Phase I Study of T Cells Expressing an Anti-CD19 Chimeric Receptor in Children and Young Adults With B Cell Malignancies
Phase: PHASE1
Conditions: ALL, B Cell Lymphoma, Leukemia, Large Cell Lymphoma, Non-Hodgkin Lymphoma
Sponsor: National Cancer Institute (NCI)

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Abbreviated Title: Phase I Anti-CD 19 CAR
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
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,
Filgrastim

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
PRÉCIS
Background:
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. 
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.
Objectives:
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.
Eligibility:
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.
Design:
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 

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
TABLE OF CONTENTS 
PRÉCIS ......................................................................................................................................2 
TABLE OF CONTENTS ............................................................................................................4 
1. 
INTRODUCTION...............................................................................................................7 
1.1 
Study Objectives...........................................................................................................7 
1.2 
Background and Rationale ............................................................................................8 
1.3 
Exploratory Analysis...................................................................................................21 
1.4 
Protocol Summary and Rationale ................................................................................22 
1.5 
Protocol Status Summary and Amendment Justification..............................................22 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT .....................................................24 
2.1 
Eligibility Criteria.......................................................................................................24 
2.2 
Screening Evaluation ..................................................................................................27 
2.3 
Registration Procedures...............................................................................................28 
2.4 
Treatment Assignment Procedures ..............................................................................29 
3 
STUDY IMPLEMENTATION..........................................................................................29 
3.1 
Study Design...............................................................................................................29 
3.2 
Apheresis for Cell Acquisition ....................................................................................30 
3.3 
Cell Processing ...........................................................................................................31 
3.4 
Anti-Neoplastic Therapy.............................................................................................32 
3.5 
Impact of Intercurrent Illness after Enrollment ............................................................32 
3.6 
Treatment Limiting Toxicity.......................................................................................32 
3.7 
Dose Escalation (Completed 6-14) ..............................................................................34 
3.8 
Dose Expansion Cohort (COMPELTED 6-14)............................................................36 
3.9 
Drug Administration ...................................................................................................37 
3.10 
Anti-CD19 CAR T Cell Infusion – Day 0 ................................................................40 
3.11 
Option for Additional Dose(s) of Anti-CD19 CAR T Cells......................................42 
3.12 
On-Study Evaluation ...............................................................................................43 
3.13 
Study Calendar........................................................................................................48 
3.14 
Protocol Accrual Suspension ...................................................................................48 
3.15 
Criteria for Removal from Protocol Therapy and Off Study Criteria ........................48 
4 
CONCOMITANT MEDICATIONS/MEASURES ............................................................49 
4.1 
Concurrent Therapy for Extramedullary Leukemia or CNS Lymphoma ......................49 

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Abbreviated Title: Phase I Anti-CD 19 CAR
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 

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
10.2 
Fludarabine .............................................................................................................72 
10.3 
Cyclophosphamide ..................................................................................................73 
10.4 
Mesna......................................................................................................................74 
10.5 
Ifosfamide ...............................................................................................................74 
10.6 
Etoposide (Vepesid, VP-16) ....................................................................................75 
10.7 
Acetaminophen (Tylenol)........................................................................................75 
10.8 
Diphenhydramine (Benadryl) ..................................................................................75 
10.9 
Intrathecal Chemotherapy........................................................................................75 
10.10 
Hydrocotisone (Cortef, Solu-cortef) NCS# 010483..................................................75 
10.11 
Cytarabine (cytosine arabinoside, AraC, Cytosar) NSC# 063878.............................76 
10.12 
Methotrexate ...........................................................................................................77 
10.13 
Allopurinol..............................................................................................................78 
10.14 
Tocilizumab (ANTI-IL6R MAB).............................................................................78 
10.15 
Filgrastim (Neupogen).............................................................................................79 
11 APPENDICES ..................................................................................................................80 
11.1 
Appendix A -Performance Status Criteria................................................................80 
11.2 
Appendix B: Study Calendar ...................................................................................81 
11.3 
Appendix C: Grading and Treatment of Acute GVHD.............................................86 
11.4 
Appendix D: Calculation of Weight for Cell Dose Calculation in Morbidly Obese 
Candidates.............................................................................................................................87 
11.5 
Appendix E: Research Sample Requirements*.........................................................88 
11.6 
Appendix F: Guidelines for Grading and Management of Suspected Cytokine Release 
Syndrome..............................................................................................................................91 
11.7 
Appendix G: Neuropsychological Test Battery........................................................92 
11.8 
Appendix H: Neuro Symptom Checklist..................................................................93 
12 REFERENCES..................................................................................................................94 

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
1. INTRODUCTION
1.1
STUDY OBJECTIVES
1.1.1
Primary 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%)
x
x 107 transduced T cells/kg (± 20%)
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)
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 
&\WRNLQH5HOHDVH6\QGURPH&56WRRISDWLHQWVUHFHLYLQJDQWL-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.

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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 post-
infusion 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.
1.2
BACKGROUND AND RATIONALE
1.2.1
Pediatric B-cell Malignancies
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-free-
survival 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.
1.2.2
Adoptive Cell Therapy using Chimeric Antigen Receptors (CARs)
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 vivo12,13. Murine models indicated that inclusion of CD28 signaling domains 
enhanced tumor cytotoxicity and persistence of CAR-transduced T cells14,15. Increasing the 

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Abbreviated Title: Phase I Anti-CD 19 CAR
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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 receptor-
modified 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.
Figure 1

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
MSGV1-FMC63-28
7026 bp
Amp¨
CD3 zeta
CD28
5' LTR
3' LTR
variable regions of FMC63
SD
SA
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 
Figure 1. Normal PBMC can be transduced with an anti-CD19 CAR. A. Linearized plasmid map of the 
MSGV1-FMC63-28 gamma-retroviral vector that encodes the FMC63-28 anti-CD19 chimeric antigen 
receptor. B. Expression of the anti-CD19 chimeric receptor can be detected on the surface of transduced T cells 
by staining with biotinylated Protein L. Plots are gated on live CD3+ lymphocytes.

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Abbreviated Title: Phase I Anti-CD 19 CAR
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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-to-
target 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 3

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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 

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Abbreviated Title: Phase I Anti-CD 19 CAR
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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 anti-
CD19 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 
IL-2
CD28
High toxicity, cell dose and 
IL2 dose lowered
NCI SB
1 x 106
2.5 x 106
None
CD28
Reduced intensity 
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, 
50% none
Children included
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

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Abbreviated Title: Phase I Anti-CD 19 CAR
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1 x 107
3 x 107
MSKCC
1 x 106
3 x 106
None
CD28
MRD patients get higher 
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 
Pennsylvania
1.5 x 109 to
5 x 109 CD3+ cells 
(Flat dosing) in 4 
split doses
None
4-1BB
dealer’s choice preparative 
regimen
University of 
Pennsylvania
1-5 x 108 CD3+ 
cells
None
4-1BB
Phase II; Unclear preparative 
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
Table 1. Clinical trials using anti-CD19-CAR strategies that are proposed or currently 
enrolling.
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.

15
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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 (CART-
19-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.

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Abbreviated Title: Phase I Anti-CD 19 CAR
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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 CD3-
zeta after transplantation. Patients will then receive 3 doses of irradiated donor-derived EBV-
lymphoblastoid 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

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Abbreviated Title: Phase I Anti-CD 19 CAR
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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 

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Abbreviated Title: Phase I Anti-CD 19 CAR
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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 well-
defined 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 anti-
CD19-CAR transduced T cells.
1.2.5
Safety Considerations
Several safety concerns regarding the infusion of large numbers of retrovirally modified tumor 
reactive T-cells should be addressed.
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 

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Abbreviated Title: Phase I Anti-CD 19 CAR
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fludarabine, cytarabine, and filgrastim (FLAG) and has been used routinely in the POB.  Risks of 
fludarabine are described above.
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.
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 vector-
mediated gene transfer into CD34+ bone marrow stem cells. In the case of retroviral vector-
mediated 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.
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 

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Abbreviated Title: Phase I Anti-CD 19 CAR
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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 

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
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.
1.3
EXPLORATORY ANALYSIS
1.3.1
Use of Adaptive sequencing for MRD monitoring

22
Abbreviated Title: Phase I Anti-CD 19 CAR
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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 allele-
specific 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.
1.4
PROTOCOL SUMMARY AND RATIONALE
The primary goal of this trial was to evaluate the safety and feasibility of administering anti-
CD19 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. 
1.5
PROTOCOL STATUS SUMMARY AND AMENDMENT JUSTIFICATION
1.5.1
Protocol Status
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 

23
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
hematopoietic stem cell transplantation.  As previously noted cytokine release syndrome (CRS) 
was the dose limiting toxicity (DLT) establishing MTD.
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.
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 
HVFDODWLRQDQGH[SDQVLRQRIWKHILUVWSDWLHQWVHQUROOHGZLWK$//HLJKWSDWLHQWVKDG
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 
SDWLHQWVZLWKERQHPDUURZEODVWV
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.
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 liveUWUDQVDPLQDVHV$/7$67EH;8/16HYHUDO
patients have leukemia involvement of their liver resulting in elevated transaminases.  In cases 
where transaminitis is attributed to disease involvement, patients with Grade 3 or less ALT, AST 
should NOT be excluded from enrolling.  As an added safety measure, we have now required 
WKDW$/7DQG$67EH;8/1SULRUWRLQIXVLQJ&$57FHOOV
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 

24
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
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.
2
ELIGIBILITY ASSESSMENT AND ENROLLMENT
2.1
ELIGIBILITY CRITERIA
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.
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 

25
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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 
DQGYHUEDODVVHQWZLOOEHREWDLQHGIRUWKRVH\HDUVRIDJHZKHQDSSURSULDWH
2.1.1.8 Clinical performance status: Patients > \HDUVRIDJH.DUQRIVN\3DWLHQWV
\HDUVRIDJH/DQVN\VFDOHVHH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 &DUGLDFIXQFWLRQ/HIWYHQWULFXODUHMHFWLRQIUDFWLRQE\08*$RUFDUGLDF05,RU
IUDFWLRQDOVKRUWHQLQJE\(&+2RUOHIWYHQWULFXODUHMHFWLRQIUDFWLRQE\
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
(Years)
Maximum Serum
Creatinine (mg/dL)

26
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018

0.8
DJH
1.0
>10
1.2
2.1.2.4 Hematologic function
x
$EVROXWHQHXWURSKLOFRXQW$1&ȝ/RUSODWHOHWFRXQWȝ/LIWKHVH
cytopenias are not judged by the investigator to be due to underlying disease (i.e. 
potentially reversible with anti-neoplastic therapy);
x
$VXEMHFWZLOOQRWEHH[FOXGHGEHFDXVHRISDQF\WRSHQLD*UDGHLILWLVGXHWRGLVHDVH
based on the results of bone marrow studies.
2.1.2.5 +\SHUOHXNRF\WRVLVEODVWVȝ/RUUDSLGO\SURJUHVVLYHGLVHDVHWKDWLQWKH
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\VWHPLFFKHPRWKHUDS\ZHHNVZHHNVIRUQLWURVRXUHDVRUUDGLDWLRQWKHUDS\
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 therapy-
related are exempt from this exclusion.
2.1.2.10 HIV/HBV/HCV Infection:

27
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
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. 

28
Abbreviated Title: Phase I Anti-CD 19 CAR
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
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 

29
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
2.4
TREATMENT ASSIGNMENT PROCEDURES
2.4.1
Cohorts
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).
2.4.2
Arms
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.
3
STUDY IMPLEMENTATION
3.1
STUDY DESIGN
This is a phase I safety/feasibility study in pediatric and young adult patients with CD19-
expressing 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. 

30
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
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 

31
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
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 CD19-
expressing 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. 
The release criteria will be based upon analyses for each dose of anti-CD19 CAR retroviral-
transduced autologous PBL and will include:
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 
(8P/
Mycoplasma2
Mycoplasma test
Negative 
RCR2
RCR-PCR
Negative
RCR5
S+L- Assay
Negative 
Sterility testing4
gram stain, culture 
Negative 
1Performed on sample from final product immediately prior to infusion if given fresh or immediately prior to cryopreservation,
results available at the time of infusion.

32
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
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.
3.5
IMPACT OF INTERCURRENT ILLNESS AFTER ENROLLMENT
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.
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. 

33
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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).
3.6.1
Definition of TLT
Adverse events that are at least possibly related to the treatment regimen (chemotherapy and/or 
anti-CD19-&$57FHOOVDQGDUH*UDGHLQVHYHULW\ZLOOEHFRQVLGHUHG7/7VZLWKWKH
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.

34
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
e.
Grade 3 transaminase, alkaline phosphatase, bilirubin or other liver function test 
elevation, provided there is rHVROXWLRQWRJUDGHZLWKLQGD\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.
3.6.2
Maximal Tolerated Dose (MTD) (COMPLETED 6-14)
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.
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 

35
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Version Date: 01/12/2018
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-CAR-
transduced 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 

36
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
3.8
DOSE EXPANSION COHORT (COMPELTED 6-14)
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 
Number of Patients with DLT
at a Given Dose Level
Escalation Decision Rule
0 out of 3
Enter up to 3 patients at the next dose level. 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.

37
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
6&7\HDUVRIDJHDQG!\HDUVRIDJH,IGLIIHUHQW07'VDUHREVHUYHGSHUVWUDWXPD
total of 12 patients per stratum will be enrolled at the MTD, attempting to balance enrollment 
EHWZHHQSDWLHQWV\HDUVRIDJHDnd > 12 years of age.
3.9
DRUG ADMINISTRATION
3.9.1
Arm 1 Chemotherapy
Each patient in Arm 1 will receive the lymphodepleting regimen as follows: (This may be given 
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 
VSHFLILFJUDYLW\
prior to infusion.
3
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 

38
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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. 
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).
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.
3.9.2.1 FLAG Chemotherapy
Drug
Dose
Supportive Care
Days
Fludarabine
25 mg/m2/d IV over 
30 min± 10 minutes
IV pre-hydration 1 hour prior 
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)

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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.
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.
1, 2, 3, 4, 5 
(Hour 5-9)
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 
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 

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Abbreviated Title: Phase I Anti-CD 19 CAR
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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 
risk of significant tumor lysis syndrome.
3.9.2.3 Treatment Modifications
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 2-
times 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.
3.10 ANTI-CD19 CAR T CELL INFUSION – DAY 0
(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

41
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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 $/7DQG$67;8/1EDVHGRQDJH- 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. 
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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Abbreviated Title: Phase I Anti-CD 19 CAR
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3.11 OPTION FOR ADDITIONAL DOSE(S) OF ANTI-CD19 CAR T CELLS
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 

43
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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.
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\HDUVRI age: ECOG 0, 1, or 2; patients < 12 years of age: Lansky 

44
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
VFDOHVHHAppendix 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).
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), 

45
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
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. Stetler-
Stevenson’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.
x
Cardiology Studies
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 

46
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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).
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.

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x
Flow cytometry for CD19 and CD22 in ALL patients and in lymphoma patients with pre-
treatment 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 post-
infusion 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.  

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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
3.13 STUDY CALENDAR
See Appendix B: Study Calendar
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.
3.15 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA
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. 

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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 off-
study. 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 ncicentralregistration-
l@mail.nih.gov.
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.
4.1
CONCURRENT THERAPY FOR EXTRAMEDULLARY LEUKEMIA OR CNS LYMPHOMA
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

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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
TIT-chemotherapy will be dosed by age as follows:
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.
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.
4.3
PROPHYLAXIS AND TREATMENT OF TUMOR LYSIS SYNDROME IN CHILDREN
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 

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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 90-
120 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. 
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.
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 (anti-
IL6R mAb) with other measures including corticosteroids or other immunosuppressive mAbs 
initiated at the discretion of the Principal Investigator and/or his designee.
5
BIOSPECIMEN COLLECTION
5.1
CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES
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:

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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. 
5.1.3
Monitoring Gene Therapy Trials: Persistence and RCR
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 anti-
CD19 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 post-
treatment 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 

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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.
5.1.4
Minimal Residual Disease (Leukemic Cells)  
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.
5.1.5
Blood and Tissue Specimen Banking
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.
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.
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.
5.2
SAMPLE STORAGE, TRACKING AND DISPOSITION
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 

54
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Version Date: 01/12/2018
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.
6
DATA COLLECTION AND EVALUATION 
6.1
DATA COLLECTION
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. 
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:

55
Abbreviated Title: Phase I Anti-CD 19 CAR
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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 
PXVWKDYHUHJUHVVHGWRQRUPDOVL]HFPLQJUHDWHVWGLDPHWHULf > 1.5 cm before 
therapy). Previously involved nodes that were 1.1 to 1.5 cm in their long axis and more 
WKDQFPLQWKHLUVKRUWD[LVEHIRUHWUHDWPHQWPXVWKDYHGHFUHDVHGWRFPLQWKHLU
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. GHFUHDVHLQVXPRIWKHSURGXFWRIWKHGLDPHWHUV63'RIXSWRRIWKHODUJHVW
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. ,IPXOWLSOHVSOHQLFDQGKHSDWLFQRGXOHVDUHSUHVHQWWKH\PXVWUHJUHVVE\LQWKH
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 mantle-
cell 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: 

5 6
A b bre vi ate d Title: P h ase I A nti- C D 1 9 C A R
Versi o n D at e: 0 1/ 1 2/ 2 0 1 8
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 ncreas 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 are 
decr easi n g i n size
3.  Gr eater t ha n or e q ual t o a 5 0 % i ncreas 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 pre 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 creas 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 rec 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 res p o ns e criteria, treat me nt o ut c o mes, a n d re 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 mia 5 8.
B o ne M arr o w Cl assifi c ati o n
 
%
blasts ( at least 2 0 0 cells c o u nte d)  
M 1  
< 5 %  
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 heral blasts ( m or p h ol o gi c), a bs e nce of e xtra 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 orat 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 pre 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)

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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.
CNS Classification
 
CSF Cell Count and Cytology 
CNS 1 
0 blasts on cytospin 
CNS 2 
< 5/μl WBCs, cytospin positive for blasts
or
TraumatLFVSLQDOWDSZLWK/5%&V
:%&/F\WRVSLQSRVLWLYHIRUEODVWVEXW
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* 

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Abbreviated Title: Phase I Anti-CD 19 CAR
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*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:%&/DQGEODVWVWKHIROORZLQJDOJRULWKm should be used to distinguish between 
CNS2 and CNS3 disease:
CSF WBC/RBC > 2X Blood WBC/RBC
6.3
PERSISTENCE CRITERIA FOR ANTI-CD19 CAR TRANSDUCED T CELLS
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.
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).
7
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN
7.1
DEFINITIONS
7.1.1
Adverse Event
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.
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

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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.
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.
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

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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.
7.2
NCI-IRB AND CLINICAL DIRECTOR REPORTING
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. 
7.2.3
NCI-IRB Reporting of IND Safety Reports
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NCI IRB.

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7.3
IND SPONSOR REPORTING CRITERIA
During the first 30 days after the subject receives investigational agent/intervention, the
investigator must immediately report to the sponsor, using the mandatory MedWatch form 3500a
or equivalent, any serious adverse event, whether or not considered drug related, including those 
listed in the protocol or investigator brochure and must include an assessment of whether there is 
a reasonable possibility that the drug caused the event. For serious adverse events that occur 
more than 30 days after the last administration of investigational agent/intervention, only report 
those that have an attribution of at least possibly related to the agent/intervention.
Required timing for reporting per the above guideline:
x
Deaths (except death due to progressive disease) must be reported via email within 24 
hours.  A complete report must be submitted within one business day. 
x
Other serious adverse events as well as deaths due to progressive disease must be 
reported within one business day
Events will be submitted to the Center for Cancer Research (CCR) at: CCRsafety@mail.nih.gov
and to the CCR PI and study coordinator.
7.4
INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) REPORTING CRITERIA
7.4.1
Serious Adverse Event Reports to IBC
The Principal Investigator (or delegate) will notify IBC of any unexpected fatal or life-
threatening experience associated with the use of the anti-CD19 CAR transduced cells as soon as 
possible but in no event later than 7 calendar days of initial receipt of the information. Serious 
adverse events that are unexpected and associated with the use of the anti-CD19 CAR transduced 
cells, but are not fatal or life-threatening, must be reported to the NIH IBC as soon as possible, 
but not later than 15 calendar days after the investigator’s initial receipt of the information.
Adverse events may be reported by using the FDA Form 3500a.
7.4.2
Annual Reports to IBC
Within 60 days after the one-year anniversary of the date on which the IBC approved the initial 
protocol, and after each subsequent anniversary until the trial is completed, the Principal 
Investigator (or delegate) shall submit the information described below. Alternatively, the IRB 
continuing review report can be sent to the IBC in lieu of a separate report.  Please include the 
IBC protocol number on the report.
7.4.2.1 Clinical Trial Information 
A brief summary of the status of the trial in progress or completed during the previous year. The 
summary is required to include the following information:
x
the title and purpose of the trial 
x
clinical site
x
the Principal Investigator
x
clinical protocol identifiers;

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Abbreviated Title: Phase I Anti-CD 19 CAR
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x
participant population (such as disease indication and general age group, e.g., adult or 
pediatric); 
x
the total number of participants planned for inclusion in the trial; the number entered into 
the trial to date whose participation in the trial was completed; and the number who 
dropped out of the trial with a brief description of the reasons 
x
the status of the trial, e.g., open to accrual of subjects, closed but data collection ongoing, 
or fully completed, 
x
if the trial has been completed, a brief description of any study results. 
7.4.2.2 Progress Report and Data Analysis 
Information obtained during the previous year's clinical and non-clinical investigations, 
including: 
x
a narrative or tabular summary showing the most frequent and most serious adverse 
experiences by body system
x
a summary of all serious adverse events submitted during the past year
x
a summary of serious adverse events that were expected or considered to have causes not 
associated with the use of the gene transfer product such as disease progression or 
concurrent medications
x
if any deaths have occurred, the number of participants who died during participation in 
the investigation and causes of death
x
a brief description of any information obtained that is pertinent to an understanding of the 
gene transfer product’s actions, including, for example, information about dose-response, 
information from controlled trials, and information about bioavailability. 
7.5
DATA AND SAFETY MONITORING PLAN
7.5.1
Principal Investigator/Research Team 
The clinical research team will meet on a regular basis when patients are being actively treated 
on the trial to discuss each patient. Decisions about dose level enrollment and dose escalation if 
applicable will be made based on the toxicity data from prior patients. 
All data will be collected in a timely manner and reviewed by the principal investigator or a lead 
associate investigator. Adverse events will be reported as required above. Any safety concerns, 
new information that might affect either the ethical and or scientific conduct of the trial, or 
protocol deviations will be immediately reported to the IRB using iRIS and to the Sponsor. 
The principal investigator will review adverse event and response data on each patient to ensure 
safety and data accuracy. The principal investigator will personally conduct or supervise the 
investigation and provide appropriate delegation of responsibilities to other members of the 
research staff. 
7.5.2
Sponsor Monitoring Plan

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As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring 
program. The CCR’s program allows for confirmation of: study data, specifically data that could 
affect the interpretation of primary study endpoints; adherence to the protocol, regulations, and 
SOPs; and human subjects’ protection. This is done through independent verification of study 
data with source documentation focusing on:
x
Informed consent process
x
Eligibility confirmation
x
Drug administration and accountability
x
Adverse events monitoring
x
Response assessment.
The monitoring program also extends to multi-site research when the CCR is the coordinating 
center.
This trial will be monitored by personnel employed by an CCR contractor. Monitors are 
qualified by training and experience to monitor the progress of clinical trials. Personnel 
monitoring this study will not be affiliated in any way with the trial conduct.
7.5.3
Safety Monitoring Committee (SMC)
This protocol will require oversight from the Safety Monitoring Committee (SMC).  Initial 
review will occur as soon as possible after the annual NCI-IRB continuing review date.   
Subsequently, each protocol will be reviewed as close to annually as the quarterly meeting 
schedule permits or more frequently as may be required by the SMC.   For initial and subsequent 
reviews, protocols will not be reviewed if there is no accrual within the review period. Written 
outcome letters will be generated in response to the monitoring activities and submitted to the 
Principal investigator and Clinical Director or Deputy Clinical Director, CCR, NCI.
8
STATISTICAL CONSIDERATIONS
8.1
GENERAL CONSIDERATIONS
All subjects who are enrolled in the study and received the planned dose of anti-CD19 CAR T 
cells will be included in the statistical summaries and analyses of safety data. All available data 
will be used and thus, missing data will not be estimated or carried forward in any statistical 
summary or analyses.
Descriptive summary statistics include the number of subjects, mean, median, percent coefficient 
of variation (%CV) (if required), standard deviation, and minimum and maximum values 
(quantitative variables) or the number of subjects and percentages by category (qualitative 
variables). Changes will be assessed as the difference between the baseline measurement and the 
appropriate post-cell infusion measurement unless otherwise specified. Baseline is defined as the 
last non-missing value prior to initiation of preparative regimen except for cytokine analyses 
where baseline is defined as the last non missing value prior to cell infusion. Individual subject 
listings of data will also be provided to allow for review of all safety parameters.

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8.2
DOSE ESCALATION DESIGN
The cell doses for patients will be weight-based. The Phase I dose-escalation design will consist 
of three cohorts of a minimum of three patients per cohort as described in Sections 3.6 and 3.7.
COMPLETED
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 dose (IBW) dose plus 50% of the 
difference between actual weight and IBW (see Appendix D).
In order to determine if there are differences in safety or feasibility related to manufacturing or 
administering anti-CD19 CAR T cells after a nonmyeloablative preparative regimen in children 
and young adults who have not undergone prior allogeneic stem cell transplant (SCT) versus 
those that have previously undergone SCT, the enrollment will be stratified for these two groups 
of subjects. For each stratum of patients, the number of anti-CD19 transduced T cells 
administered in each cohort will be:
x
Cohort 1
1 x 106 transduced T cells/kg (± 20%)
x
Cohort 2
3 x 106 transduced T cells/kg (± 20%)
x
Cohort 3 
1 x 107 transduced T cells/kg (± 20%)
Doses of anti-CD19-CAR transduced T-cells will be administered in a standard 3 + 3 dose 
escalation design until MTD is determined. After treatment of the first patient in a cohort there 
will be a two week (14 day) safety assessment prior to enrollment of the 2nd patient in each 
cohort. Subsequent patients in that cohort may be enrolled after a one week safety assessment 
period following cell infusion. Patients will be enrolled sequentially; 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. If cell growth 
limitations preclude administration of the targeted cohort cell dose, the patient will receive as 
many cells as possible, and be considered part of the lower dose cohort. If a minimum of 1.0 
x105 anti-CD19-CAR-transduced T cells per kg cannot be obtained for infusion, the patient will 
not be treated and if possible a second attempt to produce cells for infusion will be made. As per 
Section 3.7, toxicity data collected from the SCT stratum dose cohorts may be used to inform 
dose escalation decisions in the non SCT dose cohorts. For example, if 3 patients in the SCT 
stratum complete a cohort without DLT, the next patient enrolled in either stratum may proceed 
to the next dose cohort. But if 3 patients in the NON-SCT stratum complete a cohort without 
DLT, the SCT stratum must continue enrollment until 3 patients complete therapy without DLT 
prior to dose escalation. COMPLETED
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 15 
patients. 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 
SDWLHQWVZLWKSULRU6&7ZLWKRXWSULRU6&7\HDUVRIDJHDQG!\HDUVRIDJH,IGLIIHUHQW
MTDs are observed per stratum, a total of 12 patients per stratum will be enrolled at the MTD, 
DWWHPSWLQJWREDODQFHHQUROOPHQWEHWZHHQSDWLHQWV2 years of age and > 12 years of age.
COMPLETED

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8.3
SAFETY AND FEASIBILITY OF HIGH-DISEASE BURDEN VS. NON-HIGH-DISEASE BURDEN
Given the impressive response to this therapy, Amendment G allows us to explore further the 
study findings.  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.  Amendment G 
will allow us to evaluate a safe option for bringing this therapy to patients with high-burden of 
disease.  For purposes of this study, high-burden disease will be defined as M3 bone marrow 
blasts and/or presence of peripheral blood blasts on routine CBC.  We will therefore explore the 
safety and feasibility of this therapy in two groups of patients:
1. Arm 1 = Patients without high-burden disease, or who due to safety concerns require the 
cyclophosphamide/fludarabine preparative regimen 
2. Arm 2 = Patients with high-burden disease
Thirty patients are to be enrolled in arm 1 and 30 patients in arm 2 in order to have sufficient 
patients to establish a reasonably precise confidence bound on the safety of the regimen overall 
as well as the feasibility of delivering treatment to patients in arm 2.
8.3.1
Safety 
All 60 patients on arms 1 and 2 will be considered together for a safety evaluation.  Combining 
arms 1 and 2, if there are 0-1 patients in the first 10 who have a grade 4 CRS, then accrual would 
continue as planned per arm. However, if there are 2 patients within the first 10 who experience 
a grade 4 CRS then no further patients may be accrued.  If accrual beyond the initial 10 is 
permitted, then this will continue to be the case provided that the cumulative percentage of 
patients with grade 4 CRS on both arms combined is below 20%; realizing 20% or more with 
grade 4 CRS at any time would require ending accrual and amending the study to reconsider 
toxicity definitions (since they are newly implemented and not fully vetted) or revise the 
treatment regimen. The upper one sided 90% CI on 2/10 is 45.0% which would indicate possible 
consistency with a very high rate of grade 4 CRS.  Provided that these interim toxicity limits are 
not met, accrual will be permitted until 60 total patients are enrolled, 30 per arm and the overall 
fraction with grade 4 CRS will be determined. As illustrations, if 6/60 have grade 4 CRS, the 
upper one-sided 90% CI bound on this fraction is 16.9% and if the fraction ends up being as high 
as 20% at the end of 60 patients, e.g. 12/60 with grade 4 CRS, the upper 90% one-sided 
confidence bound is 28.2%.
8.3.2
Arm 2 Feasibility  
In addition to safety, establishing feasibility of administering the anti-CD19 CAR T cells within 
21 days of the planned infusion is an important goal of this study. Feasibility will be assessed 
only in Arm 2 and defined by the successful manufacturing, expansion, and administration 
(within 21 days of the planned infusion date) of the anti-CD19 CAR T cells to satisfy the dose 
level and meet the requirements of the COA.  Unless there is excessive toxicity, enroll 30 
evaluable patients on arm 2 over a period of approximately 3 years. If there are 22 or more 
patients among 30 who are able to fully receive therapy, then the probability this would be the 
case is 87.1% if the true rate of feasibility were 80% while it is only 9.4% if the true rate of 
feasibility were 60%. Thus, obtaining 22 or more patients who are able to receive treatment out 

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of 30 would demonstrate a feasibility rate more consistent with 80% or greater as opposed to 
60% or less, and thus this would be a reasonable goal.
8.4
ACCRUAL TARGET AND STUDY DURATION
Twenty subjects were enrolled to achieve MTD and expanded cohorts in the initial objectives of 
this study.  To complete Arm 1 and 2 primary objectives added in Amendment G, up to 30 
subjects will be enrolled in each arm.  The study will allow for a small number of inevaluable 
patients of 5 in each arm (patients enrolled but who cannot receive cells, either due to physical 
deterioration or withdrawal of consent during cell growth, or inability to grow adequate cells).  
Thus the maximum accrual will be 90 patients (20 previously treated + 30 in each arm [60 total] 
+ 5 inevaluable patients in each arm [10 total].  Up to a total of 180 patients may be screened to 
allow for enrollment of up to 90 patients eligible to proceed to treatment.
8.5
FEASIBILITY
In addition to safety, establishing feasibility of the creation of anti-CD19 CAR T cells within 21 
days of the planned infusion for patients (who have had prior SCT or not) is an important goal of 
this study. Feasibility will be defined by the successful manufacturing and expansion of the anti-
CD19 CAR T cells to satisfy the targeted dose level and meet the requirements of the COA. If 
after the first 6 patients per stratum have been enrolled, more than 3 are unable to have adequate 
anti-CD19 CAR T cells produced (that meet COA for infusion), accrual to that stratum in the 
study will be terminated, since the upper 90% one-sided confidence interval about 3/6 is 79.9%; 
thus, it would be unlikely that the true feasibility rate is 80% or greater, which would be 
desirable. Each stratum will be evaluated independently for feasibility. COMPLETED
8.6
SECONDARY ENDPOINTS
8.6.1
Efficacy Analyses
The best overall response will be assessed at end of study, based upon the disease assessments 
recorded during the study visits, and reported by dose cohort in terms of confirmed CR/PR, 
unconfirmed CR/PR, Hematological Activity (ALL only), SD, or PD. The rate of relapse from 
CR prior to end of study will also be summarized by dose cohort only for the subjects who 
achieve confirmed CR during the study. The overall objective response rate (CR + PR) will be 
summarized by dose cohort. The duration of objective response will be summarized for those 
subjects who achieve objective response (CR + PR). In subjects who have a confirmed response, 
duration of response will be calculated from the first date of documented response until 
progression. Unconfirmed CR or PR is defined as the first documentation of response. Overall 
survival (OS) and progression-free survival (PFS) will be assessed by dose cohort. Progression-
free survival (PFS) will be measured from the start of the preparative regimen until the 
documentation of disease progression or death due to any cause, whichever occurs first. Overall 
survival (OS) will be determined as the time from the start of the preparative regimen until death.
8.6.2
Persistence of CD19-CAR T Cell Analyses
Peripheral blood, bone marrow aspirate, and CSF will be collected and separately analyzed for 
the presence of anti-CD19 CAR T cells. The percentage of all CD3+ cells in a sample that are 
positive by flow cytometry for anti-CD19 CAR containing T cells will be analyzed and reported 
as time from T cell infusion.

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8.6.3
Cytokines
Endogenously produced IFNg, TNFa, GM-CSF, IL-6 and IL-2 will be measured by ELISA and 
reported as change from baseline. The baseline will be defined as the cytokine level just prior to 
cell infusion. Cytokine levels will also be analyzed in the context of clinical response.
8.6.4
Monitoring for CD19+ Cells
Peripheral blood, bone marrow aspirates, and CSF will be collected and analyzed for the 
presence of CD19+ cells by flow cytometry. Any CD19+ cells will be analyzed to determine 
whether these are normal B cells or leukemia blasts. The percentage of CD19+ normal B cells 
and CD19+ leukemia blasts relative to total cells analyzed in the sample will be reported and 
analyzed in relation to time from cell infusion and compared to baseline. Baseline percentage of 
CD19+ normal B cells and CD19+ leukemia blasts will be defined as the result obtained from 
the same sample type (e.g. peripheral blood, CSF, or aspirate) prior to cell infusion. 
8.6.5
Toxicity in patients with CNS disease
Patients enrolled in Arm 1 or Arm 2 after approval of Amendment G who have CNS disease will 
be pooled and analyzed for descriptive reporting of efficacy and toxicity.  This data may be used 
in an exploratory fashion to develop new questions related to use of and indications for anti-
CD19 CAR gene engineered T cells.
8.6.6
Analyses of Cognitive Testing Data
To analyze the data for the secondary cognitive objective, we will use paired t-tests to examine 
the change in scores between the baseline and post-infusion evaluation to determine if the change 
is significantly different from zero.  For the exploratory cognitive objectives, we will use 
analysis of variance with repeated measures to examine change in scores between the baseline 
and the follow-up evaluations.  In addition, correlations or analyses of variance will be computed 
to examine the relationship between cognitive scores and cytokine levels.
8.6.7
Analyses of Cardiology Studies
The effect of CAR cell therapy on cardiac function will be compared with baseline data.  
Changes in cardiac function will be correlated with levels of inflammatory markers and 
biomarkers.  Further analysis depends on the type of data obtained.
Data will be analyzed using SAS 9.4 (Cary, NC). Data will be expressed as mean (standard 
deviation).  In all analyses, the conventioQDOĮ-level of 0.05 will be used for significance testing.
9
HUMAN SUBJECTS PROTECTIONS 
9.1
RATIONALE FOR SUBJECT SELECTION
The patients to be entered in this protocol have B cell malignancies which are refractory to 
standard therapy, and limited life expectancies. Subjects from both genders and all racial/ethnic 
groups are eligible for this study if they meet the eligibility criteria. To date, there is no 
information that suggests that differences in drug metabolism or disease response would be 
expected in one group compared to another. Efforts will be made to extend accrual to a 
representative population, but in this preliminary study, a balance must be struck between patient 
safety considerations and limitations on the number of individuals exposed to potentially toxic 
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of clinical research on the other hand. If differences in outcome that correlate to gender or to 
ethnic identity are noted, accrual may be expanded or a follow-up study may be written to 
investigate those differences more fully.
9.2
PARTICIPATION OF CHILDREN
The age range of patients eligible for this trial is greater than 1 year of age but less than or equal 
to 30 years of age. Physicians, nurses, and multidisciplinary support teams of the POB, NCI and 
Clinical Center will provide patient care. The staff of the POB has expertise in the management 
of children with complex oncologic disorders and complications of therapy. Full pediatric 
support and subspecialty services are available at the NIH Clinical Center.
9.3
EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS
The experimental treatment has a chance to provide clinical benefit though this is unknown. The 
risks in the investigational portion of this treatment are detailed in Section 1.2.5. The goal of this 
study is to improve upon the number of patients who may benefit from adoptive cell therapy by 
using patients’ own transduced T-cells without the need to identify anti-tumor T-cells uniquely 
from each patient as was required in many cancer therapy protocols. The success of this effort 
cannot be predicted at this time. Because all patients in this protocol have B cell malignancies 
without curative options and limited life expectancies the potential benefit is thought to outweigh 
the potential risks. It is anticipated that this study will provide scientific information relevant to 
tumor immunotherapy. Consequently, participation of children involves greater than minimal 
risk, but presents the prospect for direct benefit for patients who are minors (45CFR 46.405).
9.3.1
Risks of Anti-CD19 CAR T Cells
The risk profile of anti-CD19 CAR cells in children is not fully known at this time. DLT on the 
dose-escalation phase of our study was CRS.  In one clinical trial in adults in the Surgery 
Branch, NCI two events of reversible grade 4 somnolence and one of grade 4 keratinize were 
attributed to the anti-CD19 CAR-transduced cells. One each event of grade 2 left ventricular 
dysfunction and grade 1 pulmonary hypotension were attributed to the anti-CD19 CAR-
transduced cells and IL-2, and both of these toxicities resolved. Three patients on this study were 
intubated and one patient underwent dialysis, for which the attribution is unknown. Aldesleukin 
will not be used in the present study.
Section 1.2.5 discusses the potential risks of this investigational therapy, including risk of 
chemotherapy, risk of autoimmunity, risk of the gene therapy component, and risk of cytokine 
storm. Transient, reversible mild effects have been observed with the administration of fresh 
cells, including chills, fever, rigors, diaphoresis, anorexia, nausea, diarrhea, headache and 
myalgias. As described in Section 1.2.4 recently reported toxicities associated with similar 
studies using anti-CD19 CAR T cells included grade 3 tumor lysis syndrome, and reversible 
laboratory abnormalities, thrombocytopenia, neutropenia, and hypogammaglobulinemia. 
9.3.2
Risk of Apheresis 
Apheresis is a safe procedure that is routinely performed in healthy children and adults. 
Participants will be closely monitored and procedures to minimize risks and prevent side effects 
are incorporated into all aspects of the protocol. The POB, DTM, and NIH CC have broad 

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expertise to adequately manage side effects, including in pediatric apheresis subjects. The 
potential risks of apheresis in this trial are as follows:
a.
The most common side effects of apheresis are pain and bruising at IV sites. A central 
venous catheter may be required. Possible side effects include pain, bleeding, bruising, 
infection, thrombosis, vascular perforation, and risks associated with the sedation used 
for placement.
b. During apheresis, mild side effects from citrate anticoagulant are common and include 
chills, numbness and tingling ("pins and needles"), anxiety, muscle cramps, and nausea. 
More serious side effects due to citrate-induced hypocalcemia are uncommon and include 
low blood pressure, seizures, weakness, and tetany. Citrate reactions rapidly resolve 
when the collection is slowed down or stopped. Prophylactic IV CaCl2 and MgSO4 
infusions may be administered to donors deemed to be at high risk of citrate toxicity. 
Risks of parenteral calcium and magnesium include extravasation necrosis and 
cardiovascular effects including bradycardia and blood pressure changes. However, side 
effects are unlikely given the low rate of infusion and use of large bore catheters for 
apheresis.
c.
Transient mild thrombocytopenia is common after apheresis, but bleeding is unlikely.
d. Dilutional anemia occurs during apheresis, but this is unlikely to be clinically significant. 
e.
Side effects of blood draws include pain and bruising, lightheadedness, and rarely, 
fainting. 
9.3.3
Risks of the chemotherapy regimens and supportive therapies
The chemotherapy agents and supportive medications used in this study are FDA approved 
agents with well-known toxicity profiles. Refer to Section 11 for summary of toxicities.
9.4
RISKS/BENEFITS ANALYSIS
Patients will be monitored frequently as both inpatients and outpatients and side effects will be 
treated promptly. These measures will help to mitigate the potential risks observed with the 
preparative chemotherapy regimen and the cell administration. The enrollment profile and dose 
escalation rules will help to ensure that patients are not exposed to unacceptable risk as a safe 
dose level is determined. 
Patients on this study may directly benefit from participation. It is anticipated that this study will 
provide scientific information relevant to tumor immunotherapy. Consequently, participation 
involves greater than minimal risk, but presents the prospect for direct benefit for patients who 
are minors (45CFR 46.405).
9.5
CONSENT AND ASSENT PROCESS AND DOCUMENTATION
Pediatric patients who meet the thorough screening for protocol eligibility will have apheresis 
conducted to obtain PBMC on this study once the protocol consent is signed. The investigational
nature and research objectives of this trial, the procedures and treatments involved and their 
attendant risks and discomforts and potential benefits, and alternative therapies will be carefully 
explained to the patient’s parents/guardians, who are asked to review it and to ask questions prior 
to agreeing to participate in this protocol. The patient and his/her parent(s) or guardian are 

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reassured that participation on trial is entirely voluntary and that they can withdraw or decide 
against treatment at any time without adverse consequences. The investigators are requesting a 
waiver from the IRB to allow only one parent to sign the informed consent to enter a child on the 
protocol. Because many patients must travel to the NIH from long distances at substantial 
expense, requiring both parents to be present for the consent process could be a financial 
hardship for many families. When necessary, the protocol will be reviewed by telephone with the 
other parent and their approval will be verified. When guardianship status of the child is 
uncertain, a social worker will be asked to investigate and, if necessary, seek documentation of 
custody status. The research nurse, principal investigator, associate investigator, or clinical 
associate is responsible for obtaining written consent from the patient. Where deemed 
appropriate by the clinician and the child’s parents or guardian, the child will also be included in 
all discussions about the trial and verbal assent will be obtained. The parent or guardian will sign 
the designated line on the informed consent attesting to the fact that the child has given assent.
9.5.1
Screening Sample Consent
The screening consent will initially be used to test samples for CD19 expression, prior to 
complete eligibility screening and enrollment. Telephone consent may be employed in order to 
screen outside samples from prospective subjects for CD19 expression. In such cases, a protocol 
investigator will review the Screening Consent form by telephone. The consent/assent signatures 
will be witnessed and a copy will be faxed and the original sent by mail to the PI. Prospective 
subjects who consent to send such samples for outside testing will NOT be registered with the 
NCI Central Registration Office unless they are subsequently enrolled on protocol. Subjects and 
their referring medical team will be notified of the results and records will be maintained with 
the protocol research files. The Principal Investigator, Associate Investigator, or their designee 
will be available to answer all questions from prospective patients or parents/guardians. 
The attached informed consent documents contain all elements required for consent.
9.5.2
Consent for minors when they reach the age of majority
When a pediatric subject reaches age 18, continued participation (including ongoing interactions 
with the subject or continued analysis of identifiable data) requires consenting of the now adult 
with the standard protocol consent document to ensure legally effective informed consent has 
been obtained. We request a waiver of informed consent for those individuals who became lost 
to follow up during their participation in the research study or who, prior to the approval of 
Amendment P were taken off study prior to reaching the age of majority.
Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d):
(1) The research involves no more than minimal risk to the subjects.
a.
Analysis of samples and data from this study involves no additional risks to 
subjects.
(2) The waiver or alteration will not adversely affect the rights and welfare of the 
subjects.
a.
Retention of these samples or data does not affect the welfare of subjects. 
(3) The research could not practicably be carried out without the waiver or alteration.
a.
Considering the length of time between the minor’s last contact with the 
research team and their age of majority, it will likely be very difficult to locate 

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them again. A significant reduction in the number of samples analyzed is 
likely to impact the quality of the research. 
(4) Whenever appropriate, the subjects will be provided with additional pertinent 
information after participation.
9.5.3
Informed consent of non-English speaking subjects
If there is an unexpected enrollment of a research participant for whom there is no translated 
extant IRB approved consent document, the principal investigator and/or those authorized to 
obtain informed consent will use the Short Form Oral Consent Process as described in MAS 
Policy M77-2, OHSRP SOP 12, 45 CFR 46.117 (b) (2), and 21 CFR 50.27 (b) (2)).  The 
summary that will be used is the English version of the extant IRB approved consent document.  
Signed copies of both the English version of the consent and the translated short form will be 
given to the subject or their legally authorized representative and the signed original will be filed 
in the medical record.
Unless the PI is fluent in the prospective subject’s language, an interpreter will be present to 
facilitate the conversation (using either the long-translated form or the short form). Preferably 
someone who is independent of the subject (i.e., not a family member) will assist in presenting 
information and obtaining consent. Whenever possible, interpreters will be provided copies of 
the relevant consent documents well before the consent conversation with the subject (24 to 48 
hours if possible). 
We request prospective IRB approval of the use of the short form process for non-English 
speaking subjects and will notify the IRB at the time of continuing review of the frequency of the 
use of the Short Form.
10 PHARMACEUTICAL INFORMATION  
10.1 RETROVIRAL VECTOR CONTAINING THE ANTI-CD19 CAR GENE 
Cells will be administered intravenously through a free-flowing IV at dose 1 X 106 anti-CD19 
CAR T cells/kg (+/- 20%) as described in Section 3.10.4.
The retroviral vector supernatant (PG13-CD19-H3) encoding a chimeric antigen receptor (CAR) 
directed against the B cell antigen, CD19, was prepared and preserved following cGMP 
conditions in the Surgery Branch Vector Production Facility (SBVPF). The retroviral vector 
utilizes the MSGV1 retroviral vector backbone and consists of 7026 bps including the 5' LTR 
from the murine stem cell virus (promoter), packaging signal including the splicing donor (SD) 
and splicing acceptor sites, FMC63-based (anti-CD19 FMC63-28) CAR protein containing a 
signal peptide (human GM-CSF receptor), FMC63 light chain variable region (FMC63 VL), 
linker peptide, FMC63 heavy chain variable region (FMC63 VH), CD28 (hinge, transmembrane 
and cytoplasmic region), and TCR zeta (cytoplasmic region), followed by the murine stem cell 
virus 3'LTR. The physical titer will be determined by RNA dot blot according to sponsor 
certificate. 
The supernate will be stored at SBPVF upon the completion of production at –80° C or shipped 
on dry ice and stored at Cryonix, Rockville, MD. Both storage facilities are equipped with 
around-the-clock temperature monitoring. Upon request, supernatant will be delivered on dry ice 
to be used in in vitro transductions of PBL. There will be no re-use of the same unit of supernate 

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for different patients. Retroviral titer has been shown to be stable after immediate thawing and 
immediate administration (coating the tissue culture wells previously coated with Retronectin). 
Handling of the vector should follow the guidelines of Biosafety Level-2 (BSL-2). The specific 
guidelines for Biosafety Level-2 (BSL-2) can be viewed at http://bmbl.od.nih.gov/sect3bsl2.htm.
10.2 FLUDARABINE
10.2.1 Description 
(Please refer to package insert for complete product Information) Fludarabine phosphate is a 
synthetic purine nucleoside that differs from physiologic nucleosides in that the sugar moiety is 
arabinose instead of ribose or deoxyribose. Fludarabine is a purine antagonist antimetabolite. 
10.2.2 How Supplied 
It will be purchased by the NIH Clinical Pharmacy Department from commercial sources. 
Fludarabine is supplied in a 50 mg vial as a fludarabine phosphate powder in the form of a white, 
lyophilized solid cake.
10.2.3 Stability 
Following reconstitution with 2 mL of sterile water for injection to a concentration of 25 mg/ml, 
the solution has a pH of 7.7. The fludarabine powder is stable for at least 18 months at 2-80C; 
when reconstituted, fludarabine is stable for at least 16 days at room temperature. Because no 
preservative is present, reconstituted fludarabine will typically be administered within 8 hours. 
Specialized references should be consulted for specific compatibility information. Fludarabine is 
dephosphorylated in serum, transported intracellularly and converted to the nucleotide 
fludarabine triphosphate; this 2-fluoro-ara-ATP molecule is thought to be required for the drug’s 
cytotoxic effects. Fludarabine inhibits DNA polymerase, ribnucleotide reductase, DNA primase, 
and may interfere with chain elongation, and RNA and protein synthesis.
10.2.4 Storage 
Intact vials should be stored refrigerated (2-8qC).
10.2.5 Administration 
Fludarabine is administered as an IV infusion in an appropriate solution over 30 minutes. To 
prevent undue toxicity the dose will be based on BSA (25 mg/m2/dose) 
10.2.6 Toxicities
At doses of 25 mg/m2/day for 3 days, the primary side effect is myelosuppression; however, 
thrombocytopenia is responsible for most cases of severe and life-threatening hematologic 
toxicity. Serious opportunistic infections have occurred in CLL patients treated with fludarabine. 
Hemolytic anemia has been reported after one or more courses of fludarabine with or without a 
prior history of a positive Coomb’s test; fatal hemolytic anemia has been reported. In addition, 
bone marrow fibrosis has been observed after fludarabine therapy. Other common adverse 
effects include malaise, fever, chills, fatigue, anorexia, nausea and vomiting, and weakness. 
Irreversible and potentially fatal central nervous system toxicity in the form of progressive 
encephalopathy, blindness, and coma is only rarely observed at the currently administered doses 
of fludarabine. More common neurologic side effects at the current doses of fludarabine include 
weakness, pain, malaise, fatigue, paresthesia, visual or hearing disturbances, and sleep disorders. 

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Adverse respiratory effects of fludarabine include cough, dyspnea, allergic or idiopathic 
interstitial pneumonitis. Tumor lysis syndrome has been rarely observed in fludarabine treatment 
of CLL. 
10.3 CYCLOPHOSPHAMIDE 
(Refer to FDA-approved package insert for complete product information)
Description: 
Cyclophosphamide is a nitrogen mustard-derivative alkylating agent. Following conversion to 
active metabolites in the liver, cyclophosphamide functions as an alkyating agent; the drug also 
possesses potent immunosuppressive activity. The serum half-life after IV administration ranges 
from 3-12 hours; the drug and/or its metabolites can be detected in the serum for up to 72 hours 
after administration. 
10.3.1 How Supplied 
Cyclophosphamide will be obtained from commercially available sources by the Clinical Center 
Pharmacy Department. 
10.3.2 Stability
Following reconstitution as directed with sterile water for injection, cyclophosphamide is stable 
for 24 hours at room temperature or 6 days when kept at 2-80C. 
10.3.3 Administration 
It will be diluted in an appropriate solution and infused over one hour. The dose will be based on 
the patient’s body weight, at 900 mg/m2/dose after fludarabine infusion on Day -2.
10.3.4 Toxicities 
Hematologic toxicity occurring with cyclophosphamide usually includes leukopenia and 
thrombocytopenia. Anorexia, nausea and vomiting, rash and alopecia occur, especially after 
high-dose cyclophosphamide; diarrhea, hemorrhagic colitis, infertility, and mucosal and oral 
ulceration have been reported. Sterile hemorrhagic cystitis occurs in about 20% of patients; 
severity can range from microscopic hematuria to extensive cystitis with bladder fibrosis. 
Although the incidence of hemorrhagic cystitis associated with cyclophosphamide appears to be 
lower than that associated with ifosfamide, mesna (sodium 2-mercaptoethanesulfonate) has been 
used prophylactically as a uroprotective agent in patients receiving cyclophosphamide. 
Prophylactic mesna is not effective in preventing hemorrhagic cystitis in all patients. Patients 
who receive high dose cyclophosphamide may develop interstitial pulmonary fibrosis, which can 
be fatal. Hyperuricemia due to rapid cellular destruction may occur, particularly in patients with 
hematologic malignancy. Hyperuricemia may be minimized by adequate hydration, 
alkalinization of the urine, and/or administration of allopurinol. If allopurinol is administered, 
patients should be watched closely for cyclophosphamide toxicity (due to allopurinal induction 
of hepatic microsomal enzymes). At high doses, cyclophosphamide can result in a syndrome of 
inappropriate antidiuretic hormone secretion; hyponatremia with progressive weight gain without 
edema occurs. At high doses, cyclophophamide can result in cardiotoxicity. Deaths have 
occurred from diffuse hemorrhagic myocardial necrosis and from a syndrome of acute
myopericarditis; in such cases, congestive heart failure may occur within a few days of the first 

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dose. Other consequences of cyclophosphamide cardiotoxicity include arrhythmias, potentially 
irreversible cardiomyopathy, and pericarditis. Other reported adverse effects of 
cyclophosphamide include headache, dizziness, and myxedema; faintness, facial flushing, and 
diaphoresis have occurred following IV administration. Mesna (sodium 2-
mercaptoethanesulphonate; given by IV injection) is a synthetic sulfhydryl compound that can 
chemically interact with urotoxic metabolites of cyclophosphamide (acrolein and 4-
hydroxycyclophosphamide) to decrease the incidence and severity of hemorrhagic cystitis. 
10.4 MESNA 
(Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891) (Please refer to the FDA-
approved package insert for complete product information)
10.4.1 Description 
Mesna will be obtained commercially by the Clinical Center Pharmacy Department and is 
supplied as a 100 mg/ml solution. 
10.4.2 Storage 
Intact ampoules are stored at room temperature. 
10.4.3 Stability 
Diluted solutions (1 to 20 mg/mL) are physically and chemically stable for at least 24 hours 
under refrigeration. Mesna is chemically stable at room temperature for 48-72 hours in D5W, 48-
72 hour in D5W/0.45% NaCl, or 24 hours in 0.9% sodium chloride. 
10.4.4 Administration
Dilute to concentrations less than or equal to 20 mg mesna/ml fluid in D5W or normal saline and 
to be administered intravenously as a continuous infusion. 
10.4.5 Toxicities
Toxicities include nausea, vomiting and diarrhea. 
10.5 IFOSFAMIDE
10.5.1 Description
Ifosfamide for injection is supplied in single-dose vials for constitution and administration by 
intravenous infusion, each containing 1 gm or 3 gms of sterile ifosfamide.  Ifosfamide is a 
chemotherapeutic agent chemically related to the nitrogen mustards and a synthetic analog of 
cyclophosphamide.
10.5.2 Supplied
Ifosfamide is commercially available and will be supplied by the Clinical Center Pharmacy 
Department.  Please refer to the package insert for full details.
10.5.3 Storage and stability
Preparation for Intravenous Administration:  Injections are prepared for parenteral use by add 
Sterile Water for Injection, USP or Sterile Bacteriostatic Water for Injection, USP to the vial and 
shaking to dissolve.

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10.5.4 Preparation for Intravenous Administration
Injections are prepared for parenteral use by add Sterile Water for Injection, USP or Sterile 
Bacteriostatic Water for Injection, USP to the vial and shaking to dissolve.
10.5.5 Toxicities
The dose limiting toxicities are myelosuppression and urotoxicity. Dose fractionation, vigorous 
hydration and a protector such as mesna can significantly reduce the incidence of hematuriar.  
Other significant toxicities include alopecia, nausea, vomiting and central nervous toxicities. 
10.6 ETOPOSIDE (VEPESID, VP-16)
10.6.1 Availability
Commercially available and will be supplied by the Clinical Center Pharmacy.
10.6.2 Toxicity
Includes nausea, vomiting, stomatitis, diarrhea, neutropenia, thrombocytopenia, and alopecia. 
Secondary AML has been associated with etoposide. Bradycardia and hypotension are 
sometimes observed with etoposide administration.
10.7 ACETAMINOPHEN (TYLENOL)
Will be given as a pre-medication. This agent will be provided by the clinical center pharmacy. 
Please refer to the package insert for complete pharmaceutical information on this product.
10.8 DIPHENHYDRAMINE (BENADRYL)
Will be given as a pre-medication IV over 10-15 minutes This agent will be provided by the 
Clinical Center Pharmacy. Please refer to the package insert for complete pharmaceutical 
information on this product.
10.9 INTRATHECAL CHEMOTHERAPY 
The following agents will be provided by the Clinical Center Pharmacy. Please refer to the 
package inserts for complete pharmaceutical information on these products.
10.10HYDROCOTISONE (CORTEF, SOLU-CORTEF) NCS# 010483
10.10.1
Source and Pharmacology
Synthetic steroid akin to the natural adrenal hormone, cortisol. It binds with steroid receptors on 
nuclear membrane, impairs cellular mitosis and inhibits protein synthesis. It is phase specific, 
killing cells primarily during S phase. It has a catabolic effect on proteins and alters the kinetics 
of peripheral blood leukocytes. It is excreted in the urine and catabolized in the liver.
10.10.2
Toxicity
The following toxicities may occur when hydrocortisone is given intrathecally: Nausea and 
vomiting, headache, pleocytosis, fever, somnolence, meningismus, learning disability, 
leukoencephalopathy.
10.10.3
Formulation and Stability

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Available as 100mg, 250mg, 500mg, and 1000mg vials for aqueous injection. In powder form, 
the drug is stable for 2 years at room temperature. After reconstitution, it is stored at room 
temperature, and should be discarded after 3 days. INTRATHECAL ADMINISTRATION: IT 
hydrocortisone should be further diluted with physiologic buffered diluents (lactated Ringer's, 
0.9% sodium chloride, Elliott’s B solution). Do not use Bacteriostatic Water to reconstitute for 
IT use, use only preservative free solutions.
10.10.4
Guidelines for Administration
Hydrocortisone will be given by intrathecal administration in an age-specified dose and will be 
mixed with the other agents to the age-specified volume. (Section 4.1)
10.10.5
Supplier
Commercially Available.
10.11CYTARABINE (CYTOSINE ARABINOSIDE, ARAC, CYTOSAR) NSC# 063878
10.11.1
Source and Pharmacology
Deoxycytidine analogue which is metabolized to ARA-CTP, a substance which inhibits DNA 
polymerase. It is S phase specific, and thus affects DNA synthesis. Rapidly catabolized by 
hepatic cytidine deaminases to AraU.
10.11.2
Toxicity
The following toxicities may occur when cytarabine is given intrathecally: Nausea, vomiting, 
headache, pleocytosis, arachnoiditis, rash, fever, somnolence, meningismus, convulsions, 
paresis, myelosuppression, ataxia, learning disability. CNS impairment may not be fully 
reversible. No further cytarabine will be administered if there is CNS toxicity (any grade) 
deemed related to cytarabine.
IV Administration: Acute DLT consists of severe leukopenia and thrombocytopenia.  Nausea 
and vomiting may be dose limiting at higher doses.  Other adverse reactions include diarrhea, 
immunosuppression, anorexia, stomatitis, oral ulceration, flu-like syndrome, fever, hepatic 
dysfunction, and alopecia. At high doses, as in this protocol, keratoconjunctivitis, dermatitis, and 
central nervous system toxicity (e.g., ataxia, somnolence, coma, dysarthria) may occur. 
Occasionally, the CNS impairment is not fully reversible. Renal impairment will enhance 
toxicity.  In the event signs of CNS toxicity, the cytarabine will be interrupted and the M.D. 
notified. In the event of signs of CNS toxicity the 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.
10.11.3
Formulation and Stability
A freeze-dried powder available in 100mg, 500mg, 1g and 2g vials. The unreconstituted form of 
the drug is stable at room temperature for at least 2 years. 
INTRAVENOUS ADMINISTRATION: The manufacturer supplies the drug as unreconstituted 
lyophilized powder in vials. With each vial, an ampule of bacteriostatic water for injection 
containing 0.945% benzyl alcohol is provided.  To prepare high dose (2 gm/m2) cytarabine 
intravenous infusions, each vial of drug should be reconstituted with 2 mL (100 mg) or 10 mL 
(500 mg) of Sterile Water for injection USP (i.e. not the manufacturer-provided diluent) which 

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contains no preservative. Begin each cytarabine dose 3.5-4 hours after completion of the 
preceding fludarabine dose.
INTRATHECAL ADMINISTRATION: IT cytarabine should be further diluted with physiologic 
buffered diluents (lactated Ringer's, 0.9% sodium chloride, Elliott’s B solution). Do not use 
Bacteriostatic Water to reconstitute for IT use, use only preservative free solutions.
10.11.4
Guidelines for Administration
Cytarabine will be given by intrathecal administration in an age-specified dose and will be mixed 
with the other agents to the age-specified volume (Section 4.1.2). If emesis occurs it usually 
occurs 4-6 hours after the intrathecal administration. If the patient has had emesis with prior 
intrathecal chemotherapy, premedications should be considered.
10.11.5
Supplier
Commercially available. See package insert for further information.
10.12METHOTREXATE
10.12.1
Source and Pharmacology
Methotrexate is an antimetabolite and antifolate agent with antineoplastic and 
immunosuppressant activities. Methotrexate binds to and inhibits the enzyme dihydrofolate 
reductase, resulting in inhibition of purine nucleotide and thymidylate synthesis and, 
subsequently, inhibition of DNA and RNA syntheses. Methotrexate also exhibits potent 
immunosuppressant activity although the mechanism(s) of actions is unclear.
10.12.2
Toxicity
The following toxicities may occur when given intrathecally: occasional headache, dizziness, 
tiredness, blurred vision or loss of balance for a few hours. Up to 15% of children may develop 
neurological changes including changes in level of consciousness, abnormal movements or 
confusion, very rarely leucoencephalopathy. 
10.12.3
Formulation and Stability
Methotrexate Sodium Injection USP is available for single use only in 20 mg and 1 gram vials, 
and will be diluted according to TIT in Section 4.1. Dilutions should be used within 24 hours if 
kept at room temperature. Unused solution should be discarded after this time in order to avoid 
risk of microbial contamination. INTRATHECAL ADMINISTRATION: IT methotrexate should 
be further diluted with physiologic buffered diluents (lactated Ringer's, 0.9% sodium chloride, 
Elliott’s B solution). Do not use Bacteriostatic Water to reconstitute for IT use, use only 
preservative free solutions.
10.12.4
Preparation
The three agents will be filtered and mixed in sterile preservative free 0.9% sodium chloride 
immediately (less than 4 hrs) prior to administration. The final concentration should be no 
greater than 1.5mg/ml for MTX and HDC, and 3 mg/ml for Ara-C.
10.12.5
Guidelines for Administration

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Methotrexate will be given by intrathecal administration in an age-specified dose and will be 
mixed with the other agents to the age-specified volume (Section 4.1.2). If emesis occurs it 
usually occurs 4-6 hours after the intrathecal administration. If the patient has had emesis with 
prior intrathecal chemotherapy, premedications should be considered.
10.12.6
Supplier
Commercially available. See package insert for further information.
10.13ALLOPURINOL
Allopurinol will be obtained by the Clinical Center Pharmacy Department. It will be used as 
prophylaxis or treatment of pediatric patients with or at high risk for Tumor Lysis Syndrome. 
Dosage is approximately 100 mg/m2/dose po TID (maximum dose 200 mg TID). The most 
common side effects include hypersensitivity, rash, nausea, vomiting, renal insufficiency, and 
hepatic dysfunction. Allopurinol should be stopped immediately if rash develops. Consult the 
package insert for a complete list of all side effects.
10.14TOCILIZUMAB (ANTI-IL6R MAB)
10.14.1
Description  
Tocilizumab is an anti-human IL-6R antibody that binds specifically to cell associated and 
soluble IL-6 receptors, thus blocking IL-6 signaling.  IL-6 is involved in T cell activation, acute 
phase protein synthesis and stimulation of hematopoietic precursors.
10.14.2
Storage and Stability 
Store unused vials refrigerated between 2 and 8 degrees C (36 and 46 degrees F). Do not freeze 
and protect from light in original package3. Diluted tocilizumab solutions may be stored at 2 to 8 
degrees C (36 to 46 degrees F) for up to 24 hours. Protect the diluted solution from light. Discard 
any unused portion remaining in the vial because the product does not contain preservatives1.
Fully diluted tocilizumab solutions are compatible with polypropylene, polyethylene, and 
polyvinyl chloride infusion bags, or with polypropylene, polyethylene, and glass infusion 
bottles1.
10.14.3
Administration
Allow tocilizumab infusion to reach room temperature prior to infusion. Administer over 60 
minutes using an infusion set. Do not administer as an IV push or bolus1. Tocilizumab should not 
be infused with other drugs in the same IV line due to a lack of physical or biocompatibility 
studies1.
10.14.4
Dosage 
4-12 mg/kg, Single dose should not exceed 800 mg.
10.14.5
Toxicities  
The major toxicity associated with Tocilizumab is immunosuppression, the risk of which is 
further increased when the agent is given with other immunosuppressants including
corticosteroids and cytotoxic chemotherapy.  The risks and benefits of treatment with 
tocilizumab should be carefully considered prior to initiating therapy in patients with chronic or 

79
Abbreviated Title: Phase I Anti-CD 19 CAR
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recurrent infection.  Patients should be closely monitored for the development of signs and 
symptoms of infection during and after treatment with tocilizumab, including the possible 
development of tuberculosis in patients who tested negative for latent tuberculosis infection prior 
to initiating therapy3.
10.15FILGRASTIM (NEUPOGEN)
10.15.1
Description  
Filgrastim is a human granulocyte colony-stimulating factor (G-CSF), produced by recombinant 
DNA technology.  Neupogen is a sterile, clear, colorless, preservative-free liquid.  See package 
insert for complete details.
10.15.2
Storage
Filgrastim should be stored at 2 C to 8 C (36 F to 46 F) and should not be frozen.
10.15.3
Administration
Filgrastim is administered intravenously or subcutaneously (under the skin) for 6 to 14 days. 
Filgrastim vials should not be shaken since the drug may be damaged, and bubbles may form 
that can prevent some of the drug from being drawn up into the syringe at the time of injection.
Toxicities
The most common side effects are nausea, vomiting, bone pain, fever, fatigue, hair loss, and 
diarrhea. Headache, weight loss, shortness of breath, mouth sores, and rash also occur. Uric acid, 
lactate dehydrogenase, and alkaline phosphatase levels may rise and spontaneously return to 
normal levels

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11 APPENDICES
11.1 APPENDIX A -PERFORMANCE STATUS CRITERIA

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
11.2 APPENDIX B: STUDY CALENDAR
Procedure
Screening 
(within 14 
days, unless 
specified)
Day -
11
Baseline 
(within 24 
hours of 
starting 
chemo 
unless 
specified)
Chemothera
py (see B1, 
B2 and B3)
Prior to 
Cell 
infusion 
(within 24 
hrs)
Day 0 
(Day of 
cell 
infusion)
During Hospitalization
Day 
1
Day 
2
Day 
3
Day 
4-7
Every 
2-4
days 
Until 
D/C
On Treatment 
28(±4) days after cell 
infusion, then 3, 6, 12 
mos, then Q 6-12 mos) 
Off 
treatment  
History and PE
X
X
X
X
X
X
X
X
X
Xi
X L
Vital signs
X
X
X
Xg
X
X
X
X
X
X
Performance Score
X
X
X
X
X
X
X
X
X
X
X
Cognitive Evaluation
X
Xn
Neuro symptom checklist
Xm
Height and weight
X
WT only
WT only 
WT only
WT only
Labs 
x CBC with diff and 
platelets
X
X
X
X
X
X
X
x PT/aPTT
X
Xe
x Chem 20b
X
X
X
X
X
X
X
x Thyroid Panel
Xe
Xo
x CD19 staining of 
malignant cells by 
immunohistochemistry 
or flow cytometry 
(anytime)
X
x Peripheral blood CD3 
count
X
x HIV, HbsAG, anti-
HCV, EBV
Xa
x IgG level
Xk
x Urine analysis, culture if 
indicated
X
X
X
X
x ȕ-HCG pregnancy test 
on all women of child-
bearing potential
Xc
Xf
ECG 
X
X
X

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
Procedure
Screening 
(within 14 
days, unless 
specified)
Day -
11
Baseline 
(within 24 
hours of 
starting 
chemo 
unless 
specified)
Chemothera
py (see B1, 
B2 and B3)
Prior to 
Cell 
infusion 
(within 24 
hrs)
Day 0 
(Day of 
cell 
infusion)
During Hospitalization
Day 
1
Day 
2
Day 
3
Day 
4-7
Every 
2-4
days 
Until 
D/C
On Treatment 
28(±4) days after cell 
infusion, then 3, 6, 12 
mos, then Q 6-12 mos) 
Off 
treatment  
ECHO, cardiac MRI or 
MUGA 
X
X
X
Ophthalmologic Exam
Xj
Xj
Correlative Research 
Studies 
x
Apheresis 
X
X (DAY 28 only)
X
x
Research Sampling
Disease Evaluation 
x CT 
chest/abdomen/pelvis 
(or other appropriate 
imaging)
X
x Lumbar punctured
X
Xe
X
x Bone marrow 
aspirate/biopsy
X
Xe
X
Treatment Regimen
x
Anti-CD19-CAR 
transduced cells
Xl
Response Evaluation
X
X
X
X
X
Adverse Events
X
X
Concomitant Medications
X
X
Research labs
X
X
X
X
X
X
X
X
X
aWithin 4 weeks prior to enrollment
bLaboratory evaluation to include; 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); creatinine clearance may be performed if the serum 
creatinine is elevated.
c within 1 week prior to enrollment
d for patients with aggressive NHL, ALL, history of leptomeningeal disease, or signs/symptoms of leptomeningeal involvement
eWithin 14 days of starting chemo preparative regimen
f Within 72 hours of starting chemo preparative regimen
g Monitor every q15 min X 1 hr, q 30 min for 2nd hour, then q 1 hr for 2 more hrs or until stable, then routinely.

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h For cell acquisition for product development
i Physical examinations annually for 5 years. After 5 years, health status data will be obtained from surviving patients via telephone contact or mailed questionnaires. The long term follow up period for 
retroviral vectors is 15 years. Persistence of CAR gene transduced cells: prior to cells, 1 week, 4-6 weeks, 3 and 6 months post cell infusion; RCR PCR prior to cell infusion; and at 3, 6, and 12 mos post 
cell infusion
j within 14 days of preparative regimen
kevery 3-6 weeks post infusion.
l cells may be infused up to: Arm 1 = 72 hours following the time of the planned infusion. Otherwise, cells may be frozen and infused up to 7 days following the time of the planned infusion, or attempts 
to infuse at a later time may be made per Section 3.5. Arm 2 = 21 days (cryopreserved) following the time of the planned infusion, or attempts to infuse at a later time may be made per Section 3.5
m on or about Day 14 ± 1 day
n at 3 month visit only (± 4 weeks)
o Thyroid panel only before day 28
LOff treatment after day 28. 
x
Off treatment all studies will be done as per day 28 (+/-4).
x
If no response (progressive disease) then no further clinical follow-up required expect for Section 3.12.5, which will be followed 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 discretion and documentation collected.  All will be collected until relapse or bone marrow transplantation. Section 3.12.5, which will be followed 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.

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
11.2.1 Appendix B 1: Arm 1
Procedure
Day prior to Cells Infusion 
-4, -3
-2
-1
Fludarabine: 25 
mg/m2/day IV
X
X
REST
Cyclophosphamide: 
900 mg/m2/day IV
X
REST
Mesna 540 
mg/m2/day IV
X
REST
11.2.2 Appendix B 2: Arm 2: FLAG
Procedure
Day prior to Cells Infusion
Day prior to 
chemotherapy
-12, -11, -10, 
-9, -8, 
From last day of 
chemo to Cell 
Infusion (DAY 0) [at 
least 2 days]
Fludarabine: 25 
mg/m2/day IV
X
REST
Cytarabine 2000 
mg/m2/day IV
X
REST
Filgrastim 
μg/kg/dose 
subcutaneously
X
X
Daily until 
ANC>1000 on 2 
consecutive days
11.2.3 Appendix B 3: Arm 2: Etoposide/ifosfamide
Procedure
Day prior to Cells Infusion
Day prior to 
chemotherapy
-12, -11, -10, -9, 
-8, 
From last day of 
chemo to Cell 
Infusion (DAY 0) [at 
least 2 days]
Etoposide 100 
mg/m2/day IV
X
REST

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Abbreviated Title: Phase I Anti-CD 19 CAR
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Ifosfamide 1,800 
mg/m2/day and 
Mesna 360 
mg/m2/dose IV
X
REST
Mesna 360 
mg/m2/dose IV 
(then PO or IV)
X (after 
ifosfamide, then 
every 3 hours X 
6 doses)
REST
Physical Exam
X
Twice weekly
Twice weekly
Chem 20:
X
Twice weekly
Twice weekly
CBC with 
differential
X
Twice weekly
Twice weekly

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
11.3 APPENDIX C: GRADING AND TREATMENT OF ACUTE GVHD
Acute GVHD
Skin
Liver
GI Tract
Upper GI
Stage
Rash
Bilirubin
Diarrhea (ml/day)
0
No rash
< 2 mg/dL
< 500 (<10/kg)
1
<25%
2.1-3.0 mg/dl
501-1,000 (10-15/kg)
severe 
nausea/vomiting
2
25-50%
3.1-6.0 mg/dl
1,001-1,500 (15-20/kg)
3
>50%
6.1-15 mg/dl
>1,501 (>20/kg)
4
desquamation
>15 mg/dl
severe pain, ileus
Glucksberg Grade*
1
1-2
0
0
2
1-3
1
1
2o
0
1
1
2s
4
0
0
3
2-4
2-4 &/or
2-4 (1 only >2)
4
3-4
2-4
2-4
IBMTR Severity Index*
A
1
0
0
0
B
2
or
1-2
or
1-2
or
1
C
3
or
3
or
3
D
4
or
4
or
4
*Assigned based on highest score 
Adapted from Rowlings PA et al: IBMTR Severity Index for grading acute graft-versus-host 
disease: retrospective comparison with Glucksberg grade. Br J Haematol 97:855,1997; Weisdorf 
DJ et al: Acute upper gastrointestinal graft-versus-host disease: clinical significance and 
response to immunosuppressive therapy. Blood 76:624, 1990.

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
11.4 APPENDIX D: CALCULATION OF WEIGHT FOR CELL DOSE CALCULATION IN MORBIDLY 
OBESE CANDIDATES
Formulation for deriving the weight to be used in targeting cell doses in morbidly obese cell 
candidates.
1.
Definition
Obesity is defined as a BMI > 30.
BMI = wgt (kg) / [hgt (M)]2 
2.
Calculation of ideal body weight is performed using the standard, published formula:
Male: 50 + 2.3(Hgt - 60) where Hgt is in inches, and the result is expressed in kg. 
ex. The ideal weight of a 5'10" male = 50 + 2.3(10) = 73 kg. 
Female: 45.5 + 2.3(Hgt - 60), where height is in inches, and the result is in kg.
3.
Calculation of the “practical weight.”
Calculate the midway point, halfway between the actual and ideal body weights (ie the 
average of the two numbers). This is the "practical weight" to be used in calculating the 
targeted cell dose.
4.
Example:
Patient’s actual weight = 143 kg.
Patient’ s actual height 173 cm = 69 in
BMI = 48
IBW formula = 50 + 2.3(9) = 70.7 kg
Midway point between 70.0 and 143 = 107 kg.
The weight we would use in targeting cell dose is 107 kg.
1
dose by weight with adjustment: 
x
IBW + 50%(Weight-IBW)
x
Practical body weight = (IDW + actual BW)/2
2
Formula
IBW (men) 
x
52 kg + 1.9 kg/inch above 5 feet
x
50 kg + 2.39 (height in inches – 60)
IBW (women)
x
49 kg + 1.7 kg/inch above 5 feet
x
45.5 kg + 2.39 (height in inches – 60)

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Abbreviated Title: Phase I Anti-CD 19 CAR
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11.5 APPENDIX E: RESEARCH SAMPLE REQUIREMENTS*
(the order of the research studies listed below reflect the priority should blood volume for research purposes become limited) Day 0 is the day of 
cell infusion
Study
Test/Lab Location
Sample Time Points
Volume/type
Tube
Notes
RCR (replication 
competent 
retrovirus)
Cell product
S+L- (product only)/
Indiana University
Prior to cell infusion or 
cryopreservation of cells
5 x 10^5 cells
Cellular product
In cryovial with 1mL freezing media 
(90% AB serum, 10% DMSO)
Frozen at -80 C
Received from CPS 
Shipped on dry ice priority 
overnight to: Lisa Duffy
Indiana University Vector 
Production Facility
980 W. Walnut St., R3-C668
Indianapolis, IN 46202
e-mail: ljwoods@iupui.edu
phone: 317-274-0323 No Sat 
or Sun Deliveries
Rapid PCR analysis 
(product only)
Location: DTM
At least 2-4 days prior to 
cell infusion
6 x 10^6 cells 
from both CAR 
transduced cells 
AND
untransduced 
control cells
Fresh in culture media in sterile tube
To be performed in DTM
Performed in CPS 
Results must be available 
prior to cell infusion
Patient Testing
RCR (replication 
competent 
retrovirus)
High-sensitivity PCR 
analysis (patient samples)/
Indiana University
1. Prior to cell infusion
2. 3 months(± 1 month)
3. 6 months (± 1 month)
4. 1 year (± 2 month)
5. Archive samples 
annually thereafter for 5 
years (+/- 3 months).        
All Samples are scheduled 
from first cell infusion.        
3 mL/ 
peripheral 
blood 
Purple top (EDTA) tube
Refrigerated at 4 C
PAX DNA tube for archival samples
If all post-therapy RCR-PCR 
tests are negative in the first 
year, future samples will be 
cryopreserved and banked.in 
Frederick
Shipped on ice pack priority 
overnight to: Lisa Duffy
Indiana University Vector 
Production Facility
980 W. Walnut St., R3-C668
Indianapolis, IN 46202
e-mail: ljwoods@iupui.edu
phone: 317-274-0323
Archival samples: Courier to 
NCI-Frederick repository 301-

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Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
846-5893 to schedule pick-up
Cell product
Storage for future 
research and 
regulatory studies 
Dr. William Kopp
SAIC/Frederick
Prior to cell infusion or 
cryopreservation of cells
10% of cellular 
product 
remaining after 
infusion
In cryovials with 10-50 x10^6 cells in 
1mL freezing media each (90% AB 
serum, 10% DMSO)
Frozen at -80 C
Received from CPS 
Courier to NCI-Frederick 
repository 301-846-5893 to 
schedule pick-up
Patient Testing
Flow Cytometry:
Bone marrow
ALL panel including 
hematagones/
Stetler-Stevenson Lab
10/6N109
301-402-1716
At time of enrollment and
At first disease restaging 
after each cycle and then at 
the at PI oncologist 
discretion in responding 
patients when available.
5 mL marrow 
aspirate
10 ml in a heparinized syringe 
This test will only be done 
when a marrow aspirate is 
clinically indicated or required 
to monitor disease status
Schedule with lab in advance 
using Microsoft Outlook 
Calendar and order test in 
CRIS
CAR T Cell 
Persistence
POB Panel -T-regs/ TBNK
NIH Clinical Pathology 
Flow Cytometry Lab.
1. Prior to cell infusion 
2. Day 1, 7, 14,(+/- 2
days) and 28 (+/- 4 days)
3. If response, repeat at 
least 3 and 6 months, 
then every 6-12 months 
until CAR negative.
6 mL and 3ml 
peripheral 
blood
One 6ml and one 3 ml Purple top tube 
(PTT) at room temperature (1 for 
flow/1 for CBC)
Schedule with clinical lab 301-
496-4879
Flow Cytometry for CAR T 
cells and subsets: 
Stetler-Stevenson Lab
10/6N109
301-402-1716
3 mL peripheral 
blood or bone 
marrow
Two 10 ml Green Sodium Heparin 
Tubes at room temperature
Schedule with Stetler-
Stevenson Lab 301-402-1716
DNA for CAR
PCR for CAR promoter 
from T cell genomic DNA/
Dr. William Kopp
SAIC/Frederick
1. Prior to cell infusion 
2. Day 1, 7, 14, (+/- 2 days) 
and 28 (+/- 4 days). If 
response, repeat at least 3 
and 6 months, then every 
6-12 months until CAR 
negative.
3 mL peripheral 
blood.
Day 28 (+/-4) 
one 3 ml 
peripheral 
blood and one 3 
ml bone 
marrow
PAX DNA  tube or a EDTA tube  
sample  at room temperature
Samples will be stored and 
batched
Courier to NCI-Frederick 
repository 301-846-5893 to 
schedule pick-up
Serum Cytokine 
Levels (including 
by not limited to 
IFN-Ȗ, IL-2, IL-6, 
and TNFĮ)
Multiplex cytokine panel
Dr. William Kopp
SAIC/Frederick
1. Prior to cell infusion 
(within 24 hours)
2. Day 0 (1 and 12 hours 
after cells), 1 (24 hrs), 2 
(48 hrs), 3 (72 hrs) +/- 4
hours
3. Days 4-14, and 28 (+/- 4
4 mL peripheral 
blood 
For scheduled samples  4 ml Green 
Sodium heparin tube. Keep 
refrigerated on patient care unit..
Cytokine storm samples will be 
refrigerated on patient unit 
 
At any time for patients 
suspected of developing 
cytokine storm, additional 
samples will be collected, 
stored in the refrigerator sent 
ASAP for analysis and 
weekend samples will be spun 

90
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
days) 
4. If response, repeat at 
least 3 and 6 months, then 
every 6-12 months until 
CAR negative.
and frozen by fellow.
Courier to NCI-Frederick 
repository 301-846-5893 to 
schedule pick-up
CD64
Flow Cytometry for CD64: 
Stetler-Stevenson Lab
10/6N109
301-402-1716
1.
Prior to cell infusion 
(Day -2, -1 OR Day 0)
2.
Post cell infusion: up 
to daily for Days 1-14
3.
Additional samples 
may be sent if the 
patient is febrile
0.5 mL from the 
Cytokine 
sample listed 
above into 
cryovial.
Mix blood well by inverting several 
times. Extract 0.5 ml from cytokine 
sample and place in cyro vial. Place 
in refrigerator. Sample is viable for 4 
hours at room temperature or 48 hours 
if refrigerated. Send to Flow 
Cytometry lab (B1B58)
Schedule with Stetler-
Stevenson Lab 301-402-1716 
Cytogenetics 
(Cyto)
Dr. Diane Arthur
37/2016
301-435-3711
At PI’s discretion.
3-5 ml of bone 
marrow 
Marrow: 0.5 ml preservative-free 
sodium heparin in capped syringe
Schedule with lab in advance and 
Flow Cytometry:
CSF
Flow Cytometry for CAR T 
cells/ 
Stetler-Stevenson Lab
10/6N109
301-402-1716
At time of enrollment and 
at each diagnostic 
(clinically indicated) LP
if performed at NIH CC 
3-8 mL CSF
Spinal fluid placed immediately in 
flow media available on 3 NE North 
Procedure Area.
Delivered immediately to 
Stetler-Stevenson Lab 
Minimal Residual 
Disease (MRD) 
for leukemia cells 
(Real Time PCR)
Evaluation of DNA for 
MRD of leukemia cells 
At time of response 
evaluation when indicated 
by PI
5 mL blood or
2 mL bone 
marrow
EDTA tube; send to POB laboratory 
for processing
Send DNA to:
Adaptive Biotechnologies Corp.
1551 Eastlake Ave E, Suite 200, 
Seattle WA 98102
ph:206-687-7708
email:  hrobins@fhcrc.org
ATTN: clonoSEQ
Serum Storage 
SAIC/Frederick
Prior to chemo, Day -1, 7, 
28 (+/-4 days)
10 ml red top 
tube peripheral 
blood
Store in refrigerator
Courier to NCI-Frederick 
repository 301-846-5893 to 
schedule pick-up
Apheresis (single 
pass) (Only if the 
subject  has 
apheresis line.**
SAIC/Frederick
Day 28 (+/-4)
Single pass
Room temperature
Courier to NCI-Frederick 
repository 301-846-5893 to 
schedule pick-up
x
Sample volume for protocol studies will be restricted. The amount of blood drawn from adult (those 18 years of age or older) for research purposes shall not exceed 10.5 mL/kg or 550 
mL, whichever is smaller, in an 8 week period. The amount of blood drawn for research purposes from pediatric patient subjects (those under 18 years of age) will not exceed 5 mL/kg 
in a single day, and no more than 9.5 mL/kg over an 8 week period.
x
**if no apheresis line one time blood draw will be done.  3cc/Kg not to exceed 50 mls. (half in sodium heparin tubes and half in red top tubes.  Send to SAIC.

91
Abbreviated Title: Phase I Anti-CD 19 CAR
Version Date: 01/12/2018
11.6 APPENDIX F: GUIDELINES FOR GRADING AND MANAGEMENT OF SUSPECTED CYTOKINE 
RELEASE SYNDROME
Table 1. Cytokine Release Syndrome Revised Grading System 
Grade 1 
Symptoms are not life threatening and require symptomatic treatment only  
e.g. fever, nausea, fatigue, headache, myalgias, malaise 
Grade 2 
Symptoms require and respond to moderate intervention   
-oxygen requirement <40%  or 
-hypotension responsive to fluids or low dose1 of one vasopressor or 
-grade 2 organ toxicity 
Grade 3 
Symptoms require and respond to aggressive intervention  
-R[\JHQUHTXLUHPHQWRU 
-hypotension requiring high dose1 or multiple vasopressors or 
-grade 3 organ toxicity or grade 4 transaminitis 
Grade 4 
Life-threatening symptoms 
-requirement for ventilator support or 
-grade 4 organ toxicity (excluding transaminitis) 
1high dose vasopressor doses shown in Table 2. 
Table 2. High-'RVH9DVRSUHVVRUVDOOGRVHVDUHUHTXLUHGIRUKRXUV 
Norepinephrine monotherapy 
PFJNJPLQ 
Dopamine monotherapy 
PFJNJPLQ 
Phenylephrine monotherapy 
PFJNJPLQ 
Epinephrine monotherapy 
PFJNJPLQ 
If on vasopressin 
19DVRSUHVVLQ1(HTXLYDOHQWRIPFJNJPLQ 
If on combination vasopressors (not 
vasopressin) 
11RUHSLQHSKULQHHTXLYDOHQWRIPcg/kg/min  
x
1VASST Trial Vasopressor Equivalent Equation: 
Norepinephrine equivalent dose = [norepinephrine (mcg/min)] + [dopamine (mcg/kg/min) ÷ 2] + 
[epinephrine (mcg/min)] + [phenylephrine (mcg/min) ÷10]

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11.7 APPENDIX G: NEUROPSYCHOLOGICAL TEST BATTERY
Domains
Measure*
Age range (yrs) Time (min)
Working Memory
NIH Toolbox List Sorting Test
3.0 - adult
7
Attention
NIH Toolbox Flanker Test
3.0 - adult
   4
Wechsler Cancellation subtest
4 - adult
   3
WRAML Letter Number
5.0 - adult
   6
Processing Speed
Wechsler Symbol Search
4 - adult
   3
Executive Function NIH Toolbox Dimensional Change Card Sort Test
3.0 – adult
   7
DKEFS Trailmaking
8 - adult
   5
DKEFS Verbal Fluency (8+ yrs)
2.5 - adult
   6
   or McCarthy Verbal Fluency (2.5-7 yrs)
Background
Background Information Form
all
   (5)
Information
(parent/observer report or adult self-report)
Neurologic
Neuro-Symptom Checklist
all
   (2)
Symptoms
(parent/observer report)
x
This full test battery will be administered at baseline and 21-28 days post-infusion for participants 2.5 
years and older.  The battery also will be administered at 3 months post-infusion if the patient returns 
to the NIH for a follow-up evaluation.  In addition, at Day 14 (+/- 1 day) post-infusion, the Neuro-
Symptom Checklist will be administered to a parent/adult observer.  
x
Other measures may be given in order to test a particular domain more thoroughly if clinically 
indicated. Also, some measures may not be administered if the participant does not appear to 
understand the task or becomes frustrated. 
x
We estimate that this test battery will take less than 1 hour for all participants and less than 
45 minutes for children under 6 years of age to complete.  

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11.8 APPENDIX H: NEURO SYMPTOM CHECKLIST

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12
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