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Version Date: 01/12/2018 Abbreviated Title: Phase I Anti-CD 19 CAR CC Protocol \#: 12-C-0112 P OSP \#: 1201-1147 IBC \#: RD-12-I-01 Version Date: 01/12/2018 NCT Number: NCT01593696 Title: Phase I Study of T Cells Expressing an Anti-CD19 Chimeric Receptor in Children and Young Adults with B Cell Malignancies NCI Principal Investigator: Nirali N. Shah, M.D.  
Pediatric Oncology Branch National Cancer Institute Bldg 10, Room 1W-5750 9000 Rockville Pike, MSC 1806 Bethesda, MD 20892-1806 Phone: (240) 760-6199 Email: shahnn@mail.nih.gov
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investigational agents

| Drug Name:                                                                           | Autologous peripheral blood lymphocytes (PBL) cultured with OKT3 and  Interleukin-2; Transduced with retroviral vector (PG13-CD19-H3)  expressing Anti-CD19 chimeric antigen receptor gene   |
|--------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| IND Number:                                                                          | 14985                                                                                                                                                                                        |
| Sponsor:                                                                             | Center for Cancer Research, NCI                                                                                                                                                              |
| Manufacturer:                                                                        | Clinical Center Department of Transfusion Medicine (DTM), Cell  Processing Section (CPS)                                                                                                     |
| Commercial Agents: Fludarabine, Cyclophosphamide, Cytarabine, Etoposide, Ifosfamide, |                                                                                                                                                                                              |  
Commercial Agents: Fludarabine, Cyclophosphamide, Cytarabine, Etoposide, Ifosfamide, Filgrastim  
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precis background objectives eligibility design

x Chimeric antigen receptors (CAR) that recognize the CD19 antigen have been constructed and are in clinical trials at several institutions. In this trial, the POB will utilize a chimeric receptor containing the signaling domains of CD28 and CD3-zeta, currently under study in the CCR in adults, for children and young adults with CD19 expressing malignancies.  
1) To determine the safety and feasibility of administering escalating doses of anti-CD19-
CAR engineered peripheral blood lymphocytes in two strata (prior allogeneic stem cell transplant [SCT] vs. no prior SCT) of children and young adults with B cell malignancies following a cyclophosphamide/fludarabine preparative regimen. COMPLETED March 2014.  
2) To determine the safety of administering cells in two groups of children and young adults with B-cell malignancies expressing CD19:
x Arm 1 - Patients without high-burden disease or patients for whom chemotherapy toxicity is a concern will receive standard preparative regimen.  
x Arm 2 - Patients with high-burden disease who receive standard chemotherapy to reduce burden, (defined as patients with ALL who have M3 bone marrow blasts and/or presence of peripheral blood blasts on routine CBC, or patients with lymphoma).  
3) To determine the feasibility of administering anti-CD19 CAR transduced T cells within 21 days of the target date in children and young adults with B-cell malignancies expressing CD19 enrolled on arm 2: Patients with high-burden disease who receive standard chemotherapy to reduce burden.  
Patients 1-30 years of age, at least 15 kg, with a CD19-expressing B-cell malignancy that has recurred after or not responded to one or more standard chemotherapy-containing regimens for their malignancy and is deemed incurable by standard therapy. Patients with a history of allogeneic SCT who meet all eligibility criteria are eligible to participate.  
x PBMC will be obtained by leukapheresis. Anti-CD19 CAR T cells will be manufactured from fresh or frozen PBMCs. On Day -7, PBMC will be enriched for CD3+ cells and cultured in the presence of anti-CD3/-CD28 beads followed by retroviral vector x In co-cultures with CD19-expressing acute lymphoblastic leukemia cells, anti-CD19-
CAR-transduced T cells show robust killing, and in xenograft models, can rapidly clear CD19-expressing ALL cell lines. supernatant containing the anti-CD19 CAR. Total culture time is approximately 7-14 days.  
x Patients will be divided into the 2 groups listed above.  
o Arm 1: Patients will begin preparative regimen comprising fludarabine 25 mg/m2 on Days –4, –3 and –2 and cyclophosphamide 900 mg/m2 on day –2.  
o Arm 2: Patients with high-disease burden will be treated with intensive standard of care chemotherapy to decrease disease burden during cell manufacturing.  
x All patients: The CD19-CAR cells will be infused on Day 0, with up to a 72h delay allowed for fresh cells or a 21 day delay if cells are cryopreserved, if needed for resolution of clinical toxicities or to generate adequate cell numbers.  
x The previously determined maximum tolerated dose (MTD) of 1 X 106 will be administered intravenously.  
x Patients will be monitored for toxicity, response and T cell persistence.
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version date 01/12/2018

| TABLE OF CONTENTS                                                                                                 |                                                                                                                                                       |                                                                                         |
|-------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------|
| PRECIS .                                                                                                          |                                                                                                                                                       |                                                                                         |
| TABLE OF CONTENTS                                                                                                 |                                                                                                                                                       |                                                                                         |
| INTRODUCTION                                                                                                      |                                                                                                                                                       |                                                                                         |
| 1.                                                                                                                | 1.1                                                                                                                                                   | STILLING AND INTERACTION                                                                |
| 1.2                                                                                                               | Background and Rationale .                                                                                                                            |                                                                                         |
| 1.3                                                                                                               | Exploratory Analysis                                                                                                                                  |                                                                                         |
| Protocol Summary and Rationale .                                                                                  |                                                                                                                                                       |                                                                                         |
| 1.4                                                                                                               |                                                                                                                                                       |                                                                                         |
| 1.5                                                                                                               | Protocol Status Summary and Amendment Justification ...............................................................2                                  |                                                                                         |
| ELIGIBILITY ASSESSMENT AND ENROLLMENT ............................................................24              |                                                                                                                                                       |                                                                                         |
| 2.1                                                                                                               | Eligibility Criteria .                                                                                                                                |                                                                                         |
| 2.2                                                                                                               | Screening Evaluation ..............................................................................................................................27 |                                                                                         |
| 2.3                                                                                                               | Registration Procedures                                                                                                                               |                                                                                         |
| 2.4                                                                                                               | T Treatment Assignment Procedures ......................................................................................................29            |                                                                                         |
| STUDY IMPLEMENTATION                                                                                              |                                                                                                                                                       |                                                                                         |
| 3                                                                                                                 | 3.1                                                                                                                                                   | S Study Design                                                                          |
| 3.2                                                                                                               | Apheresis for Cell Acquisition ...............................................................................................30                      |                                                                                         |
| 3.3                                                                                                               | CC                                                                                                                                                    |                                                                                         |
| 3.4                                                                                                               | Anti-Neoplastic Therapy .............................................................................................................32               |                                                                                         |
| 3.5                                                                                                               | Impact of Intercurrent Illness after Enrollment .....................................................................32                               |                                                                                         |
| 3.6                                                                                                               | T Treatment Limiting Toxicity ...........................................................................................................32           |                                                                                         |
| 3.7                                                                                                               | Dose Escalation (Completed 6-14) ................................................................................................34                   |                                                                                         |
| Dose Expansion Cohort (COMPELTED 6-14) ........................................................................36 |                                                                                                                                                       |                                                                                         |
| 3.8                                                                                                               |                                                                                                                                                       |                                                                                         |
| 3.9                                                                                                               | D Drug Administration ...................................................................................................................37           |                                                                                         |
| Anti-CD19 CAR T Cell Infusion - Day 0 .......................................................................40   |                                                                                                                                                       |                                                                                         |
| 3.10                                                                                                              | Option for Additional Dose(s) of Anti-CD19 CAR T Cells ............................................42                                                 |                                                                                         |
| 3.11                                                                                                              |                                                                                                                                                       |                                                                                         |
| 3.12                                                                                                              | ON-Study Evaluation ....................................................................................................................43            |                                                                                         |
| 3.13                                                                                                              | S Study Calendar .....................................................................................................................48              |                                                                                         |
| 3.14                                                                                                              | Protocol Accrual Suspension ...........................................................................................48                             |                                                                                         |
| 3.15                                                                                                              | Criteria for Removal from Protocol Therapy and Off Study Criteria ........................48                                                          |                                                                                         |
| CONCOMITANT MEDICATIONS/MEASURES ................................................................49               |                                                                                                                                                       |                                                                                         |
| 4                                                                                                                 | 4.1                                                                                                                                                   | Concurrent Therapy for Extramedullary Leukemia or CNS Lymphoma ......................49 |  
4  
| Version Date: 01/12/2018 4.2 Infection Prophylaxis 50 4.3 Prophylaxis and Treatment of Tumor Lysis Syndrome in Children 50 5 BIOSPECIMEN COLLECTION 51 5.1 Correlative Studies for Research/Pharmacokinetic Studies 51 5.2 Sample Storage, Tracking and Disposition 53 6 DATA COLLECTION AND EVALUATION 54 6.1 Data Collection 54 6.2 Response Criteria 54 6.3 Persistence Criteria for Anti-CD19 CAR Transduced T Cells 58 6.4 Toxicity Criteria 58 7 SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN 58 7.1 Definitions 58 7.2 NCI-IRB and Clinical Director Reporting 60 7.3 IND Sponsor Reporting Criteria 61 7.4 Institutional Biosafety Committee (IBC) Reporting Criteria 61 7.5 Data and Safety Monitoring Plan 62 8 STATISTICAL CONSIDERATIONS 63 8.1 General Considerations 63 8.2 Dose Escalation Design 64 8.3 Safety and Feasibility of High-Disease Burden vs. Non-High-Disease Burden 65 8.4 Accrual Target and Study Duration 66 8.5 Feasibility 66 8.6 Secondary Endpoints 66 9 HUMAN SUBJECTS PROTECTIONS 67 9.1 Rationale For Subject Selection 67 9.2 Participation of Children 68 9.3 Evaluation of Benefits and Risks/Discomforts 68 9.4 Risks/Benefits Analysis 69 9.5 Consent and Assent Process and Documentation 69 10 PHARMACEUTICAL INFORMATION 71 10.1 Retroviral Vector Containing the Anti-Cd19 Car Gene 71   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
5  
| Version Date: 01/12/2018                                                                                         |                                                                                                                                                          |                                                                                                                         |
|------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------|
| 10.2                                                                                                             | Fludarabine .........................................................................................................................................72  |                                                                                                                         |
| 10.3                                                                                                             | Cyclophosphamide .                                                                                                                                       |                                                                                                                         |
| 10.4                                                                                                             | Mesna                                                                                                                                                    |                                                                                                                         |
| 10.5                                                                                                             | Ifosfamide ...........................................................................................................................................74 |                                                                                                                         |
| 10.6                                                                                                             | Etoposide (Vepesid, VP-16) ..........................................................................................................75                  |                                                                                                                         |
| 10.7                                                                                                             | Acetaminophen (Tylenol) .............................................................................................................75                  |                                                                                                                         |
| 10.8                                                                                                             | Diphenhydramine (Benadryl) ..........................................................................................................75                  |                                                                                                                         |
| Intrathecal Chemotherapy                                                                                         |                                                                                                                                                          |                                                                                                                         |
| 10.9                                                                                                             |                                                                                                                                                          |                                                                                                                         |
| 10.10                                                                                                            | Hydrocotisone (Cortef, Solu-cortef) NCS# 010483                                                                                                          |                                                                                                                         |
| 10.11                                                                                                            | Cytarabine (cytosine arabinoside, AraC, Cytosar) NSC# 063878                                                                                             |                                                                                                                         |
| 10.12                                                                                                            | Methotrexate .                                                                                                                                           |                                                                                                                         |
| 10.13                                                                                                            | Allopurinol                                                                                                                                              |                                                                                                                         |
| 10.14                                                                                                            | Tocilizumab (ANTI-IL6R MAB)                                                                                                                              |                                                                                                                         |
| 10.15                                                                                                            | Filgrastim (Neupogen)                                                                                                                                    |                                                                                                                         |
| APPENDICES .                                                                                                     |                                                                                                                                                          |                                                                                                                         |
| 11                                                                                                               | 11.1                                                                                                                                                     | Appendix A -Performance Status Criteria .............................................................................80 |
| 11.2                                                                                                             | Appendix B: Study Calendar .........................................................................................................81                   |                                                                                                                         |
| Appendix C: Grading and Treatment of Acute GVHD....................................................86            |                                                                                                                                                          |                                                                                                                         |
| 1.3                                                                                                              | Appendix D: Calculation of Weight for Cell Dose Calculation in Morbidly Obese                                                                            |                                                                                                                         |
| 11.4                                                                                                             |                                                                                                                                                          |                                                                                                                         |
| CANDIDATES                                                                                                       |                                                                                                                                                          |                                                                                                                         |
| Appendix E: Research Sample Requirements*.....................................................................88 |                                                                                                                                                          |                                                                                                                         |
| 11.5                                                                                                             |                                                                                                                                                          |                                                                                                                         |
| 1.6                                                                                                              | Appendix F: Guidelines for Grading and Management of Suspected Cytokine Release                                                                          |                                                                                                                         |
| Syndrome                                                                                                         |                                                                                                                                                          |                                                                                                                         |
| 1.7                                                                                                              | Appendix G: Neuropsychological Test Battery ...............................................................92                                            |                                                                                                                         |
| 1.8                                                                                                              | Appendix H: Neuro Symptom Checklist..........................................................................93                                          |                                                                                                                         |
| 12                                                                                                               | REFERENCES                                                                                                                                               |                                                                                                                         |  
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1. introduction 1.1 study objectives

1.1.1.1 To determine the safety and feasibility of administering escalating doses of anti-CD19-
CAR engineered peripheral blood lymphocytes in two strata (prior allogeneic stem cell transplant [SCT] vs. no prior SCT) of children and young adults with B cell malignancies following a cyclophosphamide/fludarabine lymphodepletion regimen. The following cell dose escalation scheme will be used: **COMPLETED**
x 1 x 106 transduced T cells/kg (± 20%)  
x x 106 transduced T cells/kg (± 20%)
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x x 107 transduced t

1.1.1.2 To determine the safety of administering cells in two groups of children and young adults with B-cell malignancies expressing CD19:
x Arm 1 - Patients without high-burden disease or patients for whom chemotherapy toxicity is a concern will receive standard preparative regimen.  
x Arm 2 - Patients with high-burden disease who receive standard chemotherapy to reduce burden (defined as patients with ALL who have M3 bone marrow blasts and/or presence of peripheral blood blasts on routine CBC, or patients with lymphoma).  
1.1.1.3 To determine the feasibility of administering anti-CD19 CAR transduced T cells within 21 days of the target date in children and young adults with B-cell malignancies expressing CD19 enrolled on arm 2: Patients with high-burden disease who receive standard chemotherapy to reduce burden (defined as patients with ALL who have M3 bone marrow blasts and/or presence of peripheral blood blasts on routine CBC, or patients with lymphoma)
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1.1.2 secondary objectives

1.1.2.1 Explore whether the administration of anti-CD19-CAR engineered peripheral blood lymphocytes can mediate antitumor effects in children with B cell malignancies who 1) do NOT have high-burden disease and receive standard preparative regimen, or 2) have high-burden disease and receive standard cytotoxic chemotherapy to reduce disease burden.  
1.1.2.2 To evaluate the ability of CRS treatment algorithm to reduce the incidence of Grade 4
&\WRNLQH5HOHDVH6\QGURPH&56WRRISDWLHQWVUHFHLYLQJDQWL-CD19 CAR engineered peripheral blood lymphocytes at the maximum tolerate dose (MTD).  
1.1.2.3 Measure persistence of adoptively-transferred anti-CD19-CAR-transduced T cells in the blood and where possible the bone marrow and CSF of patients.  
1.1.2.4 To describe the toxicity of administration of anti-CD19-CAR engineered peripheral blood lymphocytes in children and young adults with CNS disease.  
1.1.2.5 To assess cognitive functioning (memory, attention, processing speed, and executive functioning [primary outcome = CogState One Card Learning score]) pre- and postinfusion to examine the cognitive effects of the anti-CD19 CAR infusion.  
1.1.2.6 Define the cardiovascular toxicity associated with CD-19 CAR therapy infusion and associated cytokine release syndrome (CRS). Explore the relationship between cardiac function, inflammatory and cardiac biomarkers and cardiac imaging to evaluate cardiac function pre- and post-CAR to determine whether specific markers could provide an early signal for subjects at risk of developing cardiac dysfunction post-infusion.  
1.1.3 Exploratory Objectives 1.1.3.1 Describe the safety, feasibility and clinical impact of administration of cryopreserved anti-CD19-CAR engineered peripheral blood lymphocytes in patients who received clinical benefit from the initial infusion. Explore the relationships between cognitive test scores and serum cytokine levels (e.g. IFN-Ȗ,/-2, IL-6, and TNFD).  
1.1.3.2 Explore whether sequence-based genomic analysis can detect minimal residual disease
(MRD) in patients who are MRD negative by flow cytometry.
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1.2 background and rationale 1.2.1

Between infancy and 15 years of age, cancer is the leading cause of death by disease in U.S.  
children1. Acute lymphoblastic leukemia (ALL) represents the most common pediatric malignancy, accounting for approximately 25% of childhood cancer. Although disease-freesurvival rates are high for children with ALL, approximately 20% still die of their underlying disease, making ALL the leading cause of cancer death in childhood. For those who relapse, long-term prognosis is poor. Even with attainment of a second remission and despite allogeneic stem cell transplant, long-term disease-free survival rates of <50% are expected2. New treatment approaches are needed to overcome resistance to standard therapy.
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1.2.2 adoptive cell therapy using

Adoptive transfer of donor T cells following bone marrow transplantation has shown activity in recurrent leukemia3,4 and adoptive therapy with tumor infiltrating lymphocytes (TIL) has shown activity in some cancers5. More recently, adoptive transfer of T cells transduced with the genes of tumor-antigen-specific T cell receptors caused regressions of melanoma and synovial cell sarcoma in some patients6. Another approach to adoptive T cell therapy is to engineer T cells to express chimeric antigen receptors (CARs). CARs are made up of a single chain variable fragment (scFv) coupled to signaling molecules that can activate T cells expressing the CAR7-9.  
The earliest preclinical work conducted with CARs was performed in the late 1980s10,11 and more recently, CARs have been designed to target many different tumor antigens and many important questions have been addressed using CARs in mouse models. Murine models have shown that syngeneic T cells transduced with retroviruses encoding CARs protected mice from tumor challenges *in vivo*12,13. Murine models indicated that inclusion of CD28 signaling domains enhanced tumor cytotoxicity and persistence of CAR-transduced T cells14,15. Increasing the number of CAR-transduced T cells administered to mice consistently enhanced tumor protection as well15-17. One murine study reported that administration of high-dose IL-2 enhanced tumor protection by CAR-transduced T cells18.  
In the last 5 years, several clinical trials of CAR based therapies have been initiated including CARs targeting carbonic anhydrase IX in kidney cancer19, anti-GD2 CARs in neuroblastoma20,21, anti-CD20-CAR T cells in B cell lymphoma, anti-Her2Neu CAR therapy22, as well as anti-CD19 CAR therapy (discussed in greater detail below). From this early clinical experience, several general conclusions are emerging. First, chimeric antigen receptor expressing T cells can mediate antitumor activity as tumor shrinkage has been demonstrated in neuroblastoma20 and lymphoma23. Second, the CAR engineered T cells can induce substantial toxicity primarily related to cytokine storm24, as on-target but off-tumor toxicity resulting from antigen expression on normal tissues19. Thirdly, antitumor effects appear to correlate with cell persistence and lymphopenia inducing therapies, such as chemotherapy administered prior to CAR therapy, which also appears to increase the incidence and degree of CAR-cell persistence. Another factor potentially modulating persistence is anti-CAR immune responses as recently demonstrated in patients with lymphoma and renal cell carcinoma19,25. Finally, recent data demonstrates that 2nd generation chimeric antigen receptor T cells that incorporate a co-stimulatory domain, such as CD28, have superior persistence and expansion compared to 1st generation CAR T cells that have only one signaling motif (usually zeta subunit of the T-Cell Receptor) 26. The historical development and composition of CARs are discussed at length in the following paper: Savoldo, B., et al., CD28 costimulation improves expansion and persistence of chimeric antigen receptormodified T cells in lymphoma patients. J Clin Invest. 121(5): p. 1822-626. Based upon these principles, we performed a dose-escalation trial of anti-CD19 CAR T cells (2nd generation) after a pre-CAR lymphodepleting regimen. 1.2.3 Development of an Anti-CD19 CAR for Clinical Adoptive T Cell Transfer CD19 was chosen as the target because it is universally expressed on pediatric pre-B ALL
cells27,28, and the only normal cells that express CD19 are B cells and perhaps follicular dendritic cells29. Importantly, CD19 is not expressed on pluripotent hematopoietic stem cells29. While destruction of normal B cells is a drawback to targeting CD19, clinical experience suggests that destruction of normal B cells is a tolerable toxicity of leukemia directed therapy. When patients receive the anti-CD20 monoclonal antibody rituximab, the number of normal B cells is severely depressed for several months30, yet patients that receive chemotherapy plus rituximab do not have an increased rate of infections when compared to patients that receive chemotherapy alone31. Similarly, patients with congenital absence of B cells have prolonged survival and acceptable quality of life as a result of intravenous infusions of IgG18.  
The anti-CD19 CAR used in this study consists of three main components: the variable regions of the anti-CD19 monoclonal antibody FMC6332, the signaling domain of the CD28 costimulatory molecule, and the signaling domain of the CD3 zeta chain33. This receptor is referred to as FMC63-28. The DNA encoding this receptor was cloned into the MSGV1 retroviral vector backbone34 to form the plasmid shown in Figure 1A. This plasmid is referred to as MSGV1-FMC63-28. For the remainder of this document the FMC63-28 receptor will be referred to as the anti-CD19 CAR.
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figure 1

A  
  
Preliminary experiments using transiently produced retroviral supernatant for transduction showed that the FMC63-28 receptor functioned well in vitro; therefore, a stable producer cell clone that produces gibbon ape leukemia virus (GALV) pseudotyped retrovirus encoding the FMC63-28 anti-CD19 CAR was generated. The supernatant from this clone was used for the experiments described in this document.  
In addition to the extensive in vitro experiments already performed34 we confirmed the suitability of the anti-CD19 CAR for use in a clinical trial targeting pediatric B-cell malignancies. Normal peripheral blood mononuclear cells (PBMC) were activated using artificial antigen presenting cells (aAPC) loaded with the anti-CD3 monoclonal antibody OKT3 and in the presence of IL-2 to activate T cells and induce their proliferation. After 48 hours of stimulation, the T cells were exposed to gamma-retrovirus encoding the anti-CD19 CAR that was spin-loaded onto a retronectin-coated plate, then cultured overnight with a second transduction performed on Day 2. Transduction efficiency was determined using biotinylated Protein L. Protein L is a recombinant protein that interacts with the constant region of kappa chains in scFv (present in anti-CD19-
CAR) or Fab fragments. Following transduction, the cultured cells were almost entirely CD3+ T
cells, with transduction efficiencies of 30-70% as measured by CAR expression. An example of staining of anti-CD19-transduced T cells with biotinylated Protein L is shown in Figure 1B. CD19-CAR transduced T cells demonstrate cytotoxicity against CD19 expressing ALL cell lines in vitro but not CD19 negative controls with an average of 50-60% cytotoxicity at effector-totarget ratios as low as 2.5:1 (Figure 2A). Anti-CD19-CAR T cells also produce significant levels of interferon-J (IFNJ), tumor necrosis factor-D (TNFD) and IL-2 specifically in response to CD19-expressing target cell lines (Figure 2B-D).  
In a pilot experiment, immunodeficient NOG (NOD/Shi-*scid*/IL-5Ȗnull) mice were injected via tail vein with one million NALM6-GL cells (human ALL stably expressing GFP and luciferase) four days prior to injection of either anti-CD19-CAR T cells or stimulated but untransduced T cells (Mock). At the time of injection of T cells, mice already had bioluminescent evidence of leukemia (Figure 3A). Surprisingly, only forty hours after T cell injection the mice that received anti-CD19-CAR T cells nearly cleared their leukemia. By Day 7, all but one of the treated mice were free of leukemia while the mice that received Mock T cells continued to progress in their disease burden (Figure 3B) and required sacrifice by Day 17.  
  
Figure 2  
Figure 4  
  
We have replicated this data using anti-CD3/anti-CD28 beads in lieu of aAPC for T cell stimulation as this is the approach we will use to expand the anti-CD19-CAR cells in this trial. PBMC will be collected, CD3+ cells will be enriched using anti-CD3/anti-CD28 beads cultured in the presence of IL-2 for 48 hours, then transduced as previously described. Anti-CD19-CAR T cells produced in this manner have equivalent transduction efficiencies as indicated by interaction with Protein L (Figure 4A). Immunodeficient NOG mice were again injected with one million NALM6-GL cells four days prior to injection of anti-CD19-CAR T cells generated using beads. Control mice received equivalent numbers of untransduced T cells (Mock) that had been stimulated with beads. All mice treated with anti-CD19-CAR T cells cleared all bioluminescent evidence of ALL within 48 hours (Figure 4B). This indicates that anti-CD19- CAR T cells are a potent therapeutic agent against human ALL and strongly supports pursuit of such therapy in human clinical trials targeting pediatric ALL. 1.2.4 Other Anti-CD19 CAR Clinical Trials Several other groups have constructed anti-CD19 CARs35. Jensen and coworkers completed a clinical trial of adoptive transfer of T cells transfected with a plasmid encoding an anti-CD19 chimeric receptor36. The T cells expressing the anti-CD19 receptor did not persist *in vivo* and did not cause objective remissions of lymphoma. Our proposed clinical trial differs from the completed trial of Jensen and coworkers in many ways. The most important difference is that our proposed trial will use a retroviral vector rather than plasmid transfection to transfer an antiCD19 receptor to T cells, thus resulting in integration and persistent (rather than transient) CAR
expression. Another potentially important difference is that the CAR we propose to utilize in our clinical trial contains the signaling component of CD28, whereas the receptor construct used by Jensen and coworkers did not include a CD28 moiety. Researchers at MSKCC conducted a clinical trial of adoptive transfer of T cells that have been transduced with retroviruses encoding an anti-CD19 receptor (NCI Clinical Trials PDQ, trial MSKCC trial \#09-114, Principal Investigator: R. Brentjens). This trial enrolled only adult ALL patients. In this trial, CAR-transduced T cells are transferred to patients without preparative chemotherapy prior to cell transfer. They have since modified their protocol to include a preparative regimen and are enrolling both children and adults with ALL (NCT01860937). Our trial differs from the trial of Brentjens and coworkers in several ways. First, we will treat a pediatric population with B-cell malignancies and will not exclude patients who have already undergone an allogeneic stem cell transplant (which comprises the majority of pediatric patients with refractory ALL). Secondly, we will include a lymphodepleting preparative regimen prior to cell infusion, which should allow for improved engraftment and function of infused T cells. Third, since we have demonstrated clearance of mild CNS leukemia in 2 patients with a single infusion of CAR T cells, we will allow enrollment of patients with any level of CNS leukemia and regardless of bone marrow relapse status. Finally, we will incorporate our recently modified CRS grading and treatment system37 in an effort to reduce the incidence of Grade 4 CRS.  
| CD19 CAR Trials Currently Proposed or Underway Trial Location Cell Dose/kg Cytokine Costim   | Notes                          |                               |           |                                                                                    |
|----------------------------------------------------------------------------------------------|--------------------------------|-------------------------------|-----------|------------------------------------------------------------------------------------|
| NCI SB                                                                                       | ~5 x 106                       | High Dose                     | CD28      | High toxicity, cell dose and                                                       |
| IL-2                                                                                         | IL2 dose lowered               |                               |           |                                                                                    |
| NCI SB                                                                                       | 1 x 106                        | None                          | CD28      | Reduced intensity                                                                  |
| 2.5 x 106                                                                                    | preparative regimen            |                               |           |                                                                                    |
| NCI ETIB                                                                                     | 0.5-10 x 106                   | None                          | CD28      | Relapse after Allo-SCT using  donor as source for T cells;  No preparative regimen |
| Baylor COM                                                                                   | 1 x 106 5 x 106 10 x 106       | None                          | 50% CD28, | Children included                                                                  |
| 50% none                                                                                     |                                |                               |           |                                                                                    |
| Baylor COM                                                                                   | 0.3-2.4 x 106                  | None but  may get  ipilimumab | CD28      | Relapse after Allo-SCT,  children included                                         |
| Baylor COM                                                                                   | 3.75 x 106 11.2 x 106 33 x 106 | None                          | CD28      | Donor multi-virus CTL  transduced post-SCT,  children included                     |
| University  College  London                                                                  | 1.6 x 106                      | None                          | None      | Donor EBV CTL post-SCT  followed by vaccination  w/EBV-LCL; children  included     |
| MSKCC                                                                                        | 3 x 106                        | None                          | CD28      | Donor EBV CTL post-SCT                                                             |  
| 1 x 107 3 x 107                       |                                                                         |                      |                               |                                                                   |
|---------------------------------------|-------------------------------------------------------------------------|----------------------|-------------------------------|-------------------------------------------------------------------|
| MSKCC                                 | 1 x 106                                                                 | None                 | CD28                          | MRD patients get higher                                           |
| 3 x 106                               | dose of cells; dose is "T  cells"; dealer's choice  preparative regimen |                      |                               |                                                                   |
| MSKCC                                 | 5 x 106 1 x 107 2 x 107                                                 | None                 | CD28                          | Cells given after  myeloablative allo-SCT;  adults only           |
| University of                         | 1.5 x 109 to                                                            |                      |                               |                                                                   |
| Pennsylvania                          | 5 x 109 CD3+ cells  (Flat dosing) in 4  split doses                     | None                 | 4-1BB                         | dealer's choice preparative  regimen                              |
| 1-5 x 108 CD3+                        |                                                                         |                      |                               |                                                                   |
| University of                         | None                                                                    | 4-1BB                | Phase II; Unclear preparative |                                                                   |
| Pennsylvania                          | cells                                                                   | regimen; Adults only |                               |                                                                   |
| Children's  Hospital of  Philadelphia | 1 x 107 1 x 108 CD3+ cells  (Flat dosing)                               | None                 | 4-1BB                         | Phase I; Children included;  dealer's choice preparative  regimen |
| Seattle  Children's  Hospital         | Unknown                                                                 | None                 | 4-1BB                         | Dealer's choice preparative  regimen; Children included           |  
At Baylor College of Medicine, Dotti, Brenner, and coworkers are conducting three clinical trials involving anti-CD19 CAR. First, two types of anti-CD19-CAR-transduced T cells, one with both the CD3-zeta and CD28 signaling moieties and one with only CD3-zeta, are administered to adult patients with indolent B cell malignancies (NCI Clinical Trials PDQ, trial NCT00586391) in a 1:1 ratio at three dose levels. The aim of the trial is to evaluate the importance of the CD28 moiety on T cell persistence and function. Recent results from this and other trials indicate that CARs with only CD3-zeta do not have significant clinical activity or persistence *in vivo* beyond a few days26. Our proposed trial differs from this trial in that our anti-CD19-CAR will exclusively have both CD3-zeta and CD28. In addition, the use of anti-CD3/-CD28 beads to expand our CD19-CAR cells is not being performed at Baylor. A second trial at Baylor involves EBV-specific T cells are first pulsed with dendritic cells and peptide for CMV and adenovirus to generate tri-viral specific T cells, which are then transduced with anti-CD19-CAR (NCI Clinical Trials PDQ, trial \#BCM- H-23627). Tri-viral anti-CD19- CAR T cells are then administered to patients with pre-B ALL, CLL, or B-cell lymphomas undergoing allogeneic hematopoietic stem cell transplantation. Generation of cells using this method takes several months. Our proposed trial differs from this trial in that we will produce cells in a few days' time thereby limiting the possibility of enrolled patients dying before cells are prepared. Also, we will include patients who have previously received an allogeneic transplant but obtain cells for transduction directly from the patient rather than the original donor. Tolerized donor derived cells obtained directly from patients who have no GVH and are off immunosuppression have less chance of generating severe GVH upon reinfusion as compared to host-naïve T cells obtained directly from the donor. Finally, our trial will incorporate a lymphodepleting preparatory regimen whereas that of Bollard and coworkers does not. The third trial at Baylor treats children after allogeneic SCT who have relapsed with leukemia using anti-CD19 CAR that contains the CD3-zeta and CD28 signaling moieties. Patients on this trial may also get the anti-CTLA4 monoclonal antibody, ipilimumab, in an effort to improve CAR T cell persistence and activity after infusion. Our protocol is open to all children regardless of bone marrow transplant status and will not use ipilimumab since we have seen dramatic complete responses in many patients without additional T cell support. Gress and colleagues with ETIB, NCI are enrolling adults who have previously had an allogeneic stem cell transplant for a B-cell malignancy and are now recurrent or refractory (NCI Clinical Trials PDQ, trial \#10-C-0054). In this trial, T cells are obtained from the patient's donor, activated with anti-CD3 (OKT3) in the presence of IL-2, and then transduced with anti-CD19- CAR. No preparative regimen is used. As is often seen in patients who receive donor lymphocyte infusions (DLI), these cells may induce significant GVH. The key difference between this and our study is that for patients who have had an allogeneic transplant we will obtain tolerized T cells directly from those patients who have no active GVH off immunosuppression. This may result in less risk for severe GVH. Further, to improve the function of CAR T cells we will administer a preparative regimen. Carl June and coworkers at the University of Pennsylvania have opened a Phase I clinical trial of anti-CD19-CAR transduced T cells in adults with CD19+ B-cell malignancies (NCI Clinical Trials PDQ, trial \#UPCC 04409). They are evaluating two different anti-CD19-CARs delivered using a lentiviral vector: one with both CD3-zeta and the costimulatory molecule 4-1BB (CART19-zeta:41BB) and one with only CD3-zeta (CART-19-zeta). The first group of patients who received only CART-19-zeta:41BB have tolerated it well and some have had significant clinical responses38,39. The intended dose range was 1 x 109 to 1 x 1010 CD3+ T cells (not on a /kg basis),
but they have had significant difficulty in growing T cells ex vivo as most of their patients had prior treatment with fludarabine for CLL. However, one patient who received only 2 x 105 CART-19-zeta:41BB cells/kg had a complete response. This provides support for potential clinical activity at the dose levels we have proposed in the present study. The use of CD28 rather than 4-1BB as costimulation for T cells, the use of a non-ablative preparative regimen prior to anti-CD19-CAR T cell infusion, and including pediatric patients are key differences between June's study and ours. Interestingly, one patient experienced grade 3 tumor lysis syndrome with symptoms of chills, low grade fever and fatigue starting 14 days after the first cell infusion, progressing to rigors, diaphoresis, anorexia, nausea and diarrhea. Laboratory values were consistent with tumor lysis syndrome with hyperuricemia, elevated LDH and elevated creatinine levels. The patient also had transient grade 1 thrombocytopenia from day 19 through day 26 and grade 1 or 2 neutropenia from day 17 through day 33. Other signs and symptoms probably related to the study treatment included grade 1-2 elevations in alkaline phosphatase and transaminases, grade 2 hypogammaglobulinemia, and constitutional symptoms, including those noted above as well as myalgias and headache. June has recently opened a Phase II clinical trial of CART-19-zeta;41BB but this is restricted to adults and the preparative regimen used, if any, is unclear. His group has not published pediatric phase I results. In collaboration with Dr. June, Stephen Grupp and colleagues are enrolling children with ALL on a Phase I trial of CART-10-zeta;41BB. Two case reports demonstrated complete responses and significant cytokine storm40. No other results have been published but dosing is based on total T cells rather than CAR T cell dose and patients receive dealer's choice preparative regimen. Persis Amrolia and colleagues at the Great Ormond Street Hospital for Children in London have opened an anti-CD19-CAR trial for pediatric patients with pre-B ALL who are undergoing allogeneic stem cell transplantation from an EBV seropositive donor (NCI Clinical Trials PDQ,
trial \#UCL/09/0050). Enrolled patients will receive 2 x 108/m2 (approx. 1.6 x 106/kg) donor derived, EBV-specific T cells modified to express an anti-CD19-CAR that contains only CD3zeta after transplantation. Patients will then receive 3 doses of irradiated donor-derived EBVlymphoblastoid cells from the cell line used to generate the CTL as a vaccine. Our proposed protocol differs significantly from Amrolia and coworkers in that our anti-CD19-CAR contains both CD3-zeta and CD28, will not be used in conjunction with allogeneic stem cell transplantation, and will not be EBV-specific. The latter feature of our trial allows for relatively rapid production of the cells while not necessarily sacrificing long-term persistence of transduced T cells *in vivo*. Rosenberg and colleagues are conducting a clinical trial of autologous CD19-CAR T cells in the NIH Clinical Center (\#09-C-0082) that employs high dose and low dose IL-2 and is limited to adult patients with B-cell lymphoma and CLL41. While the CAR cells are identical to those used in this trial, our method for growing the CAR-transduced T cells (anti-CD3/CD28 beads), the absence of IL-2 therapy and the enrollment of children differs from this trial. Eight patients in the Rosenberg study have been treated, two of whom have been treated twice. Six of seven evaluable patients treated had responses of their advanced, progressive B-cell malignancies. Cells containing the anti-CD19 CAR gene were detected in all patients post adoptive therapy, while four of the eight had elevated serum inflammatory cytokine levels (IFN gamma and tumor necrosis factor alpha [TNFD]). Toxicities included grade 3 and 4 myelosuppression, elevated liver function tests, hypermagnesemia, hypocalcemia, hypophosphatemia, hypernatremia, hyperuricemia, hyperbilirubinemia, hypotension, fatigue, febrile neutropenia, sepsis, hypoxia, dyspnea, pleural effusion, acute renal failure, and capillary leak syndrome, Additional toxicities included: reversible grade 4 somnolence (2 patients) and grade 4 creatinine (1 patient), and reversible grade 2 left ventricular dysfunction and reversible grade 1 pulmonary hypotension (1 patient). Three patients on this study were intubated for 7, 9 and 10 days, respectively. One patient underwent dialysis. One patient died 18 days after CAR-transduced T cell infusion with culture-proven influenza A, nonbacterial thrombotic endocarditis, and cerebral infarction, which were deemed unrelated to the anti-CD19 CAR-transduced cells. Results41 demonstrated that:
x Elevated IFN-gamma and TNF-alpha levels in a subset of patients occurred during the first 10 days after cell infusion; x In 3 of the 4 patients with prominent elevations of serum inflammatory cytokines, the elevations occurred 4 or more days after the last dose of exogenous IL-2 administration, suggesting their toxicities were not attributable to the IL-2, but rather to serum inflammatory cytokine levels caused by the anti-CD19 CAR transduced cell infusions; and x The severity of the toxicity experienced by patients in this trial did not correlate with the number of IL-2 doses administered, although it may have contributed to the severity, as demonstrated in a mouse model42.  
Rosenberg, et al39 conclude that anti-CD19 CAR transduced T cells can eradicate CD19+ cells in humans and that the cytokine-associated toxicities that have occurred with these cells may not be related to exogenous IL-2 administration. Again, our study will differ significantly from the Rosenberg trial by not including aldesleukin in the regimen and should therefore result in fewer or less severe side effects. However, in light of these recent findings we will closely monitor patients and attempt to correlate toxicities observed with serum cytokine levels, although this will not be available in real time. An additional study has been initiated by Kochenderfer (\#10-C-0054) using allogeneic T cells genetically modified with the same anti-CD19 CAR retroviral vector in patients with CLL. The first patient experienced dramatic regression. This patient had relapsed after HLA-matched unrelated donor hematopoietic stem cell transplantation, followed by 4 donor lymphocyte infusions (DLIs) and a second stem cell transplant from the original donor, without a response.  
He received an infusion of 6.2x107 (1x106 cells/kg) allogeneic anti-CD19-CAR-transduced T
cells derived from his unrelated transplant donor five months after the second transplant. He experienced fevers, fatigue, mild hypoxemia and intermittent mild hypotension, with increase in serum magnesium, phosphorous and uric acid, consistent with tumor lysis syndrome. In addition, he experienced a decrease in cardiac left ventricular function, which subsequently improved. The patient's blood B cell count decreased from 286 cells/mcL before the CAR-transduced T cell infusion to 0 cells/mcL 26 days after the cell infusion. CT scans revealed a greater than 50% decrease in the size of multiple lymph nodes after the CAR-transduced T cell infusion, but residual adenopathy was present. Kochenderfer is also treating adults who have relapsed after allogeneic SCT with CAR T cells that are generated from the transplant donor rather then the patient. No preparative regimen is given before cell infusion. Although responses have been small, no reported GVH has occurred to date. To summarize our responses to date in the 20 patients enrolled, all 20 were infused with CAR T cells. Our feasibility rate was 89% as 2 patients did not meet the assigned dose level but were still infused with cells. One of these received 28,000 CAR-transduced T cells/kg (3% of prescribed dose) and had stable disease. Another received 480,000 CAR+ T cells/kg (16% of prescribed dose), and experienced an MRD-negative CR, then underwent HSCT and remains disease free 13·4 months after CD19-CAR therapy. In an intent-to-treat analysis of all enrolled patients, our therapy produced a 65% Complete Response (CR) rate. Eleven had MRD negative CRs and were therefore considered for allogeneic SCT, which is standard of care for such patients. All but 2 went on to SCT and remain leukemia-free (LFS 75% median f/u 14.5 months). The remaining 2 were determined by their principle oncologist to be medically ineligible for SCT and relapsed with CD19 negative leukemia. Therefore, we determined that our CAR T cell therapy serves as an effective bridge to transplant.  
We determined the MTD to be 1 x 106 CAR transduced T cells/kg. Three grade 4 CRS have occurred on our protocol during both the dose escalation and dose expansion phases. Specifics are detailed in Section **1.2.5.4**, but briefly all adverse events in these and the remaining patients were completely reversible. Four patients required use of the anti-IL6 receptor monoclonal antibody, tocilizumab, to blunt the effects of severe CRS. Our experience has led to publication of a consensus paper on the modified grading and treatment of CRS and is incorporated in this protocol to improve safety. In summary, T cells transduced with gamma-retroviruses encoding the sequence of this CAR can specifically recognize and kill CD19 expressing acute lymphoblastic leukemia cells in vitro and in vivo in xenograft models. These anti-CD19-CAR-transduced T cells also produce the cytokines interferon-J (INFJ), tumor necrosis factor D (TNFD) and IL-2 specifically in response to CD19. In this study, we propose to conduct a clinical trial in which gamma-retroviruses encoding the anti-CD19 CAR will be used to transduce T cells in the Department of Transfusion Medicine Cell Processing Facility from patients with advanced pediatric B cell malignancies. The protocol will enroll patients at the MTD to gain more experience with using this therapy and to decrease the incidence of grade 4 CRS through the use of early intervention using welldefined criteria and by decreasing overall tumor burden in patients with more extensive disease involvement by intensifying the preparative regimen used prior to cell infusion. Subjects will receive a non-ablative, lymphocyte depleting preparatory regimen followed by infusion of antiCD19-CAR transduced T cells.
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1.2.5 safety considerations

Several safety concerns regarding the infusion of large numbers of retrovirally modified tumor reactive T-cells should be addressed.
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1.2.5.1 risk of chemotherapy

Toxicities resulting from fludarabine and cyclophosphamide in the doses proposed in the current study are well known. Such a preparative regimen is designed to decrease the number of endogenous T cells, including T regulatory cells, which may otherwise suppress anti-CD19-CAR T cell cytotoxicity, and to induce the production of homeostatic cytokines thereby allowing for better engraftment of the transferred anti-CD19-CAR T cells. The dose limiting toxicity for both fludarabine and cyclophosphamide is myelosuppression, however myelosuppressive effects are expected to be transient using the doses proposed. Other toxicities including fever, nausea, vomiting, stomatitis, diarrhea, anorexia, edema, skin rashes, myalgias, headache, agitation, and fatigue should be easily managed with appropriate supportive care. Sterile hemorrhagic cystitis occurs in about 20% of patients who receive cyclophosphamide. Severity can range from microscopic hematuria to extensive cystitis with bladder fibrosis. Mesna will be used prophylactically as a uroprotective agent, but is not effective in preventing hemorrhagic cystitis in all patients. Furthermore, hemorrhagic cystitis is unlikely given the relatively low dose of cyclophosphamide administered in this trial. Tumor lysis syndrome (TLS) following fludarabine and cyclophosphamide administration has been observed, especially in patients with advanced bulky disease. Before beginning chemotherapy, patients will be prescribed allopurinol and appropriately hydrated with close monitoring for the development of TLS. Finally, opportunistic infections (protozoan, viral, fungal, and bacterial) have been observed post-fludarabine and cyclophosphamide, especially in heavily pre-treated individuals. Patients will receive appropriate antimicrobial prophylaxis (eg, Bactrim for PCP prophylaxis) during treatment. Patients with higher disease burdens (Arm 2) will receive a standard salvage regimen in lieu of the fludarabine/cyclophosphamide lymphodepleting regimen. The regimen will consist of fludarabine, cytarabine, and filgrastim (FLAG) and has been used routinely in the POB. Risks of fludarabine are described above.
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1.2.5.2 risk of autoimmunity

As discussed above, expansion of tumor reactive cells is a desirable outcome following the infusion of antigen reactive T-cells. Some patients receiving gp100 or MART-1 reactive cells have developed vitiligo, uveitis, hearing loss and rash probably due to destruction of normal melanocytes, though these toxicities have been manageable. In addition, two patients experienced vestibular dysfunction possibly due to the transduced cells. Some of these toxicities can be explained as on-target, off-tissue effects. In a similar fashion, it is likely that anti-CD19- CAR T cells will eliminate most circulating normal B cells since they also express CD19. It is important to note that pluripotent stem cells do not express CD19 and should not be affected by this therapy and that patients who receive the B cell depleting anti-CD20 antibody, rituximab, in conjunction with chemotherapy do not experience additional toxicities over chemotherapy alone. It is conceivable, but not likely, that as leukemic blasts are lysed host antigen presenting cells may present peptides to T cells specific for ALL or that cross-react with other normal host tissues. Termed epitope spread, this phenomenon is actually desired for better immunologic control of disease but may lead to autoimmunity. No confirmed cases of autoimmunity have been encountered on the study to date.
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1.2.5.3 risk of gene therapy

Risks of gene therapy include insertional mutagenesis or emergence of replication-competent retrovirus (RCR). While insertional mutagenesis is theoretically possible using retroviral vectors, this has only been observed in the setting of infants treated for X-SCID using retroviral vectormediated gene transfer into CD34+ bone marrow stem cells. In the case of retroviral vectormediated gene transfer into mature T-cells, there has been no evidence of long-term toxicities associated with these procedures since the first NCI sponsored gene transfer study in 1989. Despite the fact that clinical data currently available suggest that the introduction of retroviral vectors transduced into mature T-cells is a safe procedure, continued follow-up of all gene therapy patients will be performed as required. The proposed protocol follows all current FDA guidelines regarding testing and follow up of patients receiving gene transduced cells. Similarly, the viral vectors used have been engineered to minimize the risk of emergence of replication competent retrovirus, but patients will be monitored according to Recombinant DNA Advisory Committee guidelines for several years following receipt of this therapy and in the case of the development of second malignancy, all efforts will be made to determine whether replication competent retrovirus has emerged.
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1.2.5.4 risk of cytokine storm

Cytokine storm is a safety concern with the use of CAR transduced adoptive cell therapy. The cytokine release syndrome (CRS) is defined clinically by nausea, headache, tachycardia, hypotension, rash and shortness of breath (CTCAE 4.0 definition) but may also result in other organ and neurologic dysfunction. Believed to be caused by the release of cytokines from cells following intense immune activation, IL-6 appears to play a major role. A rapid rise in serum cytokine levels (cytokine storm) has been associated with Systemic Inflammatory Response Syndrome (SIRS) potentially leading to multi-organ dysfunction (MOD)43. This syndrome has been described following various forms of immunotherapy, including antibody infusions (e.g.  
OKT344, Rituximab45, Campath46, anti-CD2847) but also following cellular immunotherapy.  
Fatal cytokine storm occurred in one patient in the Surgery Branch, NCI with metastatic adenocarcinoma of the sigmoid colon, receiving anti-Her2-CAR gene engineered lymphocytes and IL-2 after non-myeloablative chemotherapy24. This patient received 1010 CAR transduced cells, and shortly after infusion experienced shortness of breath, hypotension and required admission to the ICU, vasopressor support and intubation for respiratory failure. She deteriorated despite supportive care and developed liver and kidney failure, expiring within 5 days of receiving the cells. The specific cause of death was determined to be gastrointestinal hemorrhage in the setting of systemic microangiopathic injury, but this was felt to be secondary to the cytokine release syndrome. Current concepts hold that this toxicity was likely primarily related to the dose of cells administered and IL-2 administration after infusion. A second reported death occurred at Memorial Sloan Kettering Cancer Center in a phase I study in which anti-CD19 CAR T lymphocytes (autologous 19-28z+ T cells) were administered48.  
Three patients with B cell lymphomas received a single dose of 3 X 107/kg modified T cells without significant toxicity, with 2 of 3 patients experiencing transient reduction in peripheral lymphadenopathy. The fourth patient with chronic lymphocytic leukemia received cyclophosphamide (1500 mg/m2) prior to cell infusion, and six hours later experienced fever, hypotension, dyspnea, and renal failure. Thirty hours later, the patient required intubation and expired soon after when withdrawal of support was requested. Immunohistochemistry (IHC) studies revealed significant numbers of 19-28z+ T cells in the bone marrow, spleen, liver and lymph nodes. Serum cytokine analyses in patient 4 were elevated compared to the first 3 patients enrolled. Intense analysis determined the outcome was in large part mediated by cyclophosphamide induced 'cytokine storm' in a setting of preexisting subclinical infection, leading to a sepsis-like scenario and which likely enhanced the modified T cell in vivo activation. The investigators feel that these findings underscore the potency of adoptively transferred genetically modified T cells. Two additional deaths in adults were reported at Memorial Sloan Kettering Cancer Center in April 2014. Specifics have not yet been published in the medical literature but initial indications are that one had intractable seizures and the other developed cardiotoxicity both at least possibly related to CAR T cells. We have not seen any seizure activity in any of our patients, though this will be a risk especially in patients with CNS leukemia. We will therefore prophylactically start CNS leukemia patients on an anti-epileptic medication. One patient on our trial had cardiac arrest driven by cytokine storm and was successfully resuscitated. This patient was hypotensive but stable on multiple vasopressors for several days before acutely decompensating. Immediate changes to this protocol were implemented at that time aimed at better monitoring and intervening earlier in severe CRS. The current version incorporates a new CRS grading system that tolerates low-dose vasopressor support but calls for anti-cytokine therapy(ies) such as tocilizumab or corticosteroids for patients requiring more aggressive intervention or grade 3 organ dysfunction. We have found that all patients who had grade 3 or 4 CRS have had complete responses and that all of these (except 1 who had grade 3 CRS driven by dysphasia and not hypotension) had high leukemia burdens. Therefore, some degree of CRS may be tolerated in the interest of achieving a CR.  
Based on these reports, serum cytokine levels, including IFN-Ȗ,/-2, IL-6, GM-CSF and TNFD,
will be assayed serially (0, 1, 12, 24, 48, 72, 96, 120 hours, then daily until day 14 and on day 28 and in the case of symptoms consistent with cytokine storm, levels will be monitored) following infusion of anti-CD19-CAR T cells. The results of the assays will not be available in real time but if toxicity is observed following infusion, review of the cytokine levels will be undertaken to assess risk to subsequent patients. Cytokine levels will also be evaluated retrospectively for correlation with anti-tumor effects. In addition, subjects will be monitored closely after infusion of anti-CD19 CAR T cells, and if discharged prior to day 14, be required to return to clinic daily for evaluation. After day 14, subjects will return to clinic at least twice a week for up to 28 days for evaluation. Investigators will maintain a low threshold for re-admission to the hospital and cytokine storm will be assessed and managed according to **Appendix F**. In relation to the cytokine storm, recent reports from previous studies using anti-CD 19 CAR T cells have documented transient neurologic symptoms including cognitive confusion, visual hallucinations, and lack of responsiveness to commands. Moreover, prior research indicates that cytokine inflammation is associated with poorer executive functioning and memory in people with cancer49,50. For these reasons, this study will evaluate the cognitive status of patients prior to receiving their CD19 infusion and again 21 to 28 days after the infusion to assess possible changes related to this treatment. We also will evaluate cognitive functioning again for the participants who return to the NIH at 3 months post-infusion to assess later cognitive effects.
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1.2.5.5 risk of cardiac dysfunction

Cardiovascular dysfunction is a significant and yet incompletely defined aspect of CRS. The hemodynamic abnormalities associated with CRS can range from tachycardia to life-threatening hypotension. A recent trial of anti-CD19 CAR T cells documented shock requiring vasoactive infusion in 13.6% of participants51. Since the current treatment of CRS involves tocilizumab and corticosteroids, which can be cytotoxic to the infused therapeutic T cells, it is important to define the cardiovascular dysfunction associated with CRS in an effort guide therapy and ultimately define patients at high risk for cardiac sequela. Thus, the one of the secondary objectives is to define anti-CD19 CAR T cell immunotherapy related cardiac dysfunction. We will evaluate traditional cardiac biomarkers pre- and post-infusion of anti-CD19 CAR T cells in the study population. In addition, we will evaluate echocardiograms performed pre- and post-infusion to directly assess both systolic and diastolic function using 2-D and strain imaging. Strain echocardiography is a sensitive technique for myocardial function assessment and is well validated in detecting subclinical chemotherapy mediated cardiac toxicity52. Strain echocardiography is utilized to monitor patients undergoing chemotherapy since changes in myocardial strain during therapy predict progression to overt heart failure52. We will retrospectively combine cardiac biomarkers, 2-D and strain echocardiography with clinical parameters in an effort to define anti-CD19 CAR T cell mediated CRS cardiac dysfunction and evaluate if certain variables can be utilized to predict the severity of the syndrome in affected patients.
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1.3 exploratory analysis

The persistence of minimal residual disease is a poor prognostic factor in pediatric patients with acute lymphoblastic leukemia. Recent use of high-throughput sequencing (HTS) has shown that MRD testing using this technique is more sensitive at detecting low levels of leukemia in patient samples, as compared to the gold standards of multi-channel flow cytometry (MFC) or allelespecific oligonucleotide (ASO) PCR amplification53. Flow cytometry typically permits detection of recurrent or persistent disease with sensitivity on the order of 1 cell in 104, while PCR can detect disease on the order of 1 cell in 105. Various groups have tested new methods of MRD monitoring using next generation sequencing (NGS) to determine if this provides a more precise way of detecting low levels of leukemia (on the order of 1 cell in 106) that could be used predictively for relapse54-56. Adding this testing to our current CD2219 protocol can yield information on very low levels of MRD in our patients, and help us retrospectively analyze data acquired for these patients after CAR therapy. As many of the patients treated on this study proceeded to transplant, it would be useful to know what depth of remission could be attained with use of CAR therapy as a bridge to transplant. This information is extremely important to the field of CAR therapy and transplantation. These studies will be performed using samples already collected.
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1.4 protocol summary and rationale

The primary goal of this trial was to evaluate the safety and feasibility of administering antiCD19 CAR T cells after a nonmyeloablative conditioning regimen in children and young adults who have or have not undergone prior allogeneic SCT. During the dose escalation phase, no DLTs were encountered at dose level 1 in either the post-HSCT or no-HSCT cohorts. Two DLTs were encountered during the dose escalation phase at dose level 2. Subject \#7 had Grade 4 CRS and Subject \#10 had Grade 3 neurologic toxicity manifested as dyphasia in the setting of high fever and chills but remained hemodynamically stable. The symptoms resolved within 12 hours with only supportive care.  
The MTD was therefore defined as 1 x 106 CAR+ T cells/kg (dose level 1) and that dose was expanded. Two additional grade 3 and two additional grade 4 CRS were encountered in the subsequent 10 patients enrolled in the dose expansion phase. The feasibility was established in this patient population. All patients underwent apheresis and T-cell collection without difficulty. Cell therapy products met the criteria for release prior to administration in 18 of the 20 patients. Two subjects had low cell counts, which did not meet release criteria. Secondary aims include determining the persistence of infused cells and evaluating in a preliminary fashion, the clinical activity of this regimen.
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1.5 protocol status summary and

In total 53 subjects were enrolled (51 with ALL, 2 with DLBCL) on 12-C-0112. This study has since closed to enrollment but remains open for data analysis. The overall response rate for those with ALL was 62% (n=31). One of the two patients with DLBCL had an FDG-PET response but it did not meet criteria for PR due to insufficient shrinkage of tumor size. Thirty-one patients (62%) have had complete responses (CR) and 28 of these were minimal residual disease (MRD) negative CRs. Twenty-two of these patients have since gone on to receive a potentially curative hematopoietic stem cell transplantation. As previously noted cytokine release syndrome (CRS) was the dose limiting toxicity (DLT) establishing MTD.
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1.5.2 rationale for amendment f

Based upon the severity of CRS observed in patient \#14, additional monitoring and early CRS intervention was discussed with the FDA. The early treatment for CRS will include specific clinical signs and symptoms that will trigger administration of anti-IL6R moAb therapy (tocilizumab) and/or other pharmacologic therapies including etanercept (Enbrel) or IV steroids.
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1.5.3 rationale for amendment g

Based on the relationship observed between the incidence of Grade 3-4 CRS and patients with high-disease burden, additional investigation is warranted to determine if reduction of disease burden prior to administration of the anti-CD19 CAR will decrease the incidence of severe CRS,
while using an updated assessment/treatment algorithm for CRS. During the phase 1 dose HVFDODWLRQDQGH[SDQVLRQRIWKHILUVWSDWLHQWVHQUROOHGZLWK$//HLJKWSDWLHQWVKDG
bone marrow blasts. Excluding one patient who received less cells than dose level 1 (< 1 x 106 transduced T cells/kg), 4 out of the 7 patients treated experienced a clinical response AND had grade 3 or 4 CRS. The remaining 3 patients did not have severe CRS and did not have a clinical response. Of the three patients who experienced a grade 4 CRS seen to date, all occurred in SDWLHQWVZLWKERQHPDUURZEODVWV It is also important to note that when the toxicity and response was correlated with the patients who had peripheral blood blasts during eligibility screening, 15/18 patients with ALL had blasts in peripheral blood detected by flow cytometry. This measure of peripheral blood blasts was therefore not a good indicator of who would experience severe CRS. Rather, four (4) patients had blasts in peripheral blood detected on routine CBC. Three of these did not have severe CRS or respond. The one that did respond (\#14) had cardiac arrest. Further, of the 5 non-responders who were infused with adequate cell dose, 3 had blasts on CBC at eligibility screening. Therefore, for purposes of this study, high-burden disease (Arm 2) will be defined as M3 bone marrow blasts and/or presence of peripheral blood blasts on routine CBC.
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1.5.4 rationale for amendment h

No significant liver toxicities related to CAR T cells have been seen to date. Currently, eligibility requirements dictate that liveUWUDQVDPLQDVHV$/7$67EH;8/16HYHUDO patients have leukemia involvement of their liver resulting in elevated transaminases. In cases where transaminitis is attributed to disease involvement, patients with Grade 3 or less ALT, AST should NOT be excluded from enrolling. As an added safety measure, we have now required WKDW$/7DQG$67EH;8/1SULRUWRLQIXVLQJ&$57FHOOV Three patients have received second infusions of CAR T cells after having an MRD+ response to the first infusion or relapsed after attaining an MRD negative remission, and none of these have responded to the second infusion and none had toxicity. All 3 patients were treated with the lymphodepleting regimen (fludarabine/cyclophosphamide) prior to their second cell infusion. In some, anti-CD19 CAR T cell immune responses have developed which may limit the efficacy of a second dose. This amendment allows for more flexibility in choosing the chemotherapy regimen prior to second cell infusions to include FLAG and etoposide/ifosfamide regimens already included in the protocol (Arm 2). These are standard of care regimens for ALL that does not increase the risk of the CAR T cells. In fact, we found that disease burden directly correlates with severity of CRS. Since these patients seeking second treatment will have responded to the first infusion, and therefore have lower disease burdens, their risk of severe CRS should be lower as well.
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1.5.5 rationale for amendment p

Cardiovascular dysfunction associated with CAR infusion and CRS has not been well studied. Cardiology studies including echocardiograms, and laboratory investigations will be retrospectively analyzed in an effort to define anti-CD19 CAR T cell mediated CRS cardiac dysfunction and evaluate if certain variables can be utilized to predict the severity of the syndrome in affected patients. Additionally, MRD assessments using sequencing was shown to be advantageous in the pre-transplant setting56 thus we will incorporate this as an exploratory objective to retrospectively compare this method of detection as compared to gold standard flow cytometry in patients who achieved MRD negativity by flow cytometry.
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2 eligibility assessment and enrollment

Under no circumstances are subjects who enroll once in this study permitted to be reenrolled for a second time (e.g., into a later cohort). Study subjects who have relapsed or treatment refractory ALL or NHL may enroll as defined by the following inclusion and exclusion criteria.
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2.1.1 inclusion criteria

2.1.1.1 Patient must have a CD19-expressing B cell ALL or lymphoma and must have relapsed or refractory disease after at least one standard chemotherapy and one salvage regimen. In view of the PI and the primary oncologist, there must be no available alternative curative therapies and subjects must be either ineligible for allogeneic stem cell transplant (SCT), have refused SCT, or have disease activity that prohibits SCT at this time.  
2.1.1.2 CD19 expression must be detected on greater than 15% of the malignant cells by immunohistochemistry or greater than 30% by flow cytometry in a CLIA approved test in the Laboratory of Pathology, CCR, NCI, NIH or from the referring institution or reference laboratory. The choice of whether to use flow cytometry or immunohistochemistry will be determined by what is the most easily available tissue sample in each patient. In general immunohistochemistry will be used for lymph node biopsies, flow cytometry will be used for peripheral blood and bone marrow samples.  
2.1.1.3 Patients must have measurable or evaluable disease at the time of enrollment, which may include any evidence of disease including minimal residual disease detected by flow cytometry, cytogenetics, or polymerase chain reaction (PCR) analysis.
2.1.1.4 Greater than or equal to 1 year of age (and at least 15 kg) and less than or equal to 30 years of age.  
2.1.1.5 Adequate absolute CD3 count estimated to be required to obtain target cell dose based on discussion with DTM apheresis and Cell Processing Section, DTM.  
2.1.1.6 Subjects with the following CNS status are eligible only in the absence of neurologic symptoms suggestive of CNS leukemia, such as cranial nerve palsy x CNS 1, defined as absence of blasts in cerebral spinal fluid (CSF) on cytospin preparation, regardless of the number of WBCs;  
x CNS 2, defined as presence of < 5/uL WBCs in CSF and cytospin positive for blasts, or >
5/uL WBCs but negative by Steinherz/Bleyer algorithm x CNS3 with marrow disease who has failed salvage systemic and intensive IT
chemotherapy (and therefore not eligible for radiation)
x Patients with isolated CNS relapse will be eligible if they have previously been treated with cranial radiation (at least 1800 cGy)
2.1.1.7 Ability to give informed consent. For subjects <18 years old their legal guardian must give informed consent. Pediatric subjects will be included in age appropriate discussion DQGYHUEDODVVHQWZLOOEHREWDLQHGIRUWKRVH\HDUVRIDJHZKHQDSSURSULDWH
2.1.1.8 Clinical performance status: Patients > \HDUVRIDJH.DUQRIVN\3DWLHQWV
\HDUVRIDJH/DQVN\VFDOHVHH**Appendix A** for conversion). Subjects who are unable to walk because of paralysis, but who are upright in a wheelchair will be considered ambulatory for the purpose of calculating the performance score.  
2.1.1.9 Patients of child-bearing or child-fathering potential must be willing to practice birth control from the time of enrollment on this study and for four months after receiving the preparative regimen.  
2.1.1.10 Females of child-bearing potential must have a negative pregnancy test because of the potentially dangerous effects on the fetus.  
2.1.1.11 &DUGLDFIXQFWLRQ/HIWYHQWULFXODUHMHFWLRQIUDFWLRQE\08*$RUFDUGLDF05,RU
IUDFWLRQDOVKRUWHQLQJE\(&+2RUOHIWYHQWULFXODUHMHFWLRQIUDFWLRQE\ ECHO.  
2.1.1.12 Patients with history of allogeneic stem cell transplantation are eligible if at least 100 days post-transplant, if there is no evidence of active GVHD (See **Appendix C**) and no longer taking immunosuppressive agents for at least 30 days prior to enrollment.  
2.1.2 Exclusion Criteria
Subjects meeting any of the following criteria are not eligible for participation in the study. 2.1.2.1 Recurrent or refractory ALL limited to isolated testicular disease.  
2.1.2.2 Hepatic function: Inadequate liver function defined as total bilirubin > 2x upper limit of normal (ULN) (except in the case of subjects with documented Gilbert's disease > 3x ULN) or transaminase (ALT and AST) > 20x ULN based on age- and laboratory specific normal ranges;
2.1.2.3 Renal function: Greater than age-adjusted normal serum creatinine (see Table below) and a creatinine clearance < 60 mL/min/1.73 m2.  
| Age     | Maximum Serum      |
|---------|--------------------|
| (Years) | Creatinine (mg/dL) |  
| "     | 0.8   |
|-----|-------|
| DJH"     | 1.0   |
| >10 | 1.2   |  
2.1.2.4 Hematologic function x $EVROXWHQHXWURSKLOFRXQW$1&ȝ/RUSODWHOHWFRXQWȝ/LIWKHVH
cytopenias are not judged by the investigator to be due to underlying disease (i.e. potentially reversible with anti-neoplastic therapy);
x $VXEMHFWZLOOQRWEHH[FOXGHGEHFDXVHRISDQF\WRSHQLD*UDGHLILWLVGXHWRGLVHDVH
based on the results of bone marrow studies.  
2.1.2.5 +\SHUOHXNRF\WRVLVEODVWVȝ/RUUDSLGO\SURJUHVVLYHGLVHDVHWKDWLQWKH
estimation of the investigator and sponsor would compromise ability to complete study therapy; 2.1.2.6 Pregnant or breast-feeding females; 2.1.2.7 Recent prior therapy:
x 6\VWHPLFFKHPRWKHUDS\ZHHNVZHHNVIRUQLWURVRXUHDVRUUDGLDWLRQWKHUDS\
weeks prior to apheresis; Exceptions:
a. There is no time restriction in regard to prior intrathecal chemotherapy provided there is complete recovery from any acute toxic effects of such; b. Subjects receiving hydroxyurea may be enrolled provided there has been no increase in dose for at least 2 weeks prior to starting apheresis (see Section 4.1);
c. Patients who relapse while receiving standard ALL maintenance chemotherapy will not be required to have a waiting period before entry onto this study provided they meet all other eligibility criteria; d. Subjects receiving steroid therapy at physiologic replacement doses only are allowed provided there has been no increase in dose for at least 2 weeks prior to starting apheresis; e. For radiation therapy: Radiation therapy must have been completed at least 3 weeks prior to enrollment, with the exception that there is no time restriction if the volume of bone marrow treated is less than 10% and also the subject has measurable/evaluable disease outside the radiation port.  
2.1.2.8 Other anti-neoplastic investigational agents currently or within 30 days prior to apheresis
(i.e. start of protocol therapy);
2.1.2.9 Subjects must have recovered from the acute side effects of their prior therapy, such that eligibility criteria are met. Cytopenias deemed to be disease-related and not therapyrelated are exempt from this exclusion.  
2.1.2.10 HIV/HBV/HCV Infection:
a. Seropositive for HIV antibody. (Patients with HIV are at increased risk of lethal infections when treated with marrow-suppressive therapy. Appropriate studies will be undertaken in patients receiving combination antiretroviral therapy in the future should study results indicate effectiveness.)
b. Seropositive for hepatitis C or positive for Hepatitis B surface antigen (HbsAG).  
2.1.2.11 Monoclonal antibody therapy administered within 30 days of the agent prior to apheresis; 2.1.2.12 Uncontrolled, symptomatic, intercurrent illness including but not limited to infection, congestive heart failure, unstable angina pectoris, cardiac arrhythmia, psychiatric illness, or social situations that would limit compliance with study requirements or in the opinion of the PI would pose an unacceptable risk to the subject; 2.1.2.13 Second malignancy other than in situ carcinoma of the cervix, unless the tumor was treated with curative intent at least two years previously and subject is in remission; 2.1.2.14 History of severe, immediate hypersensitivity reaction attributed to compounds of similar chemical or biologic composition to any agents used in study or in the manufacturing of the cells (i.e. gentamicin).  
2.1.3 Recruitment Strategies The following recruitment strategies will be employed to elicit potential candidates for this trial:
1. Listed on clinical trials.gov; 2. Listed in PDQ; 3. In addition, patients from POB clinic who are eligible for participation will be offered participation in this study Prior to distribution of any recruitment materials, such materials will be submitted to the NCI IRB for review.
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2.2 screening evaluation

Within 2 weeks (except as specified otherwise) prior to enrollment: 2.2.1 Clinical evaluation 2.2.1.1 Complete physical examination, including vital signs, noting in detail the exact size and location of any lesions that exist.  
2.2.1.2 Height, weight 2.2.1.3 Performance status determination 2.2.2 Laboratory studies 2.2.2.1 Evaluation for HIV seropositivity to consist of ELISA and, if positive, confirmation by Western blot within 4 weeks. The investigator, in the event of a positive finding, will make appropriate counseling available.  
2.2.2.2 Evaluation for Hepatitis B surface antigen (HbsAG) and anti-HCV antibodies, within 4 weeks.
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version date 01/12/2018

2.2.2.3 Creatinine Clearance: A measured 24 hour urine creatinine clearance test may be performed if the serum creatinine is elevated, and the measured value will be recorded in the CRF and may be used to qualify the subject for study participation.
2.2.2.4 ȕ-HCG pregnancy test on all women of child-bearing potential (within one week) 2.2.2.5 Peripheral blood CD3 count. 2.2.2.6 PT/PTT 2.2.2.7 General Tests: The following will be obtained during the screening process:  
2.2.2.8 Chem 20: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate),
Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid; CRP
2.2.2.9 CBC with differential and platelet count 2.2.2.10 Urinalysis and culture, if indicated 2.2.2.11 CD19 staining of malignant cells by immunohistochemistry or flow cytometry (testing is permitted to be conducted at any time since most recent disease presentation).  
2.2.2.12 Additional Tests: The PI may order additional tests in some subjects if needed to fully assess clinical status and obtain baseline results. Examples of such tests include: fibrinogen, haptoglobin, cholesterol, triglyceride, immunoglobulin levels, and viral serology or PCR for cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), and Epstein-Barr virus (EBV).  
2.2.3 Disease Evaluation (method determined by patients' type and location of disease): 2.2.3.1 Imaging Studies such as Chest CT and appropriate imaging methods of any sites relevant to the subject's disease (e.g., PET/CT).
2.2.3.2 Bone marrow aspirate, including flow cytometry, and biopsy 2.2.3.3 Spinal fluid for cell count and cytospin 2.2.4 Other Tests 2.2.4.1 ECG 2.2.4.2 Echocardiogram, Cardiac MRI,or MUGA
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2.3 registration procedures

Screening: Authorized staff will register patients signing the screening consent for analysis of CD19 expression with the Central Registration Office (CRO) (301-402-1732) by completing the screening portion of the eligibility checklist. Treatment Enrollment Procedures: Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) within 24 hours of signing consent. A registration Eligibility Checklist from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov. After confirmation of eligibility at Central Registration Office, CRO staff will call Department of Transfusion Medicine (DTM) Cell Processing Service (CPS) to advise them of the acceptance of the patient on the protocol prior to the release of any investigational agents. Verification of Registration will be forwarded electronically via e-mail to the research team. A recorder is available during non-working hours.
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2.4 treatment assignment procedures

| Number   | Name     | Description                                                                                                                                                                                                                                   |
|----------|----------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1        | Cohort 1 | Patients without high-burden disease or patients for  whom chemotherapy toxicity is a concern will receive  standard preparative regimen.                                                                                                     |
| 2        | Cohort 2 | Patients with high-burden disease who receive standard  chemotherapy to reduce burden, (defined as patients  with ALL who have M3 bone marrow blasts and/or  presence of peripheral blood blasts on routine CBC, or  patients with lymphoma). |  
| Number   | Name   | Description                                                                                                                                                                         |
|----------|--------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1        | Arm 1  | Lymphodepleting regimen of Fludarabine and  Cyclophosphamide.                                                                                                                       |
| 2        | Arm 2  | Intensive standard of care chemotherapy, in lieu of the  lymphodepleting chemotherapy regimen, to decrease  tumor burden in preparation for the administration of  the CAR T cells. |  
2.4.3 Arm Assignment Patients in Cohort 1 will be directly assigned to Arm 1. Patients in Cohort 2 will be directly assigned to Arm 2.
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3 study implementation 3.1 study

This is a phase I safety/feasibility study in pediatric and young adult patients with CD19expressing B cell ALL or lymphoma who have relapsed or refractory disease after at least one standard chemotherapy and one salvage regimen. All patients enrolled on this trial have been deemed incurable by standard therapies. Autologous PBMC will be obtained by leukapheresis and transduced with anti-CD19-CAR retroviral vector.  
Patients will be enrolled into one of two arms: Arm 1: Patients without high-burden of disease or patients requiring the cyclophosphamide/fludarabine preparative regimen for safety concerns; Arm 2: Patients with high-burden of disease (defined as patients with ALL who have M3 bone marrow blasts and/or presence of peripheral blood blasts on routine CBC, or patients with lymphoma).  
All eligible patients will undergo apheresis and anti-CD19 CAR T cells will be manufactured from fresh or frozen PBMCs. PBMCs previously collected by DTM and cryopreserved may be used so long as the subject has not had a hematopoietic stem cell transplant between the time of PBMC collection and enrollment on this protocol. PBMCs collected by an extramural institution may not be used. Patients in Arm 1 will receive the lymphodepleting chemotherapy regimen outlined in Sections 3.4 and **3.9.1**. Patients in Arm 2 will be treated with intensive standard of care chemotherapy, in lieu of the lymphodepleting chemotherapy regimen, to decrease tumor burden in preparation for the administration of the CAR T cells. Choice of chemotherapy will be individualized since patients will have previously received a variety of treatment regimens and may have received different lifetime anthracycline doses or may have experienced significant toxicities with certain agents, i.e. asparaginase. Only standard of care chemotherapy agents will be used. Preferred regimens are given in Section 3.9 but patients may be treated with another standard regimen not listed if these are determined to pose an unacceptable medical risk to the patient. No investigational therapy will be administered for disease control. At least 2 days must pass between the last dose of chemotherapy (excluding IT chemotherapy) and administration of the anti-CD19 CAR T cells. CAR T cells will be cryopreserved on day 7 of culture unless the target dose has not been reached or to accommodate scheduling purposes. In this case, the PI after discussion with CPS will determine if the culture is likely to expand further given more culture time. If not, cells will be cryopreserved despite the dose level not being met and will be administered to the patient on schedule. If cells are deemed likely to expand further, the culture time may be extended up to 14 days total. All patients will receive anti-CD19 CAR T cells no sooner than 10 days after culture initiation (at least 2 days after that last dose of chemotherapy). Patients will be evaluated after treatment for toxicity, antitumor effects and for persistence and functionality of transduced T cells. Toxicity assessment of neurologic symptoms will include cognitive confusion, visual hallucinations, and lack of responsiveness to commands. In addition, this study will evaluate the cognitive status of patients prior to receiving their CD19 infusion and again after the infusion to assess possible changes related to this treatment.
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3.2 apheresis for cell acquisition

Patients will undergo a 2-5 blood volume apheresis, as estimated by recipient weight and target cell harvest dose in the Department of Transfusion Medicine (DTM). A target cell number extrapolated based upon the expected expansion and planned cell dose of transduced T cells/kg will be collected. Citrate anticoagulant will be used in subjects greater than 18 kg. For subjects less than 18 kg, low-dose heparin may be added. Prophylactic intravenous CaCl2 and MgSO4 infusions may be administered by the DTM physician per standard operating procedure. Bilateral peripheral venous access will be used whenever possible. Alternatively, a temporary central venous catheter may be placed for collection and supportive care should significant toxicity occur. The CVL will be inserted by Critical Care or Interventional Radiology staff with the assistance of anesthesiology as indicated. For patients weighing between 15 and 25 kg, red blood cell priming may be used to prevent dilutional anemia and increase the collection efficiency. The decision will be made on a case-by-case basis after discussion between the primary investigator, pediatric medical care, and DTM teams, and the patient's parent.
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3.3 cell processing

Fresh or cryopreserved PBMCs will be prepared on approximately Day -11. CD3+ cells will be enriched using anti-CD3/anti-CD28 beads and cultured in the presence of IL-2 in order to stimulate T-cell growth. After two days of stimulation T cells will be transduced by exposure to supernatant containing the anti-CD19 CAR retroviral vector in bags coated with RetroNectin®
(Takara Biomedicals, Japan) to facilitate the interaction between the viral particles and the activated cells. Expansion of these transduced cells will be supported with IL-2. CD19-CAR transduction efficiency will be determined by FACS analysis for the CAR using the anti-idiotype monoclonal antibody. Successful CAR gene transfer for each transduced PBL population will be defined as >15% CAR positive cells. In some patients, the peripheral blood might be contaminated with large numbers of CD19expressing malignant cells. However, the anti-CD3/anti-CD28 magnetic beads will provide a selection step for the non-leukemic T cell population. Further, ALL blasts are not likely to survive ex vivo under these culture conditions and culture duration and will not be expanded by the anti-CD3/CD28 stimulation. This was evident in the results of the validation run using cells from a patient with CLL. In previous testing performed for this study, the CD19 expressing malignant cell results was 0.  
| Test                                                                                                                                                                       | Method                       | Criteria                          |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|-----------------------------------|
| Cell viability1                                                                                                                                                            | Trypan blue exclusion        | •60%                                   |
| Cell number1                                                                                                                                                               | Cell counter                 | within 20% of planned  dose level |
| % CAR+ cells2                                                                                                                                                              | Anti-idiotype flow cytometry | -                                   |
| % CD19 positive cells1                                                                                                                                                     | Flow cytometry               | <5%                               |
| Endotoxin1                                                                                                                                                                 | Gel Clot                     | "(8P/                                   |
| Mycoplasma2                                                                                                                                                                | Mycoplasma test              | Negative                          |
| RCR2                                                                                                                                                                       | RCR-PCR                      | Negative                          |
| RCR5                                                                                                                                                                       | S+L- Assay                   | Negative                          |
| Sterility testing4                                                                                                                                                         | gram stain, culture          | Negative                          |
| 1 Performed on sample from final product immediately prior to infusion if given fresh or immediately prior to cryopreservation, results available at the time of infusion. |                              |                                   |  
The release criteria will be based upon analyses for each dose of anti-CD19 CAR retroviraltransduced autologous PBL and will include:
2Performed on a sample collected between Day –4 and Day –1. Results are available at time of infusion. 4Gram stain is performed on final product prior to infusion and is available at the time of infusion, cultures will be sent from Day
–2 product at the time of infusion if given fresh or at time of cryopreservation, and results will be in process at the time of infusion, therefore they may not be definitive.  
5Sample collected from the final product prior to infusion. Results will not be available before cells are infused into the patient.  
Any prepared cells not required for infusion or for research or regulatory purposes will be cryopreserved by standard DTM techniques and will be made available should the subject be eligible for a second infusion as outlined in Section **3.11**.
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3.4 anti-neoplastic therapy

Patients must have evaluable disease at the time of protocol enrollment. Given the correlation in the original 20 patients treated on this study between the incidence of severe CRS and amount of disease burden, patients with high-burden disease (Arm 2: M3 bone marrow blasts and/or presence of peripheral blood blasts on routine CBC) will receive standard of care chemotherapy as described in Section 3.9. Patients in Arm 1 will receive fludarabine/cyclophosphamide preparative regimen on Days -4 through -2.
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3.5 impact of intercurrent illness

Should a subject develop a serious or life-threatening condition or infection after enrollment but before anti-CD19-CAR T cells are administered, the subject will be treated for their condition as clinically indicated and administration of anti-CD19-CAR T cells will be delayed. Cells will be maintained in culture for up to a total culture time of 14 days while attempts are made to correct the condition. Cells may be infused within this period only if the PI determines that administration of the cells does not pose any additional risk to the subject. Otherwise, the cells will be cryopreserved and, if clinically indicated, the subject may receive them up to 21 days from the planned infusion date, and be considered evaluable. If the patient is able to receive cells within 21 days of the planned original dose on Day 0, administration should follow the procedure as outlined in Section **3.10** and the patient will remain evaluable for toxicity and efficacy. Alternatively, if > 21 days has transpired prior to subject recovery, the subject has the option of receiving the cells on at a later point once the condition has resolved, but these patients will be replaced on study and not considered evaluable for toxicity or response endpoints. Clinical response in these patients will be analyzed and may be reported descriptively.
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3.6 treatment limiting toxicity

Adverse events that are considered disease-related (not suspected of relationship to anti-CD19- CAR T cells) will not be considered treatment-limiting toxicities. Only those AEs suspected to be related to pre-infusion chemotherapy and/or anti-CD19-CAR T cells (any component of the treatment regimen) will be used in the definition of TLT. Toxicities occurring after initiation of the chemotherapy preparative regimen but prior to anti-CD19 CAR cell infusion, will primarily be attributable to the chemotherapy administration or disease, if not extraneous causes. After cell infusion, toxicities will be evaluated for temporal and causal relationship to chemotherapy versus cell infusion. Some symptoms may overlap and attribution will not be clearly definable, in which case, toxicities will be attributed as possibly related to both chemotherapy and cell infusion. Toxicities will be attributed to the T cells if: 1) they were NOT present before T cell infusion; OR 2) they increase in Grade in temporal association with the T cell infusion; AND 3) they are not clearly explained by other factors. Patients who are fully assessable are those that have completed the chemotherapy regimen and received the anti-CD19 CAR T cell infusion. Chemotherapy-related toxicities experienced by patients who are unable to receive anti-CD19 CAR T cells (See Section 8.3 for the definition of inevaluable patients) will be considered in the definition of TLT. See Section 7.1 Causality for definitions of 'suspected'. The definition of TLT in these studies uses NCI's Common Terminology Criteria for Adverse Events (CTCAEv4.0).
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3.6.1 definition of tlt

Adverse events that are at least possibly related to the treatment regimen (chemotherapy and/or anti-CD19-&$57FHOOVDQGDUH*UDGHLQVHYHULW\ZLOOEHFRQVLGHUHG7/7VZLWKWKH following additional criteria or exceptions: 3.6.1.1 Hematological Toxicity:  
x Subjects with normal, Grade 1 or Grade 2 Hematologic Parameters at baseline
(independent of transfusion) and cytopenias NOT due to Bone Marrow Involvement by Disease o Any Grade 4 hematological toxicity (with the exception of lymphopenia) lasting greater than 30 days will be considered a TLT. Lymphocyte count and subsets will not be considered in the definition of TLT.
x Subjects with abnormal blood counts (Grades 1 through 4) at baseline due to bone marrow involvement by disease  
o These subjects will be considered non-evaluable for hematologic TLT but evaluable for all other aspects of the study.
3.6.1.2 Non-hematological Toxicity:
x Any Grade 3 or greater, non-hematological toxicity will be considered a TLT with the following **exceptions**:
a. Tumor lysis syndrome, including associated abnormalities (eg, electrolytes, uric acid, renal function)  
b. Grade 3 low electrolyte levels that are correctable and asymptomatic. Grade 3 hypoalbuminemia. Such individuals should receive supplementation as indicated.  
c. Hypocalcemia toxicity grade should be assigned based on the calcium level corrected for degree of hypoalbuminemia according to the following formula: For every albumin decrease of 1 gm/dL a total calcium increase of 0.2 mmol/L is to be made; d. Subjects on anticoagulant therapy or with pre-existing coagulopathy with abnormal coagulation parameters.  
e. Grade 3 transaminase, alkaline phosphatase, bilirubin or other liver function test elevation, provided there is rHVROXWLRQWRJUDGHZLWKLQGD\V
f. Grade 3 or 4 fever; g. Grade 3 or 4 infection or neutropenic fever unless subjects have normal blood counts at baseline, and infection is not considered likely related to the chemotherapy regimen and relationship to anti-CD19-CAR T cells is suspected (Note: Grade 4 infection uncontrolled for > 7 days will be considered TLT.);
h. Toxicities occurring within 24 hours post cell infusion related to cell infusion,
(including Grade 3 and 4 allergic reaction) that are reversible to a grade 2 or less within 8 hours with up to two doses of acetaminophen 15 mg/kg/dose (to a max adult dose of 650 mg) or up to two doses of diphenhydramine 1 mg/kg up (to a max adult dose of 50 mg).  
i. Grade 3 or 4 transient (< 72 hours) serum hepatic enzyme abnormalities. j. Grade 3 nausea and/or anorexia. k. Grade 3 cytokine release syndrome (**Appendix F**) will not constitute TLT.
x A Grade 2 non-hematologic toxicity of > 30 days duration will constitute a TLT (with the exceptions noted above in Section **3.6.1.2**).  
x In patients with history of prior SCT, any histologically proven acute GVHD grade 2 or higher (See **Appendix C**) within 30 days of receiving the anti-CD19 CAR cells will be considered TLT.
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3.6.2 maximal tolerated dose (mtd)

The MTD is a dose level immediately below the level at which the enrollment is stopped for a stratum due to a DLT, as explained specifically below:  
x If **more than one subject** in the first three subjects included in a dose level experience DLT as defined above, MTD will have been exceeded.  
x If DLT develops in one of the 3 subjects included in a cohort, the cohort will be then expanded up to six:
If 2 or more of these 6 included patients develop DLT, the MTD will have been exceeded. If no additional subject develops a DLT, the MTD will not have been exceeded and the next dose level can be administered after the four week safety assessment period of the last patient at this dose level.  
If MTD is exceeded at any dose level, three subjects will be added to the immediate lower dose level, unless it has been previously expanded to six. If less than 2 of 6 subjects develop DLT at that level, it will be defined as the MTD.
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3.7 dose escalation (completed 6-14)

Cell dose will be body weight-based. In patients with BMI equal to or less than 30, the dose of anti-CD19-transduced T cells for each cohort will be based on doses calculated using actual body weight. Patients with BMI greater than 30 will have doses calculated using ideal body weight (IBW) plus 50% of the difference between actual weight and IBW (see **Appendix D** for calculation). There will be a Phase I dose-escalation design in two strata with three dose cohorts. One stratum will enroll subjects who have previously undergone allogeneic stem cell transplant (SCT), and the other stratum will include subjects who have not undergone SCT. The number of anti-CD19- CAR transduced T cells transferred for each dose level cohort will be:  
| Dose Escalation Schedule   |                                          |
|----------------------------|------------------------------------------|
| Dose Level                 | Dose of anti-CD19-CAR transduced T cells |
| Cohort Level 1             | 1 x 106 transduced T cells/kg (± 20%)    |
| Cohort Level 2             | 3 x 106 transduced T cells/kg (± 20%)    |
| Cohort Level 3             | 1 x 107 transduced T cells/kg (± 20%)    |  
Each dose cohort will initially include a minimum of 3 patients. Patients per stratum will initially be enrolled sequentially. A two week (14 days) safety assessment period will follow regimen completion (defined as infusion of anti-CD19 CAR cells) of the first patient in each cohort. Subsequent patients in that cohort may be enrolled after a one week (7 day) safety assessment period. Four weeks must elapse after completion of cell infusion in the final patient in a cohort to allow for safety assessment before accruing patients to the next dose cohort level. Therefore, enrollment will not proceed to a higher dose level until all patients have been treated in the prior cohort and the last patient treated on the completed cohort has been observed for at least 4 weeks after infusion of anti-CD19 CAR cells. Toxicity data collected from the prior SCT stratum dose cohorts may be used to make dose escalation decisions in the non-SCT dose cohorts. For example, if 3 patients in the prior SCT stratum complete a cohort without DLT, the next patient enrolled in either strata may proceed to the next dose cohort. But if 3 patients in the NON-SCT stratum complete a cohort without DLT, the prior SCT stratum must continue enrollment until 3 patients complete therapy without DLT prior to dose escalation. If cell growth limitations preclude administration of the number of cells targeted for the assigned cohort level, the patient will receive as many cells as possible and the patient will be enrolled in the appropriate cohort for the number of cells infused, allowing for an additional three patients to be enrolled per cohort due to cell growth limitations. If a DLT occurs in an additional patient entered at a lower dose due to cell growth limitations, accrual will continue at the previously planned dose level for subsequent patients. If a minimum of 1x105/kg anti-CD19-CARtransduced T cells cannot be obtained for infusion, the patient will still receive the cell infusion, as the effective dose of cells is not well defined, and the patient will be evaluable for feasibility but not analyzed for MTD, although toxicities will be assessed and reported separately. If the third dose level in each stratum is completed without DLT, an MTD may not be determined. This will be considered the 'highest cell dose' studied, and will be the dose level that will be studied further in the expansion cohort. Alternatively, if no toxicity or clinical activity is observed after completion of the 3rd dose cohort, consideration may be given to adding additional dose cohorts in a protocol amendment. Dose escalation will follow the rules outlined in the Table below.  
| Number of Patients with DLT                                               | Escalation Decision Rule                                                                                                                                                                                                                                                                                                                                                                                                            |
|---------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| at a Given Dose Level 0 out of 3                                          | Enter up to 3 patients at the next dose level. If 0 out  of 3 occurs in the prior SCT stratum, both strata may  dose escalate. If 0 out of 3 occurs in the NON-SCT  stratum, enrollment must continue in the prior SCT  stratum until 0 out of 3 (or 1 out of 6) patients  demonstrate DLT.                                                                                                                                         |
| > 2                                                                       | Dose escalation to that stratum will be stopped. The  other stratum may continue to dose escalate  independently. This dose level will be declared the  maximally administered dose (highest dose  administered) for that stratum. Up to three (3)  additional patients from that stratum 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 from this stratum at this  dose level. x If 0 of these 3 patients experience DLT, proceed  to the next dose level. x 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. At least 6 patients must be entered at  this dose level.                                                                                                                                                                                                                                                                                                            |
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3.8 dose expansion cohort (compelted

To gain further experience with the safety, feasibility and clinical activity of the anti-CD19 CAR T cells in this patient population, the MTD (or highest cell dose studied) will be expanded to a total of 15 patients (i.e., 9 additional patients at the MTD). If the MTD or highest cell dose studied is the same for both strata (prior SCT vs. NO prior SCT) one expansion cohort will be studied and efforts will be made to include a balance of patients with prior SCT, without prior 6&7\HDUVRIDJHDQG!\HDUVRIDJH,IGLIIHUHQW07'VDUHREVHUYHGSHUVWUDWXPD total of 12 patients per stratum will be enrolled at the MTD, attempting to balance enrollment EHWZHHQSDWLHQWV\HDUVRIDJHDnd > 12 years of age.
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3.9 drug administration 3.9.1 arm

Each patient in Arm 1 will receive the lymphodepleting regimen as follows: (This may be given as an inpatient or outpatient as necessary).  
| as an inpatient or outpatient as necessary). Drug Dose   | Supportive Care                                                                                             | Chemo  Days   |    |
|----------------------------------------------------------|-------------------------------------------------------------------------------------------------------------|---------------|----|
| Fludarabine                                              | 25 mg/m2 per day IV  infusion in 50mL of 0.9%  sodium chloride over 30  minutes± 10 min, daily for  3 days. | 1,2,3         |    |
| Cyclophosphamide                                         | 900 mg/m2 per day IV  infusion over 60 minutes ±  10 min, once on Day 3  after fludarabine                  | IV pre-hydration 2 hours ±  30 min prior to  cyclophosphamide and for  at least 8 hours after with  0.45% sodium chloride and  5% dextrose (or other  composition appropriate for  the clinical situation) at a  rate of at least 90  mL/m2 /hour. Hydration  after cyclophosphamide  will include Mesna as  described below. Urine  VSHFLILFJUDYLW\" prior to infusion.               | 3  |
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3.9.1.1 mesna

Mesna will be administered at a dose of 540 mg/m2 per day by continuous IV infusion on day -2.  
The first 180 mg/m2 will be mixed with the cyclophosphamide in an appropriate amount of fluid based on the hydration rate. Immediately upon completion of this initial infusion, mesna at a dose of 360 mg/m2 in 0.45% sodium chloride and 5% dextrose (or other composition appropriate for the clinical situation) will be infused over 8 hours after completion of the cyclophosphamide. 3.9.1.2 Hydration with Cyclophosphamide IV hydration will be initiated 2 hours ± 30 min prior to cyclophosphamide using 0.45% sodium chloride with 5% dextrose (or other composition appropriate for the clinical situation). Hydration may be temporarily interrupted to give fludarabine or cyclophosphamide but should continue until at least 8 hours after the cyclophosphamide dose has been completed. 3.9.1.3 Adjusting hydration/electrolytes during preparative regimen. During chemotherapy, IV furosemide (0.5 - 1 mg/kg/dose to a maximum of 20 mg per dose) may be administered as needed to maintain normal urine output and fluid balance. All patients should receive at least maintenance fluids (1440mL/m2/day), orally and/or intravenously unless medically contraindicated. Patients deemed at high risk for tumor lysis will receive additional hydration; see Section **4.3.1** for fluid hydration guidelines. Hydration will be adjusted to maintain fluid balance, urine output and urine specific gravity. In addition, serum electrolyte levels will be monitored every 12 hours and IV fluid content including potassium chloride supplementation will be adjusted to maintain normal serum electrolyte levels.
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3.9.1.4 anti-emetics

Routine anti-emetic prophylaxis and treatment should be employed. Corticosteroids may not be used (except for physiologic replacement as outlined in Section **2.1.2.8**, or in IT chemotherapy as outlined in Section **4.1.1**).
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3.9.2 arm 2 chemotherapy

Standard of care chemotherapy will be administered to patients as inpatient and will be used prior to cell infusion in patients in Arm 2 to reduce the disease burden while depleting endogenous lymphocytes in preparation for cell infusion. The choice of chemotherapy agents will be determined by the research team based on the patient's prior therapies, e.g. total lifetime anthracycline doses, responses to prior regimens, and toxicities from prior regimens. Only FDA approved chemotherapy agents will be used. Efforts will be made to treat patients with one of the two regimens outlined below. At least 2 days must pass after the last dose of chemotherapy prior to cell infusion in Arm 2. Supportive care medications will be administered as per standard of care requirements for the agents administered. Standard chemotherapy will be given for 1 cycle (5 days for the two regimens outlined below), and cells will be administered as soon as possible, but no sooner than 2 days after the last dose of chemotherapy.  
| Drug               | Dose                                                                                                                                                                                                                                                                | Supportive Care               | Days   |
|--------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------|--------|
| Fludarabine        | 25 mg/m2 /d IV over                                                                                                                                                                                                                                                 | IV pre-hydration 1 hour prior |        |
| 30 min± 10 minutes | to fludarabine daily for 5 days  with 0.9% NaCl and KCl 10  meq/liter at a rate of 90  ml/m2 /hour to a maximum rate  of 100 ml/hour. Following  fludarabine infusion, restart  fluid at a rate of 90  ml/m2 /hour to a maximum rate  of 100 ml/hour continue until | 1, 2, 3, 4, 5  (Hour 0-1)     |        |  
| Version Date: 01/12/2018                                                                    | cytarabine infusion begins.                                                                                                                                                                                                  |                                                         |                                                                                                                                                                     |                           |
|---------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------|
| Cytarabine (Ara-C)                                                                          | 2000 mg/m2 /d IV over  4 hrs± 30 minutes, 4  hrs after fludarabine  begins                                                                                                                                                   | a.                                                      | Corticosteroid ophthalmic  drops 2 drops to each eye  every 6 hours starting  prior to first dose and until  24 hours after the last dose  of cytarabine completed. | 1, 2, 3, 4, 5  (Hour 5-9) |
| b.                                                                                          | Begin cytarabine doses 3.5  hours after completion of  the preceding fludarabine.                                                                                                                                            |                                                         |                                                                                                                                                                     |                           |
| c.                                                                                          | Infuse in 250 ml of D5W.                                                                                                                                                                                                     |                                                         |                                                                                                                                                                     |                           |
| d.                                                                                          | IV post-hydration x 4  hours daily for 5 days with  0.9% NaCl and KCl 10  meq/liter at a rate of 90  ml/m2/hour to a maximum  rate of 100 ml/hour.                                                                           |                                                         |                                                                                                                                                                     |                           |
| e.                                                                                          | In the event of signs of  CNS toxicity, cytarabine  infusion will be  interrupted and the M.D.  notified. No further  cytarabine will be  administered if there is  CNS toxicity (any grade)  deemed related to  cytarabine. |                                                         |                                                                                                                                                                     |                           |
| Filgrastim                                                                                  | 5 μg/kg/dose  subcutaneously daily starting one day prior  to starting FLAG                                                                                                                                                  | Administer daily until  ANC>1000 on 2 consecutive  days | 0 and continue  until criterion  is met                                                                                                                             |                           |
| IV hydration composition may be modified to compensate for electrolyte abnormalities or for |                                                                                                                                                                                                                              |                                                         |                                                                                                                                                                     |                           |  
IV hydration composition may be modified to compensate for electrolyte abnormalities or for risk of significant tumor lysis syndrome.  
3.9.2.2 Etoposide/Ifosfamide Regimen Prior to chemotherapy, patients should receive appropriate hydration so that urine flow is 2 ml/kg/hr for at least one void or one hour prior to initiation of chemotherapy.  
| Drug      | Dose                  | Supportive Care   | Days   |
|-----------|-----------------------|-------------------|--------|
| Etoposide | 100 mg/m2 /dose IV in | 1, 2, 3, 4, 5     |        |  
| D5W or NS IV over 1  hour± 10 min daily X  5 days                                           | (Hour 0-1)                                                                                                                                                                                                           |                                                                                                      |                            |
|---------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------|----------------------------|
| Ifosfamide                                                                                  | 1,800 mg/m2 /dose and  Mesna 360  mg/m2/dose IV in 100  mL D5W over 1 hour  ± 10 min daily X 5  days                                                                                                                 | Recommended hydration to be  maintained at 125 ml/m2 /hr  during days of ifosfamide  administration. | 1, 2, 3, 4, 5  (Hour 1-2)  |
| Mesna                                                                                       | 360 mg/m2 /dose IV  over 15 minutes ± 5  min immediately after  ifosfamide infusion,  then every 3 hours for  6 additional doses PO  or IV over 15 ± 5 min  minutes daily on days  1 through 5 (total of  35 doses). | Adequate oral hydration  (minimum 500 mL PO q 4-6 hr) must be maintained.                            | 1, 2, 3, 4, 5  (Hour 2-20) |
| IV hydration composition may be modified to compensate for electrolyte abnormalities or for |                                                                                                                                                                                                                      |                                                                                                      |                            |  
IV hydration composition may be modified to compensate for electrolyte abnormalities or for risk of significant tumor lysis syndrome.  
3.9.2.3 Treatment Modifications
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hematuria

For microscopic hematuria with <50 RBC/HPF in urine, continue cyclophosphamide or ifosfamide at full dose. For microscopic hematuria with >50 RBC/HPF initiate IV hydration at 2times maintenance rate and initiate bladder irrigation with normal saline 100-150 ml/m2/hr prior to cyclophosphamide/ifosfamide; initiate bladder irrigation with subsequent courses. For gross hematuria discontinue cyclophosphamide and ifosfamide until the hematuria has resolved and then follow the guidelines for microscopic hematuria with >50 RBC/HPF (hydration and bladder irrigation). If the gross hematuria recurs or does not resolve after 2 weeks, discontinue all use of cyclophosphamide and ifosfamide.
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3.10 anti-cd19 car t cell

(may be delayed up to Day 21 for clinical issues or cell growth limitations.) 3.10.1 Cell Infusion Criteria Subjects must meet the following criteria in order for cells to be infused (based on labs obtained within 24 hrs of cell infusion): 3.10.1.1 Anti-CD19-CAR T cells must have met release criteria (Section 3.3) 3.10.1.2 Subject has no evidence of hemodynamic instability  
3.10.1.3 Subject has not developed symptoms concerning for new, systemic infection or condition that in the opinion of the PI may pose an unacceptable risk to the subject 3.10.1.4 There is no evidence of uncontrolled, significant tumor lysis syndrome prior to cell infusion
3.10.1.5 At least 2 days have elapsed since the last dose of chemotherapy in Arm 2.  
3.10.1.6 Liver transaminase $/7DQG$67;8/1EDVHGRQDJH- and laboratory specific normal ranges
If these criteria are not met, measures will be taken to resolve the underlying condition(s) and if successful cells may be infused up to 21 days following the time of the planned infusion, or attempts to infuse at a later time may be made per Section 3.5.  
3.10.2 Inpatient and outpatient care post-cell infusion Patients will be admitted for cell infusion to NIH in accordance with CC and nursing policies. Patients will remain hospitalized until stable after cell infusion. Once discharged, patients must remain outpatient in close proximity to the NIH for daily evaluations until Day 14, and then twice weekly evaluations until Day 28.
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3.10.3 premedications

Patients will receive the following medications 30-60 minutes prior to cell infusion:  
x Diphenhydramine 0.5 - 1 mg/kg/dose (maximum 50 mg/dose) PO or IV over 10-15 minutes; x Acetaminophen 15 mg/kg/dose (maximum 650 mg/dose) PO.  
3.10.4 Cell Administration
Cells will be administered at a dose of 1 X 106 anti-CD19 CAR T cells/kg (+/- 20%). Cells are delivered to the patient care unit by a staff member from the DTM CPS. Prior to infusion, the cell product identity label is double-checked by two authorized staff (MD or RN), an 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 (IV) over 10-20 minutes ± 10 min via non-filtered tubing, gently agitating the bag during infusion to prevent cell clumping. 3.10.5 Required monitoring during cell infusion:  
3.10.5.1 Monitoring will include vital signs (blood pressure, heart rate, respiratory rate) prior to infusion, every 15 minutes ± 5 min for 1 hour after the start of infusion, every 30 minutes ± 10 min for the second hour and then hourly ± 10 min for an additional 2 hours or until stable, and more frequently if clinically indicated. Temperature will be evaluated at least hourly.
3.10.5.2 Supplemental oxygen will be available at the bedside.  
3.10.5.3 If an allergic or other acute reaction occurs, studies appropriate for investigation of a transfusion reaction will be performed (urinalysis, CBC, Coomb's test). Acute reactions will be treated according to institutional standards of care.
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3.11 option for additional dose(s)

On the day cells are cryopreserved, up to 6 aliquots sufficient for additional dosing will be cryopreserved for potential future use by the Subject. The remaining cells will be cryopreserved and used for research. The second and any subsequent doses will be administered at dose 1 X
106 anti-CD19 CAR T cells/kg (+/- 20%). If the subject experienced treatment limiting toxicities
(TLT) from prior dose, the second and any subsequent doses will be a half-log less at 3 X 105 anti-CD19 CAR T cells/kg (+/- 20%). Subjects will have the option for additional infusions of anti-CD19-CAR T cells (including a preparative chemotherapy regimen selected based on available standard chemotherapy regimens (prioritizing FLAG and Etoposide/ifosfamide), toxicity assessment and research blood sampling) if the following criteria are met: 3.11.1 Eligibility Criteria for subsequent cell infusions in subjects who did not experience TLT
after first infusion:
3.11.1.1 Response to previous infusion: Subjects who had a PR, or SD with clinical benefit may elect to receive another infusion of cells. Subjects that initially had a CR may only receive a second dose if evaluable disease recurs. Clinical benefit is indicated by an improvement in the subject's health status (e.g., decreased transfusion requirement, improved cytopenias, decrease in number of blasts not sufficient to reach a PR, improved performance status or quality of life, etc.).  
3.11.1.2 At least 28 days have passed since the previous cell infusion. 3.11.1.3 Any toxicity regardless of causality to the cell infusion after the previous anti-CD19 CAR cell infusion must resolve to a grade 2 or less or return to baseline levels.  
3.11.1.4 Subjects must meet all the initial eligibility criteria as outlined in Section 2, 3.11.1.5 An adequate number of cryopreserved cells must be available: 3.11.2 Eligibility Criteria for subsequent cell infusions in subjects who experienced TLT after the first infusion:
3.11.2.1 Subsequent doses will be reduced to a lower dose level.  
3.11.2.2 Disease burden must be reduced according to the following:
1. Improvement from M3 to M2 or less marrow classification, or 2. Improvement from M2 to M1 or less marrow classification, or 3. For patients beginning with M1 marrow, a decrease of at least 50% in their disease as determined by flow cytometry (MRD).  
3.11.2.3 Response to previous infusion: Subjects who had a PR, or SD with clinical benefit may elect to receive another infusion of cells. Subjects that initially had a CR may only receive additional dose if evaluable disease recurs. Clinical benefit is indicated by an improvement in the subject's health status (e.g., decreased transfusion requirement, improved cytopenias, decrease in number of blasts not sufficient to reach a PR, improved performance status or quality of life, etc.).  
3.11.2.4 At least 28 days have passed since the previous cell infusion. 3.11.2.5 Any toxicity, regardless of causality to the cell infusion after the anti-CD19 CAR cell infusion must resolve to a grade 2 or less, or return to baseline levels.  
3.11.2.6 Subjects must meet all the initial eligibility criteria as outlined in Section 2.  
3.11.2.7 An adequate number of cryopreserved cells must be available. 3.11.3 All patients receiving additional doses will undergo chemotherapy prior to cell infusion based on disease burden as described in Section 3.9.
3.11.4 On day 0, the cellular product will be thawed and administered IV immediately at a rate of approximately 10-15 ml/min or as tolerated based on volume status and/or DMSO toxicity. Do not exceed 20 ml of product per kg body weight (if 10% DMSO) or 40 ml of product per kg body weight (if 5% DMSO) so that per 24-hour period total DMSO administered is less than 1 mL/kg/day.  
3.11.5 Any subject who receives subsequent doses of anti-CD19 CAR T cells will be observed for 28 day post cell infusion and post-infusion monitoring will be the same as for the 1st infusion and all toxicities, including secondary reactions, will be recorded and reported.
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3.12 on-study evaluation

3.12.1 Prior to chemotherapy regimen (within 24 hours unless otherwise specified) 3.12.1.1 Complete physical examination, including, weight and vital signs  
3.12.1.2 Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate),
Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric Acid)
3.12.1.3 Thyroid panel (within 14 days) 3.12.1.4 C-reactive protein (CRP) 3.12.1.5 CBC with differential and platelet count 3.12.1.6 PT/PTT (within 14 days) 3.12.1.7 Disease staging (within 14 days)
x Flow cytometry of peripheral blood for CD19 determination, includes site density, absolute blast count x All patients will have lumbar puncture for examination of cerebral spinal fluid (at time of screening evaluation).  
3.12.1.8 ȕ-HCG pregnancy test on all women of child-bearing potential (within 72 hours of starting chemotherapy)
3.12.1.9 Ophthalmologic Exams: For subjects with a history of prior allogeneic stem cell transplantation, ophthalmologic examinations (slit-lamp and fundoscopic) will be performed prior to apheresis and 4 weeks after cell infusion. These examinations will be performed for safety monitoring and will not be used to disqualify the subject for study participation unless active GvH is identified.  
3.12.1.10 3DWLHQWV\HDUVRI age: ECOG 0, 1, or 2; patients < 12 years of age: Lansky
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vfdohvhhappendix a)

3.12.1.11 Baseline Cognitive Evaluation (within 30 days prior to Day 0): A brief battery of cognitive tests evaluating memory, attention, processing speed, and executive functions
(less than one hour to complete) will be administered to participants prior to anti-CD19- CAR T cell infusion (see **Appendix G**). A parent/adult observer also will complete a brief symptom checklist assessing the severity and duration of any neurologic symptoms over the past week (less than 15 minutes to complete).
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3.12.2 during chemotherapy (arm 2)

Evaluations conducted during standard of care chemotherapy in Arm 2 will be according to the package insert recommendations or best clinical practice procedures associated with the chemotherapy regimen administered. These evaluations will be individualized to the patients receiving chemotherapy in Arm 2 but at a minimum will include once or twice per week: 3.12.2.1 Physical exam and vital signs 3.12.2.2 Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate),
Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Uric Acid, CRP)
3.12.2.3 CBC with differential and platelet count. 3.12.3 Prior to cell infusion (within 24 hours unless otherwise noted) 3.12.3.1 Complete physical examination, including performance status, weight and vital signs 3.12.3.2 Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate),
Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric Acid, CRP)
3.12.3.3 CBC with differential and platelet count 3.12.3.4 Urinalysis 3.12.3.5 Serum Cytokine Levels (pre-infusion) 3.12.3.6 Detection of RCR by PCR and persistence of anti-CD19-CAR T cells (Section **5.1.3**
and **Appendix E**) as a baseline before cell infusion (within 72 hours of cell infusion)
3.12.4 During and After Cell Infusion: 3.12.4.1 Vital signs will be monitored as per Section **3.10.5**, and then routinely (every 4 hours)
unless otherwise clinically indicated.  
3.12.4.2 Serum Cytokine Levels: 1, 12 and 24 hours following infusion (See **Appendix E**). 3.12.5 Monitoring Day 1 to Day 28 (including Day 28) 3.12.5.1 Daily history and physical exam with vital signs and performance status through Day 14, then at least twice weekly until Day 28.  
3.12.5.2 Serum cytokine levels Days 1(24hr ±4 hours), 2 (48 hr ±4 hours), 3 (72 hr ±4 hours),
then daily on days 4-14 and Day 28 (± 3 days) following cell infusion. Levels should also be obtained daily if clinical symptoms of cytokine release syndrome develop.  
3.12.5.3 Twice weekly laboratory analysis: CBC with differential, LDH, SGPT (ALT), SGOT
(AST), alkaline phosphatase, bilirubin (total and direct), BUN, creatinine, electrolytes, calcium, magnesium, phosphorus, uric acid, albumin, CRP, and urinalysis 3.12.5.4 Post-Infusion Neurologic Symptom Checklist (Day 14 +/- 1 day) (**Appendix H** ): A
parent/adult observer for each participant will be asked to complete a brief symptom checklist to assess the severity and duration of any neurologic symptoms during the past week (about 2 minutes to complete).  
3.12.5.5 Research Testing x Persistence/Expansion of anti-CD19-CAR T cells:
i. Peripheral blood for flow cytometry [POB Panel] or TBNK in 3 mL PTT to be sent to NCI Flow Cytometry Lab at baseline and on Days 1, 7, 14, and 28 ± 3 days. If there is a response, repeat monthly for 2-3 months, then every 3-6 months as needed.  
ii. Peripheral blood for flow cytometry for CAR persistence at D-1, Day -2, or Day -
3and on Days 1, 7, 14 and 28 ± 3 days in 3 mL PTT: to be sent Dr. StetlerStevenson's Lab. If there is a response, repeat monthly for 2-3 months, then every 3-6 months as needed. CD 64 will be collected from the serum cytokine specimens according to **Appendix E**.  
iii. CSF (3-8 mL in spinal fluid collection tube) for flow cytometry each time intrathecal chemotherapy is administered after cell infusion (maximum of 5 doses) to be sent to Dr. Stetler-Stevenson's Lab. IT chemotherapy will be administered in an isovolumetric manner (See Section 4.1); therefore, CSF removed for this purpose will be utilized to determine if Anti-CD19-CAR T cells are present in the CSF.  
i. Echocardiograms were performed at baseline, during CRS, and at 1-month restaging ii. Cardiology studies, troponin, BNP, and CK/CK-MB were obtained at baseline, during cytokine release syndrome, and at 1-month restaging.  
3.12.6 Analysis of Disease: Day 28+/-4 (method determined by patients' type and location of disease)
3.12.6.1 Imaging Studies such as Chest CT and appropriate imaging methods of any sites relevant to the subject's disease (e.g., PET/CT).  
3.12.6.2 Bone marrow aspirate and biopsy with flow cytometry for B cell leukemia/lymphoma markers will be performed in all patients: Day 28+/-4 3.12.6.3 In ALL patients and in lymphoma patients with pre-treatment peripheral blood involvement with lymphoma, flow cytometry: Day 24-32.  
3.12.6.4 In addition, an LP will be performed for staging with the option to administer intrathecal chemotherapy. Intrathecal therapy will NOT be administered until at least Day 24 (i.e., during the first re-staging evaluation performed at Day 28 +/- 4) unless the x Cardiology Studies patient is suspected to have progressive disease and IT chemotherapy is needed to reduce the risk of seeding the CSF with leukemia from the diagnostic procedure. Subsequent intrathecal therapy will be given no more frequently than monthly unless CNS leukemia involvement is confirmed. Further, intrathecal chemotherapy will NOT be administered in any patient who develops CNS toxicity related to CAR T cells until at least 2 weeks after complete resolution of neurologic toxicity. In cases where CNS toxicity is due to progression of CNS leukemia IT chemotherapy may be administered. CSF will be evaluated by flow cytometry for disease and CAR T cell presence.  
3.12.6.5 Post-Infusion Cognitive Evaluation (Day 21 - 28): A brief battery of cognitive tests evaluating memory, attention, processing speed, and executive functions (less than one hour) will be re-administered to patients after the anti-CD19-CAR T cell infusion (see Appendix G) between Day 21 and Day 28. In addition, a parent/adult observer for each participant will complete a post-infusion neurologic symptom checklist (about 2 minutes to complete).
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3.12.7 evaluations after day 28

After Day 28, if the patient is clinically stable, they may be discharged to the care of their primary oncologist and return to NIH as determined by the following protocol evaluations or changes to their clinical condition, at the discretion of the PI. All protocol designated disease response evaluations will be performed at NIH, unless otherwise specified. After Day 28, patients will be evaluated (+/- two week) at 2, 3, 6, 9, and (+/- 4 weeks) 12 months after cell infusion and then, every 6 months during the second year, and then annually, except as noted below, or as clinically indicated. At a minimum, evaluations will include: 3.12.7.1 History and Physical examination including vital signs  
3.12.7.2 Laboratory Assessment: Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT,
AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric Acid),
complete blood count, IgG levels (every 3-6 weeks).  
3.12.7.3 Toxicity assessment, including evaluation of visual symptoms and if changes have occurred from baseline, i.e. changes in visual acuity, an ophthalmologic consult will be performed.  
3.12.7.4 Disease Evaluation (based on etiology and location of disease):
x Scans and x-rays: Chest CT and appropriate imaging of any sites relevant to the subject's disease. When applicable, PET scans will be performed 3 and 6 months after cell infusion and then as clinically indicated.  
x Bone marrow aspirate and biopsy with flow cytometry for B cell leukemia/lymphoma markers will be performed in all patients.
x Flow cytometry for CD19 and CD22 in ALL patients and in lymphoma patients with pretreatment peripheral blood involvement with lymphoma. Repeat evaluations will be performed as indicated above until off study criteria are met.  
x In addition, a diagnostic LP with the option for administration of intrathecal chemotherapy (per Section 4.1) will be performed at each restaging, and as clinically indicated. CSF will be evaluated by flow cytometry for disease and CAR T cell presence.  
3.12.7.5 Post-Infusion Follow-up Cognitive Evaluation (3 months +/- 4 weeks): A brief battery of cognitive tests evaluating memory, attention, processing speed, and executive functions (less than one hour to complete) will be re-administered to patients if they return for a 3-month visit (+/- 4 weeks) after the anti-CD219-CAR T cell infusion (see Appendix G). In addition, a parent/observer for each participant will complete a postinfusion neurologic symptom checklist (less than 15 minutes to complete).
3.12.7.6 Persistence/Expansion of Anti-CD19-CAR T cells:
i. PCR: 1-3 mL of blood in a PAXGene or a EDTA tube to be sent to NCI/Frederick until negative by flow cytometry ii. Peripheral blood for flow cytometry: to be sent to NIH Clinical Pathology Flow Cytometry Lab until negative by flow cytometry.  
iii. CSF (3-8 mL in flow media) for flow cytometry each time a diagnostic LP is performed to be sent to (NCI Flow Cytometry ab) NIH Clinical Pathology Flow Cytometry Lab if performed at the NIH CC and schedule permits.  
3.12.7.7 Detection of RCR per Section **5.1.4**. 3.12.7.8 Long-term follow up of patients receiving gene transfer (on this study or on a long-term follow-up protocol):
Physical examinations with medical history will be performed and documented annually for 5 years following cell infusion to evaluate long-term safety on this protocol or on 15-C-0028: Follow-Up Evaluation for Gene-Therapy Related Delayed Adverse Events after Participation in Pediatric Oncology Branch Clinical Trials. This long term follow up may be performed by the local health care provider who will submit documentation of H&P, with the necessary blood samples for RCR to POB NIH. After 5 years, health status data will be obtained from surviving patients via telephone contact or mailed questionnaires annually and blood will be requested to be sent to NIH annually for 10 more years for storage in the event of a new cancer, wherein RCR studies will be performed. The total follow up period for retroviral vectors is 15 years. 3.12.8 Off Treatment Follow up Evaluations AFTER Day 28 Subjects will be followed per the following x Off treatment all studies will be done as per day 28 (+/-4). x If no response and no ongoing toxicity (i.e. progressive disease) then no further clinical follow up required except for ongoing toxicity follow up and evaluations contained in Sections **3.12.6** and **3.12.7**, which will be collected for 15 years.  
x If response, results and documentation for physical examinations, vital signs, CBC with diff, Chem 20, and IGG levels, will be collected. Bone marrows/lumbar punctures will be done at the primary oncologist's discretion and documentation collected. All will be collected until relapse or bone marrow transplantation or other therapeutic interventions are initiated. The evaluations in Sections **3.12.6** and **3.12.7** will be collected for 15 years.  
3.12.9 Follow up Off-Study
Once a patient develops PD or goes on to other therapies, patients will be followed for Gene therapy follow up **ONLY** as per Section **5.1.3**, on this protocol or on 15-C-0028: *Follow-Up* Evaluation for Gene-Therapy Related Delayed Adverse Events after Participation in Pediatric Oncology Branch Clinical Trials protocol per FDA regulations. 3.12.9.1 Gene therapy follow up (See Section **5.1.3** and **Appendix E**)
a. Persistence/Expansion of Anti-CD19-CAR T cells b. Detection of RCL
c. Long-term follow up for toxicity assessment
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3.14 protocol accrual suspension

The study will be halted pending discussions with the FDA and NCI IRB if any of the following conditions are met throughout the study implementation:
a. Development of EBV lymphoma or polyclonal lymphoproliferative disease (PLPD).  
b. Grade 4 cytokine release syndrome in **more than** 2 subjects in the protocol (both cohorts).  
c. Any Grade 5 event at least possibly related to the research product.
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3.15 criteria for removal from

3.15.1 Criteria for removal from protocol therapy Patients will be taken off treatment (and followed until off-study criteria are met) for the following:
x Treatment limiting toxicity (TLT) due to cell infusion [unless criteria for subsequent infusions are met]. The definition of TLT is in Section **3.6.1**.  
x Grade 3 autoimmunity that involves vital organs (heart, kidneys, brain, eye, liver, colon, adrenal gland, lungs).  
x Grade 3 or 4 toxicity due to cell infusion (reaction to cellular product or infusion reaction) that is not reversible to a grade 2 or less within 8 hours with acetaminophen and/or diphenhydramine as outlined in Section 3.6, the patient will not have the option of receiving a second infusion of cells.
x General or specific changes in the patient's condition render the patient unacceptable for further treatment on this study in the judgment of the investigator.  
x Progressive disease (unless criteria are met for additional doses of cells)
3.15.2 Off-Study Criteria x Patient withdrawal from protocol (in which case the reason will be documented, if possible)
x Death x Conclusion of the 15 years of follow up, or subject enrolls in long-term follow up protocol of patients receiving gene transfer.  
3.15.3 Off Protocol Therapy and Off-Study Procedure Authorized staff must notify Central Registration Office (CRO) when a subject is taken offstudy. A Participant Status Update Form from the website
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistrationl@mail.nih.gov.
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4 concomitant medications/measures

Concomitant medications to control side effects of therapy will be given. Patients who require transfusions will receive irradiated blood products. Serotonin antagonists may be administered for nausea and vomiting as prophylaxis prior to chemotherapy and as needed. Additional antiemetics will be administered as needed for nausea and vomiting uncontrolled by serotonin antagonists. Antibiotic coverage for antimicrobial prophylaxis and central venous catheters may be provided at the discretion of the investigator. If patients require steroid therapy beyond physiologic replacement and for purposes other than to control CRS they will be taken off treatment.
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4.1 concurrent therapy for extramedullary

Patients with CNS leukemia or lymphoma are allowed. Concurrent craniospinal radiation will not be allowed. Concurrent therapy or prophylaxis for CNS leukemia or lymphoma consisting of standard intrathecal chemotherapy will be allowed as clinically indicated in patients with ALL or pediatric NHL at times of restaging spinal taps. Intrathecal chemotherapy will NOT be given until at least Day 24 (i.e., during the first re-staging evaluation performed at Day 28 +/- 4) unless the patient is suspected to have progressive disease and IT chemotherapy is needed to reduce the risk of seeding the CSF with leukemia from the diagnostic procedure. Subsequent intrathecal therapy will be given no more frequently than monthly unless CNS leukemia involvement is confirmed. Further, intrathecal chemotherapy will NOT be administered in any patient who develops CNS toxicity related to CAR T cells until at least 2 weeks after complete resolution of neurologic toxicity. 4.1.1 Regimen The standard intrathecal chemotherapy regimen used to prevent or manage CNS relapse will be employed. Individual agents may be eliminated from the standard triple intrathecal (TIT)- chemotherapy regimen based on clinical contraindications. 4.1.2 Dose  
| Age (years)   | MTX (mg)   | HDC (mg)   | ARA-C (mg)   | Volume (ml)   |
|---------------|------------|------------|--------------|---------------|
| <1            | 7.5        | 7.5        | 15           | 5             |
| 1             | 8          | 8          | 16           | 6             |
| 2             | 10         | 10         | 20           | 7             |
| 3-8           | 12         | 12         | 24           | 8             |
| >9            | 15         | 15         | 30           | 10            |  
MTX - methotrexate; HDC - Hydrocortisone; ARA-C - cytarabine 4.1.3 Administration Delivery should be isovolumetric (ml CSF out = ml drug in) via lumbar puncture (LP) in the lateral decubitus position. Patients should remain in prone or Trendelburg position for 30 minutes post LP to facilitate drug circulation throughout the CNS. Leucovorin will be given as 10 mg/m2/dose (rounded up to the nearest 5 mg increment to a maximum dose of 15 mg) PO or IV x 2 doses, 24 hours and 30 hours after intrathecal methotrexate.
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4.2 infection prophylaxis

Dosing of pediatric patients for infection prophylaxis will be in accordance with NIH Clinical Center Blood & Marrow Transplant Consortium guidelines (http://intranet.cc.nih.gov/bmt/clinicalcare/guidelines.shtml). Special Statement about IVIG: Since anti-CD19-CAR T cells have been demonstrated in other studies to also eliminate normal B cells, serum IgG levels will be monitored before cell infusion and every 3-6 weeks after infusion. If IgG < 500, IVIG will be administered at a dose of 500 mg/kg with the appropriate pre-medications and per institutional guidelines unless clinically contraindicated.
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4.3 prophylaxis and treatment of

Pediatric patients at greatest risk are those with high disease burden (e.g. bulky disease) and high cell turnover (e.g. elevated uric acid, LDH). 4.3.1 Regimen Subjects deemed to be at high risk of tumor lysis syndrome should begin allopurinol at a dose of approximately 100 mg/m2/dose po TID (maximum dose 200 mg TID). This should be started at least 8 hours prior to the first dose of the preparative regimen and continued until disease burden is reduced (e.g. peripheral blasts clear) or it is apparent that no tumor lysis has developed.  
Additional suggested monitoring and supportive care for subjects deemed to be at high risk of tumor lysis syndrome:  
x Hydration: Starting at least 6 hours prior to initiating preparative regimen, and continuing for at least 24 hours after cell infusion, IV fluids should be administered at a rate of 90120 ml/m2/hour (1.5 - 2 times maintenance) to maintain urine specific gravity <1.010 and normal urine output. Potassium should be avoided. Then IV+PO should continue at 90/m2/hour (1.5 times maintenance) until disease burden is reduced (e.g. peripheral blasts clear) or it is apparent that no tumor lysis has developed after at least 72 hours x For subjects judged to be at high risk for uric acid nephropathy, consider the use of rasburicase rather than allopurinol.
For subjects with renal insufficiency, consult Nephrology.
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4.3.2 blood product support

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 Hb >8.0 gm/dl, and plts >10,000/mm3. All blood products with the exception of the lymphocyte product will be irradiated. Leukocyte filters will be utilized for all blood and platelet transfusions to decrease sensitization to transfused WBC's and decrease the risk of CMV infection.
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4.3.3 hydroxyurea

Patients receiving hydroxyurea are eligible providing the dose has been stable for at least two weeks prior to starting apheresis. Hydroxyurea must be discontinued at least 24 hours prior to the start of the first dose of fludarabine. 4.3.4 Treatment of Cytokine Storm Cytokine storm should be monitored closely according to the algorithm detailed in **Appendix F**.  
If treatment is indicated according to the algorithm it should be initiated with tocilizumb (antiIL6R mAb) with other measures including corticosteroids or other immunosuppressive mAbs initiated at the discretion of the Principal Investigator and/or his designee.
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5 biospecimen collection 5.1 correlative

5.1.1 Post cell infusion evaluations for T-cell persistence: Samples will be drawn as outlined in **Appendix E** and Section **3.12** for evaluation of CAR-T cell persistence. 5.1.2 Immunological Testing When appropriate, apheresis will be performed or the maximum amount of blood allowed to be drawn in a single day based on the patient's weight will be obtained and PBMC will be isolated through whole blood by purification using centrifugation on a Ficoll cushion. Aliquots of these PBMC will be:
a. Cryopreserved for immunological monitoring of cell function, b. Subjected to DNA and RNA extraction for PCR analysis of vector copy number estimation c. Lymphocytes will be tested directly and following in vitro culture. Direct immunological monitoring will consist of quantifying CD3+ T cells that express the anti-CD19 CAR by FACS analysis using the anti-idiotype antibody to the CAR. Site density of the CD19 antigen will be quantified on any blasts present using flow cytometry techniques.
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5.1.3 monitoring gene therapy trials

a. Immunological monitoring using both anti-idiotype antibody staining to detect CARs on the surface of T cells and CD19-specific cytokine production to detect functional antiCD19 T cells will be used to quantitate persistence of T cells in the blood. Persistence will be determined at about Day 1, 7, 14, and 28 after cell infusion, then monthly (+/- 7 days) for 2-3 additional months, and then every 3-6 months thereafter until no CAR T cells are detected. In addition, CAR-transduced T cell might be quantitated by using specific PCR assays capable of detecting the unique DNA sequence for each retroviral vector engineered T-cell.  
b. Patients' blood samples will be obtained and undergo analysis for detection of RCR by PCR prior to cell infusion (at baseline) and at 3, 6, and 12 months post cell administration. Blood samples will be archived annually thereafter if all previous testing has been negative with a brief clinical history. If a patient dies or develops neoplasms during this trial, efforts will be made to assay a biopsy sample for RCR. If any posttreatment samples are positive, further analysis of the RCR and more extensive patient follow-up will be undertaken, in consultation with the FDA. RCR PCR assays detect the GaLV envelope gene and are performed under contract by the Indiana University Vector Production Facility. The results of these tests are maintained by the contractor performing the RCR tests and by the Pediatric Oncology Branch research team.  
c. Due to the nature of these studies, it is possible that expansion of specific T-cell clones will be observed as tumor reactive T-cells proliferate in response to tumor antigens. Therefore, care will be taken to track T-cell persistence both immunologically and molecularly. Blood samples (5-10 mL) for persistence of CAR transduced cells will be obtained prior to cell infusion (baseline), at 1, 7, 14, and 28 days (+/- 4 days), then so long as a response continues to be demonstrated, every month (+/- 7 days) for 2-3 months then every 3-6 months until off-study criteria are met. If any patient has more than 5% persistence of CAR gene transduced cells at month 6 (by anti-idiotype antibody staining) the previously archived samples will be subjected to techniques that would allow the identification of clonality of persisting CAR gene transduced cells. Such techniques may include T cell cloning or LAM-PCR. If a predominant or monoclonal T cell clone derived from CAR gene transduced cells is identified during the follow-up, the integration site and sequence will be identified and subsequently analyzed against human genome database to determine whether the sequences are associated with any known human cancers. If a predominant integration site is observed, the T cell cloning or LAM-PCR test will be used at an interval of no more than three months after the first observation to see if the clone persists or is transient. In all instances where monoclonality is persistent and particularly in instances where there is expansion of the clone, regardless of whether or not the sequence is known to be associated with a known human cancer, the subject should be monitored closely for signs of malignancy, so that treatment, if available, may be initiated early.
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5.1.4 minimal residual disease (leukemic

Patients who are anticipated to be MRD negative by flow cytometry, may have one additional sample collected at the time of disease evaluation (5 mL of blood or 2 mL of bone marrow in EDTA tube) to evaluate MRD. Samples will be sent immediately to the laboratory where DNA will be extracted and sent to Adaptive Biotechnology Corporation in Seattle where next generation DNA sequencing will be used to identify residual leukemia. Clinical parameters considered when selecting patients for sample submission include clinical course (i.e. presence of cytokine storm), decrease in circulating blasts (if any) or B-cells, or presence of CAR T cells by flow cytometry on periodic measurements of blood (as described in Immunologic Testing). As of Amendment P, all patients who attained flow cytometric MRD negativity following CAR infusion will have their samples sent (de-identified and only if available) to Adaptive to evaluate for MRD using next generation DNA sequencing to identify residual leukemia.
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5.1.5 blood and tissue specimen

Blood and tissue specimens from consenting participants collected in the course of this research project may be banked and used in the future to investigate new scientific questions related to this study. However, this research may only be done if the risks of the new questions were covered in the consent document. If new risks are associated with the research (e.g., analysis of germ line genetic mutations) the principal investigator must amend the protocol and obtain informed consent from all research subjects. Any new use of samples will require prospective IRB review and approval.
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5.1.6 minimal residual disease detection

DNA samples from blood and/or bone marrow will be stripped of identifiers and coded, and may be sent to Adaptive Biotechnology Corporation in Seattle where next generation DNA sequencing will be used to identify residual leukemia.
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5.1.6.1 t-cell analysis

Samples of blood and/or bone marrow stripped of identifiers and coded, may be sent to Adaptive Biotechnology Corporation in Seattle. T cells will be analyzed for clonality by sequencing the T cell receptors.
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5.2 sample storage tracking and

Blood and tissue collected during the course of this study will follow the handling procedures established by the Central Repository for NCI/Frederick. Blood and tissue processed by the Department of Transfusion Medicine (DTM), Clinical Center will comply with the Standard Operating Procedures of DTM. All samples (blood or tissue) are tracked by distinct identification labels that include a unique patient identifier and date of specimen collection. All cryopreserved samples are tracked for freezer location and storage criteria. All samples are stored in monitored freezers/refrigerators either in the Central Repository for NCI/Frederick or in the investigator's laboratory at specified temperatures with alarm systems in place. All samples (blood or tissue) are documented in a secure central computer database with identification and storage location, with computer backup according to established standards for the Central Repository for NCI/Frederick. At the completion of this protocol, samples will be stored for future use under a repository protocol. Any new use of samples will require an IRB approved protocol or approval of an exemption from IRB review. Otherwise, specimens will be disposed of in accordance with the environmental protection laws, regulations and guidelines of the Federal Government and the State of Maryland. Any new uses of samples collected during the course of this trial must be reviewed and approved by the NCI IRB. Any loss or unintentional destruction of the samples will be reported to the IRB. 5.2.1 Pediatric Research Samples Research blood sample aliquot size will be minimized for patients < 18 years of age and the total amount restricted to a maximum of 3 ml/kg per draw and 7 ml/kg per 6-week period (maximum 450 ml). In the event that blood draws are limited due to patient size, research studies will be performed in order of priority as listed in **Appendix E**. Small volume apheresis collections will not be included in this calculation since such is performed isovolumetrically with minimal red cell loss.
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6 data collection and evaluation

The PI will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D) and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant.  
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH
Intramural Records Retention Schedule as applicable. Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, the IRB will be notified.
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6.2 response criteria

Note that response criteria depend on the tumor type described below: For the purposes of this study, patients should be re-evaluated for response as outlined in Section 3.12.6. In addition to a baseline scan, confirmatory scans should also be obtained 4 weeks following initial documentation of objective response. 6.2.1 Response Criteria Lymphoma (Adapted from: Cheson BD, et al: Revised response criteria for malignant lymphoma. J Clin Oncol 2007; 25 :579-86)57 6.2.1.1 Complete Response (CR): CR requires all of the following: Complete disappearance of all detectable clinical evidence of disease and disease-related symptoms if present before therapy.  
1. Typically, FDG-avid lymphoma (large cell, mantle cell and follicular lymphomas are all typically FDG-avid): in patients with no pretreatment PET scan or when the PET scan was positive before therapy, a post-treatment residual mass of any size is permitted as long as it is PET negative.  
2. Variably FDG-avid lymphomas/FDG avidity unknown: in patients without a pretreatment PET scan, or if a pretreatment PET scan was negative, all lymph nodes and nodal masses PXVWKDYHUHJUHVVHGWRQRUPDOVL]HFPLQJUHDWHVWGLDPHWHULf > 1.5 cm before therapy). Previously involved nodes that were 1.1 to 1.5 cm in their long axis and more WKDQFPLQWKHLUVKRUWD[LVEHIRUHWUHDWPHQWPXVWKDYHGHFUHDVHGWRFPLQWKHLU short axis after treatment.  
3. The spleen and/or liver, if considered to be enlarged before therapy on basis of physical exam or CT scan, must be normal size on CT scan and not be palpable on physical examination and nodules thought to represent lymphoma must no longer be present.
4. A bone marrow aspirate and biopsy is performed only when the patient had bone marrow involvement with lymphoma prior to therapy or if new abnormalities in the peripheral blood counts or blood smear cause clinical suspicion of bone marrow involvement with lymphoma after treatment. The bone marrow aspirate and biopsy must show no evidence of disease by morphology or if indeterminate by morphology it must be negative by immunohistochemistry. The biopsy core sample must be a minimum of 20 mm in length.  
6.2.1.2 Partial Response (PR): PR requires all of the following:
1. GHFUHDVHLQVXPRIWKHSURGXFWRIWKHGLDPHWHUV63'RIXSWRRIWKHODUJHVW
dominant nodes or nodal masses. Dominant nodes or nodal masses should be clearly measurable in at least 2 perpendicular dimensions, should be from different regions of the body if possible and should include mediastinal and retroperitoneal nodes if possible.  
2. No increase in size of nodes, liver or spleen and no new sites of disease.  
3. ,IPXOWLSOHVSOHQLFDQGKHSDWLFQRGXOHVDUHSUHVHQWWKH\PXVWUHJUHVVE\LQWKH
SPD. There must be a > 50% decrease in the greatest transverse diameter for single nodules.  
4. Bone marrow is irrelevant for determination of a PR. If patient has persistent bone marrow involvement and otherwise meets criteria for CR the patient will be considered a PR.  
5. Typically FDG-avid lymphoma: for patients with no pretreatment PET scan or if the PET
scan was positive before therapy, the post-treatment PET scan should be positive in at least one previously involved site. Note: in patients with follicular lymphoma or mantlecell lymphoma, a PET scan is only indicated in patients with one or at most two residual masses that have regressed by 50% on CT scan.  
6.2.1.3 Progressive Disease (PD): Defined by at least one of the following:  
1. of t he pr o d ucts of at least t w o l y m p h n o des, or if a si n gle n o de is i n v ol ve d at least a 5 0 % i ncr eas e i n t he pr o d u ct of t he dia meters of t his o ne  
n o de.  
2. A p pear a n ce of a ne w lesi o n gr eater t ha n 1. 5 c m i n a n y a xis e ve n if ot her lesi o ns ar e decr easi n g i n size 3. Gr eater t ha n or e q ual t o a 5 0 % i ncr eas e i n size of s ple nic or he patic n o d ules 4. At least a 5 0 % i ncr eas e i n t he l o n gest dia meter of a n y si n gle pr e vi o usl y i de ntifie d n o de m ore t ha n 1 c m i n its s h ort a xis.  
5. Lesi o ns s h o ul d be P E T p ositi ve i n t y pi call y F D G-a vi d l y m p h o mas u nless t he lesi o n is t o o s mall t o be detecte d b y P E T ( < 1. 5 c m i n its l o n g a xis b y C T)  
6. 2. 1. 4 Sta ble Dis eas e ( S D): Neit her s ufficie nt s hri n ka ge t o q ualif y f or P R n or s uffi cie nt i n cr eas e t o q ualif y f or P D. P E T s h o ul d be p ositi ve i n t ypi call y F D G-a vi d l y m p h o mas.  
Fl o w c yt o metri c, m ole c ular or c yt o ge neti c st u dies will n ot be us e d t o deter mi ne res p o ns e.  
6. 2. 2 Res p o ns e Criteria f or A L L  
* M o difie d fr o m: C h es o n B D, et al. Re vis e d r ec o m me n dati o ns of t he Inter nati o nal W or ki n g Gr o u p f or dia g n osis, sta n dar dizati o n of r es p o ns e criteria, tr eat me nt o ut c o mes, a n d r e p orti n g sta n dar ds f or t her a pe utic trials i n ac ute m yel oi d le u ke mia5 8
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b o ne m arr

M 1 < 5 %  
blasts ( at least 2 0 0 cells c o u nte d)  
M 2 5 - 2 5 %  
M 3 > 2 5 %  
6. 2. 2. 1 C o m plet e Res p o nse ( C R)  
1. M 1 marr o w, a bs e nce of peri p her al blasts ( m or p h ol o gi c), a bs e nce of e xtr a me d ullar y sites of dis eas e, peri p her al bl o o d ne utr o p hil c o u nt > 1, 0 0 0/ L a n d platelet c o u nt >  
1 0 0, 0 0 0/ L. T his para meter will be t he re q uisite criteri o n f or C R. T he f oll o wi n g a d diti o nal para meters will be re p orte d as e x pl or at or y fi n di n gs.  
2. M or p h ol o gi c C R wit h i nc o m plete bl o o d c o u nt re c o ver y ( C Ri): A b o ve C R criteria wit h o ut s pecifie d bl o o d c o u nts.  
3. C yt o ge n eti c C R ( C R c yt o): I n a d diti o n t o a b o ve C R criteria, re versi o n t o n or mal kar y ot y pe f or t h os e wit h pr e vi o usl y dete ct e d c yt o ge netic a b n or malit y.  
4. M ole c ular C R ( C R m ole c): I n a d diti o n t o a b o ve C R c criteria, n or mali zati o n of pr e vi o usl y detect e d m ole c ular c yt o ge neti c a b n or malit y.  
6. 2. 2. 2 Partial R es p o ns e ( P R)
1. M2 marrow and a decrease in the percentage of marrow blasts by at least 50%, absence of peripheral blasts (morphologic), absence of extramedullary sites of disease.
6.2.2.3 Hematological Activity (HA) Does not meet the criteria for CR or PR with any of the following:
1. At least a 50% decrease in the percentage of marrow blasts  
2. At least a 50% decrease in the absolute peripheral blast count 3. Improvement of the peripheral blood neutrophil count to > 1,000/PL or platelet count to
> 100,000/PL
6.2.2.4 Stable Disease (SD)
1. Does not meet the criteria for CR, PR, HA, or PD
6.2.2.5 Progressive Disease (PD)  
1. Worse marrow classification (i.e., M status) with at least a 50% increase in the percentage of marrow blasts.
Or  
2. No change in marrow classification (i.e., M status), but a 50% or greater increase in absolute peripheral blast count or extent of extramedullary disease
6.2.2.6 CNS Classification CNS is the most common site of extramedullary disease in ALL. The following table lists the CNS disease classification. This classification should be used when evaluating the patient's overall response to treatment.
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cns classification

| CSF Cell Count and Cytology   |                                              |
|-------------------------------|----------------------------------------------|
| CNS 2                         | < 5/μl WBCs, cytospin positive for blasts or |  
CNS 1 0 blasts on cytospin  
TraumatLFVSLQDOWDSZLWK/5%&V :%&/F\WRVSLQSRVLWLYHIRUEODVWVEXW negative by Steinherz/Bleyer algorithm CNS 3 5/μl WBCs, cytospin positive for blasts or Traumatic spinal tap ZLWK/5%&V cytospin positive for blasts, and positive Steinherz/Bleyer algorithm*
*Steinherz/Bleyer algorithm method of evaluating traumatic lumbar punctures: If the patient has leukemic cells in the peripheral blood and the lumbar puncture is traumatic and FRQWDLQV:%&/DQGEODVWVWKHIROORZLQJDOJRULWKm should be used to distinguish between CNS2 and CNS3 disease: CSF WBC/RBC > 2X Blood WBC/RBC
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6.3 persistence criteria for anti-cd19

The absolute number of persisting anti-CD19-CAR-transduced T cells will be calculated by multiplying the absolute peripheral blood lymphocyte (PBL) count by the percentage of PBL that express both CD3 and the anti-CD19 CAR. The percentage of PBL that express both CD3 and the anti-CD19 CAR will be determined by a flow cytometry assay that involves staining with anti-CD3 and anti-idiotype antibody that is specific for the scFv on the extracellular portion of the anti-CD19 CAR. This analysis will be performed on Days 1, 7, 14, and 28 (+/- 4 days) then, so long as the patient continues to have a response or achieve CR, monthly (+/- 7 days) for 2-3 additional months then every 3-6 months after cell infusion. The 4 week time point is the only primary analysis that will be used for the primary evaluation of the number of CD3+ cells that express the anti-CD19 CAR.
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6.4 toxicity criteria

The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP website at (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc_40).
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7 safety reporting requirements/data and

Any untoward medical occurrence in a human subject, including any abnormal sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the subject's participation in research, whether or not considered related to the subject's participation in the research.
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7.1.2 suspected adverse reaction

Suspected adverse reaction means any adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, 'reasonable possibility' means there is evidence to suggest a causal relationship between the drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than adverse reaction, which means any adverse event caused by a drug. 7.1.3 Unexpected adverse reaction An adverse event or suspected adverse reaction is considered "unexpected" if it is not listed in the investigator brochure or is not listed at the specificity or severity that has been observed; or, if an investigator brochure is not required or available, is not consistent with the risk information described in the general investigational plan or elsewhere in the current application. "Unexpected" also refers to adverse events or suspected adverse reactions that are mentioned in the investigator brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are not specifically mentioned as occurring with the particular drug under investigation.
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7.1.4 serious

An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a Serious Adverse Event or if it compromises the safety, welfare or rights of subjects or others. 7.1.5 Serious Adverse Event An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following:
x Death, x A life-threatening adverse drug experience x Inpatient hospitalization or prolongation of existing hospitalization  
x Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions x A congenital anomaly/birth defect. x Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.  
7.1.6 Disability
A substantial disruption of a person's ability to conduct normal life functions.
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7.1.7 life-threatening adverse drug experience

Any adverse event or suspected adverse reaction that places the patient or subject, in the view of the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it does not include a reaction that had it occurred in a more severe form, might have caused death. 7.1.8 Protocol Deviation (NIH Definition) Any change, divergence, or departure from the IRB-approved research protocol. 7.1.9 Non-compliance (NIH Definition) The failure to comply with applicable NIH Human Research Protections Program (HRPP) policies, IRB requirements, or regulatory requirements for the protection of human research subjects. 7.1.10 Unanticipated Problem Any incident, experience, or outcome that:
x Is unexpected in terms of nature, severity, or frequency in relation to
(a) the research risks that are described in the IRB-approved research protocol and informed consent document; Investigator's Brochure or other study documents, and (b) the characteristics of the subject population being studied; AND
x Is related or possibly related to participation in the research; AND  
x Suggests that the research places subjects or others at a *greater risk of harm* (including physical, psychological, economic, or social harm) than was previously known or recognized.
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7.2 nci-irb and clinical director

7.2.1 NCI-IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths The Protocol PI will report in the NIH Problem Form to the NCI-IRB and NCI Clinical Director:
x All deaths, except deaths due to progressive disease x All Protocol Deviations x All Unanticipated Problems x All non-compliance Reports must be received within 7 days of PI awareness via iRIS 7.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review The protocol PI will report to the NCI-IRB:
1. A summary of all protocol deviations in a tabular format to include the date the deviation occurred, a brief description of the deviation and any corrective action.  
2. A summary of any instances of non-compliance 3. A tabular summary of the following adverse events:
x All Grade 2 **unexpected** events that are possibly, probably or definitely related to the research; x All Grade 3 and 4 events that are possibly, probably or definitely related to the research; x All Grade 5 events regardless of attribution; x All Serious Events regardless of attribution.  
NOTE: Grade 1 events are not required to be reported.
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7.2.3 nci-irb reporting of ind

Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported to the NCI IRB.