bone marrow or a lymphoma mass by flow cytometry or immunohistochemistry. The sample used for this expression analysis can come from any time prior to enrollment on the protocol. The sample can be a fresh biopsy or paraffinfixed slides. For all types of Hodgkin lymphoma any level of documented CD19 or CD20 expression at any time is adequate for enrollment; this requirement might be dropped later in the trial.  
- Electrocardiogram (ECG) - MRI of the brain
- PET only if necessary, to document measurable malignancy. If possible, PET scans previously performed after the most recent lymphoma treatment will be obtained, so that a new PET at NIH can be avoided.  
- Cardiac echocardiogram - CT scan of neck, chest, abdomen, and pelvis only if necessary, to document measurable malignancy. If possible, CT scans previously performed after the most recent lymphoma treatment will be obtained, so that new CTs at NIH can be avoided.  
- Bone marrow biopsy with flow only if necessary, to document measurable CD19+ and/or CD20+ malignancy for enrollment purposes. If not necessary for enrollment, the bone marrow biopsy can be delayed until the baseline evaluation.  
- A biopsy, possibly CT-guided might be needed for confirmation of lymphoma diagnosis and to confirm antigen expression for eligibility purposes. This biopsy will not be performed if biopsy material can be obtained from a prior biopsy conducted before protocol enrollment.  
- Lumbar Puncture is required for patients with Burkitt's lymphoma only. - Venous assessment for Apheresis (will be performed within 6 months before apheresis.  
Does not need to be repeated if no further apheresis needs to take place.)
- Laboratory Evaluation o Antibody screen for Hepatitis B and C; HIV, HTLV-I/II, T. cruzi (Chagas agent),
West Nile, and syphilis (RPR)
o Chemistries: (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)
o Hematological profile: CBC with differential o PT/PTT o Glucose-6-phosphate dehydrogenase (G6PD) deficiency screening o Thyroid Stimulating Hormone, T3**, T4**
o Serum cortisol o Serum or urine pregnancy test for female participants of childbearing age (in the absence of prior hysterectomy).
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2.3 participant registration and status

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

| Number   | Name     | Description                                                                                                      |
|----------|----------|------------------------------------------------------------------------------------------------------------------|
| 1        | Cohort 1 | Phase I dose escalation cohort for patients  with B-cell lymphoma or CLL                                         |
| 2        | Cohort 2 | Phase I dose escalation cohort for patients  with Hodgkin lymphoma                                               |
| 3        | Cohort 3 | Patients enrolled at the MTD or maximum  administered dose of CAR-T cell after  MTD of CAR-T cell is established |  
| Number   | Name                                                         | Description                                                                                                           |
|----------|--------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------|
| 1        | Conditioning  chemotherapy plus CAR  T-cells dose escalation | All patients will be receiving escalating dose of Anti-CD19 and  anti-CD20 CAR T cells/kg + conditioning chemotherapy |
| 2        | Conditioning  chemotherapy plus CAR  T-cells expansion phase | MTD dose or maximum administered dose of Anti-CD19 and  anti-CD20 CAR T cells/kg + Conditioning chemotherapy          |
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arm assignment

Note: As of approval of Amendment A, Cohort 2 will open only after at least 6 patients with Bcell malignancies are treated without incidence of Guillain-Bare syndrome Patients in cohort 1 and cohort 2 will be directly assigned to arm 1, each with its own dose escalation conducted separately.  
Patients in cohort 3 will be directly assigned to arm 3
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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 a bicistronic construct that leads to expression of a fully-human anti-CD19 CAR and a fully-human anti-CD20 CAR by the same T cells. The cell dose will be escalated unless occurrence of dose-limiting toxicities limits further escalation or until the maximum tolerated dose level is reached. The protocol will enroll patients with B-cell malignancies that are resistant to standard therapies. Patients will be evaluated for general health with an emphasis on detecting cardiac and neurological abnormalities. An assessment of CD19 and CD20 expression will be an important part of the eligibility screening. Patients enrolled on the study will undergo leukapheresis, and CAR-expressing T cells will be generated by transducing the patient's T cells with a lentivirus encoding the CAR construct. Patients will receive a conditioning chemotherapy regimen of cyclophosphamide 500 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. Three days after the end of the conditioning chemotherapy, patients will receive a single infusion of CAR-expressing T cells in the inpatient setting. Patients will then be evaluated for toxicity, and malignancy will be staged at 1, 2, 3, 4, 6, 9, and 12 months after the infusion.  
A small number of subjects may be eligible for re-enrollment if a patient is removed from the protocol BEFORE completing protocol therapy or if a patient is eligible for re-treatment.Retreatment will only be administered after dose finding is complete and preliminary safety and efficacy of the product has been established. At that point, re-treatment can be considered on a case-by-case basis. Patients for re-enrollment would be required to meet all eligibility criteria at the time of re-enrollment. Patients will be assigned a new sequential study number for the reenrollment study period. Any cryopreserved 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 44 Version Date:May 27, 2020 3.1.2 Protocol schema
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3.1.3 dose limiting toxicity

Dose-limiting toxicities are defined as the following toxicities that are possibly, probably, or definitely attributable to protocol interventions and occurring between the first protocol treatment through 28 days after the CAR T-cell infusion.  
- Grade 3 toxicities possibly or probably related to Hu1928-Hu20BBCAR T cells and lasting more than 9 days, with the exceptions listed below. Symptomatic seizure activity is included as a grade 3 toxicity. This must be deemed by a neurologist to be true seizure activity. All suspected seizures must be evaluated by a neurologist.  
- Grade 4 toxicities possibly or probably related to the study interventions with the exceptions listed below 3.1.3.1 Exceptions
- The following specific toxicities will not be dose-limiting toxicities:
- Neutropenia (ANC<500/µL) lasting continuously 9 days or less - Anemia (Hgb<8 g/dL) lasting continuously 9 days or less - Thrombocytopenia that is not transfusion-dependent - Grade 3 thrombocytopenia - Transfusion-dependent thrombocytopenia lasting 30 days or less
- All cytopenias except neutropenia, anemia, or thrombocytopenia  
- Hypotension (defined as systolic blood pressure <90 mm Hg) requiring treatment with vasopressors (norepinephrine dose of >3 mcg/minute or equivalent, doses less than or equal to 3 mcg/minute are not a DLTs) for 72 continuous hours or less.  
- Fever
- Hypophosphatemia - Grade 4 elevation in alanine aminotransferase, aspartate aminotransferase, or bilirubin that resolves to Grade 3 or less within 3 days or less
- QT interval increases not associated with ventricular arrythmias - Grade 4 creatinine kinase elevation that resolves to Grade 3 or less within 4 days or less - Grade 4 prothrombin time (PT) or partial thromboplastin time (PTT) that resolves to Grade 3 in 3 days or less with no evidence of clinically-significant bleeding or thrombosis  
- Asymptomatic electrolyte disturbances regardless of grade
46 Version Date:May 27, 2020 3.1.4 Dose Escalation The trial will be a dose-escalation with 4 dose levels based on the patient's **actual** bodyweight.  
For the following dose escalation plan, CAR+ T cells are defined as CD3+CAR+ cells as measured by flow cytometry according to Department of Transfusion Medicine (DTM) SOPs. The number of Hu1928-Hu20BB CAR-expressing T cells transferred for each dose level will be as follows:  
| Dose Escalation Plan-Cohort 1 B-cell lymphoma or CLL                                                                                                                                                                                                                                                                                                                                                                       | or lymphocyte   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---|
| predominant Hodgkin lymphoma or Gray-zone lymphoma Dose Level Dose of anti-CD19 and anti-CD20 CAR T cells Level -1 0.3x106 CAR+ T cells per kg of recipient bodyweight Level 1 0.66x106 CAR+ T cells per kg of recipient bodyweight Level 2 2.0x106 CAR+ T cells per kg of recipient bodyweight  Level 3 6.0x106 CAR+ T cells per kg of recipient bodyweight  Level 4 10.0x106 CAR+ T cells per kg of recipient bodyweight |   |  
| Dose Escalation Plan-Cohort 2 Classical Hodgkin lymphoma Dose Level Dose of anti-CD19 and anti-CD20 CAR T cells  Level -1 0.3x106 CAR+ T cells per kg of recipient bodyweight Level 1 0.66x106 CAR+ T cells per kg of recipient bodyweight Level 2 2.0x106 CAR+ T cells per kg of recipient bodyweight  Level 3 6.0x106 CAR+ T cells per kg of recipient bodyweight  Level 4 10.0x106 CAR+ T cells per kg of recipient bodyweight   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
The trial consists of 2 cohorts, one for B-cell malignancies and the other for Hodgkin lymphoma. Each cohort has a separate but identical dose escalation scheme because toxicities for patients with B-cell malignancies might be significantly different than toxicities for Hodgkin lymphoma patients. One factor that is different is that Hodgkin lymphoma masses are made up of cells that are in large part negative for CD19; in contrast, B-cell lymphoma masses usually have CD19 expression on all or almost all cells. Dose escalation will proceed independently for each cohort. The delay between patients treated and the decision to escalate to a higher dose level is specific for each cohort. For example, if a patient is treated on Cohort 1, a different patient could be treated on Cohort 2 immediately. Also, if criteria have been met for escalation from Dose Level 1 to Dose Level 2 on Cohort 1, Cohort 2 cannot escalate to Dose Level 2 until criteria for escalating to Dose Level 2 are met specifically by patients enrolled on Cohort 2. Each dose level will include a minimum of 3 patients. All cell doses will be cryopreserved cells thawed just before infusion. All infusions will be preceded by the fludarabine and cyclophosphamide conditioning regimen. The percentage of CAR+ T cells and the number of total cells to infuse to obtain the indicated numbers of CAR+ T cell will be determined prior to cryopreservation. There will be a minimum of 16 days between the CAR T-cell infusion of a patient and the start of the conditioning chemotherapy regimen for the next patient on the same cohort. This will cause a minimum 21-day delay between sequential CAR T-cell infusions. **The**
only exception to this rule is that we will wait at least 28 days between treating the first and second patients on each dose level treated on this protocol **for each cohort**. There will also be a 28-day wait from the time of cell infusion to the last patient on a dose level of a cohort until treatment start (which means starting the chemotherapy) of the first patient on the next dose level of that same cohort. If sufficient cells cannot be grown to meet the criteria for the first dose of cells for the assigned 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, enrollment can start on the next higher dose level. Should 1 of 3 patients experience a dose limiting toxicity on a particular dose level, three more patients would be treated at that dose level to confirm that no greater than 1/6 patients have a DLT prior to proceeding to the next higher level. If 1/6 patients have a DLT at a particular dose level, accrual can proceed to the next higher dose level. 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 maximum tolerated dose. The maximum tolerated dose is the dose at which a maximum of 1 of 6 patients has a DLT. If 2 DLTs occur on dose level 1, accrual will proceed at dose level -1, as indicated in Table 2. After a maximum tolerated dose is defined for a cohort, additional patients can be treated on this cohort. Up to 10 total additional recipients can be treated with the MTD after an MTD is established; therefore, the maximum number of patients treated on this protocol is 68. In addition, we will allow 20 additional patients to allow the trial to be completed as planned if some patients who enroll are not treated due to reasons such as rapid clinical deterioration requiring cancellation of protocol participation, so total allowed enrollment will be 88 subjects. If cell growth limitations preclude administration of the maximum tolerated dose, the patient will receive as many cells as possible up to the maximum tolerated dose. If it proves to be technically impossible or impractical to achieve the higher dose levels due to cell production constraints and a maximum tolerated dose has not been reached or a maximum tolerated dose is not reached due to DLTs not occurring, the highest achievable dose level will be declared the maximum administered dose, and up to 10 additional patients will be treated with the maximum administered dose. Dose escalations will follow the rules outlined in the Table below.  
Table 6  
| Number of Patients with  DLT  at 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 highest dose  level below the maximally  administered dose | This is the MTD and is generally the recommended  phase 2 dose unless ≤1 out of 6 DLTs occurred at the  maximally administered dose, in which case, the  maximally administered dose will be the  recommended phase 2 dose                                                                                                                                                                                      |
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3.2 pre-treatment phase 3.2.1 leukapheresis

The patient will undergo a 15 to 20-liter leukapheresis (generally, 15 liters will be processed to target a yield of 6-10 x109 mononuclear cells) 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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3.2.2 hu1928-hu20bb car-expressing t-cell preparation

After cells are obtained by apheresis, further cell processing to generate CAR-expressing T cells will occur in the DTM according to standard operating procedures (SOPs). Either freshlycollected 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 the initial cell production and 1 complete back-up cell production 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: 240-858-3755for cryopreservation at 200 to 300 million PBMC per vial. The anti-CD3 monoclonal antibody OKT3 will be used to stimulate T-cell proliferation. The cells will be transduced by exposing them to replication-incompetent gamma-retroviruses encoding the CAR by using DTM SOPs. The cells will continue to proliferate in culture. CAR T cells will be cryopreserved between day 7 and day 9 of culture. CAR+ T cells will be determined by flow cytometry in accordance with DTM SOPS will be quantitated by flow cytometry. Sufficient cells will be cryopreserved for a second dose that will be one dose-level higher (up to a maximum of 10x106 CAR+ T cells/kg) than the first dose. Cryopreserved cells will be used for all infusions.  
Ten vials of the infused cells will be cryopreserved for research use and stored in the Surgery Branch Cell Production Facility SB-CPF. Each vial will contain 20 million cells. Before cryopreservation, the percentage of T cells expressing the CAR will be determined by flow cytometry, and this percentage of CAR+ T cells will be used in calculating the total number of cells to be cryopreserved in a single-infusion bag to meet the dose requirements of the doseescalation plan described in Section **3.1.4** As noted above, cells for potential 2nd doses will also be cryopreserved. For the first 2 dose levels, third infusions will also be cryopreserved at this time. In addition to flow cytometry, further testing of the cells will take place prior to infusion to evaluate for microbial contamination, replication-competent lentiviruses, 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 **3.11.2**,
any remaining cryopreserved cells from this protocol will be coded-linked and used for research or discarded after approval of the Principal Investigator of this protocol.
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3.3 drug administration

Conditioning chemotherapy and CAR T-cell administration-this can be either inpatient or outpatient 50 Version Date:May 27, 2020 3.3.1 Overall summary of the treatment plan  
| Drug             | Dose                                                                                                              | Days                                |
|------------------|-------------------------------------------------------------------------------------------------------------------|-------------------------------------|
| Cyclophosphamide | 500 mg/m2 IV infusion over                                                                                        | Daily x 3 doses on days -5, -4,     |
| 30 minutes       | -3                                                                                                                |                                     |
| Fludarabine      | 30 mg/m2 IV infusion over 30  minutes administered  immediately following the  cyclophosphamide on day -5, -4, -3 | Daily x 3 doses on days -5, -4,  -3 |
| CAR T cells      | Variable.                                                                                                         | Infuse on day 0                     |  
3.3.1.1 Detailed treatment plan 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 one hour before chemotherapy (I.V. ondansetron can be substituted). Consider aprepitant in addition to ondansetron for nausea prophylaxis. Patients should be provided with anti-emetics such as lorazepam and prochlorperazine to use at home.  
Next, on days -5, -4, and -3, cyclophosphamide at a dose of 500 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 a creatinine clearance **calculated by the CKD-EPI equation**
less than 80 ml/minute/1.73 m2of body surface are, the daily dose of fludarabine will be reduced by 20%.  
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 anti-emetics. To minimize bladder toxicity, patients should increase normal oral fluid intake to at least 2 L/day. Day 0: CAR T cells will be administered. Premedication for the cell infusion will be given approximately 30 to 45 minutes prior to the infusion. The premedications are acetaminophen 650 mg orally and diphenhydramine 12.5 mg IV. Cells will be 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 20-30 minutes. Details of the infusion procedure for nursing are included in Appendix Appendix D: Infusion Instructions Days 1 to 14: Mandatory hospitalization for observation and treatment as necessary. In addition, patients are required to stay within 60 minutes driving time from the Clinical Center until day 16 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.  
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 70 kg and over in weight. Filgrastim will be given daily and then discontinued as soon as the absolute neutrophil count recovers to 2000/microliter. 3.4 **DOSE MODIFICATIONS/DELAY**
Patients may have a delay in planned treatment if they have active infections defined as infections causing fevers or infections requiring intravenous 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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3.5 potential repeat treatment

- **No repeat treatments will be administered until dose-finding is complete and**
preliminary safety and efficacy of the product has been identified. At that point, retreatment can be considered on a case-by-case basis **after consultation with the FDA.**
- The chemotherapy component of repeat treatments will be initiated a minimum of 8 weeks after the most recent prior CAR T-cell infusion. There is no maximum time interval between treatments.  
- Patients with any response except progressive disease or continuing complete remission after an initial or second CAR T-cell infusion are potentially eligible for a repeat treatment consisting of conditioning chemotherapy followed by an infusion of CAR T cells.  
- Patients experiencing a DLT with any prior treatment on this protocol will not be eligible for re-treatment.  
- To be eligible for repeat treatments, patients must meet the same eligibility requirements listed in Section 2.1.  
o Uniform CD19 or CD20 expression or uniform expression of both CD19 and CD20 needs to be documented on the malignant cells after previous CAR T-cell infusions.  
o The patients must undergo screening evaluation as listed in Section 2.2 except infectious disease serology is not required to be repeated unless clinically indicated. Follow-up testing for retreatment will be the same as for the first treatment. A maximum of 2 total treatments can be administered to any one patient, and at least 8 weeks must elapse between each cell infusion.  
o Patients with Grade 2 or worse neurologic toxicity will not be retreated until neurologic toxicity improves to Grade 0 or 1.  
- The dose of CAR T cells administered during repeat treatments will be the maximum tolerated or maximum administered dose that will be determined by the dose escalation schedule. No patients will be retreated until the maximum tolerated or administered dose is determined.
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3.6 protocol stopping rules

- If no responses of PR or CR occur after 2 patients are treated on the highest dose level for both Cohort 1 and Cohort 2, 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 (**3.1.4**).  
- A death on study not attributable to progressive malignancy within 30 days of a cell infusion will be a cause for a pause to accrual pending amendment of the protocol approved/reviewed by the FDA and IRB.  
- If 2 or more subjects during the dose escalation phase of a given cohort experience a Grade 4 dose limiting toxicity possibly or probably or definitely 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.
- After 3 patients have been accrued to Cohort 2, enrollment to Cohort 2 only must be temporarily halted while a safety assessment by the Safety Monitoring Committee and the IRB. This is described in Section **7.5.1**.  
- If by the time 3 patients have been treated on the highest dose level, no patient on Cohort 2 has had either a complete remission lasting at least 6 months or progression-free survival lasting at least 9 months after CAR T-cell infusion, enrollment on Cohort 2 will cease.  
- If at any time more than 1 total DLT attributable to CAR T cells in the first 30 days after infusion occur on Cohort 2, and no complete remissions have occurred on Cohort 2, accrual to Cohort 2 will halt to reassess the safety of the product pending amendment of the protocol approved/reviewed by the FDA and IRB.  
- Aside from the 3 points above, continued accrual on Cohort 2 will be determined by the dose-escalation scheme in Section **3.1.4**.
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3.7 protocol evaluation 3.7.1 baseline

If results of these tests do not satisfy eligibility criteria, per PI discretion patients' treatment can be delayed or discontinued. An exception to this is that a platelet count of 50,000 or more is considered adequate to start chemotherapy The following tests must be completed within 14 days of the start of the conditioning chemotherapy regimen:  
- 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 another device. Non-valved PICC lines are greatly preferred over valved PICC lines for this protocol.  
- Physical exam with vital signs and oxygen saturation - CT scan of neck, chest, abdomen, and pelvis
- PET of the torso for all patients except patients with CLL - Bone marrow aspirate and biopsy: specifically ask for CD19 and CD20 immunohistochemistry staining of the bone marrow biopsy. Flow cytometry must be performed. The bone marrow biopsy must take place at some time after the patient's most recent malignancy treatment. If a bone marrow biopsy was performed at the NIH as part of protocol screening within 4 weeks of the start of treatment, it does not have to be repeated unless necessary for staging.  
- Peripheral blood flow cytometry for patients with CLL - G-banding cytogenetics on the bone marrow for all patients.
- 250 microgram cosyntropin stimulation test if suspicious for adrenal insufficiency based on low serum sodium or high serum potassium or hypotension or a low serum cortisol or a history of adrenal insufficiency or other clinical indication  
- 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. Twelve CPT tubes (8 mL each) of blood will be collected within 3 days 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 initiation of the chemotherapy. Send to Surgery Branch Cell Production Facility
- In addition to the CPT tubes, 16 mL of blood will be collected within 3 days prior to the start of the chemotherapy.to obtain serum for research purposes (2 SST tubes, 8 mL per tube) Send to Figg lab. For pick-up page 102-11964. For help call 240-760-6180.**The**
following tests must be completed within 7 days of the start of the conditioning chemotherapy regimen:
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- tbnk (t b and

- 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 (to be repeated on the first day of the chemotherapy at the discretion of the PI)
- Serum quantitative immunoglobulins - ABO typing
- CBC with differential (to be repeated on the first day of the chemotherapy at the discretion of the PI)  
- PT/PTT - 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)
- Fibrinogen
3.7.2 Studies to be performed on Day 0 and during the mandatory 14-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 then approximately every 4 hours unless otherwise clinically indicated.  
- Daily physical exam - CBC twice daily from day 0 until day 9 with differential once 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.)
- C-reactive protein (CRP) daily while hospitalized.
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- tbnk on the day

- Chemistries twice daily starting from day 0 to day 9. After day 9, chemistries once daily 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/GPT, AST/GOT, 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 after the infusion
- Other tests will be performed, as clinically indicated.  
- **Day 0 Research Blood:** 1 SST tube will be drawn on the morning of CAR cell infusion prior to infusion of CAR T cells.  
- **Post Infusion Research blood:** Every Monday, Wednesday, and Friday during hospitalization. Thisstarts on the first Monday, Wednesday, or Friday after the day of CAR T-cell infusion and lasting up to 14 days after infusion of 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).  
- **Additional Post-infusion research blood:** 1 SST tube will be drawn on the first Sunday after CAR T-cell infusion. This tube may be stored refrigerated on the nursing unit and processed first thing Monday morning at the latest. If the Monday is a holiday, Sunday research blood will not be drawn.  
- Research blood CPT tubes need to be sent to the Surgery Branch Cell Production Facility.  
Research blood SST tubes need to go to Figg lab. Page 102-11964 for pick-up. Call 240760-6180 for immediate pick-up.
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 business day.
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3.7.3 post-infusion outpatient evaluation

Patients will be seen at the NIH in follow-up to evaluate disease status and late problems related to CAR T-cell infusion at the following time-points: +14 (+/- 1 day), +30 (+/- 5 days), +60 (+/- 7 days), +90 (+/- 7 days), +120 (+/- 7 days), and at 180 (+/- 14 days), 270 (+/- 14 days), and 365 (+/- 30 days) after CAR T-cell infusion. After 12 months, the patient will be seen approximately every 6 months (+/- 30 days) up to three years; subsequently, patients will be seen annually (+/- 30 days) up to 5 years post infusion. In addition, follow-up visits are required at the NIH Clinical Center on days 16 and 21 after infusion for neurologic checks.  
At all outpatient follow-up visits unless otherwise noted, 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. CPT tubes need to be sent to the Surgery Branch Cell Productin Facility.  
- 1 SST tube (8 mL) of Research Blood will be obtained for serum collection.Research blood SST tubes need to go to Figg lab. Page 102-11964 for pick-up. Call 240-760-6180 for immediate pick-up. Note: after the first year **of follow-up, research blood will be**
reduced to 4 CPT tubes (32 mL total) during required protocol visits, and these CPT
tubes should be sent to the Surgery Branch Cell Production Facility. Research blood will not be collected after the 5-year follow-up.  
- CT scan of neck, chest, abdomen, and pelvis and/or PET scan at outpatient follow-up appointments starting 1 month after infusion (as necessary to stage malignancy only). All lymphoma patients should get a PET scans at 1 month after infusion. PET scans should continue at each outpatient scheduled evaluation, except the day 120 (4-month) evaluation until CR is obtained. When CR is obtained, PET scans should cease. CLL patients should not get PET scans only CT scans at each outpatient visit in the study calendar (Table 3.8 except the day 120 (4-month) follow-up visit.  
- For CLL, obtain peripheral blood flow cytometry at all outpatient follow-up visits.  
- Patients may need 1 biopsy to confirm lymphoma progression. Patients may also undergo a maximum of 1 biopsy after CAR T-cell infusion to obtain material to study interactions between CAR T cell and the lymphoma microenvironment. This biopsy could occur either during the inpatient stay or during the outpatient phase. CT-guidance might be needed for these biopsies.  
- For lymphoma, post-treatment bone marrow biopsies are needed only to document CR in patients in CR at all other sites and with pre-treatment bone marrow lymphoma involvement. For CLL, post-treatment bone marrow biopsies will only be done to document CR if the patient is in CR at all other sites. Aspirate must be sent for flow cytometry to the lab of Dr. Maryalice Stetler-Stevenson. CD19 and CD20 staining must be requested for the flow cytometry. CD19 and CD20 immunohistochemistry should also be requested on the bone marrow biopsies. 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: 240-858-3755
- 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)  
- TBNK
- Blood for serum quantitative immunoglobulins - Serum C-reactive protein if clinically indicated  
- Gene-therapy-specific follow-up must be carried out as described in section 3.9
57 Version Date:May 27, 2020  
| 3.8                                                                                                                                                                                                      | STUDY CALENDAR           | Inpatient               | Follow up               | Every 6 months after  day 365(+/- 30 days)  up to 3 years then  annually (+/- 30 days)  up to 5 years post CAR  cells.   |                          |                          |    |    |    |    |    |    |    |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------|-------------------------|-------------------------|--------------------------------------------------------------------------------------------------------------------------|--------------------------|--------------------------|----|----|----|----|----|----|----|
| Clinical Assessments                                                                                                                                                                                     | Post  Cells  while  inpt | Day  +90  (+/- 7  days) |                         |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| Day+14  (+/- 1  day)                                                                                                                                                                                     | Day+30  (+/- 5  days)    | Dayq +60 (+/-  7 days)  | Day  +120 (+/-  7 days) | Day  +180 (+/-  14 days)                                                                                                 | Day +270  (+/- 14  days) | Day +365  (+/- 30  days) |    |    |    |    |    |    |    |
| Screening                                                                                                                                                                                                | Baseline                 | Day 0                   |                         |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| Procedures a                                                                                                                                                                                             |                          |                         |                         |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| History and PE                                                                                                                                                                                           | X                        | Xs                      | X                       | Xk                                                                                                                       | X                        | X                        | X  | X  | X  | X  | X  | X  | X  |
| Vital signs, O2 saturationr                                                                                                                                                                              | X                        | X                       | X                       | Xk                                                                                                                       | X                        | X                        | X  | X  | X  | X  | X  | X  | X  |
| Height, Weight                                                                                                                                                                                           | X                        |                         |                         |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| Performance Score (ECOG)                                                                                                                                                                                 | X                        |                         |                         |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| Lesion Location (If applicable)                                                                                                                                                                          | X                        |                         |                         |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| Pathology Confirmation                                                                                                                                                                                   | X                        |                         |                         |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| Laboratory Assessments CBC with differential                                                                                                                                                             | X                        | X                       | X                       | Xk                                                                                                                       | 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                       | Xk                                                                                                                       | X                        | X                        | X  | X  | X  | X  | X  | X  | X  |
| Serum C-reactive Protein                                                                                                                                                                                 | X                        | X                       | Xk                      | Xd                                                                                                                       | Xd                       | Xd                       | Xd | Xd | Xd | Xd | Xd | Xd |    |
| PTT/PT                                                                                                                                                                                                   | X                        | X                       | X                       | Xk                                                                                                                       | X                        | X                        | X  | X  | X  | X  | X  | X  | X  |
| Fibrinogen                                                                                                                                                                                               | X                        | X                       | Xk X                    |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| TBNK                                                                                                                                                                                                     | X                        | X                       | (Day 7                  | X                                                                                                                        | X                        | X                        | X  | X  | X  | X  | X  | X  |    |
| only)                                                                                                                                                                                                    |                          |                         |                         |                                                                                                                          |                          |                          |    |    |    |    |    |    |    |
| Quantitative immunoglobulins                                                                                                                                                                             | X                        | X                       | X                       | X                                                                                                                        | X                        | X                        | X  | X  | X  |    |    |    |    |  
58 Version Date:May 27, 2020  
| Inpatient                                                                                                        | Follow up               | Every 6 months after  day 365(+/- 30 days)  up to 3 years then  annually (+/- 30 days)  up to 5 years post CAR  cells.   |                         |                          |                          |                          |    |    |    |
|------------------------------------------------------------------------------------------------------------------|-------------------------|--------------------------------------------------------------------------------------------------------------------------|-------------------------|--------------------------|--------------------------|--------------------------|----|----|----|
| Post  Cells  while  inpt                                                                                         | Day  +90  (+/- 7  days) |                                                                                                                          |                         |                          |                          |                          |    |    |    |
| Day+14  (+/- 1  day)                                                                                             | Day+30  (+/- 5  days)   | Dayq +60 (+/-  7 days)                                                                                                   | Day  +120 (+/-  7 days) | Day  +180 (+/-  14 days) | Day +270  (+/- 14  days) | Day +365  (+/- 30  days) |    |    |    |
| Screening                                                                                                        | Baseline                | Day 0                                                                                                                    |                         |                          |                          |                          |    |    |    |
| Procedures a                                                                                                     |                         |                                                                                                                          |                         |                          |                          |                          |    |    |    |
| Thyroid stimulating Hormone,  T3                                                                                 | X                       |                                                                                                                          |                         |                          |                          |                          |    |    |    |
| Anti-CMV, HSV, EBV, t.cruzi, toxoplasma                                                                          | X                       | Xf                                                                                                                       |                         |                          |                          |                          |    |    |    |
| Antibody screen for Hepatitis B  and C; HIV, HTLV-I/II, T. cruzi  (Chagas agent), West Nile, and  syphilis (RPR) | X                       | Xf                                                                                                                       |                         |                          |                          |                          |    |    |    |
| Serum Cortisol                                                                                                   | X                       |                                                                                                                          |                         |                          |                          |                          |    |    |    |
| G6PD                                                                                                             | X                       |                                                                                                                          |                         |                          |                          |                          |    |    |    |
| Urinalysis (culture prn)                                                                                         | X                       | X                                                                                                                        |                         |                          |                          |                          |    |    |    |
| ABO typing                                                                                                       | X                       | X                                                                                                                        |                         |                          |                          |                          |    |    |    |
| B2-HCG (serum or urine in women of childbearing                                                                  | X                       | X                                                                                                                        |                         |                          |                          |                          |    |    |    |
| potential) Disease Assessments CT scan of neck chest,  abdomen, pelvis                                           | XC                      | Xg                                                                                                                       | Xg                      | Xg                       | Xg                       | Xg                       | Xg | Xg | Xg |
| PET Scan                                                                                                         | XC                      | Xh                                                                                                                       | Xh                      | Xh                       | Xh                       | Xh                       | Xh | Xh | Xh |
| Bone marrow aspirate/biopsy  with flow                                                                           | XC                      | Xi                                                                                                                       | Xi                      | Xi                       | Xi                       | Xi                       | Xi | Xi | Xi |
| Peripheral blood flow  cytometry                                                                                 | XC                      | Xl                                                                                                                       | Xl                      | Xl                       | Xl                       | Xl                       | Xl | Xl | Xl |
| Lumbar Puncture (Burkitts  only)                                                                                 | Xe                      |                                                                                                                          |                         |                          |                          |                          |    |    |    |
| Other Specific Assessments Documentation of CD19  and/or CD20 expression by                                      | X                       |                                                                                                                          |                         |                          |                          |                          |    |    |    |
| malignancy                                                                                                       |                         |                                                                                                                          |                         |                          |                          |                          |    |    |    |
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59 version datemay 27 2020

| Inpatient                                                  | Follow up             | Every 6 months after  day 365(+/- 30 days)  up to 3 years then  annually (+/- 30 days)  up to 5 years post CAR  cells.   |                         |                          |                          |                          |    |    |    |    |    |    |
|------------------------------------------------------------|-----------------------|--------------------------------------------------------------------------------------------------------------------------|-------------------------|--------------------------|--------------------------|--------------------------|----|----|----|----|----|----|
| Brain MRI                                                  | X                     | Post  Cells  while  inpt                                                                                                 | Day  +90  (+/- 7  days) |                          |                          |                          |    |    |    |    |    |    |
| Day+14  (+/- 1  day)                                       | Day+30  (+/- 5  days) | Dayq +60 (+/-  7 days)                                                                                                   | Day  +120 (+/-  7 days) | Day  +180 (+/-  14 days) | Day +270  (+/- 14  days) | Day +365  (+/- 30  days) |    |    |    |    |    |    |
| Screening                                                  | Baseline              | Day 0                                                                                                                    |                         |                          |                          |                          |    |    |    |    |    |    |
| Procedures a                                               |                       |                                                                                                                          |                         |                          |                          |                          |    |    |    |    |    |    |
| 250 microgram cosyntropin test                             | Xd                    |                                                                                                                          |                         |                          |                          |                          |    |    |    |    |    |    |
| EKG, echocardiogram                                        | X                     |                                                                                                                          |                         |                          |                          |                          |    |    |    |    |    |    |
| Central                                                    | Venous                | catheter                                                                                                                 |                         |                          |                          |                          |    |    |    |    |    |    |
| placement                                                  | X                     |                                                                                                                          |                         |                          |                          |                          |    |    |    |    |    |    |
| Venous Assessment for Apheresis                            | Xp                    |                                                                                                                          |                         |                          |                          |                          |    |    |    |    |    |    |
| Ongoing Evaluation RCR (replication competent  retrovirus) | X                     | X                                                                                                                        | X                       | X                        | Xj                       |                          |    |    |    |    |    |    |
| Research Blood                                             | Xm                    | Xn                                                                                                                       | Xn                      | Xo                       | Xo                       | Xo                       | Xo | Xo | Xo | Xo | Xo | Xo |
| Adverse Events                                             | X                     | X                                                                                                                        | X                       | X                        | X                        | X                        | X  | X  | X  | X  | X  |    |  
Note: In addition, follow-up visits are required at the NIH clinical center on days 16 and 21 after infusion for neurologic checks.  
a. see section 2.2 and section 3.7 for details b. see section 3.3 for details of testing during hospitalization c. PET or CT (not both) performed as disease appropriate only if needed to establish measurable disease; in addition, bone marrow biopsy, lumbar puncture, and peripheral blood flow cytometry will only be performed if needed to document measurable malignancy. If possible, imaging studies should be obtained from outside facilities from time-points after the patient's last course of lymphoma therapy prior to protocol enrollment.  
d. If clinically indicated e. Lumbar punctures will also need to be repeated after treatment if clinically indicated f. 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 g. CT scan for lymphoma and CLL patients at the indicated time-points including within 14 days of chemo start (before treatment start)
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60 version datemay 27 2020

h. PET for all patients at baseline except for CLL patients (With 14 days of start of chemo). at indicated time-points including within 14 days of chemo start (before treatment start) for all lymphoma patients. No PET scans should be done for CLL patients unless CLL is transformed to DLBCL. As soon as CR is established, PET scans should cease.  
i. All patients need a pre-treatment baseline bone marrow biopsy **(G-banding cytogenetics included).** Post-treatment bone marrow biopsies are only done if useful for staging. CLL patients only need post-treatment bone marrow biopsies if in CR at all other sites. Lymphoma patients only need bone marrow biopsies post-treatment if the pre-treatment bone marrow was positive and the patient is in CR at all other sites.  
j. RCR blood collection will only continue after the 1 year time-point if a previous RCR test has been positive, see section 3.9 for details k. See section **3.7.2** for details on labs during inpatient l. Only CLL
m. Baseline Research Blood (See section **3.7.1**)
n. Inpatient Research Blood. One SST tube of research blood is to be collected on the morning of cell infusion. One SST tube of research blood is to be drawn on the first Sunday after CAR T-cell infusion. Research blood is also drawn on Mon-Wed-Fri up to Day 14 after CAR T-cell infusion while inpatient. (See section **3.7.2**)
o. Follow up Research Blood (See section **3.7.3**)
p. Valid for 6-month before apheresis collection. Does not have to be performed is cells were previously collected. q. Assessment may be performed remotely per PI discretion (outside labs will be accepted and symptoms assessment may be performed via telephone) s. This assessment may include a neurology consult if clinically indicated. r. +/- 15 minutes
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3.9 gene-therapy-specific follow-up 3.9.1 clinical

Long-term follow up of patients receiving gene transfer is required by the FDA and must continue even after the patient comes off the study for another follow-up. Physical examinations will be performed and documented annually for 5 years following cell infusion to evaluate long-term safety. Physical exams can be performed by other physicians if clinic notes are obtained and retained in SB. A complete blood count should be done at these physician visits for the first 5 years after infusion. 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. 3.9.2 Testing for persistence of CAR transduced cells Persistence of CAR transduced cells will be assessed by quantitative PCR and/or flow cytometry at 1, 2, 3, 6 and 12 months after cell infusion or untilthey are detectable at levels <0.10% of PBMC or until a stable or decreasing level of CAR T cells is present at least 3 years after infusion. 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.
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3.9.3 replication competent retrovirus (rcr)

Patients' blood samples will be obtained for analysis for detection of replication competent retroviruses (RCR) by GALV-specific 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..  
- In case of detection of replication-competent retrovirus, the following actions will be taken:
a. Immediately report the finding of RCR to the FDA, the NCI IRB, the NIH Institutional Biosafety Committee, and the Indiana University Vector Production Facility (where the vector was made).  
b. Repeat the S+L- and RCR PCR on the infused cells for the patient in question. Repeat the RCL PCR on the sample that was found to be positive.
c. Have the patient come to the NIH for a clinic visit. Perform a complete history and physical exam. Draw blood for a complete blood count with differential, flow cytometry to assess T, B, and NK cell numbers in the blood, repeat RCR PCR, perform standard HIV and HTLV-I screening, repeat PCR to assess for the presence of CARexpressing T cells in the blood. Perform a bone marrow biopsy with flow cytometry and assess the bone marrow for the presence of CAR-expressing T cells.  
d. If no abnormalities requiring intervention are found after evaluating the patient, the patient should return monthly for a history, physical, CBC, and repeat RCR PCR tests on the blood.
3.10 COST AND COMPENSATION
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3.10.1 costs

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. 3.10.2 Compensation Subjects will not receive compensation for participation in this study. 3.10.3 Reimbursement This study offers subject reimbursement or payment for travel, lodging and/or meals while participating in the research. The amount, if any, is guided by NIH policies and guidelines. On this study, the NCI will cover the cost for some of the expenses. Some of the costs may be paid directly by the NIH and some may be reimbursed to the subject. Someone will work with subjects to provide more information.
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3.11 criteria for removal from

Prior to removal from study, effort must be made to have all subjects complete a safety visit:  
- If no CAR-T cells administered: visit should occur approximately 30 days after the last dose of study chemotherapy treatment.  
- If CAR-T cells given: visit should occur approximately 60 days after the last dose of CART-cells.
3.11.1 Criteria for removal from protocol therapy Note that the treatment consists of a conditioning chemotherapy regimen followed by a T-cell infusion. Off-treatment criteria applies to eligibility for potential repeat treatments; and also, cancellations of treatment for toxicity arising any time before the cell infusion. Patients will be taken off treatment for the following:
- Any DLT that 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 treatment can proceed if not, the patient will be off-study.  
- The patient receives any other treatment for their malignancy (including corticosteroids at a dose higher than 5 mg/day of prednisone or equivalent) except the planned protocol treatment within 3 weeks of the start of the initial protocol treatment or if the patient receives any treatment for their malignancy at any time after their initial CAR T-cell infusion. If a patient receives corticosteroids in doses greater than 5 mg/day of prednisone or an equivalent dose of another corticosteroid within 2 weeks of the start of protocol treatment, the treatment will need to be delayed or cancelled.
- General or specific changes in the patient's condition render the patient unacceptable for further treatment on this study in the judgment of the Principal Investigator.  
- Participant requests to be withdrawn from active therapy - Investigator discretion - Positive pregnancy test 3.11.2 Off-study Criteria Patients will be taken off study for the following (all patients coming off study must 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 a safety visit 30 day after last administration of study treatment (only for patients who have received chemotherapy but will not receive a cell infusion)
- The patient requests to be withdrawn from the study - There is significant patient noncompliance - Death
- Investigator discretion - Development of progressive or relapsed malignancy after the CAR T cell infusion in patients not desiring or not eligible for re-treatment on this protocol.  
- The patient receives any anti-malignancy therapy after the CAR T-cell infusion except for repeat treatments on this protocol.  
- Taking corticosteroids for any reason (Except to treat CAR cell related toxicities) after CAR T-cell infusion at a dose higher than 5 mg/day of prednisone or equivalent dose of another corticosteroid.  
- 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. In this case, treatment will be delayed until the patient meets eligibility criteria, or if the patient will never likely meet eligibility criteria, the patient will be removed from the study. An exception to this is that a platelet count of 50,000 or more is considered adequate to start chemotherapy. This platelet level is slightly lower than that required for initial protocol enrollment.
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4 concomitant medications/measures

All routine and appropriate supportive care (including blood products) will be provided during this study, as clinically indicated, and in accordance with the standard of care practices. Clinical judgment should be utilized in the treatment of any adverse event experienced by the patient.
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4.1 antibiotic prophylaxis

- 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 400 mg/dL will receive intravenous immunoglobulin replacement as needed to maintain an IgG level above 400 mg/dL . 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 38.3 C 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.
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4.3 anti-emetics

Anti-emetics will follow current literature such asASCO and/or NCCN (except that corticosteroids will be avoided).