Phase I/II Study of Autologous (Central Memory/Naïve) CD8+ T Cells That Have Been Transduced to Express a WT1-Specific T Cell Receptor for Treatment of AML
Phase: PHASE1
Conditions: Acute Myeloid Leukemia, EBV-Positive Neoplastic Cells Present, HLA-A*0201 Positive Cells Present, Blasts Under 5 Percent of Bone Marrow Nucleated Cells, Elevated WT1
Sponsor: Fred Hutchinson Cancer Center

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FRED HUTCHINSON CANCER RESEARCH CENTER (FHCRC)
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE (UW)
Current Version: 05/15/2019  
(Previous Version: 03/21/2019) 
PROTOCOL 9296
TITLE OF PROTOCOL: PHASE I/II STUDY OF AUTOLOGOUS (CENTRAL 
MEMORY/NAÏVE) CD8+ T CELLS THAT HAVE BEEN TRANSDUCED TO 
EXPRESS A WT1-SPECIFIC T CELL RECEPTOR FOR TREATMENT OF AML
Investigator
Professional Title
Telephone Number
Aude Chapuis, MD 
Principal Investigator
Assistant Member, FHCRC
Assistant Professor, UW
(206) 667-4369
Daniel Egan, MD
Affiliate Investigator
Elihu Estey, MD 
Member, FHCRC
Professor, UW
(206) 288-7176
Merav Bar, MD
Affiliated Investigator, FHCRC
Ann Woolfrey, MD
Associate Member, FHCRC
Professor, UW
(206) 667-4453
Ann Dahlberg, MD
Assistant Member, FHCRC
Assistant Professor, UW
(206) 667-1959
Cecilia Yeung, MD
Assistant Member, FHCRC
(206) 288-7104
Katherine Tarlock, MD
Acting Instructor, UW
(206) 667-7121
Philip D. Greenberg, 
MD
Member and Head, Program in Immunology, 
FHCRC 
Professor, UW 
(206) 667-4462
Biostatistican:  
Ted Gooley, PhD
(206) 667-6533
Research Nurse: 
Elizabeth Barnum
(206) 667-3792
Study Coordinator:
Ana Radu  
(206) 667-7782
Emergency 24-hour phone: (206) 598-8902 – ask for provider on call
Pediatric participants - Cancer and Blood Disorders Center, Seattle Children’s Hospital: 
206-987-2106; Emergency Number (Nights/Weekends): 206-987-2000; ask for the
Oncologist on call.
IRO received 09/09/20
FHCRC IRB Approval  
OCT 08 2020 
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Table of Contents
TITLE OF PROTOCOL: PHASE I/II STUDY OF AUTOLOGOUS (CENTRAL MEMORY/NAÏVE) 
CD8+ T CELLS THAT HAVE BEEN TRANSDUCED TO EXPRESS A WT1-SPECIFIC T CELL 
RECEPTOR FOR TREATMENT OF AML ................................................................................. 1 
2 
INTRODUCTION............................................................................................................ 6 
3 
BACKGROUND............................................................................................................. 8 
3.A.  Prognosis and current treatment options for AML ..................................................................... 8 
3.B.  Rationale for Treating AML with WT1-specific CD8+ T Cells ................................................... 9 
3.C.  Rationale for treating AML patients in CR with minimal residual disease or incomplete 
recovery of peripheral blood counts, irrespective of cytogenetic risk ...................................... 11 
3.D.  Rationale for Using Autologous CD8+ T Cells Transduced with a Lentiviral Vector to 
Express a High-affinity WT1-specific TCR for Patients with High Risk AML ............................ 13 
3.E.  Rationale for Employing a Third Generation Lentiviral Vector to Transduce Autologous 
Well-Differentiated CD8 T Cells ................................................................................................................ 15 
3.F.  Rationale for Transducing Autologous Cell Populations Derived from Long-lived 
Memory or Naïve Cells to Express the High-affinity WT1-specific TCR ..................................... 17 
3.G.  Rationale for Comparison of TCRC4-transduced cells derived from TN- and TCM-subsets 
with those derived from EBV-specific cells .......................................................................................... 18 
3.H.  Rationale for Lymphodepleting Chemotherapy Prior to T Cell Therapy ......................... 20 
3.I. Safety Concerns ....................................................................................................................................... 21 
3.J.  Previous/Ongoing Human Experience with TCRC4 .................................................................... 23 
3.K. Rationale for the proposed cell dose ............................................................................................. 37 
3.L. The Use of IL-2 in Adoptive T Cell Therapy .................................................................................. 37 
3.M.  Justification for using HTTCS for determining cellular subpopulations ......................... 39 
3.N.  Rationale for Enrollment of Pediatric Patients ......................................................................... 41 
3.O.  Rationale for modification of eligibility criteria to include patients with “high-risk” AML 
(who do not undergo transplantation), regardless of MRD status or presence of post-
induction incomplete count recovery .................................................................................................... 41 
3.O.1.  High-risk cytogenetics or molecular classification .............................................................. 41 
3.O.2.  Molecular minimal residual disease (MRD) ........................................................................... 42 
4 
STUDY OBJECTIVES...................................................................................................45 
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4.A.  Primary Objectives .............................................................................................................................. 45 
4.B.  Exploratory Objectives ....................................................................................................................... 45 
5 
STUDY ENDPOINTS ....................................................................................................46 
5.A.  Primary Endpoints ............................................................................................................................... 46 
5.B.  Exploratory Endpoints ....................................................................................................................... 46 
6 
STUDY DESIGN ...........................................................................................................47 
6.A.  Study Overview ..................................................................................................................................... 47 
6.B.  Stage 1, Cohort #1 ................................................................................................................................. 50 
6.C.  Stage 1, Cohort #2 ................................................................................................................................. 52 
6.D.  Stage 1, Cohort #3 ................................................................................................................................ 54 
6.E.  Stage 2 ....................................................................................................................................................... 56 
7 
PATIENT SELECTION..................................................................................................57 
7.A.  Eligibility for Enrollment ................................................................................................................... 57 
7.B.  Exclusion Criteria for Enrollment .................................................................................................. 59 
7.C.  Eligibility for Apheresis/blood collection ................................................................................... 60 
7.D.  Eligibility for Treatment with TCRC4-transduced CD8+ cells ................................................ 60 
7.E.  Exclusion for Treatment with TCRC4-transduced CD8+ cells ................................................. 61 
7.F.  Eligibility for Observation Arm ........................................................................................................ 62 
8 
CONSENTING ..............................................................................................................62 
9 
PROTOCOL REGISTRATION ......................................................................................63 
10 
PROCEDURE TO OBTAIN PBMC FOR GENERATION OF THERAPEUTIC T CELLS63 
11 
GENERATION OF WT1-SPECIFIC CD8+ T CELLS......................................................64 
12 
HANDLING OF T CELL PRODUCTS BEFORE INFUSION..........................................64 
13 
OTHER STUDY AGENTS.............................................................................................64 
13.A. Interleukin-2 ........................................................................................................................................ 64 
13.B. Cyclophosphamide ............................................................................................................................. 65 
13.C. Fludarabine ........................................................................................................................................... 65 
14 
PLAN OF TREATMENT................................................................................................65 
14.A. Screening Consent .............................................................................................................................. 65 
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14.B. Leukapheresis Visit ............................................................................................................................ 65 
14.C. Lymphodepletion Chemotherapy ................................................................................................. 66 
14.D. T Cell Infusion ...................................................................................................................................... 66 
14.E.  Low-dose S.C. IL-2 ............................................................................................................................... 67 
15 
EVALUATION...............................................................................................................67 
15.A.  Patient Evaluation during the Treatment Consent Visit ...................................................... 67 
15.B.  Patient Evaluation at Each Planned Visit .................................................................................. 68 
15.C. Evaluation of WT1 expression by leukemic cells .................................................................... 68 
15.D.  Patient Evaluation During T cell Infusions ............................................................................... 68 
15.E. Clinical and Laboratory Evaluation for Toxicity (Primary endpoint).............................. 68 
15.F. Feasibility Assessment (Primary Endpoint) ............................................................................. 69 
15.G.  Evaluation of Persistence and/or Efficacy of Adoptively Transferred T Cells (Primary 
Endpoint) .......................................................................................................................................................... 70 
15.H.  Efficacy Assessment (Exploratory Endpoint) .......................................................................... 72 
15.I. TN, TCM and TEBV Migration to Tumor Sites (Exploratory Endpoint) ................................. 72 
15.J. Assessment of the Functional Capacity of Transferred Cells (Exploratory Endpoint) 73 
15.K.  Evaluation for Long Term Effects of Treatment with Lentivirus-Transduced T Cells73 
16 
MANAGEMENT OF TOXICITIES AND COMPLICATIONS...........................................75 
16.A.  Toxicity Grading ................................................................................................................................. 75 
16.B Regimen-related Toxicity ................................................................................................................. 75 
16.C.  Definition and Management/Evaluation of Non-hematologic and Hematologic 
Toxicities........................................................................................................................................................... 76 
16.D.  Management of Symptoms During T cell Infusions ............................................................... 79 
16.E. Management of Severe Cytokine Release Syndromes (or Cytokine Storm) .................. 80 
16.F.  Management of Severe Treatment-related Toxicities .......................................................... 81 
16.G.  Concomitant Therapy ....................................................................................................................... 82 
16.H.  Off-study Criteria ............................................................................................................................... 82 
17 
MANAGEMENT OF T CELL INFUSIONS IN PATIENTS WHO RELAPSE OR 
PROGRESS DURING T CELL THERAPY ...............................................................................82 
18 
OPTIONS FOR FURTHER TREATMENT AFTER COMPLETION OF STUDY THERAPY
83 
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19 
TARGETED/PLANNED ENROLLMENT.......................................................................83 
20 
GUIDELINES FOR ADVERSE EVENTS REPORTING.................................................84 
20.A.  Reporting of Adverse Events (AEs) .............................................................................................. 84 
20.B.  Reporting to the FDA ....................................................................................................................... 84 
20.C.  Definitions ............................................................................................................................................ 85 
21 
DATA AND SAFETY MONITORING PLAN ..................................................................86 
21.A.  Primary Monitoring .......................................................................................................................... 86 
21.B.  Monitoring Plan .................................................................................................................................. 86 
21.C.  Monitoring the Progress of the Trial and the Safety of Participants ............................... 86 
22 
RECORDS ....................................................................................................................88 
23 
STATISTICAL CONSIDERATIONS ..............................................................................88 
23.A. Analysis of Safety/Toxicity (Primary Endpoint) ..................................................................... 88 
23.B. Analysis of the Feasibility of infusing TCRC4 Transduced T Cells (Primary Endpoint)91 
23.C. Analysis of persistence in blood or bone marrow of TCRC4-transduced cells............... 91 
23.D.  Assessment of efficacy (Exploratory Endpoint) ..................................................................... 92 
24 
ADMINISTRATIVE CONSIDERATIONS.......................................................................93 
24.A.  Institutional Review Board ............................................................................................................ 93 
24.B.  Termination of Study ........................................................................................................................ 93 
25 
REFERENCES.....................................................ERROR! BOOKMARK NOT DEFINED. 
26 
APPENDICES...............................................................................................................94 
APPENDIX A............................................................................................................................94 
APPENDIX B............................................................................................................................95 
APPENDIX C............................................................................................................................96 
APPENDIX D ……………………………………………………………………………………………97
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INTRODUCTION 
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, accounting for 
approximately 80 percent of cases in this group. Despite advances in diagnosis and therapy, the 
outcome of AML patients remains poor, with less than 30% 5-year overall survival (OS) on
average.1,2 Among patients who initially achieve a complete remission with induction therapy, the 
vast majority will ultimately relapse and die of disease-related complications.3 Allogeneic HCT 
dramatically reduces rates of disease relapse, and thus offers a potentially curative approach for 
many high-risk patients, yielding ~30-50% prolonged disease free survival (DFS).4,5 However, 
many high-risk patients, due to age, comorbidity or lack of an available donor, are not candidates 
for HCT and, thus, alternative consolidative approaches are needed.  
WT1 is expressed in cells of the developing genitourinary system as well as in early 
hematopoietic or CD34+ progenitor cells, but notably absent in mature leukocytes 6. WT1-specific 
cytotoxic T lymphocytes (CTL) in vitro and in vivo can recognize and kill leukemia cells that
express abnormally high levels of WT1, without affecting normal cells that express lower levels of 
WT1.7-13 Our lab has developed methods to isolate and expand high avidity CD8+ CTL clones 
specific for WT1 from normal donors.14 In a clinical trial for patients with high-risk leukemias 
undergoing allogeneic hematopoietic cell transplantation (HCT), eleven patients were infused with
WT1-specific CD8+ T cell clones generated from each patient’s matched donor.15 However, the 
anti-tumor efficacy was limited in part by variability in the avidity of the clones that could be 
isolated from each donor. This led to the identification and isolation of a high-affinity WT1-specific 
T cell receptor (TCR), denoted TCRC4, for use in a protocol for post-transplant treatment of 
leukemia patients.
To date, 25 AML patients have been treated with cells expressing the TCRC4 after HCT; the
preliminary data suggest this approach is safe, as patients have not experienced significant 
toxicities, including no evidence of damage to organs expressing physiologic levels of WT1
despite the long-term persistence of the T cells in most patients.15 Furthermore, we have
encouraging data supporting the potential efficacy of WT1-specific CD8+ T cells in preventing 
post-transplant relapse, in that we have observed a 92% relapse-free survival in 13 high-risk 
patients at a median of 23 months follow-up post-transplant. By comparison, patients who receive 
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myeloablative transplants from HLA-identical siblings demonstrate a relapse rate of ~25% at 
three months and ~40% at 1 year post-transplant.16-20
The proposed study is a Phase I/II trial aimed at treating up to 35 patients with AML who achieve 
morphologic remission following induction therapy, and who are determined to be at extremely 
high risk for relapse, but who either are not candidates for, or elect against, HCT following 
standard consolidation chemotherapy. Enrolled patients will receive adoptively transferred 
autologous T cells genetically modified to express TCRC4. We will assess the safety and feasibility 
of this approach, the ability of the cells to persist and localize to the bone marrow, and the 
potential clinical efficacy of WT1-specific T cells in clearing minimal residual disease (MRD),
reducing relapse, and improving survival outcomes. We predict that both initial and long-term 
clinical responses will correlate with the persistence of a functional population of antigen-specific 
CTL. Patients in morphologic CR who have MRD detectable by flow cytometry, or who have
incomplete recovery of peripheral blood counts have an estimated rate of relapse as high as 90% 
by 1 year, 21-23 and therefore represent an appropriate high-risk population to be considered for 
inclusion in the study. A contemporaneous cohort of high-risk patients who do not receive the 
study intervention, but who similarly are not candidates for HCT, will be followed prospectively to 
allow for an assessment of efficacy based on comparisons of relapse, DFS and OS. The expected 
high rate of relapse by 1 year, without HCT, suggests that we will be able to draw meaningful 
conclusions regarding the efficacy of our study intervention within only a few years of treating 
patients.20,22-28
Although studies have demonstrated that transferred T cells of the central memory (TCM) subtype 
can provide enhanced protective immunity in vivo,29 increased clonal expansion and anti-tumor
activity of transferred murine CD8+ CTL have also been observed when effector cells were 
derived from the naïve pool (TN) as compared to the TCM pool.30  Thus, the nature of the T cell
that would be the most effective source of CTL for adoptive tumor therapy remains controversial. 
In this study, we plan to evaluate the feasibility of generating and co-infusing WT1-specific CD8+ T
cells derived from the TN and TCM subset, directly compare their persistence, localization to the 
bone marrow, and potential function of CTL against residual leukemia. Additionally, distinct 
memory subpopulations may have distinct advantages because of their specificity, such as EBV-
specific memory T cells, which are generally enriched for T cells of the TCM phenotype and have 
the potential advantage of being intermittently triggered and expanded in vivo from transient viral 
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reactivation. Thus, we will also assess the feasibility of generating and infusing EBV-specific T 
cells that have been rendered WT1-specific. 
BACKGROUND
3.A.  Prognosis and current treatment options for AML
As noted above, AML is the most common acute leukemia in adults, accounting for approximately 
80 percent of cases in this group. Despite advances in diagnosis and therapy, the outcome of 
AML patients remains poor, with average 5-year overall survival (OS) of 30%.1,2,20,31 The median 
age at diagnosis is >65 years, and outcomes are even poorer for older patients, with 5-year OS of 
only 5-10%.32 Survival outcomes are generally better in pediatric AML patients, overall, although 
those with poor-risk features continue to demonstrate high rates of relapse and poor survival. 33,34
Patient age, cytogenetic and molecular classification, and performance status remain the most 
well-established predictors of response to therapy.35-37 Thus, younger patients and those with 
favorable cytogenetics are most likely to achieve a morphologic CR, defined by less than 5% 
blasts in the bone marrow, absence of extramedullary disease, and recovery of neutrophil counts 
to 1,000/mm3 and platelet counts to 100,000/mm3.38 However, for patients who do initially 
respond well to chemotherapy by achieving CR, relapse remains a persistent problem. For those 
who receive consolidation chemotherapy alone as post-remission therapy, the 5-year cumulative 
incidence of relapse ranges from 35% in those with favorable risk cytogenetics to 80% in those 
with unfavorable cytogenetics.3  
As a result of the graft versus leukemia effect mediated by alloreactive T cells,39,40 allogeneic HCT 
dramatically reduces rates of disease relapse, and thus offers a potentially curative approach for 
many patients, albeit at the expense of considerable morbidity and transplant-related 
mortality.25,26,28,41 Allogeneic HCT after an initial CR yields prolonged DFS in roughly 30-50% of
AML patients.5,42 In a meta-analysis of prospective trials including more than 6000 younger 
patients (age < 60 years) receiving HCT in first complete remission, a statistically significant 
improvement in OS has been demonstrated in unfavorable-risk patients (HR 0.69) and 
intermediate-risk patients (HR 0.76).5 Several retrospective analyses suggest that the survival 
benefit associated with HCT, albeit with reduced-intensity conditioning, likely extends to older 
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patients, as well.43-45 Thus, it is generally accepted that HCT be considered in medically fit, high-
risk patients in first CR who have a suitable donor. 
Unfortunately, even with the development of reduced-intensity regimens and alternative donor 
options, many patients, particularly older patients with significant comorbidities, remain ineligible 
for HCT.3 At our institution, it was reported that of 212 newly diagnosed patients who were 18-75
years of age, 116 (67%) underwent HCT.46 The remaining patients did not receive HCT because 
of age, comorbidity, or lack of a suitable donor. In older patients, who represent the majority of 
those not undergoing HCT, DFS with chemotherapy alone as post-remission therapy is in the 
range of 20-35% at 2 years, 43-45 and patients with selected high risk features have an even poorer 
DFS of approximately 10% at 1 year without HCT.21-23,47 Therefore, alternative strategies are 
needed for patients not receiving a HCT. The development of strategies for targeting tumor-
associated antigens that bypass the patient’s tolerance mechanisms may provide a potentially 
effective therapeutic approach. Adoptively transferred autologous T cells genetically modified to 
express a high affinity TCR that recognizes an antigen expressed by AML cells represents one 
such strategy. 
3.B. Rationale for Treating AML with WT1-specific CD8+ T Cells
The WT1 gene, located at 11p13q, encodes a 52-54 kDa protein transcription factor containing 
four C-terminal DNA-binding zinc fingers.48 WT1 plays an important role in cellular proliferation 
and differentiation in normal hematopoiesis. In vitro and in vivo studies in mice have shown that 
WT1 has multiple functions, including gene transactivation, gene repression and RNA binding.49.
,WLVLQYROYHGLQWKHUHJXODWLRQRIJHQHVHQFRGLQJJURZWKIDFWRUVHJ7*)ȕ&6)-1), growth factor 
receptors (e.g. insulin-R, IGF-1R, EGFR), transcription factors (e.g. EGR, c-Myc, Pax2, Dax-1, 
and Sry) and anti-apoptotic molecules (bcl-2 and bcl-xl).48,50,51 During embryogenesis, WT1 plays 
a critical role in the development of the genitourinary tract, spleen, and mesothelial structures,52
but WT1 expression after birth is limited to very low levels primarily in kidney podocytes, testes 
sertoli cells, ovarian granulosa cells, mesothelial cells of the lung, the uterus and fallopian tubes, 
and CD34+ hematopoietic progenitor cells.52-55
The WT1 gene was originally identified as a tumor suppressor gene in pediatric kidney cancers, 
but it subsequently became evident that the role of WT1 is more context dependent and has a 
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broader role in the oncogenesis of hematological malignancies, including AML, and solid tumors,
in which overexpression promotes oncogenesis rather than suppresses tumor formation.
Overexpression of the WT1 gene is detected by reverse transcription polymerase chain reaction 
(RT-PCR) in many leukemias, including ~80-100% cases of adult and pediatric AML, 
myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML), and in solid tumors 
including breast, ovarian, and pancreatic and lung cancers.56-58 WT1 is also mutated in 
approximately 10% of AMLs, which can lead to overexpression.59,60 High levels of WT1 
expression have been correlated with a poor prognosis in leukemia patients.61,62 Because WT1 
promotes proliferation and oncogenicity, loss of expression is disadvantageous for tumor that 
overexpress the gene, leading to growth arrest and apoptosis, which reduces the risk of outgrowth 
of antigen-loss variants if targeting this protein immunologically.63
The ubiquity of WT1 in leukemia has led to the testing of vaccines aimed at employing the host 
immune system to mount a T cell response to this antigen. However, the development of effective 
cancer vaccines has progressed poorly, and a comprehensive review of cancer vaccine trials 
involving 440 patients disappointingly revealed an objective clinical response rate of <3%, 
highlighting the substantive obstacles to generating an effective anti-tumor immune response in 
cancer patients.64,65 Similarly, although vaccines targeting WT1 have produced clear antitumor
responses in some patients, including complete remissions in patients with AML, without toxicity 
to normal tissues, most patients failed to benefit clinically, reflecting in part the induction of 
responses of low magnitude and/or low avidity due to the restricted repertoire to this self-protein 
and the limited immunogenicity of current vaccine regimens.66-71
Adoptive T cell therapy with CD8+ CTL specific for antigens expressed by AML cells, but absent 
or expressed at only low levels by normal hematopoietic progenitor cells, has the potential to 
selectively eliminate leukemia cells. This protocol will examine the activity of autologous T cells 
that are rendered reactive with WT1 by introduction of a high affinity WT1-specific TCR (TCRC4)
that recognizes the WT1126-134 epitope (RMFPNAPYL) in the context of the class I major 
histocompatibility complex (MHC) HLA-A*0201 molecule. This epitope is highly conserved and
expressed in all described WT1 isoforms. As no coding mutations of this epitope have been 
reported, it is considered a promising immunotherapeutic target.72
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3.C. Rationale for treating AML patients in CR with minimal residual disease or incomplete recovery of 
peripheral blood counts, irrespective of cytogenetic risk
It has become increasingly evident that the quality of remission achieved with first induction is a 
major predictor of outcomes, and may be more important prognostically than are conventional 
pre-treatment patient and disease characteristics such as age or cytogenetic classification. Multi-
parameter flow cytometry (MPFC) techniques are able to detect aberrant immunophenotypes in
as low as one leukemic cell in as many as 100,000 events (0.001%), and have been widely 
integrated into standard clinical practice for detection of minimal residual disease (MRD). Between 
60-90% of adult and pediatric patients found to be MRD-positive at the time of CR will relapse 
within 1-2 years, compared to roughly 25-40% of those who are MRD-negative.21,23,24,47,73 In the 
United Kingdom MRC AML12 trial, 3-year event-free survival of pediatric patients with MRD 
at end of induction was only 15%, compared to 85% in MRD-negative patients.74 The
association of MRD and relapse remains statistically significant even after correction for other 
factors, including age and cytogenetics, and has been shown to be predictive even when MRD is 
present following induction and prior to consolidation chemotherapy. However, these reported 
rates of relapse with MRD are often confounded by the inclusion of patients who undergo 
allogeneic HCT. In a Seattle cohort of patients, ages 18-80, and found to be MRD-positive at the 
time of CR, the observed rate of relapse was between 80-90% at 1 year in those who did not 
undergo HCT. 22,23,47 Similarly, an Italian group reported that 80% of MRD-positive patients who 
did not undergo HCT relapsed at a median of only 5 months. 21
In addition, recent evidence suggests that patients who attain a morphologic CR but with 
incomplete recovery of neutrophil counts - less than 1,000/mm3 (CRi) or platelet counts less than 
100,000/mm3 (CRp) - have a substantially increased risk of relapse and markedly poorer survival 
than those with CR and adequate recovery of peripheral blood counts, independent of other 
prognostic variables.20,47,75 Indeed, CRi has been designated by an International Working Group 
as a separate category of response in clinical trials. 38 In pediatric patients, a platelet threshold of 
ȝOZLOOEHXVHG to define CRp, as per consensus guidelines for evaluating pediatric 
response to therapy.76 In the above Seattle cohort, of which approximately half underwent an 
allogeneic HCT, the risk of relapse increased from 20% to 80% at 3 years in those with CRi/CRp 
compared to those with CR and good recovery of peripheral counts.22 When analysis was 
restricted to only those patients who did not undergo HCT, the negative impact of CRi/CRp was 
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even more profound, with a 90% rate of relapse at 1 year. 47 As shown in Figure 1, below, the 
expected rate of relapse for patients with either CRi/CRp or MRD not undergoing HCT was 
approximately 90% at 1 year (Figure 1, below). 
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Figure 1. Cumulative incidence of relapse in patients not undergoing HCT, stratified by 
presence or absence of MRD as determined by flow cytometry and by peripheral blood 
count recovery (CR versus CRi/CRp)
3.D.  Rationale for Using Autologous CD8+ T Cells Transduced with a Lentiviral Vector to Express a
High-affinity WT1-specific TCR for Patients with High Risk AML
Adoptive transfer of donor-derived ex vivo-expanded WT1-specific CD8+ CTLs in patients with 
AML can potentially bypass the limitations encountered during vaccination by increasing the 
number and quality of T cells targeting WT1. Our lab has developed methods to generate high 
avidity CD8+ T cell responses specific for WT1 by primary in vitro sensitization of TN cells from 
healthy donors.14 We completed a clinical trial in allogeneic HCT patients with relapsed leukemia 
in which WT1-specific CD8+ T cell clones were generated and expanded from each patient’s 
normal matched donor.77 Although we successfully generated WT1-specific CD8+ CTL clones 
from >85% of donors, this effort was very time consuming, commonly requiring 12-16 weeks, 
which can be problematic for leukemia patients who may need therapy more promptly, and the 
avidity of the T cells generated from each donor for the WT1 epitope presented in MHC class I 
was variable, with the lower avidity responses exhibiting less reactivity in vitro, which likely limited 
in vivo anti-tumor activity in those patients.
Isolating and transferring a high affinity TCR into primary T cells has the potential to efficiently 
impart specificity as well as high avidity for a cell expressing the desired target antigen, and 
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makes it possible to rapidly and reproducibly generate tumor-reactive T cells for therapy. Genes 
HQFRGLQJWKHĮDQGȕFKDLQVRID7&5specific for a candidate tumor antigen have been shown to 
be adequately expressed in T cells and to mediate antitumor effects in patients.78
One potential limitation of TCR-transduced T cells is that the introduced TCR chains can 
inappropriately pair with the endogenous TCR chains, producing TCRs with unpredicted and 
potentially auto-reactive specificities, as well as reducing expression of the correctly paired chains 
and decreasing the avidity compared to the original “donor” T cell. However, our lab demonstrated 
that introducing a point mutation into each of the TCR chains, to LQVHUWDF\VWHLQHLQWRWKH&ĮDQG
WKH&ȕGRPDLQV, created an interchain disulfide-bond that resulted in preferential pairing of the 
LQWURGXFHGĮDQGȕFKDLQV, reduced mismatching with endogenous TCR chains, and increased
expression compared to the endogenous TCR chains. Transduced T cells had higher avidity than
cells transduced to express chains without the mutation.79
Another modification that has proven beneficial for increasing TCR transgene expression is codon 
optimization. Redundancy in the genetic code allows some amino acids to be encoded by more 
than one codon, but certain codons are less ‘optimal’ for translation than others, due in part to the 
relative availability of matching transfer RNA (tRNA).80 0RGLI\LQJWKH7&5ĮDQGȕJHne
sequences to encode each amino acid with the optimal codon for human gene expression, as well 
as eliminating mRNA instability motifs and cryptic splice sites, has significantly enhanced 
expression of introduced 7&5ĮDQGȕJHQHs.81  
The WT1-specific TCR being used in this protocol (TCRC4) was selected after screening >1000 T 
cell clones isolated from the repertoires of >70 normal healthy individuals for avidity, as reflected 
by increased WT1-specific cytolytic activity for targets expressing decreasing levels of WT1 and 
by enhanced binding of peptide/MHC tetramers. To minimize the potential risk of on-target off-
tissue toxicity (i.e., recognition of the few normal tissues that express physiologic low levels of 
WT1, the selected TCRC4 was purposefully isolated from the peripheral repertoire of a healthy 
HLA*0201+ donor, guaranteeing that the TCR had undergone negative selection during thymic 
development and that TCRC4 expressing T cells did not mediate an autoimmune process in the 
periphery of the donor.
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TCRC4 was inserted into a third-generation lentiviral construct for expression in human cells and 
codon-optimized to achieve high-level protein expression. The final construct was engineered as 
DVLQJOHRSHQUHDGLQJIUDPHFRQVLVWLQJRIWKH7&5ȕDQG7&5ĮFKDLQVVeparated by a 2A element 
from the porcine teschovirus (P2A) to ensure coordinated gene expression,82,83 and incorporated 
complementary cysteine residues following introduction of point mutations in the constant 
GRPDLQVRIWKH7&5ĮDQG7&5ȕJHQHVWRSURPRWH appropriate inter-chain pairing of the 
introduced TCR chains.79   
3.E.  Rationale for Employing a Third Generation Lentiviral Vector to Transduce Autologous Well-
Differentiated CD8 T Cells
The theoretic risk of insertional mutagenesis with retroviral vectors became realized during two X-
linked severe combined immunodeficiency gene therapy trials in which five patients developed 
acute lymphoblastic leukemia, shown to reflect transactivation of either the LMO2 or CCND2 gene 
by the retroviral insert in transduced CD34+ hematopoietic stem cells (HSCs). This highlighted the 
propensity of gamma-retroviral vectors (Ȗ-RV) to preferentially insert near genes that are actively 
transcribed, which may be particularly problematic in transduced HSC that express genes that 
confer self-renewal capacity and a proliferative/survival advantage and are then transplanted into 
a setting in which they are driven to extensively expand.84  
By contrast, the risks of insertional oncogenesis or other vector-related cellular toxicities are 
extremely low when targeting peripheral blood T cells that lack the same self-renewal capacity of 
HSC, with no significant toxicities reported to date in any clinical trial. As described below, this risk 
can be further decreased with the use of a third generation self-inactivating (SIN) lentivirus (LV), 
in which the promoter regions of the long terminal repeats (LTRs) have been truncated and the 
insert, such as a TCR gene, is expressed under control of an internal murine stem cell virus 
(MSCV)-based promoter.85
Insertional oncogenesis was not observed in the long-term results from three clinical trials 
evaluating Ȗ-RV-engineered T cells for patients with HIV.86 The transduced cells persisted long 
term, in some cases >10 years, and clinical monitoring of the patients at yearly intervals for 
cumulatively >540 patient-years of observation has not detected evidence of retroviral 
genotoxicity. In more recent trials with less extensive retrospective data, no toxicities attributable 
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to the administration of T cells transduced to express HLA*0201-restricted MART1 or gp100-
specific TCRs using the MSGV1-based RV were observed in 33 patients with metastatic 
melanoma. The MSGV1-based RV uses the same promoter from the LTR of MSCV that will be 
used in the lentiviral vector in our trial.87,88 The MSGV1-based Ȗ-RV was also used to transduce 
autologous T cells to express an NY-ESO-1-specific TCR that was infused in 17 patients with 
metastatic synovial sarcoma or melanoma89 and to transduce cells to express a carcinoembryonic 
antigen (CEA)-specific TCR in T cells that were infused into three patients. Again, no insertional 
mutagenesis events were identified.  
Transcriptionally active enhancer/promoter elements may influence expression of cellular genes 
at a distance from the insertion site, independently of the vector type used to introduce the gene 
(RV vs LV; LTR-based or SIN).90 However, Ȗ-RVs have a predilection toward integrating in the 
immediate proximity of transcription start sites and deoxyribonuclease I hypersensitivity sites,91
increasing the probability that the viral LTR transcriptional enhancer will interfere with gene 
regulation and potentially activate cancer-causing genes.92 In contrast, LVs commonly insert into 
introns, and thus are more likely to integrate further away from the transcription start sites of 
active transcription units, making them less likely to induce transcriptional activation.93 In vivo
genotoxicity assays based on the transplantation of transduced tumor-prone Cdkn2a-/- murine 
hematopoietic progenitor cells have also directly compared the effect of promoter location within 
Ȗ-RVs and SIN lentiviral constructs on the oncogenic potential. Placing the strong LTR-based 
spleen focus-forming virus promoter as an internal promoter in a construct containing a SIN LTR 
rather than as a component of the LTR further reduced the propensity for insertional mutagenesis 
and lymphoid tumors.94  
Despite the risk of insertional mutagenesis being extremely low, it remains justified to initially 
examine safety/toxicity and potential efficacy of therapy with transduced T cells in patients with
AML. The potential toxicities are listed in the protocol consent form, and will be discussed with 
patients as a part of the consent process. Eligible patients who elect to enroll on the protocol will 
be monitored and managed for potential toxicities as per current guidelines, which are outlined in 
Section 15 and Section 16. Stopping rules will also be applied, as described in Section 23. 
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3.F. Rationale for Transducing Autologous Cell Populations Derived from Long-lived Memory or Naïve 
Cells to Express the High-affinity WT1-specific TCR
Establishing a persistent functional population of antigen-specific CTL after adoptive transfer will
likely be necessary to eliminate AML cells and prevent recurrence. Previous trials have often been 
limited by the inability of transferred T cells to expand and persist post- transfer. The in vivo fate of 
transferred T cells is dependent in part on the intrinsic properties of the T cells from which the 
infused cells are derived,95,96 and we can choose the T cell type used for adoptive cell therapy. 
Conventional CD8+ T cells can be divided into naïve and antigen-experienced, memory T cell
subtypes (TN, TM). TM cells can be further divided into TCM and effector memory T cell (TEM)
subsets, which have distinct transcriptional programs associated with defined characteristics, 
including homing, phenotype, and function.97  When TEM cells are stimulated in vitro, they 
differentiate largely into short-lived effector cells that kill targets, but generally have limited 
proliferative capacity and fail to persist for long periods of time after transfer.95,97-99
Studies of transferred purified CD8+ T cell subsets in a murine virus infection model revealed that 
transferred TCM provide enhanced protective immunity from in vivo challenge compared to TEM 96. 
Although TCM cells expand and also differentiate into effector cells in response to in vitro
stimulation, adoptive transfer studies in non-human primates revealed that TCM–derived effectors 
had been imprinted and retained some of the beneficial properties of their parent TCM cell, in 
particular the capacity for self-renewal, which translates into improved in vivo persistence and 
response to antigen challenge.95
Increased persistence after transfer was also observed with murine CD8+ CTLs derived from the 
naïve pool when these cells were primed in the presence of the Ȗc-chain cytokine, Interleukin-21 
(IL-21).100,101  In both murine and human studies, adding IL-21 with Interleukin-15 to T cell cultures 
during the in vitro priming of antigen-specific CD8+ T cells has been shown to not only induce 
greater expansion and to prevent apoptosis of the cells responding to antigen stimulation, but to 
also lead to in vitro generation of CD8+ T cells that are CD28hi, reflecting a less terminally 
differentiated phenotype.101,102 We have also shown in humans that WT1-reactive CTL clones 
derived from naïve donors and primed in the initial presence of IL-21 expressed significantly 
higher levels of CD27, CD28, and CD127 prior to infusions than cells not exposed to IL-21, which 
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is again consistent with a less differentiated phenotype.77 Following infusion, these CTL clones 
demonstrated enhanced capacities for in vivo persistence and proliferation compared to CTL 
clones generated in the absence of IL-21. These preliminary results in a limited number of 
patients suggest that CTL clones derived from a naïve subset and primed in vitro in the presence 
of IL-21 are a valid therapy alternative to TCM-derived CTL, and may have some potential 
advantages as a cell source. 
In this study, we will evaluate the feasibility of generating genetically-engineered WT1-specific 
CD8+ CTL derived from TN and TCM subsets, and compare their in vivo persistence, localization to 
the bone marrow, and function in eliminating leukemia cells. Patients enrolled in this trial will 
initially receive a mix of sorted CD8+ T cells from the TN subset and the TCM subsets, both of 
which will have been subsequently transduced with TCRC4. Cells derived from the TN subset will 
be additionally exposed to IL-21 at the time they are stimulated. Prior to transfer, the endogenous 
TCR usage of both population subsets will be determined by high throughput TCRȕ sequencing 
(HTTCS)103,104,105. This can then serve as a ‘bar code’ of cells derived from either the TN or TCM
populations.106 The trial will evaluate and compare the in vivo behavior of these two T cell
populations after they are infused, determining respective frequencies, persistence, function, and
localization to the bone marrow. These results will be correlated with the clinical outcomes of the 
patients.
3.G.  Rationale for Comparison of TCRC4-transduced cells derived from TN- and TCM-subsets with 
those derived from EBV-specific cells
Our clinical experience with donor-derived TCRC4-transduced CD8+ cells in a high-risk leukemia 
population after allogeneic transplant consists of cells derived from virus-specific (EBV- and CMV-
specific) T cells on FHCRC Protocol 2498. In this trial, in vivo expansion and persistence was 
greatly increased in patients who received cells derived from donor EBV-specific compared to 
cells derived from CMV-specific cells. Transduced EBV-specific cells persisted at higher 
frequencies (sometimes up to 50% of CD8+ T cells) for long periods of time (>1 year). Analysis of 
EBV- and CMV-specific populations in the donors revealed that surface markers associated with 
long lived memory (CD28, CD127, CD62L and CCR7) were expressed at significantly higher 
levels on EBV-specific compared to CMV-specific cells, consistent with a less-differentiated (more 
central memory-like) phenotype (Figure 2, unpublished data). Comparatively, markers associated 
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with exhaustion/activation (TIM3, 2B4, and KLRG1) were decreased on EBV-specific cells 
(Figure 3, unpublished data) compared to the CMV-specific subset. This suggests EBV-specific 
cells, because of their specificity and/or phenotypic state, may also have characteristics that 
render them a favorable CD8+ T cell subset to transduce with TCRC4 and subsequently transfer.  
Figure 2. Differential expression of memory markers on donor EBV- and CMV-specific 
T-cells
Figure 3. Differential expression of activation/exhaustion markers on donor EBV- and
CMV-specific T-cells
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In the proposed trial, we plan to first demonstrate safety of autologous, polyclonal TN and TCM-
derived cells transduced with TCRC4, and then ultimately broaden the components of the 
investigational product to include transduced CD8+ T cells derived from an autologous EBV-
specific population. The modified design will allow for comparison of persistence of EBV-specific T 
cells (which, as described above, commonly express TCM characteristics, but also may get 
boosted in vivo by host EBV antigens) with both TN- and TCM-derived cells within individual 
patients, allowing for investigation into whether an EBV-specific TCR is responsible (necessary) 
and/or contributes to the observed T cell persistence in treated patients. Although it is likely that in 
some patients a subset of autologous, polyclonal TCM cells selected for transduction will, in fact, 
be EBV-specific, the proportion is expected to be small. Monitoring of WT1/EBV bi-specific cells 
based on their ability to bind WT1- and EBV-specific tetramers will allow for determination of the 
proportion of TCRC4-transduced cells derived from the EBV-specific subset.   
3.H.  Rationale for Lymphodepleting Chemotherapy Prior to T Cell Therapy
Transfer of T cells into lymphopenic hosts results in enhanced cell expansion of T cells with a
memory phenotype and with enhanced effector function.99,107 The favorable impact of 
lymphodepleting conditioning on survival and efficacy of transferred T cells has been observed in 
animal models,108-111 donor lymphocyte infusions post-stem cell transplant,112 and in numerous 
clinical trials with adoptive T cell transfer in melanoma and other cancers.113-115 There are several 
proposed mechanisms by which lymphodepletion augments T cell expansion and function. These 
include removal of the potential competition with endogenous T cells for homeostatic cytokines 
such as IL-7 and IL-15, as evidenced by the fact that proliferation is reduced in a dose-dependent 
manner when “irrelevant” T cells are infused along with a T cell population of interest.116
Lymphodepleting chemotherapy may also significantly decrease CD4+ CD25+ regulatory T cells, 
which have been shown to suppress in vitro function of tumor-reactive T cells.117,118
Our clinical experience with TCRC4-transduced cells on FHCRC Protocol 2498 is limited to 
patients who have received T cells following post-transplant engraftment and count recovery, and 
has not included lymphodepleting chemotherapy, as there was concern about the adverse effects 
of chemotherapy on engraftment in the early post-transplant period. In the proposed trial, we plan 
to first demonstrate safety of autologous, polyclonal TN and TCM-derived cells transduced with 
TCRC4 in an initial cohort of leukemia patients, and in the subsequent cohort investigate the 
impact of lymphodepleting chemotherapy on safety, persistence and possibly efficacy. For the 
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proposed trial, a combination of cyclophosphamide and fludarabine will be employed, as has been 
demonstrated in several clinical trials, with proposed doses for our study in line with the current 
CD19 CAR T-cell trial FHCRC protocol 2639, as well as ongoing pediatric studies
(FHCRC/Seattle Children’s PLAT-02 protocol) .112,114,119-121
3.I. Safety Concerns
3.I.1 Pre-clinical murine model for targeting WT1
Low level WT1 expression can be detected in normal kidneys, testes, ovary, pleura, pericardium, 
the uterus and fallopian tubes, and CD34+ hematopoietic progenitor cells 55. Thus, targeting cells 
that express WT1 raises the concern of on-target/off-tissue toxicities. As WT1 expression in 
normal and malignant tissues is comparable between mice and humans, we have in part
addressed these concerns by developing a mouse model in which on-target toxicities can be 
assessed using the highest affinity murine WT1-specific TCR that we could isolate from the 
normal repertoire, as well as variants of this TCR that we mutated to achieve an affinity ~200-500
fold higher than can be isolated from the normal repertoire. Mice infused with CD8+ T cells 
isolated from the peripheral pool and transduced with either the naturally isolated or mutated high 
affinity TCRs did not demonstrate any toxicity in WT1 expressing organs, and the transduced 
T cells functioned normally in vivo, responding to immunization with a recombinant vector 
expressing WT1 and recognizing WT1+ tumor cells, with no evidence of activation from 
recognition of normal cells.122
The safety of murine T cells transduced to express the enhanced affinity TCRs in this study raised 
the question of why these TCRs were not detected in the normal repertoire. Therefore, we 
transduced these TCRs into HSC and examined thymic T cell development and maturation. We 
found that, unlike developing T cells expressing the naturally isolated TCR, developing T cells 
expressing the enhanced affinity TCRs do indeed undergo negative selection in the thymus. A
fraction of these cells survive negative selection and emigrate from the thymus, but exhibit down-
regulation of the TCR and/or CD8, resulting in peripheral T cells with lower avidity than T cells 
expressing the naturally isolated TCR. These results suggest that negative selection in the 
thymus is overprotective for WT1-reactive T cells; thus, the risk of on target/off-tissue toxicities to 
WT1-expressing organs after infusion of T cells transduced with a WT1-specific TCR derived from 
a T cell clone isolated from a naturally occurring peripheral T cell should be low.  
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3.I.2 
Previous clinical experience: targeting WT1 with adoptive transfer of donor-derived 
CD8+ T cell clones into patients after HCT 
Patients who were treated in our previous trial with escalating doses of donor-derived WT1-
specific CD8+ T cell clones showed no evidence of damage to organs expressing physiologic 
levels of WT1, including no hematopoietic suppression or graft failure, renal failure, pleuritis, 
pericarditis, abdominal pain (from splenic capsule or ovaries) and/or testicular or ovarian pain.77  
The “parental” WT1-specific CD8+ T cell clone C4, from which the TCR to be used in this trial was 
derived, was isolated from a healthy donor expressing HLA A*0201. This TCR was chosen to
maximize therapeutic potential and minimize the potential risk of on-target/off-tissue toxicity, as 
the TCRC4 represents the highest affinity TCR that we identified from the naïve peripheral 
repertoire of >70 HLA*0201+ donors. Thus, the T cell expressing this TCR had undergone 
negative selection during thymic development. 
The functional avidity of CD8 T cells, as defined by the ability to recognize and lyse target cells 
expressing limiting amounts of cognate antigen in vitro, is influenced by the multi-parametric 
binding of the effector and target cell populations. Although determined largely by the affinity of 
the expressed TCR, expression of co-stimulatory and adhesion molecules and their ligands make 
substantive contributions. The functional avidity of the parental C4 clone, as well as cell products 
transduced with the TCRC4, were assessed by lysis of T2 B-LCL cells (Figure 4, below), a
transporter associated with antigen processing (TAP)-deficient B lymphoblastoid cell line, pulsed 
with decreasing concentrations of WT1126-134 peptide. The TCRC4-transduced CD8 T cells 
exhibited a functional avidity similar to the best WT1-specific CTL clones infused into patients in 
our previous study77 and to the parental C4 clone from which the TCR was derived. Of note, a
subset of the clones with similar high avidities persisted long-term (>100 days), without toxicity.   
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Figure 4. Avidity of products to WT1-
pulsed T2 B-LCL
3.J.  Previous/Ongoing Human Experience with TCRC4
TCRC4 is currently being employed in the clinic to transduce T cells for therapy of patients with 
high-risk leukemias who have undergone allogeneic HCT and either are at a high risk of disease 
recurrence or have already relapsed, in a phase I/II clinical trial (protocol FHCRC #2498) 
designed primarily to evaluate the safety and efficacy of intra-patient escalating doses. For the 
current trial, TCRC4 is being introduced into HLA-A*0201-restricted Epstein–Barr virus (EBV)- or 
cytomegalovirus (CMV)-specific donor cells. The use of virus-specific cells serves three purposes: 
1) it restricts the antigen specificity of the endogenous TCRs expressed by the substrate cells to a 
known foreign pathogen, decreasing the likelihood of transducing donor-derived T cells that 
recognize host antigens; 2) it removes the possibility of transducing TN cells that contribute 
disproportionally to GVHD following HCT 123,124; and 3) it ensures the transduced T cells will 
contain a large fraction of T cells derived from the TCM cell pool, which have properties that 
enhance persistence after transfer. 
3.J.1  FHCRC #2498 Study Schedule
Initially, this protocol planned for each patient to receive a total of 4 infusions following a dose-
escalation schedule: 1 x 109 cells/m2, 3.3 x 109 cells/m2, 1 x 1010 cells/m2, and 1 x 1010 cells/m2
with this last dose followed by low-dose subcutaneous (s.c.) Interleukin-2 (IL-2) at 2.5 x 105 IU 
BID x 14 days, administered to enhance the survival of transferred T cells.125 The trial started out 
as a two-armed study, with patients who had no detectable disease after HCT treated on the 
‘Prophylactic Arm’ (Arm 1). For safety concerns, the first 3-6 patients (Arm 1/Stage 1) were
planned to receive infusions separated by a 28-day interval between infusions. If no unexpected 
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toxicities were detected, the interval between infusions was planned to be reduced to 14 days 
(Arm 1/Stage 2). Patients with relapsed disease after HCT were planned to receive infusions on
the ‘Treatment Arm (Arm 2). The first 3-6 patients (Arm 2/Stage 1) were planned to receive the 
same infusion doses as Arm 1/Stage 1, but these were planned to be separated by a shorter 
interval to reach higher and potentially therapeutic doses in a more limited time. Again, if no 
toxicities were observed, subsequent patients were to begin with a higher dose of cells (3.3 x 109
cells/m2), separated by a 14-day interval. The initial treatment plan is shown in Figure 5, below. 
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Figure 5. Initial treatment plan of protocol 2498, Arms 1 and 2
Because the dose-escalation schedule was well tolerated and no severe or unexpected toxicities 
were observed in the first 7 patients treated with up to 1010 TCRC4-transduced cells/m2, both the 
FDA and the FHCRC IRB approved discontinuation of the dose-escalating schedule in May 2014 
such that all patients prospectively enrolled (irrespective of their disease burden post-transplant) 
have been receiving a first dose of 1010 cells/m2 on day 0, and a second infusion of 1010 cells/m2
at least 14 days after the first infusion, or later if the persistence of the cells from the initial infusion 
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is >3% of total CD8+ cells. The second infusion is followed by 14 days of s.c. low-dose IL-2
(Figure 6). 
Figure 6. Revised treatment plan of protocol 2498, irrespective of assigned Arm
3.J.2. Patient characteristics on Protocol 2498
As of December 1, 2016, 25 patients with high-risk AML received a total of 48 doses of donor-
derived virus-specific cells (Tables 1A and 1B). Thirteen patients received prophylactic treatment 
on Arm 1 and twelve patients were treated on Arm 2 due to detectable disease post-HCT. Overall, 
33 of the 48 T-cell infusions represented the maximum target dose of 1 × 1010 WT1-specific 
CTL/m2, and, of those, 11 infusions were followed by low-dose s.c. IL-2. 
Table 1A. Patient characteristics on Protocol 2498, Arm 1
M = male; F = female; AML = acute myeloid leukemia; y = years; HCT = hematopoietic cell 
transplantation; CR = complete response; PB = peripheral blood; MRD= minimal residual disease
Pt.
Age/
Gender
Disease
EBV/ 
CMV
Arm
Comments
3
49
M
AML with complex cytogenetics, 
received HCT after 2nd CR
EBV
1
Poor T cell persistence, remains in 
remission 43 months after transplant
10
64
M
high-risk AML, received HCT in CR1
No evidence of disease after HCT.
EBV
1
Persistent T cells, in remission 30
months after HCT.
11
59
F
High risk AML, received HCT in CR
No evidence of disease after HCT.
EBV
1
Persistent T cells, remains in remission 
36 months after HCT.
12
55
F
HCT at CR1, high risk AML. No 
evidence of disease after HCT
EBV
1
Persistent T cells, remains in remission 
33 months after transplant
13
59
F 
high-risk AML, HCT in CR1, no 
evidence of disease after HCT
EBV
1
Persistent T cells, remains in remission 
30 months after transplant
16
65
M 
MDS-> AML, 5.5% blasts at HCT.
EBV
1
Poor T cell persistence, remains in 
remission 28 months after transplant
17
30
M
Extramedullary AML, MLL gene-
rearrangement.
EBV
1
Poor T cell persistence, remains in 
remission 26 months after transplant
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18
47
M
Relapsed AML in CR2
EBV
1
Persistent T cells, remains in remission 
19 months after transplant
19
77
F 
Relapsed AML in CR2
EBV
1
Persistent T cells, remains in remission 
18 months after transplant
21
59
F 
AML with complex cytogenetics, 
FLT3-ITD mutation. 
EBV
1
Persistent T cells, remains in remission
13 months after transplant
22
30
M 
AML with complex cytogenetics
EBV
1
Relapsed disease (extramedullary and 
cytogenetic relapse in marrow) at 8 month
after transplant; currently in remission afte
local and reinduction systemic therapy
24
57
M
AML in CR2; MRD at time of SCT
EBV
1
Persistent T cells, remains in remission
10 months after transplant
25
67
F
MDS -> AML, poor-risk cytogeneticsEBV
1
Persistent T cells, remains in remission
4.5 months after transplant
Table 1B. Patient characteristics on Protocol 2498, Arm 2
M = male; F = female; AML = acute myeloid leukemia; y = years; HCT = hematopoietic cell 
transplantation; CR = complete response; PB = peripheral blood; MRD= minimal residual disease
Pt.
Age/
Gender
Disease
EBV/ 
CMV
Arm
Comments
1
56
M 
AML, relapse with para-spinal 
chloroma 5 years after 1st 
myeloablative HC
EBV
2
Disease progression. Removed from 
study before 4th infusion
2
51
F 
AML, 2nd HCT for relapse 
9 years after 1st myeloablative 
HCT. Entered HCT with 16% 
leukemia blasts and blasts were 
again detectable after 2nd HCT
CMV
2
Normalization of counts and decreased 
abnormal blast population to 0.006%
after T cell infusions.
4
25
M 
AML, relapse with medullary and 
extra medullary disease after 2nd 
HCT
EBV
2
Patient remained in CR for 1 year after 
infusion, then relapsed with 
extramedullary disease when WT1+ 
CD8+ cells measured at >3% of total 
CD8 cells. Disease progressed despite 
2nd infusion.
5
49
M 
AML, disease progression after 
HCT (70%  PB blasts at T cell 
infusion)
EBV
2
Disease progression. Off study at 6 
days, and expired 12 days after 1st 
infusion.
6
20
F 
AML, second HCT for relapse 
2 years after 1st myeloablative 
HCT
CMV
2
Poor T cell persistence (0.04% at day 
173), in CR 24 months after HCT.
7
33
F 
AML, MRD after myeloablative 
HCT
EBV
2
Progressive disease. Received 
Azacitidine after 1 T cell infusion.
8
63
F 
AML, received HCT in CR2. MRD 
early after transplant
EBV
2
Persistent T cells, disease progressed 
after infusion, and now receiving 
systemic therapy.
9
67
F
AML, received HCT in CR2. 
Relapse early after transplant
CMV
2
Poor T cell persistence, progressive 
disease.
14
17
M 
AML, 2nd HCT for relapse 5 
years after first HCT.
EBV
2
Disease progression despite persistent 
T cells; received azacitidine.
15
69
F 
MDS -> AML, second HCT for 
relapse 1 year after 1st HCT
CMV
2
Disease progression despite persistent 
T cells.
20
5
F 
Relapsed AML after transplant; 
active disease (chloromas); MLL 
gene-rearrangement.
EBV
2
Disease progression despite persistent 
T cells; receiving re-induction 
chemotherapy
23
75
M 
Relapsed AML after transplant
EBV
2
CR at time of T cell infusion; continued 
complete remission
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3.J.3. Persistence of TCRC4-transduced cells on Protocol 2498
Patients who received cells generated from EBV-specific cells generally demonstrated a high-
level of persistence. Of the 14 patients who received transduced EBV-specific cells and could be 
followed beyond 4 weeks after their latest infusion, 10 had persistent frequencies >3% (range 3-
60% of CD8 T cells) for an average duration of 44 weeks (range 3-85 weeks) (Figures 7A and 
7B, below) representing an average maximum of 215 cells/microliter (range 10-989 
cells/microliter). The remaining 4 patients who received transduced EBV-specific cells exhibited 
frequencies <3% within 1-14 days. Patient 4, who received the lowest dose (1×109), had cells 
detectable in the blood at frequencies of 4% to 8% of total CD8+ T cells until 14 months after 
infusion, with a decrease to <3% by 16 months after infusion. Until adequate long-term safety is 
formally demonstrated, patients cannot receive additional T cell infusions if their WT1-specific 
CD8+ T cell frequency from the previous dose LVRIWRWDO&'+ T cells. Therefore, at this time, 
Patients 8, 10, 11, 12, 18, 19 and 20 have not received a second infusion. Of the 4 pts (Pts 2, 6, 9 
and 15) who received CMV-transduced cells, Pt 2 demonstrated persistence of the TCRC4-
transduced cells for 49 weeks after the final infusion at frequencies well below 1%. Pts 6 and 9 
had no persistence beyond 4 and 2 weeks, respectively, after their last infusion. Pt 15, however, 
had persistence of the TCRC4-transduced cells above 7%, with the last follow-up 68 
days after infusion.
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Figure 7A. Observed frequencies of infused WT1-specific T cells (% CD8+ cells) in Arm 1 
patients.    
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Figure 7B. Observed frequencies of infused WT1-specific T cells (% CD8+ cells) in Arm 2 
patients.    
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3.J.4 Toxicities observed on protocol #2498
All AEs were evaluated starting from the time of the first infusion to 30 days after the patients had 
taken the last dose of s.c. IL-2 for the first 7 patients then only AEs that were above or equal to a 
Grade 3 were recorded for subsequent patients. AEs that were deemed possibly, probably, or 
likely related were collected and graded according to the National Cancer Institute Common 
Terminology Criteria for Adverse Events version 4.0 (NCI CTCAE v4.0) (Table 2). 
Table 2. Adverse events
Categories
NCI CTCAE v4.0
Grade
1 
Grade
2 
Grade
3 
Grade
4 
Cytokine Release 
Syndrome
Fever
4
3
2
Chills
2
1
1
Generalized aches/pain/headache
10
3
Fatigue
1
1
Tachypnea
4
Hypotension
5
2
4
Sinus tachycardia
3
2
Sinus bradycardia
1
Nausea
3
3
Vomiting
1
Diarrhea
1
Hematological 
Abnormalities
Lymphopenia
3
5
8
1
Anemia
1
3
Thrombocytopenia
1
2
3
Lymphocyte count increased
2
Chemistry 
Abnormalities
ALT increased
6
2
2
AST increased
8
2
Alkaline phosphatase increased
4
1
Creatinine increased
3
Hypoalbuminemia
4
Hypocalcemia
7
Hypomagnesemia
1
Hyponatrema
5
Hypokalemia
3
Miscellaneous
Dry eyes
2
Decreased respiratory rate
4
Maculopapular Rash
1
Expected transient symptoms of cytokine release syndrome (CRS) were observed, as this 
syndrome is associated with activation of large numbers of antigen-specific CTL transferred into 
patients with targets expressing the antigen, or low-dose s.c. IL-2, or both. Specifically, 9 patients 
H[SHULHQFHGIHYHUV&ZLWKRUZLWKRXWaccompanying chills, including 2 patients with fever 
> 40.0°C following the infusion. Blood cultures were negative for bacterial or fungal growth in all 
cases. Many patients also experienced grade 2 generalized aches, tachycardia or bradycardia, 
and digestive tract symptoms. With immediate management by administration of the antihistamine 
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diphenhydramine and acetaminophen, plus the narcotic meperidine for chills, all symptoms 
resolved within 24 hours. The most severe CRS was observed in two patients who experienced 
transient grade 3 hypotension during the T cell infusion that rapidly responded to i.v. fluids. A
grade 1 decrease in respiratory rate was observed in 2 patients during the 24 hours after the T
cell infusions, but was associated with administration of meperidine for CRS. All side effects were 
managed in the outpatient General Clinical Research Center (GCRC) or on the general hospital 
ward without ICU support. 
Importantly, we have not observed any on-target/off-tissue toxicities in any of the 20 patients who 
received WT1-specific T cell infusions. Specifically, there has not been any observed graft failure, 
renal failure, nephrotic-range proteinuria, pleuritis, pericarditis, or testicular or ovarian pain in any 
of the study cohort. The hematological abnormality most commonly encountered was 
lymphopenia, which is a predictable, transient side effect of T cell infusions presumably reflecting 
redistribution of peripheral lymphocytes.15,126 The temporary drop in total lymphocyte counts 
returned to pre-infusion levels within 7 to 11 days in all patients. Three cases of grade 3 anemia 
and 2 cases of lymphocytosis were observed during the time the patient was receiving T cell
infusions, but did not occur immediately after infusions and the relationship to the T cell infusions 
remains unclear. Thrombocytopenia is very common in the first year after allogeneic transplant, 
127 and to date all patients treated on this study started the T cell infusion with existing 
thrombocytopenia. Overall, we observed a general upward trend in platelet counts during 
infusions (average platelet counts before infusion: 78 000/μl, after infusions: 140 000/μl), but 3 of 
6 evaluable patients had transient drops in platelet counts immediately after infusions most of 
which did not reach levels associated with a toxicity grade (Figure 8, below). 
Grade 1 and 2 transient electrolyte and liver function abnormalities were also observed. One 
patient developed transient grade 2 transaminitis directly after the third T cell infusion, and this 
resolved without treatment within 7 days. The relationship of other observed metabolic 
abnormalities to the T cell infusions could not be excluded, but were clinically perceived to more 
likely reflect concurrent and common post-HCT etiologies, including side effects resulting from 
medications (e.g., electrolyte abnormalities are common side effects of calcineurin inhibitors and 
antifungals; elevated liver enzymes are a common side effect of antifungals), poor nutritional 
status (hypoalbuminemia), and secondary infections. 
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Figure 8. Kinetics of platelet counts after infusions (Pt. 2)
One patient (Pt 6) without any prior GHVD was diagnosed at 10 months after transplant, which 
was 4 months after her final WT1-specific T cell infusion, with grade III late acute GVHD affecting 
liver (stage III), GI tract and skin, as well as chronic GVHD involving oral mucosa and eyes. 
Symptoms and transaminitis responded favorably to steroids. A second patient (Pt 13) was 
diagnosed 13 months after transplant, which was 10 months after her final WT1-specific T cell 
infusion, with grade III late acute GVHD affective liver (stage III). These are the only cases of 
grade III/IV GVHD observed in our study population, and notably WT1-specific T cells were near 
undetectable (0.04% of CD8+ cells) or undetectable, respectively, at the time of diagnosis making 
any association with the WT1-specific T cells unlikely.
One patient (Pt 16), with a prior history of acute GI GVHD, was diagnosed with chronic GVHD 
affecting liver (stage I, transaminitis only) and oral mucosa at 7 months after HCT, which was 3.5 
months after WT1-specific T cell infusion. As above, WT1-specific T cells were undetectable at 
the time of the transaminitis, making any association with WT1- T cells unlikely. A patient (Pt 11) 
with a history of acute GVHD involving the skin and GI tract had a GVHD flare in the setting of 
tapering tacrolimus at 3 months following WT1-specific T cell infusion. Two patients (Pts 2 and 10) 
developed chronic GVHD after treatment with WT1-specific T cells, one of whom had a prior 
history of acute GVHD. 
  
One patient (Pt 8) was diagnosed with acute GVHD affecting the skin and GI tract at 2.5 months 
after HCT, which was 7 days after receiving her first infusion of WT1-specific T cells.  Symptoms 
Platelet count x10
3
/ȝO
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were mild and responded to prednisone.  This patient also had a prior history of cryptogenic 
organizing pneumonia (COP) occurring before transplant, and was diagnosed with a flare of COP
at 6.5 months after HCT, which was 4 months after infusion of WT1-specific T cells. Initially 
responsive to steroids, she had a repeat flare of COP 4 months later while tapering prednisone, 
and required continued steroid therapy.  
3.J.5 Efficacy observed on protocol #2498
Arm 1: Thirteen patients with high-risk leukemia entering HCT (Table 3, below), and who had no 
detectable disease after HCT, received WT1-specific cells within a median of 104 days post-HCT.
With a maximum follow-up of 849 days and a median of 407 days after HCT, all patients are alive 
in CR and have not relapsed to date (Figure 9). For comparison, patients undergoing HCT with 
high-risk disease have an ~30% chance of relapse at 1 year.16-20 This suggests the infusion of 
WT1-specific cells in the post-HCT setting may prevent leukemia recurrence. 
Arm 2: Eleven patients with high-risk leukemia entering HCT with detectable disease after HCT 
had a higher cumulative risk based on adverse factors compared to pts treated on Arm 1 (Table 
4, below). Patients received T cell infusions at a median of 77 days after HCT. Although 2 of 11 
patients remain free of disease, with a median follow-up of 206 days after the first T cell infusion, 
44% are still alive (Figure 10, below). For comparison, patients who relapse within 6 months after 
HCT have <10% chances of being alive at 1 year.128 This suggests WT1-specific T-cells may 
confer a survival advantage despite the absence of complete clearance of relapsed AML post 
HCT. 
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Table 3. Cumulative risk factors for patients on Arm 1
All patients have received EBV/WT1 bi-specific cells.
Figure 9: Progression-Free Survival of Arm 1 since HCT
Pt.
>CR1
Cytogenetics
refractory 
(>1cycle to 
achieve CR)
Disease at 
HCT
MDS->AML 
or 
secondary 
AML
Chloroma
3
X
X
10
X
X
11
X
X
12
X (FLT3+)
MRD (cyto)
13
X
X
16
5.5% blasts
X
17
X (MLL)
X
18
X
19
X
21
X (FLT3+)
22
X
24
X
X
25
X
X
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Table 4. Cumulative risk factors for patients on Arm 2
Pt.
>CR1
Cyto-
genetics
Refractory 
(>1cycle to 
achieve CR)
Disease at HCT Secondary 
AML
Chloroma
2nd
Transplant
1
X
X
X
X
2
X
X
X
16% blasts
X
4
X
X
X
0.02% blasts
X
X
5
X
X
X
42% blasts
X
6
X
X
X
X
7
X
X
X
3.8% by cyto
8
X
X
X
0.01% blasts
X
9
X
X
no counts
14
X
X
X
0.02% blasts
X
X
15
X
X
X
X
20
X
X
X
23
X
X
X
Figure 10: Overall survival of Arm 2 since 1st CTL infusion
3.J.6 Protocol #2498: Conclusions to date
To date, WT1-specific T cells were detectable for a cumulative total of 471 weeks in 25 treated 
patients, and at frequencies reaching 60% of total CD8 and 998 cells/ȝO. No durable toxicities to 
tissues expressing physiological levels of WT1 (including cells of the hematopoietic, urogenital 
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and renal systems, pleura, or pericardium) were detected during the monitoring period. Therefore, 
adoptive transfer of doses up to 1 × 1010 cells/m2 of donor-derived virus-specific T cells 
transduced with the WT1-specific TCRC4 did not appear to injure normal tissues expressing 
physiologic levels of WT1, did not cause acute GVHD, and CRS symptoms induced by the 
infusion of high numbers of antigen-specific cells were readily managed in this preliminary series. 
In the proposed trial, patients will receive a first dose of 1 x 109 cells/m2 and then potentially a 
second dose of 1 x 1010 cells/m2 followed by low-dose s.c. IL-2. Although we cannot formally 
exclude the possibility of inducing tissue toxicity in the proposed trial, there is no evidence for 
such toxicity in our ongoing trial with the same TCR or in our previous trial with WT1-specific CD8 
CTL clones. 
3.K. Rationale for the proposed cell dose
Safety of adoptive transfer of doses up to 1 × 1010 cells/m2 of donor-derived virus-specific cells 
transduced with the WT1-specific TCRC4 has been demonstrated. However producing such a high
cell number is very expensive and takes several weeks. Therefore, in this study we would like to 
evaluate the potential persistence and function of smaller cell doses. Thus, we will start with 
infusion at a dose of 1 × 109 cells/m2 with intra-patient dose escalation up to 1 × 1010 cells/m2. In 
order to evaluate persistence, the second dose will be held if the transferred cells persist at a level 
of > 5% of the total CD8+ cells and at an absolute number of >30 cells/ȝO.   
3.L. The Use of IL-2 in Adoptive T Cell Therapy
In our prior clinical studies, adoptively transferred CTL clones exhibited a median survival of 6.6 
+/- 0.8 days in the absence of IL-2. The addition of a 14-day course of low-dose IL-2 (250,000 
U/m2 twice daily) following T cell infusion yielded a median CTL survival of 16.8 +/-1.6 days, which 
was significantly longer than that observed in infusions administered to the same patient without 
IL-2 125. It was apparent, however, that the in vivo frequency of transferred CTL began to drop 
before the end of the 14-day course, suggesting continuing IL-2 administration beyond this time 
may be nonproductive or may even be counter-productive, as it could concurrently enhance
expansion of CD4 T-regulatory cells.
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IL-2 is a cytokine secreted by activated T cells, and has a crucial role in the generation of an 
effective immune response. IL-2 promotes the activation and proliferation of antigen-specific 
T cells via the high affinity three-FKDLQĮȕȖ,/-2 receptor, ZLWKWKHĮ-chain induced following TCR 
triggering. High concentrations of IL-2 can also activate resting natural killer (NK) cells and T cells 
via the constitutively expressed intermediate affinity two-FKDLQȕȖ,/-2 receptor. Studies in 
cancer patients have evaluated therapies associated with IL-2 activity, including administration of 
IL-2 alone, of adoptively transferred lymphokine-activated killer (LAK) cells generated by culture in 
high dose IL-2, and of tumor infiltrating lymphocytes (TIL) expanded by culture with IL-2.129-131
These studies included administration of high doses (up to 7.2 x 105 units/kg every 8 hours) of i.v. 
or s.c. IL-2. Although clinical responses were observed in a fraction of patients with renal cell 
carcinoma or melanoma, severe toxicity was a significant problem. The IL-2 plasma levels 
obtained following high dose IL-2 administration exceed the concentrations needed to saturate 
intermediate affinity IL-2 receptors, and can therefore induce non-specific widespread activation of 
NK cells and T cells that can likely mediate most/all of the observed toxicities via cytokine release 
and/or lysis of normal cells. As CD8+ T cells activated in vivo by specific recognition of a target 
antigen are induced to express high affinity IL-2 receptors and are therefore responsive to very 
low concentrations of IL-2, the doses necessary to augment survival and in vivo persistence of 
antigen-specific transferred T cells may be much lower than the doses that induce toxicity.
Studies in cancer patients and HIV seropositive individuals have investigated the immuno-
modulatory effects of administering lower doses of IL-2. Doses of 1.25 x 105 to 5 x 105 U/m2/day 
administered s.c. are well tolerated for up to 84 days.132 In cancer patients, there is little evidence 
for anti-tumor activity from administration of low dose IL-2 alone. However, studies from our group 
in melanoma patients do suggest that the persistence, in vivo function, and anti-tumor activity of 
adoptively transferred CD8+ melanoma-specific CTL clones can be significantly enhanced by daily 
s.c. administration of low doses of IL-2 for 14 days following T cell transfer.133
The dose of IL-2 that will be used in this study was chosen based on its predicted ability to 
saturate high affinity IL-2 receptors and sustain CD8+ CTL activity/survival with minimal toxicity.125
Patients previously treated with donor-derived CD8+ T cell clones after HCT 77 and patients
treated on BB-IND 15130 – FHCRC Protocol 2498, all received the same dosing of IL-2 planned 
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for this trial. IL-2 has been well tolerated, with no evidence of induction of GVHD from non-specific 
T cell activation. 
  
3.M. Justification for using HTTCS for determining cellular subpopulations
Next-generation sequencing techniques, so-FDOOHGKLJKWKURXJKSXW7&5ȕFKDLQVHTXHQFLQJ
(HTTCS) as demonstrated by Robins et al, may be used to accurately sequence the sampled 
TCR repertoire.103-105 In the proposed study, transduced T cells will each contain a unique, 
endogenous T cell receptor that will be quantifiable by HTTCS in the generated immunotherapy 
product. In essence, this “bar coding” of the varied T cell clonotypes allows for post-infusion 
assessment of clonal dynamics within a single patient. This ability to track the individual fates of 
cells comprising the infused polyclonal cells with HTTCS has been demonstrated following 
infusion of MART1-specific T cells in a melanoma population. Indeed, the sum of MART1-specific 
TCR clonotypes was shown to correlate well with the overall proportion of MART1-specific T cells 
as assessed by multimer staining.134 Furthermore, tracking individual TCR sequences made it 
possible to identify the emergence in a patient of a single immuno-dominant clonotype within 3 
months after infusion of the cells (Figure 11). In our proposed study, HTTCS will be performed 
initially on the generated TN and TCM subsets, allowing for accurate measurement of TN-derived 
and TCM-derived cells over time. In a preliminary analysis using a 1:1 mixture of WT1-specific TN
and TCM cells in 3 lung cancer patients, the endogenous TCR composition of the TN and TCM
products was assessed. Unique TCR sequences (comprising at least >0.01% of each potential 
infusion product) accounted for 89-94% of total TN sequence reads and 88-97% of total TCM
sequence reads (Figure 12). Thus, HTTCS should allow accurate discrimination between the 
daughter cell derivatives of these two populations in the vast majority of cases, with the only 
exceptions being if immunodominant clonotypes evolve in vivo that are derived from the small % 
of overlapping sequences that need to be excluded from the analysis. 
Figure 11. Frequency and dynamics of MART1-specific TCR clonotypes in a melanoma 
patient, as determined by HTTCS.
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A) Relative frequencies of 325 
clonotypes in the infusion 
product at baseline. B)
Dynamic in vivo frequencies of 
the infused TCR sequences.
Sum of clonotypes (blue solid 
circles) was concordant with 
the proportion of MART1-
specific T cells as measured by 
multimer staining (black solid 
circles). Despite 325 
clonotypes being detected in the starting donor population, nearly half of the infusate was 
derived from 3 clonotypes – tracking of individual TCR seqs (colored lines) revealed that these 
3 represented >90% of cells detected 7 days after infusion, and 1 immunodominant clonotype 
was persistent 168 days after infusion (red line).
Figure 12. Unique and overlapping TCR sequences in TN and TCM subsets after generation 
of WT1-specific T cells for use in 3 lung cancer patients 
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3.N.  Rationale for Enrollment of Pediatric Patients
In children and adolescents, AML accounts for approximately 20% of acute leukemias. Although
outcomes are better in pediatric patients overall, with 60-65% of patients with intermediate risk 
achieving long-term survival,33 patients with selected high-risk features, including defined high-risk 
genomic aberrations or persistent disease, continue to demonstrate poor survival with less than 
50% OS at 5 years.34,135,136 The indication for HCT in non-high risk pediatric AML remains 
controversial, although retrospective analysis of Children’s Oncology Group data suggests a 
survival benefit with HCT in pediatric patients in first CR classified as having intermediate risk 
disease.137 It remains unknown whether similar long-term outcomes might be observed with 
deferment of HCT until relapse. As with adults, the prognostic impact of MRD at the end of 
induction has been demonstrated in pediatric patients, with ~30% DFS in MRD-positive patients 
compared to 65% in those without detectable disease.136 $WKLJKHUOHYHOVRI05'DWWKH
end of induction, only 15% of patients are alive at 3 years without disease.74 Thus, as per the 
practice of the Seattle Children’s pediatric oncology group, HCT is generally indicated in pediatric 
patients with MRD at end of induction.58,138-141 Therefore, we will consider pediatric patients with
MRD or incomplete count recovery, but who cannot undergo HCT, as eligible to receive the study 
intervention. 3DWLHQWVPXVWEHNJDVSDWLHQWVZLWKORZHUZHLJKWZRXOGEHLQFDSDEOHRI
providing high volume and frequent blood samples for monitoring and analysis. While the number 
of pediatric patients with these high-risk features who cannot proceed to transplant is admittedly 
expected to be small, the very poor prognosis justifies consideration of the study intervention in 
this group.
3.O.  Rationale for modification of eligibility criteria to include patients with “high-risk” AML (who do not 
undergo transplantation), irrespective of MRD status or presence of post-induction incomplete count 
recovery
3.O.1. High-risk cytogenetics or molecular classification
As of July 2018, a modification in the inclusion criteria has been made to allow for enrollment of a 
larger population of patients with “high-risk” AML who do not undergo allogeneic transplantation,
regardless of presence of MRD or poor count recovery. The clinical practice of incorporating 
cytogenetic and molecular data (e.g. complex karyotype, or FLT3-ITD mutation) to risk-stratify 
patients with AML is long-standing and well-validated. Acknowledging that cytogenetic and 
molecular risk classifications change over time, the 2017 European LeukemiaNet guidelines are 
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hereby being considered as a recent and reliable reference for current reference; these widely-
accepted guidelines are now incorporated into our determination for high-risk disease features 
that should be considered for eligibility for our protocol.142 We propose to include, in our trial, 
these other AML patients who, despite lack of MRD or CRi, remain at a very high risk for disease 
relapse without transplant.
Because of the risks of morbidity and mortality associated with SCT, there remains some 
controversy as to who should or should not undergo SCT. That being said, it is generally accepted 
that patients with “adverse risk” AML disease should be considered for SCT due to its curative 
potential, and because of the overall poor outcomes in this population.142 In support of this 
practice, a 2009 meta-analysis of many prospective trials showed that relapse is reduced and 
long-term survival is improved with SCT compared to chemotherapy alone in patients with 
adverse risk AML.5 The median time to relapse in such “adverse risk” patients who do not 
undergo SCT is reported to be approximately 12 months, which is actually quite similar to the rate 
of relapse in the original study population for this study (AML patients with MRD or CRi). Notably, 
the rate of relapse in patients with adverse cytogenetic or molecular features has been reported at 
70-90% by 2 years in multiple publications.3,44,143,144 Additionally, patients with an initial disease 
remission but who develop a subsequent relapse have a very poor prognosis, with an estimated 
survival of only 5-10% at 2 years, with death mainly due to disease relapse.145,146 Therefore, such 
“high-risk” patients who do not undergo SCT, regardless of MRD or count recovery, are 
considered to have similar risks for disease relapse to the initially proposed study population, and 
their inclusion in the study should improve accrual, while maintaining the ability to observe (within 
1-2 years) any impact of WT1-specific T cells in reducing AML relapse as compared to the 
proposed contemporaneous “observation” cohort that does not undergo SCT and that does not 
receive the study intervention.  
3.O.2.  Cytogenetic and molecular minimal residual disease (MRD) as a “high-risk” criterion
In addition to the established prognostic significance of cytogenetic abnormalities at the time of 
diagnosis, it has become increasingly apparent that the persistence of detectable leukemic-associated 
cytogenetic abnormalities (so-called “cytogenetic MRD”) is also negatively associated with survival and 
remission outcomes. In one of the earliest retrospective studies to explore the impact of cytogenetic 
MRD, Marcucci et al found a cumulative incidence of relapse of 100% by 3 years, and an associated 
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3-year disease-free survival of zero, in patients with an abnormal karyotype at the time of morphologic 
remission.147 Importantly, high rates of relapse were observed across all cytogenetic risk groups. 
Similarly dismal outcomes were observed in a subsequent, larger retrospective analysis by Chen et al, 
showing that while allogeneic transplant was able to abrogate to some degree the adverse effect of 
cytogenetic MRD on relapse-free and overall survival, those patients with cytogenetic MRD (as 
defined by a persistently abnormal karyotype) and who did not undergo allogeneic transplant had a 
relapse-free survival of around 10% at 2 years.148 Because of the markedly increased sensitivity of 
fluorescence in-situ hybridization (FISH) techniques in detecting persistent cytogenetic abnormalities, 
the possibility is raised that MRD detected by FISH alone may detect much lower levels of residual 
disease that may not be as strongly associated with relapse; indeed, the limited published literature,
largely involving small numbers of patients, has found mixed results on this matter.149-153 Thus, MRD 
by FISH alone will not be considered as a “high-risk” criterion in the present study.
Similarly, the landscape of molecular MRD is too complicated as to warrant prophylactic WT1-
specific T cell immunotherapy in all patients with any type of “molecular MRD”. Indeed, the ELN 
MRD Working Party recommends against the use of Fms-like tyrosine kinase internal tandem 
duplication (FLT3-ITD), FLT3-TKD, NRAS, KRAS, IDH1, IDH2, and MLL-PTD as single markers 
of MRD because they are all prone to frequent losses or gains, or may otherwise be detected in 
healthy individuals at low levels.154 Furthermore, mutations in TET2, DNMT3A and ASXL1 are still 
detectable during remissions of > 50% of patients, and are not clearly associated with relapse.155
However, there are clearly situations in which molecular MRD has been shown to be a strong risk 
factor for disease relapse, some of which are extremely relevant in a “non-transplant” AML 
population, including those patients with disease that is historically classified as “favorable” risk 
(e.g. core-binding factor AML or patients with normal karyotype and mutations in NPM1) and,
thus, who tend not to be referred for allogeneic transplantation. Core-binding factor AML is 
defined by the demonstration of the specific gene rearrangements CBFB/MYH11 and AML1/ETO
(the latter also known as RUNX1/RUNX1T1), and of note these specific gene rearrangements 
correlate with inv(16) and t(8;21) cytogenetics, respectively. Recent analyses show that a subset 
of this “favorable” risk population is, nevertheless, at significant risk for relapse if molecular MRD 
is detected after therapy. 
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Several authors have shown that persistence of a relative or absolute copy number of abnormal 
transcripts (as compared to a control gene such as ABL) are highly correlated with relapse.5,156-158
As an example, Yin et al demonstrated a 90% relapse risk in patients with persistently detectable 
inv(16) at greater than 10 absolute copies (compared to 10^5 copies of control gene ABL) in the 
marrow following at least 4 cycles of consolidation.158 Of note, because the normalized copy 
number (NCN) method of reporting within the SCCA system is based on a 1:1 ratio of CB:ABL as 
opposed to a 1 CBFB/MYH11 : 10^5 ABL ratio, as was reported in the study by Yin et al, this 
would correlate with a markedly increased risk in any patient with CBFB > 0.001 on the SCCA-
specific NCN scale at any time after 4 cycles of consolidation. Similarly in that same study by Yin 
et al, a 100% risk of relapse without transplantation was observed in patients with > 500 copies of 
RUNX1/RUNX1T1 (correlating with a value of 0.050 NCN in the SCCA system) at any time after 4 
cycles of consolidation.
The log reduction in core-binding factor transcripts is also increasingly used in clinical practice to 
determine risk of relapse. Stentoft et al found that the log reduction in CBFB/MYH11 and
AML1/ETO at the time of post-induction disease restaging was also strongly predictive of relapse 
risk.159 8VLQJDWKUHVKROGRIORJUHGXFWLRQYHUVXVORJUHGXFWLRQin either blood or marrow as 
a risk stratification, the observed rate of relapse was essentially 100% by 2 years in patients (who 
did not undergo a stem cell transplant) if a < 2 log reduction in the abnormal transcript was 
achieved after induction chemotherapy.  
The persistence of detectable mutated NPM1 transcripts also appears to correlate with risk of 
relapse, and monitoring of NPM1 in the marrow or blood is recommended by the European 
LeukemiaNet guidelines. However, since the impact of NPM1 MRD positivity impacts survival and 
rate of relapse significantly less than those with MRD based on detection of core-binding factor 
transcripts or other groups outlined in the study (with NPM1 MRD, the 4-year cumulative 
incidence of relapse is more in the range of 50%), this group will not be considered as an eligibility 
criterion.160-162
Thus, we propose here that the following additional MRD categories be considered as “high-risk” 
for leukemic relapse in a non-transplant population and an acceptable eligibility criterion for our 
study: 1) cytogenetic MRD, as defined by a disease-specific abnormal karyotype at any point 
during a complete (morphologic) remission; 2) a normalized copy number (NCN) of > 0.001 for 
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CBFB/MYH11 or a normalized copy number (NCN) of > 0.050 for AML1/ETO (RUNX1/RUNX1T1)
after at least 4 cycles of consolidation chemotherapy; or 3) a < 2 log reduction in either 
CBFB/MYH11 or AML1/ETO (RUNX1/RUNX1T1) in the bone marrow at the time of post-induction 
disease restaging (immediately after 1-2 cycles of induction therapy). 
STUDY OBJECTIVES
4.A. Primary Objectives
1. Determine the safety / potential toxicities associated with treating high-risk AML patients 
with autologous CD8+ T cells (polyclonal TN and TCM cells; TEBV that have been genetically-
modified to express a high affinity WT1-specific TCR (TCRC4).
2. Determine the feasibility of reproducibly treating high-risk AML patients with autologous 
CD8+ T cells (polyclonal TN and TCM cells; TEBV cells) that have been genetically-modified to 
express a high affinity WT1-specific TCR (TCRC4).
3. Determine and compare the in vivo persistence in blood and at the primary tumor site (e.g. 
bone marrow, chloroma) of transferred autologous CD8+ T cells (polyclonal TN and TCM cells; 
TEBV cells) that have been genetically-modified to express a high affinity WT1-specific TCR 
(TCRC4).
4.B.  Exploratory Objectives
1. Determine whether adoptively transferred autologous TCRC4-transduced CD8+ cells have 
antitumor activity in patients with acute myeloid leukemia.
1a.       In patients with measurable MRD at the time of infusion of TCRC4-transduced 
CD8+ cells, changes in leukemic tumor burden will be measured by morphology, flow 
cytometry, cytogenetics/FISH and/or molecular testing at baseline and after infusion of T 
cells (see Appendix C for response criteria).
1b.       In all patients (those with or without measurable tumor burden prior to T cell 
transfer, including patients who convert to MRD-negative status during consolidation), the 
probability of relapse, disease-free survival and overall survival of patients receiving 
TCRC4-transduced CD8+ cells will be compared with patients in the observation arm.
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2. Determine and compare the migration to the primary tumor site of subsets of the 
adoptively transferred autologous TCRC4-transduced CD8+ T cells (polyclonal TN and TCM
cells; TEBV cells).
3. Determine and compare the in vivo functional capacity of transferred polyclonal 
autologous TCRC4-transduced CD8+ TCM ,TN cells and TEBV CD8+ cells.
STUDY ENDPOINTS
5.A. Primary Endpoints  
1.    Evidence and nature of toxicities according to NCI CTCAE Version 4.0 (see Section 
15.E) 
2.    Feasibility of generating TCR-transduced TN and TCM subsets for adoptive 
immunotherapy in a high-risk AML population (see Section 15.F).
3.    Comparison of the relative frequencies and duration of persistence of adoptively 
transferred TCRC4-transduced CD8+ polyclonal TCM and TN cells, and of TCRC4-transduced
TEBV CD8+ cells in blood and at the primary tumor site(s) (see Section 15.G).
5.B. Exploratory Endpoints
1.    Patients with measurable MRD at the time of infusion of TCRC4-transduced CD8+ cells:
decrease in blast counts in blood or marrow (by morphology and flow cytometry), and/or
decrease in disease burden as detected by cytogenetics/FISH or molecular testing (see 
Appendix C for response criteria). 
2.    Patients with or without detectable MRD: comparison of probability of relapse, disease-
free survival (DFS) and overall survival (OS) to patients in the observation arm (see Section 
15.H).
3.    Relative frequencies at primary tumor sites of TCRC4-transduced CD8+ polyclonal
TCM and TN cells and EBV-specific (TEBV) cells compared to peripheral blood (see Section 
15.I).
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4.    Relative maintenance of functional capacity and potential acquisition of phenotypic 
characteristics associated with T cell exhaustion in transferred WT1+ cell populations 
TN compared to TCM  compared to TEBV (see Section 15.J).
STUDY DESIGN
6.A.  Study Overview 
The proposed study is a Phase I/II trial aimed at treating patients with AML who are at high risk for 
relapse after induction and standard consolidation chemotherapy. As previously described, most
newly-diagnosed AML patients who initially respond to induction therapy will nevertheless 
ultimately relapse, mostly within the first 1-2 years. Furthermore, while it is generally accepted that 
HCT be considered as a potentially curative consolidative therapy for medically fit patients without 
favorable risk disease in first CR, many patients remain ineligible for HCT, either because of age, 
comorbidity, or lack of a suitable donor. Patients with detectable MRD at the time of CR, and
patients with incomplete count recovery (CRi/CRp) represent a population at highest risk for 
disease relapse, particularly without allogeneic HCT and, thus, represent an appropriate target 
population for whom to consider an alternative strategy for reducing relapse. The estimated 
relapse rate in such a population is approximately 90% at 1 year.21-23,47  
The proposed study will explore the safety and efficacy of adoptively transferred autologous T 
cells genetically modified to express a high affinity TCR that recognizes the WT1 antigen that is 
highly expressed in leukemic blasts. The high rate of relapse by 1 year that is observed without 
HCT suggests that we will be able to draw meaningful conclusions regarding the efficacy of our 
study intervention within only a few years of treating patients.20,22-28  
Newly diagnosed non-M3 AML patients who meet enrollment criteria (see Section 7.A.) will be 
enrolled on the study. Ideally, screening will be prior to initiation of induction therapy and 
enrollment will be prior to initiation of consolidation therapy, although patients will remain eligible 
for this study after treatment. Rapid HLA typing with PCR-based methods to determine the 
presence of HLA-A*0201 will be performed at the time of screening (unless HLA typing has 
already been completed). Leukemic cells will be assessed for expression of WT1in bone marrow 
or peripheral blood. Increased expression of WT1 will be determined by either of two methods: if 
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the number of copies of WT1 divided by the number of copies of ABL x 104 is > 250 for bone 
marrow, or > 50 for peripheral blood, based on the reported expression of WT1 in normal blood 
and marrow samples and the associated negative prognostic impact of increased levels163; or by  
immunohistochemical staining (IHC) in blasts as compared to adjacent normal myeloid and 
erythroid precursors, as determined by a Fred Hutchinson/Seattle Cancer Care Alliance 
pathologist. Patients with demonstrated increased expression of WT1 will be considered eligible 
for the study treatment or the observation cohort. Patients who are HLA-A*02:01 (confirmed by 
sequence analysis) will undergo leukapheresis or large volume blood draw after recovery of blood 
counts following systemic therapy.  
The proposed study will aim to treat up to 35 AML patients at highest risk for relapse without HCT,
as defined by presence of MRD by flow cytometry at time of remission and/or by incomplete 
recovery of neutrophil or platelet counts. We will assess the safety and feasibility of administering 
autologous WT1-specific T cells as post-remission AML therapy in this group.TCRC4 , for which 
our clinical experience is described in Section 3.J., will also be used in this protocol. A
contemporaneous cohort of similarly high risk patients who do not receive the study intervention, 
but who are also not candidates for HCT, will be followed prospectively to allow for an 
assessment of efficacy based on comparisons of relapse, DFS and OS.
Patients will undergo at least one cycle of consolidation therapy, at the discretion of their treating 
physician. After recovery, patients who meet treatment criteria (see section 7.D.) will receive the 
study intervention of infused autologous WT1-specific T cells. The time required to generate T 
cells is 15 to 20 days and, therefore, patients may go on to receive consolidation therapy. Given 
this logistical limitation in administering transduced T cells, and because several published reports 
highlight the adverse prognostic impact of MRD when detected by flow cytometry at the end of 
induction (prior to consolidation), otherwise-eligible patients who convert to an MRD-negative 
status during courses of consolidation therapy will remain eligible to receive the study 
treatment.21-23,47,136,164 Patients who are not HLA-A*02:01 will be moved to the observation arm 
and treated per standard treatment plans or other clinical trials as per the discretion of their 
treating physician (Figure 13). Patients who undergo hematopoietic stem cell transplantation will 
be excluded from analysis in both the intervention arm and observation arm.  
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Figure 13. Study Schema 
The proposed study will be conducted in two stages. In the first stage, we will sequentially treat 
three cohorts of 5 patients each with autologous TCRC4-transduced T cells derived from: 1.) TN
and TCM at a 1:1 ratio without conditioning chemotherapy; 2.) TN and TCM at a 1:1 ratio with 
lymphodepleting chemotherapy and 3.) TN, TCM and TEBV cells at a 1:1:1 ratio with 
lymphodepleting chemotherapy. This approach is designed to determine the most promising plan 
of treatment with regards to safety, T cell persistence, and possibly efficacy. In stage 2, an
additional 20 patients will be treated using what is determined to be the best/safest treatment 
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approach from stage 1, as described in Section 23.A.2. Because patients will receive a mix of 
polyclonal CD8+ TN and TCM cells and TEBV T cells transduced with TCRC4, an aliquot of the 
transduced CD8+ TN and TCM and TEBV T cells will be taken for HTTCS analysis to characterize 
the composition of endogenous TCR genes, and allow distinction of each population during 
longitudinal monitoring following in vivo infusion. Monitoring of WT1/EBV bi-specific cells will also 
be based on the ability to bind WT1- and EBV-specific tetramers, which will additionally allow for 
determination of the subset origin of TCRC4-transduced cells derived from the EBV-specific 
subset.  
6.B.  Stage 1, Cohort #1  
1.  First infusion of WT1-specific T cells, cohort #1 
In stage 1 of the study, 5 patients (cohort #1) will initially receive a 1:1 mix of polyclonal CD8+ TN
and TCM cells transduced with TCRC4 (Figure 14). The infusions will be administered without
lymphodepleting chemotherapy, consistent with the approach utilized in FHCRC Protocol 2498. 
Approximately 4 weeks after the last cycle of consolidation chemotherapy (depending on a given 
patient’s clinical situation, preference of primary treating physician, and availability of the T cell 
product) patients will receive a first T cell infusion at a total target dose of 1 x 109 WT1-specific 
cells/m2 (consisting of 0.5 x 109 each TN and TCM-derived cells). If the actual cell dose for either T
cell subset is less than the target dose, or if cells from either subset cannot be generated, infusion 
of all available cells will be allowed.
2.  Second infusion of WT1-specific T cells, cohort #1 
The subsequent infusion will be held until the transferred cells persist at a level of < 5% of the 
total CD8+ FHOOVDQGDWDQDEVROXWHQXPEHURIFHOOVȝO. Thus, after a minimum of 3 weeks, an 
additional (second) infusion may be given at a total target dose of 1 x 1010 WT1-specific cells/m2 
(consisting of 0.5 x 1010 each TN and TCM-derived cells). 
3.  IL-2 administration
The second infusion will be followed by low-dose s.c. IL-2 250,000 IU/m2 twice daily x 14 days 
Patients or caregivers will be instructed on how to self-administer the drug. After the two planned 
infusions and IL-2 administration, patients will have completed the study treatment. 
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4.  Timing of disease restaging with bone marrow sampling
Bone marrow evaluation will be performed within four weeks before the first infusion and
approximately four weeks after the second infusion (approximately 14 days after completion of IL-
2). Due to high persistence of infused T cells, however, the second infusion may be delayed for 
weeks to months. Therefore, if the interval between the first and second infusion is > 4 weeks, a 
repeat marrow evaluation will be performed at that time to assess disease response to the initial 
treatment. ,IWKHWLPHLQWHUYDOEHWZHHQ7FHOOLQIXVLRQVLV 10 weeks, a repeat bone marrow 
evaluation will also be performed within four weeks prior to the second infusion, in order to allow 
for restaging prior to re-treatment.   
Figure 14. Plan of Treatment (Stage 1, Cohort #1)
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6.C. Stage 1, Cohort #2  
1.  First infusion of WT1-specific T cells, cohort #2 
We will proceed with cohort #2 if the treatment intervention from the first cohort (administration of 
a 1:1 mix of polyclonal CD8+ TN and TCM cells) is deemed to be acceptably safe, based on toxicity 
stopping rules as defined in Section 23. To assess whether T cell persistence (and possibly 
efficacy) is augmented with lymphodepleting chemotherapy, the 5 patients in cohort #2 will be 
treated with a course of lymphodepleting chemotherapy prior to receiving a 1:1 mix of polyclonal 
CD8+ TN and TCM cells transduced with TCRC4 (Figure 15). The suggested lymphodepletion 
regimen will consist of cyclophosphamide (300 mg/m2) and fludarabine (30 mg/m2) daily for 3 
days (days -4 to -2). Dose reductions or omission of chemotherapy will be allowed as per the 
patient’s clinical situation and discretion of the PI or treating attending physician.
Patients will begin the conditioning regimen approximately 4 weeks after the last cycle of 
consolidation chemotherapy (depending on the patient’s clinical situation, the preference of 
primary treating physician, and the availability of the T cell product). TCRC4-transduced T cells will 
be infused at 36-96 hours after the last dose of chemotherapy, though delayed infusion of T cells 
at > 96 hours post-conditioning will be allowable if there are clinical or logistical issues preventing 
infusion in the specified window. Patients will receive a total target dose of 1 x 109 WT1-specific 
cells/m2 (consisting of 0.5 x 109 each TN and TCM-derived cells). If the actual cell dose for either T 
cell subset is less than the target dose, or if cells from either subset cannot be generated, infusion 
of all available cells will be allowed.
2.  Second infusion of WT1-specific T cells, cohort #2 
As with cohort #1, the subsequent infusion will be held until the transferred cells persist at a level 
of < 5% of the total CD8+ cells and at an absolute number of <30 cells/ȝl. Thus, after a minimum 
of 3 weeks, an additional (second) infusion may be given at a total target dose of 1 x 1010 WT1-
specific cells/m2 (consisting of 0.5 x 1010 each TN- and TCM-derived cells). If the actual cell dose 
for any T cell subset is less than the target dose, or if cells from either subset cannot be 
generated, infusion of all available cells will be allowed. As with the first infusion, patients will 
again be treated with a course of lymphodepleting chemotherapy prior to receiving a 1:1 mix of 
polyclonal CD8+ TN and TCM cells transduced with TCRC4. The suggested lymphodepletion 
regimen will consist of cyclophosphamide (300 mg/m2) and fludarabine (30 mg/m2) daily for 3 
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days (days -4 to -2). Dose reductions in chemotherapy will be allowed as per the patient’s clinical 
situation and discretion of the PI or treating attending physician. While it may seem 
counterproductive to administer chemo-therapy prior to the second planned infusion, when up to 
5% of CD8+ cells may be WT1-specific, this study design strategy will allow the opportunity to 
evaluate persistence and efficacy at the 1.0 x 109 cells/m2 dose level, potentially rendering 
obsolete the higher dose infusion of 1.0 x 1010 cells/m2 which is associated with considerably 
longer production times and higher treatment costs.
3.  IL-2 administration
The second infusion will be followed by low-dose s.c. IL-2 at 250,000 IU/m2 twice daily x 14 days.
After the two planned infusions and IL-2 administration, patients will have completed the study 
treatment. 
4.  Timing of disease restaging with bone marrow sampling
Bone marrow evaluation will be performed within six weeks prior to the first T cell infusion and
approximately four weeks after the second infusion (approximately 14 days after completion of IL-
2). Due to high persistence of infused T cells, however, the second infusion may be delayed for 
weeks to months. Therefore, if the interval between the first and second infusion is > 4 weeks, a 
repeat marrow evaluation will be performed at that time to assess disease response to the initial 
treatment. ,IWKHWLPHLQWHUYDOEHWZHHQ7FHOOLQIXVLRQVLV 10 weeks, a repeat bone marrow 
evaluation will also be performed within four weeks prior to the second round of lymphodepleting 
chemotherapy and second infusion, in order to allow for restaging prior to re-treatment. 
Figure 15. Plan of Treatment (Stage 1, Cohort #2) 
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6.D.  Stage 1, Cohort #3 
1.  First infusion of WT1-specific T cells, cohort #3 
We will proceed with the third cohort of patients if the treatment intervention, including 
lymphodepleting chemotherapy, is deemed to be acceptably safe, based on toxicity stopping rules 
as defined in Section 23. To assess whether the long-term persistence of WT1-specific T cells on 
FHCRC Protocol 2498 is possibly dependent on the infusion of modified T cells that are EBV-
specific, such that the persistence could reflect exposure to EBV antigen, the next 5 patients 
(cohort #3) will receive a 1:1:1 mix of polyclonal CD8+ TN and TCM and TEBV T cells transduced 
with TCRC4, with lymphodepleting chemotherapy (Figure 16). The suggested lymphodepletion 
regimen will consist of cyclophosphamide (300 mg/m2) and fludarabine (30 mg/m2) daily for 3 
days (days -4 to -2). Dose reductions in chemotherapy will be allowed as per the patient’s clinical 
situation and discretion of the PI or treating attending physician.
As with cohort #2, patients will begin the conditioning regimen approximately 4 weeks after the 
last cycle of consolidation chemotherapy (depending on the patient’s clinical situation, the 
preference of primary treating physician, and the availability of the T cell product). TCRC4-
transduced T cells will be infused at 36-96 hours after the last dose of chemotherapy, though 
delayed infusion of T cells at >96 hours post-conditioning will be allowable if there are clinical or 
logistical issues preventing infusion in the specified window. Patients will receive a total target 
dose of 1.5 x 109 WT1-specific cells/m2 (consisting of 0.5 x 109 each TN and TCM-derived cells and 
TEBV cells). If the actual cell dose for any T cell subset is less than the target dose, or if cells from 
any subset cannot be generated, infusion of all available cells will be allowed.
If the addition of lymphodepleting chemotherapy in cohort #2 is associated with unacceptable 
toxicity, as defined in Section 23, cohort #3 will proceed with a 1:1:1 mix of polyclonal CD8+ TN
and TCM and TEBV  T cells transduced with TCRC4 without lymphodepleting chemotherapy.
2.  Second infusion of WT1-specific T cells, cohort #3 
As with the previous cohorts, the subsequent infusion will be held until the transferred cells persist 
at a level of <5% of the total CD8+ cells and at an absolute number of <30 cells/ȝl. Thus, after a 
minimum of 3 weeks, an additional (second) infusion may be given at a total target dose of 1.5 x
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1010 WT1-specific cells/m2 (consisting of 0.5 x 1010 each TN- and TCM-derived cells and TEBV  
cells). If the actual cell dose for any T cell subset is less than the target dose, or if cells from either 
subset cannot be generated, infusion of all available cells will be allowed. As with the first infusion, 
patients will again be treated with a course of lymphodepleting chemotherapy prior to receiving a 
1:1:1 mix of polyclonal CD8+ TN and TCM and TEBV T cells transduced with TCRC4. The suggested 
lymphodepletion regimen will consist of cyclophosphamide (300 mg/m2) and fludarabine (30 
mg/m2) daily for 3 days (days -4 to -2). Dose reductions in chemotherapy will be allowed as per 
the patient’s clinical situation and discretion of the PI or treating attending physician.
3.  IL-2 administration
The second infusion will be followed by low-dose s.c. IL-2 at 250,000 IU/m2 twice daily x 14 days.
After the two planned infusions and IL-2 administration, patients will have completed the study 
treatment. 
4.  Timing of disease restaging with bone marrow sampling
Bone marrow evaluation will be performed within four weeks of the initiation of lymphodepleting 
chemotherapy given immediately prior to T cell infusion, and approximately four weeks after the 
second infusion (approximately 14 days after completion of IL-2). Due to high persistence of 
infused T cells, however, the second infusion may be delayed for weeks to months. Therefore, if 
the interval between the first and second infusion is > 4 weeks, a repeat marrow evaluation will be
performed at that time to assess disease response to the initial treatment. If the time interval 
EHWZHHQ7FHOOLQIXVLRQVLV 10 weeks, a repeat bone marrow evaluation will also be performed 
within four weeks prior to the second round of lymphodepleting chemotherapy and second 
infusion, in order to allow for restaging prior to re-treatment. 
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Figure 16. Plan of Treatment (Stage 1, Cohort #3) 
6.E.  Stage 2 
After 15 patients (across 3 study cohorts in stage 1) have received at least 1 infusion each, and if 
the study treatment has been shown to be safe (as outlined in the statistical plan, Section 23), we 
will start enrolling patients on Stage 2 of the proposed study. The study team will convene with the 
Data Safety Monitoring Board in order to determine the most promising plan of treatment with
regards to safety, T cell persistence, and efficacy (again, as outlined in the statistical plan, 
Section 23). In stage 2, an additional 20 patients will be treated using the selected treatment 
approach, for a planned total of 35 patients treated. 
1.  First infusion of WT1-specific T cells, Stage 2  
All patients will receive a mix of TCRC4-transduced cells derived from polyclonal CD8+ TN and TCM
+/- TEBV T cells, as determined upon review of safety, T cell persistence and potentially available 
efficacy data from Stage 1, as outlined in Section 23.A.2. Unless determined to be unacceptably 
safe in Stage 1, lymphodepleting chemotherapy will be administered prior to T cell therapy, 
consisting of cyclophosphamide and fludarabine, as detailed above in stage 1.  
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Patients will begin the conditioning regimen approximately 4 weeks after the last cycle of 
consolidation chemotherapy (depending on the patient’s clinical situation, the preference of 
primary treating physician, and the availability of the T cell product). TCRC4-transduced T cells will 
be infused at 36-96 hours after the last dose of chemotherapy, though delayed infusion of T cells 
at >96 hours post-conditioning will be allowable if there are clinical or logistical issues preventing 
infusion in the specified window. Patients will receive a total target dose of 1.0 – 1.5 x 109 WT1-
specific cells/m2 (consisting of a mix of TCRC4-transduced cells derived from polyclonal CD8+ TN
and TCM +/- TEBV  T cells, as determined by the study team and DSMB). If the actual cell dose for 
any T cell subset is less than the target dose, or if cells from any subset cannot be generated, 
infusion of all available cells will be allowed.
2.  Second infusion of WT1-specific T cells, Stage 2  
The subsequent infusion will be held until the transferred cells persist at a level of <5% of the total 
CD8+ cells and at an absolute number of <30 cells/ȝl. Thus, after a minimum of 3 weeks, an 
additional (second) infusion may be given at a total target dose of 1.0 – 1.5 x 1010 WT1-specific 
cells/m2 (consisting of a mix of TCRC4-transduced cells derived from polyclonal CD8+ TN and TCM
+/- TEBV) T cells, as determined by the study team and DSMB). If the actual cell dose for any T 
cell subset is less than the target dose, or if cells from either subset cannot be generated, infusion 
of all available cells will be allowed. 
3.  IL-2 administration
Low-dose s.c. IL-2 at 250,000 IU/m2 twice daily x 14 days will follow the 2nd infusion. After the two 
planned infusions and IL-2 administration, patients will have completed the study treatment. 
4.  Timing of disease restaging with bone marrow sampling
Bone marrow assessment will similarly be performed as described above in Section 6B.
PATIENT SELECTION
7.A.  Eligibility for Enrollment  
1. Patients with (non-M3) acute myeloid leukemia (AML).
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2. 3DWLHQWVPXVWEHNJDVSDWLHQWVZLWKORZHUZHLJKWwould be incapable of providing
high volume and frequent blood samples for monitoring and analysis.  
3. Patients or parents/legal guardian must be able to give informed consent.
4. Patients must be able to provide blood and marrow samples and to undergo the 
procedures required for this protocol.
5. Elevated expression of WT1 in pre-treatment bone marrow or peripheral blood by either of 
two methods:
a. Increased expression of WT1 determined if the number of copies of WT1 divided by 
the number of copies of ABL x 104 is > 250 for bone marrow, or > 50 for peripheral 
blood.163;
b. Demonstration of WT1 overexpression will be determined by immunohistochemical 
staining (IHC) in blasts as compared to adjacent normal myeloid and erythroid 
precursors, as determined by a Fred Hutchinson/Seattle Cancer Care Alliance 
pathologist.
6. Demonstration of disease response to induction chemotherapy, in that patients must have 
achieve a morphologic remission (marrow that is at least 10% cellular with < 5% blasts on 
morphologic review) after 1-2 induction cycles, regardless of minimal residual disease or 
CRi/CRp status. 
7. Determination of “high-risk” disease. Subjects must meet one of the determinants of “high-
risk disease”, in terms of being at very high risk for relapse without allogeneic stem cell 
transplant, as per one of the follow criteria: 
a. A designation of “adverse” risk disease at the time of diagnosis, as defined by 
cytogenetic and molecular abnormalities specifically outlined in the 2017 European 
LeukemiaNet (ELN) guidelines for diagnosis and management of AML.142 These 
patients will meet “high-risk” designation, regardless of minimal residual disease or 
CRi/CRp status. 
b. Relapsed leukemia. Patients with cytogenetic or molecular classification other that 
adverse risk by ELN who go on to demonstrate disease relapse after a minimum 
duration of remission of 6 months, but who then attain a second complete remission 
with repeat induction chemotherapy, as defined above in Section 7.A.6. These 
patients will meet “high-risk” designation, regardless of minimal residual disease or 
CRi/CRp status.
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c. Minimal residual disease, as defined by having detectable disease by one of the
following criteria , but otherwise being in morphologic remission, as per Section 7.A.6. 
i.
MRD by flow cytometry at any time after induction chemotherapy or during 
consolidation chemotherapy, when patients are otherwise classified as being in 
morphologlic remission as per Section 7.A.6, and as defined by any abnormal 
myeloid blasts identified by flow cytometric analysis.
ii.
Cytogenetic MRD, as defined by a disease-specific abnormal karyotype at any 
point in patients who are otherwise in morphologic remission, as per Section 
7.A.6
iii.
Molecular minimal residual disease (MRD) with one of the following markers, 
as specified below, in patients who are otherwise in morphologic remission, as 
per Section 7.A.6: 
1. a normalized copy number (NCN) of > 0.001 for CBFB/MYH11 or a 
normalized copy number (NCN) of > 0.050 for AML1/ETO
(RUNX1/RUNX1T1) after at least 4 cycles of consolidation 
chemotherapy  
2. a < 2 log reduction in either CBFB/MYH11 or AML1/ETO 
(RUNX1/RUNX1T1) in the bone marrow at the time of post-induction 
disease restaging (immediately after 1-2 cycles of induction therapy)
d. &5L&5SDVGHILQHGE\QHXWURSKLOFRXQWȝO&5LDQGRUSODWHOHWFRXQW
ȝO&5S, but otherwise being in morphologic remission, as per Section 
7.A.6,QSHGLDWULFSDWLHQWVDSODWHOHWWKUHVKROGRIȝOZLOOEHXVHGDVSHU
consensus pediatric response criteria.76
8. HLA-A*02:01 expression must be present for patient to be on treatment arm, HLA*A02:01 
expression absent in patients designated to observation arm. 
7.B. Exclusion Criteria for Enrollment
1. Active autoimmune disease (e.g. systemic lupus erythematosus, vasculitis, infiltrating lung 
disease, inflammatory bowel disease) in which possible progression during treatment would 
be considered unacceptable by the investigators.  
2. Previous allogeneic HCT.
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3. Any condition or organ toxicity deemed by the Principal Investigator (PI) or the attending 
physician to place the patient at unacceptable risk for treatment on the protocol.
4. Pregnant women, nursing mothers, men or women of reproductive ability who are 
unwilling or unable to use effective contraception or abstinence. Women of childbearing 
potential must have a negative pregnancy test within two weeks prior to enrollment and 
initiation of treatment. 
5. Clinically significant and ongoing immune suppression including, but not limited to:
systemic immunosuppressive agents such as cyclosporine or corticosteroids (at an equivalent 
dose of 0.5 mg prednisone/kg per day, or higher), chronic lymphocytic leukemia (CLL),
uncontrolled human immunodeficiency virus (HIV) infection (untreated or detectable viral load 
within 3 months of enrollment). 
6. Acute promyelocytic leukemia (M3 leukemia, per French-American-British classification)
7.C. Eligibility for Apheresis/blood collection
1. HLA-A*02:01 expression
7.D.  Eligibility for Treatment with TCRC4-transduced CD8+ cells
1. Response to therapy and completion of at least one cycle of consolidation therapy, and
with disease status meeting one of the aforementioned “high-risk” criteria at the time of post-
induction disease restaging as already outlined in Section 7.A.6, above.
2. Hematologic recovery from induction and other post-remission therapy (ANC > 200/ȝl,
platelet count > 20,000/ȝl) at the time of the study intervention
3. No plan for allogeneic stem cell transplantation within 3 months.
4. Elevated expression above baseline of WT1 in bone marrow or peripheral blood. 
5. Additionally, patients treated in stage 1, cohort #3 must be EBV seropositive, given the 
inclusion of T cells derived from an EBV-specific subset in this group
6. Continued morphologic remission (<5% blasts in the marrow, no circulating blasts or 
known extramedullary relapse) within 6 weeks of receiving the study intervention (specified as 
T cell infusion for cohort 1, or the start of lymphodepleting chemotherapy for cohorts 2 and 3). 
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7.E.  Exclusion for Treatment with TCRC4-transduced CD8+ cells
1. Unable to generate antigen-specific WT1-specific CD8+ T cells for infusions. However, if a 
lower than planned number of cells is available, the patient will have the option to receive the 
generated WT1-specific T cells. 
2. Systemic steroids should be stopped 2 weeks before the start of treatment. Topical and 
inhaled steroids are allowed.   
3. Symptomatic and refractory central nervous system (CNS) leukemia.  
4. Complete blood count (CBC) profile prior to treatment:
x
Absolute neutrophil count (ANC) <2ȝO
x
Platelets <2ȝO  
If a patient meets other treatment eligibility but otherwise demonstrates delayed or poor 
recovery of peripheral blood counts to the above neutrophil and/or platelet thresholds, then 
treatment with the T cell intervention will be allowed if: 
a) Neutrophil and/or platelet counts remain below the thresholds after a period of at least 
6 weeks from last systemic chemotherapy; OR neutrophil and/or platelet counts remain 
below the thresholds in the setting of a maintenance therapy, such as midostaurin); 
and
b) The patient has detectable leukemia (e.g. flow cytometry positive or MRD by FISH or 
molecular testing); and
c) The P.I. or treating physician documents that the likely cause of cytopenias is 
underlying disease as opposed to another cause (e.g. medication).
5. Ongoing JUDGHFDUGLDFSXOPRQDU\UHQDOJDVWURLQWHVWLQDORUKHSDWLFWR[LFLWLHV
according to NCI CTCAE version 4 toxicity criteria.
6. .DUQRIVN\SHUIRUPDQFHVWDWXVVFRUHDJH\HDUVRU/DQVN\SOD\VFRUHDJH years) 
 (Appendix A) 
7. Medical or psychological conditions that, according to the PI, would make the patient 
unsuitable candidate for cell therapy.
8. Pregnancy or breast-feeding. Women of childbearing potential must have a negative 
VHUXPRUXULQHȕ-hCG pregnancy test result within 14 days before the first dose of WT1-
specific T cell infusions. Woman of non-childbearing potential will be defined as being 
postmenopausal greater than one year or who have had a bilateral tubal ligation or 
hysterectomy. All recipients of WT1-specific T cells will be counseled to use effective birth 
control during participation in this study and for 12 months after the last T cell infusion.
9. Treatment with alemtuzumab or other T cell-depleting antibodies within 6 months of T cell 
therapy.
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10. Documented new infection within 24 hours of T cell infusion, or concern for new infection 
as suggested by an oral temperature >38.2°C within 24 hours of T cell infusion.  
a. In patients who have T cells delayed because of development of fever (oral 
temperature >38.2°C) and who subsequently become afebrile (38.2°C or less) for 24 
hours without a documented infection, T cells may be administered.
b. In patients with a documented new infection within 24 hours of planned T cell infusion, 
they may go on to receive T cells after administration of directed antibiotic therapy and
if they subsequently remain afebrile (38.2°C or less) for at least 24 hours, and if it is 
deemed clinically appropriate by the PI.
c. Pre-existing infections requiring chronic maintenance therapy (e.g. chronic HBV or 
treated bacterial infections) are not an exclusion for T cell infusion as long as patients 
are on appropriate antimicrobial therapy for at least 1 month (e.g. for chronic hepatitis 
B or C viral infection) and who remain afebrile and without symptomatic evidence for 
uncontrolled chronic infection within 24 hours of T cell infusion. Patients should also 
have a negative HIV test by viral load within 3 months of treatment. 
7.F.  Eligibility for Observation Arm
1. The patient meets all of the eligibility criteria for enrollment, as per Sections 7.A and 7.B,
but lacks expression of HLA-A*0201 as is needed to be enrolled on the treatment arm and 
for apheresis, as per Sections 7.A and 7.C.
CONSENTING
A consent conference will be held with eligible patients. The PI or a delegated representative will 
discuss this study and alternative treatments available for AML. All known risks and potential 
hazards of treatment with WT1-specific transduced memory or naïve polyclonal CD8+ T cells and
low-dose s.c. IL-2 will be discussed. Informed consent will be obtained from the patient using 
forms approved by the FHCRC Institutional Review Board (IRB). After the consent is signed, the 
patients’ HLA-A type will be evaluated and leukemia cells will be evaluated for WT1 expression.
HLA-A*02:01+ patients with elevated WT1 expression will undergo a leukapheresis or large 
volume peripheral blood collection to obtain peripheral blood mononuclear cells (PBMC) used to 
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generate the cellular product. Patients will be moved to the observation arm of the study if they do
not express HLA-A*02:01, if they do not meet treatment criteria (as defined in sections 7D and 
7E), or if WT1-specific T cells cannot be generated.  
PROTOCOL REGISTRATION
Patients will be assigned to the protocol by the Trial Coordinator who will register the patients with 
the Registration Office, (206) 667-4728, between 8:30 am and 4:00 pm, Monday through Friday. 
After hours, the Registration Office can be reached by paging (206) 995-7437. 
PROCEDURE TO OBTAIN PBMC FOR GENERATION OF THERAPEUTIC 
T CELLS
The preferred source of PBMCs will be a non-granulocyte colony-stimulating factor (GCSF)-
mobilized 6 or 12 liter leukapheresis which will be performed after determination of eligibility and 
recovery from cytoreductive therapy. Patients will not be considered for leukapheresis if they have 
a medical condition precluding leukapheresis, but may undergo leukapheresis at a later time if 
condition resolves.  Exclusions may include:
x
Active infection. 
x
Recent hepatitis exposure, hepatitis A or B antigenemia, or hepatitis C antibody 
positivity. 
x
Pregnancy or nursing. 
x
HIV or HTLV infection. 
Leukapheresis (or peripheral blood draw) for collection of autologous T cells may be repeated if 
there are an insufficient number of T cells available for either the first or second (higher dose) 
infusion of modified T cells, as determined by the PI/study team and with the consent of the 
patient. 
If the patient is unable or unwilling to undergo leukapheresis, adults may alternatively have 120-
200 mL of peripheral blood drawn for generation of the T cells. Children < 18 years old will have 
at least 50 mL of peripheral blood drawn for generation of the T cells, based on weight. If weight 
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does not allow for 50 mL of peripheral blood, then the patient must be excluded as this would 
compromise our ability to generate a product. PBMC may be cryopreserved and used to generate 
T cells for this study.
GENERATION OF WT1-SPECIFIC CD8+ T CELLS
All products administered will be derived from the peripheral blood lymphocytes of a patient with 
an established diagnosis of AML, contain autologous transduced WT1-specific CD8+ T cells
derived from TCM and TN cells, and will be generated using clinical grade reagents according to 
Standard Operating Procedures (SOPs) in the Good Manufacturing Practice (GMP)-grade Cell 
Processing Facility (CPF) of the FHCRC. Methods employed to generate and qualify products for 
infusion are outlined in BB-IND 15130 (Sponsor, Dr. Aude Chapuis). T cells demonstrating 
antigen-specificity will be further qualified using mycoplasma, fungal, and bacterial sterility testing.
HANDLING OF T CELL PRODUCTS BEFORE INFUSION
For each infusion dose, the gene-modified cell product will be formulated at the desired cell dose 
in a final volume of 75 - 250 ml. The final product will be prepared and labeled according to SOPs 
in the CPF. After release from the CPF, the cell product will be transported to the infusion facility
by a protocol delegated staff member. During the time of transportation the cell product will be 
kept in a cooler with a cool pack. A research nurse or designee will then administer the cells to the 
patient over 1 to 4 hours (as described in Section 14.C.).
OTHER STUDY AGENTS
13.A. Interleukin-2  
IL-2 will be initiated within 6 hours of the second T cell infusion (second T cell infusion at a dose of 
1.0 x 1010 cells/m2), at a dose of 250,000 U/m2 s.c. twice daily x 14 days. The patient or the 
caregiver will be instructed on s.c. self-administration.
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13.B. Cyclophosphamide 
CY 300 mg/m2 will be administered intravenously daily for 3 total doses (on days -4 to -2) prior to 
administration of T cells. Standard practice policy guidelines will be followed, and its 
administration will be completed at least 36 hours prior to the T cell infusion. Dose reductions will
be allowed as per the patient’s clinical situation and discretion of the PI or treating attending 
physician.
13.C. Fludarabine 
Fludarabine at 30 mg/m2 will also be administered daily for 3 total doses (on days -4 to -2) prior to 
administration of T cells. Its administration will be completed at least 36 hours prior to the T cell 
infusion. Dose reductions will be allowed as per the patient’s clinical situation and discretion of the 
PI or treating attending physician.
PLAN OF TREATMENT
14.A. Screening Consent
After signing the informed consent for screening, patients will be screened for their eligibility, and 
HLA-A typing and analysis of WT1 expression will be performed. If the patient is HLA-A*02:01+
and has high WT1 expression, a leukapheresis (or large volume blood draw) may be scheduled 
after recovery of patients’ counts after chemotherapy. Patients who do not express HLA-A*02:01 
but who otherwise meet study criteria will be moved to the “observation arm” of the study. Those 
found to be eligible for treatment will be consented for the study during a subsequent consent 
visit. Pediatric patients who consent to the study will continue to be treated by the pediatric 
Hematology/Oncology service at Seattle Children’s, whereas it is planned that adult patients who 
consent to the study will be managed by the Immunotherapy service at the Seattle Cancer Care 
Alliance.
14.B. Leukapheresis Visit
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Patients will undergo leukapheresis following cytoreductive chemotherapy. Adult patients will 
preferably undergo the leukapheresis procedure in the outpatient setting (5th floor Apheresis Unit)
at the Seattle Cancer Care Alliance, unless clinical circumstances dictate that leukapheresis must 
be done as an inpatient at the University of Washington Medical Center. Pediatric patients will 
undergo the leukapheresis procedure at Seattle Children’s (Ambulatory Nursing Infusion Unit or 
inpatient setting, if clinically indicated). Leukapheresis will be performed after recovery from 
treatment (ANC >500/ȝO, platelet > 30,000/ȝO) or alternatively a large volume blood draw will be 
performed, as outlined in Section 10. Separate consent for the leukapheresis procedure will be 
obtained, as per Seattle Cancer Care Alliance, University of Washington Medical Center, and 
Seattle Children’s policies.
14.C. Lymphodepletion Chemotherapy
Patients who enroll to be treated on the study, other than the initial 5 patients treated on cohort 
#1, will receive lymphodepleting conditioning chemotherapy as outlined in the Study Design 
(Section 6). Pediatric patients meeting treatment eligibility will receive chemotherapy under the 
continued care of the pediatric Hematology/Oncology service at Seattle Children’s (Ambulatory 
Nursing Infusion Unit). It is planned that adult patients meeting treatment eligibility will receive 
chemotherapy in the outpatient setting (Immunotherapy Clinic or 5th floor Infusion Unit) at the 
Seattle Cancer Care Alliance under the care of the Immunotherapy service.    
14.D. T Cell Infusion
For adults, T cell infusions will be given in the outpatient setting (Immunotherapy Clinic or 5th floor 
Infusion Unit) at the Seattle Cancer Care Alliance, or alternatively at the University of Washington 
(UW) if the study subject is an inpatient at the UWMC yet meets criteria for treatment (Sections 
7D and 7E) at the time of T cell infusion. Adult patients may also be admitted for infusion and 
overnight observation at the Translational Research Unit (TRU) at the UWMC, at the discretion of 
the study team. Pediatric patients will receive T cell infusions in the Ambulatory Nursing Infusion 
Unit located at Seattle Children’s or alternatively in the inpatient setting at Seattle Children’s if the 
study subject is an inpatient yet meets criteria for treatment (Sections 7D and 7E) at the time of T 
cell infusion. For adults, T cells will be infused intravenously at a rate of 250 cc/hour through an 
18- or 20-gauge catheter inserted into a peripheral vein or through a central catheter. For children, 
T cells will be infused intravenously over 1-4 hours through an 18- or 20-gauge catheter inserted 
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into a peripheral vein or through a central catheter. The infusion bag will be gently mixed during 
the infusion. Subjects will have vital signs and oxygen saturation measured by pulse oximetry 
obtained at the start of the infusion, every 15 minutes during the infusion, at the end of the 
infusion, and hourly for 2 hours following the infusion. Intravenous fluids will be administered post-
infusion at the discretion of the PI or treating physician. If no adverse events requiring more 
intensive monitoring or continued hospitalization have occurred, the patients will be released two 
hours following the infusion or at the completion of prescribed intravenous fluids, whichever is 
later (see Section 16). Patients will contact the clinic or overnight medical provider with any fever 
> 100.5oC or other concerning symptoms in the post-infusion period, and will be triaged according 
to standard clinical practice. 
14.E. Low-dose S.C. IL-2 
After cell infusions of 1.0 x 1010 cells/m2, patients will receive twice daily s.c. injections of 
recombinant IL-2 at a dose of 2.5 x 105 U/m2 every 12 hours for 14 days (28 doses) with the first 
dose starting between 2-4 hours after completing the T cell infusion. Patients and/or caregivers 
will be trained (if possible) to self-administer the subsequent IL-2 doses as an outpatient. IL-2
therapy will be discontinued in any patient developing grade 3 or greater treatment-related 
toxicities (see Section 16) while receiving daily s.c. IL-2 injections.
EVALUATION
See Protocol Appendix B for a summary of patient evaluation before, during, and after T cell 
therapy. The dates listed on the study calendar are approximate as it is anticipated many patients 
will reside out of the area and may not always be able to follow the precise time points as dictated 
by the protocol.  
15.A.  Patient Evaluation during the Treatment Consent Visit
Patients will have had a history taken, a physical exam and blood draw performed. Results of
previous marrow examination will be reviewed. Eligibility for enrollment will be checked.
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15.B. Patient Evaluation at Each Planned Visit
Patients will have a history taken, a physical exam with vital signs performed, and a
comprehensive chemistry panel and complete blood count obtained at planned visits including 
prior to the T cell infusion, 1 and 7 days after each infusion, and weekly up to 4 weeks after each 
infusion; urinalysis will be obtained prior to the T cell infusion, and at 14 and 28 days after each 
infusion (Appendix B), or as otherwise clinically indicated.
15.C. Evaluation of WT1 expression by leukemic cells
A two mL (in EDTA) aliquot for research will be obtained with each bone marrow aspirate the 
patient undergoes after enrollment. If the patient has circulating leukemic blasts in peripheral 
blood, 10 mL (in EDTA) of peripheral blood may also be obtained at time of enrollment (pre-
treatment). WT1 expression will be assessed by PCR or by immunohistochemical (IHC) staining 
in pre-treatment peripheral blood or bone marrow samples. Peripheral blood and bone marrow 
specimens may also be collected at selected time points following therapy, as per Section 15.G, 
for analysis of WT1 expression in residual or relapsed leukemic cells. 
15.D. Patient Evaluation During T cell Infusions
Blood pressure, heart rate, temperature, respiratory rate and O2 saturation (by pulse oximetry) will 
be recorded prior to initiation of infusion, every 15 minutes during the 1 to 4 hour T cell infusion, 
and then hourly for 2 hours following the T cell infusion. AEs will be managed by standard medical 
practice (see Section 16). 
15.E. Clinical and Laboratory Evaluation for Toxicity (Primary endpoint)
The period of monitoring for treatment-related toxicity will start with the first day of 
lymphodepletion chemotherapy (or in those patients in stage 1, cohort 1 who do not receive 
chemotherapy such monitoring will start with the first T cell infusion), and continue for 3 months 
after each infusion, as outlined in Section 15.E.1. Subsequently, patients will continue to be 
monitored for evidence of long term effects of treatment with lentivirus-transduced T cells on at 
least an annual basis, as outlined in our plan for Long Term Follow-up in Section 15.K. The 
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evaluation days listed in Appendix B are approximate and it is anticipated that patients who 
reside outside the Seattle area would not always be able to follow the exact time points as 
dictated by the protocol. In addition, frequency and duration of monitoring may be increased if 
indicated based on the patient’s clinical condition and/or duration of cells persistence.
15.E.1 General Toxicity Assessment
All *UDGH Adverse Events (AEs) will be recorded and graded according to the NCI CTCAE 
v4.0 up to 4 weeks after each infusion. Thereafter, unexpected, related and serious adverse 
events (SAEs) will be recorded through the end-of-study (see Section 15.I.). To evaluate for 
potential toxicities, patients will undergo a history and physical exam and laboratory evaluations 
(comprehensive chemistry panel and complete blood count) immediately prior to infusions, and on 
days +1, +7, +14, +21 and +28, and at 2 months and 3 months after each infusion; urinalysis will 
be obtained prior to the T cell infusion, and at 14 and 28 days after each infusion. Although the AE 
reporting period is designated to end at 4 weeks following each given T cell infusion, the study 
team will attempt to obtain and review any internal or external clinical or laboratory records at 
approximately 2 and 3 months post T cell infusion, and then at quarterly intervals thereafter, in 
order to monitor for any SAEs that might develop outside the specified AE reporting period. 
Additionally, the persistence of T cells will be assessed every 3 months. Following the last 
infusion, patients will be monitored, as above. Adjustment of the timing of toxicity assessment 
may be indicated per patient’s clinical situation and at the discretion of the PI or designee. 
Monitoring for treatment-related toxicity will be discontinued if a patient becomes eligible and 
undergoes allogeneic HCT, or if the patient receives other systemic therapy for AML.
15.F. Feasibility Assessment (Primary Endpoint)
All screened patients will be assessed for expression of HLA A*0201, degree of WT1 expression
cytogenetic and molecular features, and burden of disease following induction and consolidation 
therapy. In eligible patients, the proportion of those for whom a T-cell product can be generated 
from PBMC and time required to generate the product will be tracked. The feasibility of admin-
istering autologous WT1-specific T cells as post-remission AML therapy will be evaluated as:
1. The proportion of subjects who undergo leukapheresis or large volume blood draw who 
are ultimately determined to be eligible to receive T-cells. 
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2. The proportion of subjects who sign the treatment consent and undergo a leukapheresis or 
large volume blood draw, from which we are and are not able to generate a product. 
3. The proportion of subjects who sign the treatment consent, have a T cell product 
generated, and ultimately receive the study intervention. 
15.G.  Evaluation of Persistence and/or Efficacy of Adoptively Transferred T Cells (Primary Endpoint)
 
 
15.G.1 Collection of Blood Sample
For adults and children >50kg, 60 ml of blood with heparin (green top) and 10 ml of blood in a 
serum separator (gold top) will be drawn prior to infusions (including at the time of screening 
and/or prior to treatment with the study intervention) and at selected time points following each 
infusion per Section 15.E (see also Appendix B, blood draw for research). The pre-specified 
time points are approximate, with some flexibility due to patient condition or scheduling, and the 
PI may request additional research specimens in the interval between infusions as is necessary to 
determine the persistence level of residual WT1-specific T cells and therefore eligibility for the 
subsequent infusion. The PI may also request additional blood samples for toxicity monitoring or 
persistence testing during the Long Term Follow Up period, as clinically indicated. For children 
weighing 30-50kg, 30 ml of blood with heparin (green top) and 5 ml of blood in a serum separator 
(gold top) will be drawn; and for children weighing 15-30kg, 1 ml/kg of blood with heparin (green 
top) and 3 ml of blood in a serum separator ( gold top) will be drawn per schedule. All blood 
samples will be kept at room temperature and sent to FHCRC Research Cell Bank, Eastlake 
Building, E1-305.  An aliquot of the blood collected will be used to evaluate in vivo persistence of 
infused T cells, including the derivation from the TN and TCM subsets (see Section 15.G.3).  
15.G.2 Collection of Bone Marrow and Extramedullary Samples
Patients will undergo bone marrow evaluation within 6 weeks prior to the first T cell infusion to 
evaluate pre-treatment disease status and endogenous WT1 specific CTL responses, and also 
approximately 14 days after completion of IL-2 to assess disease response to the treatment 
intervention, or as clinically indicated per the treating physician. If high persistence of infused T 
cells is detected (5% of CD8+ cells), resulting in delay of the second infusion  4 weeks, a 
repeat marrow evaluation may be performed to assess disease response to the initial treatment. If 
WKHWLPHLQWHUYDOEHWZHHQ7FHOOLQIXVLRQVLVZHHNVDUHSHDWERQHPDUURZHYDOXDWLRQZLOODOVR
be performed within six weeks prior to the second infusion, in order to allow for restaging prior to 
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re-treatment. Bone marrow evaluation in addition to, or outside of, these specified time points will 
be allowed as clinically indicated, as per the patient’s treating physician.
Bone marrow samples, as per the approved methodk may be also obtained, if patients have 
signed screening consent 9296R, re-treatment marrow already undergoing a marrow procedure 
for non-research reasons, if a patie, if  may be obtained in advance of planned treament, once a 
subject consent on 9296R.
For any bone marrow evaluation during the study period, we request the following specimens for 
research in addition to any marrow samples sent for clinical purposes: 
x
Approximately 7 ml of bone marrow aspirate (5 ml for patients 15-30 kg) in heparin (green
top) tubes should be sent to FHCRC Research Cell Bank, Eastlake Building, E1-305 to be 
evaluated for the presence of adoptively transferred T cells by WT1 and/or EBV HLA-
A*0201 peptide/multimer analysis and for other immunological parameters.
In patients with surgically accessible disease (disease that is accessible by needle or core biopsy, 
a malignant aspirate, or tumor that is surgically excised) in whom enough material can be 
obtained, single cells suspensions will be prepared to assess for T cell persistence and/or WT1 
expression in the leukemia cells. All samples will be kept at room temperature and sent to the
FHCRC Research Cell Bank, Eastlake Building, E1-305. 
15.G.3.  Assessment of Overall, TCM, TN and TEBV Cell Persistence
Transferred TCRC4 CTL (overall persistence) will be enumerated by staining with TCRC4-binding 
WT1 multimer in PBMC obtained at each time-points for as long as infused cells can be detected 
by multimer-binding (limit of detection approximately 0.03% of total CD8+ T cells). DNA will also 
be isolated from blood and bone marrow samples and analyzed by PCR for WPRE as a marker of 
the presence of transduced T cells. HTTCS will then be used to characterize the presence and 
relative frequencies of individual T cell clonotypes derived from the TN, TCM and TEBV subsets in 
the blood, bone marrow and/or extramedullary sites. HTTCS will be performed from samples 
drawn at day +7 (the peak of the response), at day +28 (establishment of short-term memory), at 
3 months (long-term memory), and every 3 months thereafter while transduced cells are still 
detectable by multimer analysis. Clonotype frequencies >0.01% of CD3+ T cells will be summated 
to determine relative frequencies of TN-, TCM- and TEBV-derived at different time points. Because 
the sum of frequencies for each subset will likely still be >0.01% at 3-6 months, a dominant
population (TN, TCM and TEBV) will be defined as one in which the sum of its clonotype frequencies 
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reaches >80% of detectable transferred clonotypes. The time to disappearance of a transferred
subpopulation will be defined as the time-point when the sum of transferred clonotype frequencies 
from that subset reaches 0.01% of CD8+ T cells (see Section 23.C). Persistence of transferred 
TCRC4 WT1-specific T cells by multimer analysis and HTTCS will be performed in a similar 
fashion on specimens from any extramedullary sites, as they are available.
15.H.  Efficacy Assessment (Exploratory Endpoint)
To assess the potential anti-leukemic effect of adoptively transferred T cells, bone marrow will be 
obtained to evaluate disease status by morphology, flow cytometry, and if appropriate by 
cytogenetics and molecular studies before and after treatment.  For patients with a history of 
extramedullary disease, appropriate radiographic studies will be used to assess for treatment 
response or recurrence. Efficacy will be assessed by comparing outcomes of the study cohort 
with those in the non-treatment (observation) arm, as well as by determining treatment response 
in patients with measurable disease at the time of treatment.
15.H.1. For patients with measurable MRD: Patients with MRD detectable by flow cytometry at 
the time of study treatment will be evaluated at subsequent time points for eradication or reduction 
of MRD, as per response criteria described in Appendix C. Although patients will be enrolled to 
the study based on the presence of MRD by flow cytometry, some patients will also be expected 
to have MRD detectable by cytogenetics, FISH or PCR; therefore, appropriate patients will also 
be evaluated for evidence of eradiation or reduction in cytogenetic and molecular MRD, as per 
response criteria described in Appendix C. 
15.H.2. The probability of relapse, disease-free survival and overall survival in the entire cohort of 
patients receiving the study intervention will be determined in comparison to those in the 
observation cohort (see Section 23D). The probability of relapse, disease-free survival and
overall survival in both will also be determined in the subgroups of patients who are enrolled with 
MRD, as well as for the subgroup with CRi/CRp.  
15.I. TN, TCM and TEBV Migration to Tumor Sites (Exploratory Endpoint)
Localization of transferred TCRC4 TN, TCM and TEBV cells to tumor sites will be evaluated in 
marrow samples obtained after infusions as well as in extra-medullary samples if available. DNA 
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will be isolated from all samples to determine the presence and frequency of TN, TCM and TEBV
cells by HTTCS. To assess for the preferential localization of TN/TCM/ TEBV, results will be 
compared to pre-treatment and concurrent PBMC values and, where possible, to pre-infusion 
extra-medullary tumor sample values. If enough material is obtained, single cell suspensions will 
be prepared and isolated by multimer staining to determine the phenotype and function of 
transferred CTL (see Section 15.J)  
15.J. Assessment of the Functional Capacity of Transferred Cells (Exploratory Endpoint)
Persisting cells in blood, bone marrow and, if possible, tumor tissue, will be assessed for the 
surface expression of markers of central memory T cells (for example expression of CD28, CD27, 
CD127, CD62L and CCR7), markers associated with recent cell activation (for example CD69, 
CD25, CD107 and CD137), and markers associated with prolonged activation/exhaustion (for 
example PD1, TIM-3, LAG-3, CD160, 2B4, KLRG-1 and CD57). Results will be compared the 
original infusion products. The function of persisting TCRC4 cells will be assessed by measuring
intracellular IFNJ, TNFD and IL-2 in response to stimulate with the WT1 epitope. Intra-nuclear Ki-
67 expression will be used as an indication of in vivo proliferation. CFSE dilution will be used to 
assess ex vivo proliferative capacity in response to the WT1 epitope, and will be compared to the 
infusion product, as described165,166. HTTCS will be used to determine the origin and proportions 
of the persisting cells (TN/TCM/ TEBV). The function, proliferation and proliferative capacity of T 
cells will be correlated with the type of persisting cells (TN/TCM/ TEBV).
15.K. Evaluation for Long Term Effects of Treatment with Lentivirus-Transduced T Cells
15.K.1 Testing for Replication Competent Lentivirus (RCL)
In compliance with FDA Guidance, “Supplemental Guidance on Testing for Replication 
Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up
of Patients in Clinical Trials Using Retroviral Vectors” (As of November 28, 2006, the same rules 
were stated to apply to lentiviral vectors), efforts will be made to obtain blood samples to test for 
replication competent lentivirus (RCL) at the following time points: Pretreatment, at 3, 6 and 12 
months for the first year, and annually for up to 15 years. If all post-treatment assays are negative 
during the first year, subsequent yearly samples may be discontinued or may be obtained and be 
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archived for future analysis, and analyzed if clinical or scientific events make this indicated. 
Samples will be archived with appropriate safeguards to ensure long-term storage using a system 
that allows for the prompt linkage and retrieval of the stored samples with the medical records of 
the patient and the production lot records. If any post-treatment samples are positive, further 
analysis of the RCL and more extensive patient follow-up will be undertaken, in consultation with 
the Center for Biologics Evaluation & Research (CBER). 
15.K.2  Long-Term Follow-Up 
Efforts will be made to follow patients for 15 years, in compliance with the FDA Guidance, “Gene 
Therapy Clinical Trials - Observing Subjects for Delayed Adverse Events” (November 28, 2006). 
As per this guidance, viruses that have a potential to integrate, including lentiviruses, “present 
sufficient risk that long-term follow-up (LTFU) observations are necessary to mitigate long-term 
risks to subjects receiving these vectors." The patients on this study will have follow-up clinical 
visits on the same time points as testing for RCL occurs (pretreatment, at 3, 6 and 12 months for 
the first year, and annually thereafter if possible). At clinical follow-up visits, patients will be 
examined for clinical evidence suggestive of a potential lentiviral disease, such as cancer, 
neurologic disorders, and new onset hematologic disorders. Additionally, samples will be collected 
to determine levels of gene-modified cells in peripheral blood for up to 15 years as recommended
by FDA regulations. Suspect clinical symptoms or findings as noted above will trigger performing 
RCL analysis of archived samples and/or attempting to obtain additional samples, in consultation 
with CBER. 
At clinic visits, patients will undergo physical examination and laboratory testing including CBC 
with differential, comprehensive chemistry panel and assays for levels of gene-modified cells. 
Details of all exposures to mutagenic agents and other medications will be ascertained and 
recorded in case histories. Physicians will also be asked to record the emergence of new clinical 
conditions, including new malignancies, new incidence or exacerbation of a pre-existing 
neurologic disorder, new incidence or exacerbation of a prior rheumatologic or other autoimmune 
disorder, and new incidence of a hematologic disorder. Study subjects and health providers will
be asked to cooperate in reporting delayed adverse events, including unexpected illnesses and 
hospitalizations. In general, most patients will be seen by their primary doctors in their local area,
but the FHCRC in collaboration with Seattle Cancer Care Alliance (SCCA) will be available to 
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assist in the LTFU of participants in this clinical trial. If patients die or develop neoplasms, an
effort will be made to assay for RCL and lentiviral integration in a biopsy sample of the neoplastic 
tissue or the pertinent autopsy tissue.
MANAGEMENT OF TOXICITIES AND COMPLICATIONS
16.A. Toxicity Grading 
 
Toxicity grading will be evaluated according to the current guidelines in NCI CTCAE v4.0.80 The
full text of the NCI CTCAE is available online at: http://evs.nci.nih.gov/ftp1/CTCAE/About.html. 
16.B Regimen-related Toxicity 
If the patient develops excessive toxicity attributable to one or more component of the regimen, 
the patient will not receive additional study therapy and therapy with corticosteroids will be given if 
clinically indicated (see Section 16.E and Section 16.F). If there is evidence of excessive 
unexpected treatment-related toxicity, the study stopping rules will apply (see Section 23.A). 
Excessive toxicity will be considered when a non-pre-existing grade 3 or higher toxicity develops 
after the start of treatment with the following exceptions: 
16.B.1. Expected toxicities attributable to T cell infusions and considered exceptions to criteria 
for discontinuation include: 
i.
Cytokine Release Syndrome (CRS)  grade 3, including but not limited to asthenia, flu-like 
symptoms, myalgia, lymphopenia, and rigors. 
ii.
Skin rash/erythroderma (grade 3 toxicity) lasting for <7 days. 
iii.
Hypoxemia requiring continuous oxygen, but not mechanical ventilation or intubation, 
lasting <72 hours.
iv.
Fever XSWR!Û&WKDWUHVROYHVZLWKLQ hours of the T cell infusion.
v. 
Hypotension responsive to intravenous hydration and lasting for <72 hours, but not 
requiring pressor support. 
vi.
Grade 4 lymphopenia (lymphocytes < 200) that resolves within 3 weeks to baseline levels 
(pre-therapy).
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vii.
Single laboratory values out of normal that according to the investigator do not have a 
FOLQLFDOFRUUHODWHDQGUHVROYHWRJUDGHZLWKLQGD\VZLWKDGHTXDWHPHGLFDO
management. 
16.B.2. Expected toxicities attributable to low-dose s.c. IL-2 considered exceptions to criteria for 
discontinuation include: 
i.
Flu-like symptoms (headache, muscle ache, joint ache). 
ii.
Mild fever. 
iii.
Redness, pain, and swelling at injection site. 
iv. 
Drop in blood pressure determined by physician to require intravenous fluids.
v. 
Cardiac aUUK\WKPLDVJUDGH
vi.
Single laboratory values out of normal that according to the investigator do not have a 
FOLQLFDOFRUUHODWHDQGUHVROYHWRJUDGHZLWKLQGD\VZLWKDGHTXDWHPHGLFDO
management. 
16.B.3. Expected toxicities attributable to cyclophosphamide and fludarabine conditioning
considered exceptions to criteria for discontinuation include: 
i.
Nausea, vomiting or diarrhea that resolves within 7 days. 
ii. 
Lymphopenia (lymphocytes <200) that resolves within 3 weeks to baseline levels (pre-
therapy).
iii.
Thrombocytopenia (platelets <20,000) that resolves within 3 weeks to baseline levels (pre-
therapy)
iv.
Grade 4 neutropenia (neutrophils <500) that resolves within 3 weeks to baseline levels 
(pre-therapy).
a. *Note that febrile neutropenia constitutes an expected toxicity in a proportion of
patients treated with lymphodepleting chemotherapy. However, this will not be 
considered an exception to criteria for treatment discontinuation, as consideration 
to altering the lymphodepletion chemotherapy regimen will be made if stopping 
rules are met because of febrile neutropenia. For the purpose of this study, febrile 
neutropenia is defined as ANC<500/mm3 with a temperature of 38.3oC.  
16.C. Definition and Management/Evaluation of Non-hematologic and Hematologic Toxicities 
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16.C.1  Definition of Dose-Limiting Toxicities  
Although unlikely based on prior experience, it is possible that the infusion of TCRC4-transduced 
CD8+ T cells will result in toxicities related to the recognition of normal tissues expressing WT1. It 
is anticipated that such symptoms and signs will occur within hours to 4 weeks after completion of 
the T cell infusion, though patients will continue to be followed for potential toxicities for 12 months 
after the last infusion, as described in Section 15.E and Section 15.K.  
16.C.2 Non-hematologic toxicity requiring treatment discontinuation 
This is defined as any grade 3 or 4 non-hematologic toxicity (CTCAE 4) that is deemed to be 
probably or definitely caused by infusion of the study treatment (i.e. not attributable to infection, 
recurrent/progressive AML, toxicity from prior therapies, or any identifiable cause other than 
conditioning chemotherapy, T cell infusion or IL-2 administration) that occurs at any time point 
after the start of therapy on this study. It is expected that the onset of non-hematologic toxicities 
will be within four weeks after completion of the last T cell infusion although patients will be 
followed beyond four weeks for potential toxicities, as described in Section 15.E and Section 
15.K. In patients who are still within the 12 month window of toxicity monitoring, but who no longer 
have any detectable WT1-specific T cells, new adverse events will not be attributed to WT1-
specific T cells and will not be considered for treatment discontinuation.
16.C.3 Management of Non-hematologic toxicity 
Any toxicities potentially caused by the T cell infusion and not attributable to a non-infusion related 
cause, occurring within 4 weeks of study treatment and requiring treatment discontinuation, will be 
managed by supportive care and steroids or tocilizumab at the discretion of the attending and the 
PI. This is outlined in Section 16.E. and Section 16.F.
16.C.4 Hematologic toxicity requiring treatment discontinuation 
This is defined as any new or recurrent onset of thrombocytopenia (platelets < 20,000/mm3) or 
otherwise grade 4 hematologic toxicity (blood/bone marrow CTCAE 4) that occurs at any time 
point after the first T cell infusion such as neutropenia (ANC < 500/mm3) lasting > 2 weeks in 
patients not receiving chemotherapy and lasting  3 weeks in patients receiving chemotherapy, in 
the context of new bone marrow aplasia (< 5% cellularity), and which is attributed to the study 
treatment (i.e. not associated with prior chemotherapy, tumor progression in the bone marrow, 
infection, medications, or any identifiable cause other than T cell infusion or IL-2 administration).
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In patients with incomplete count recovery (CRi/CRp) prior to the study intervention, we will 
consider grade 4 hematologic toxicity requiring treatment discontinuation as a sustained drop in 
ANC below 20% of the starting measurement and/or in platelets below 20% of the starting 
measurement lasting > 3 weeks and which is attributed to the study treatment. In patients who are 
still within the 12 month window of toxicity monitoring, but who no longer have any detectable 
WT1-specific T cells, new adverse events will not be attributed to WT1-specific T cells and will not 
be considered for treatment discontinuation.
16.C.5 Management of hematologic toxicity  
If a patient develops new onset hematologic toxicity lasting at least 2 weeks in patients not 
UHFHLYLQJFKHPRWKHUDS\DQGODVWLQJZHHNVLQSDWLHQWVUHFHLYLQJFKHPRWKHUDS\, defined herein 
as ANC <500/mm3 or platelets <20,000/mm3 in a patient with pretreatment laboratory values of 
ANC >1000/mm3 and platelets >100,000/mm3, and which cannot be explained by alternative 
cause such as known disease progression, infection, or other medications, a bone marrow 
sample will be obtained. As mentioned above, in patients with incomplete count recovery 
(CRi/CRp) prior to the study intervention, we will consider grade 4 hematologic toxicity requiring 
treatment discontinuation as a sustained drop lasting at least one week in ANC below 20% of the 
starting measurement and/or in platelets below 20% of the starting measurement.  
a. If the bone marrow shows evidence of aplasia, corticosteroids will be started as described 
in Section 16.F. and study treatment discontinued. G-CSF may be administered at the 
discretion of the medical team.
b. If the bone marrow does not reveal aplasia or disease progression, and if no other clear 
etiology for marrow suppression is found, patients will be observed for seven more days. If 
by that time there is no improvement of bone marrow function and no clear etiology for 
marrow suppression is still detected, corticosteroids will be started as described in
Section 16.F and study treatment discontinued. G-CSF may be administered at the
discretion of the medical team.
c. If bone marrow cannot be obtained and no alternative cause of hematologic toxicity is 
identified by the seventh consecutive day of neutropenia or thrombocytopenia, 
corticosteroids will be started as described in Section 16.F. and study treatment 
discontinued. G-CSF may be administered at the discretion of the medical team.
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It is expected that onset of hematologic toxicities will be within four weeks after completion of 
study therapy, although patients will be followed beyond four weeks for potential toxicities as 
described in Section 15.E and Section 15.K. 
16.D. Management of Symptoms During T cell Infusions 
Immediate reactions to infusions (i.e. defined as those occurring either during the first 24 hours 
following T cell infusion) might occur due to release of cytokines from T cells stimulated by the 
recognition of targets. Mild transient symptoms have been observed with LAK, TIL cell and
antigen-specific T cell clones. 
16.D.1. Milder reactions (i.e. <grade 3 CTCAE v.4 or less severe than specified below) 
Including symptoms such as: 
x
Fever, chills, fatigue. 
x
Dyspnea, chest tightness, or myalgia. 
x
Alteration in vital signs such as: 
- 
Lowering of blood pressure (BP)EXWZLWKV\VWROLF%3PP+JRU mmHg 
below baseline. 
- 
Tachycardia, but with heart rate (HR) 5RU50 above baseline.
- 
Tachypnea, but with respiration rate (RR) PLQRUDERYe baseline.
- 
Hypoxemia, but O2 VDWXUDWLRQRQURRPDLURUIDOOIURPEDVHOLQH
x
Skin changes such as erythema or urticaria. 
Suggested Management may include decreasing the rate of infusion and/or appropriate 
supportive care such as:
x
Acetaminophen or Demerol for fever and chills. (All subjects who develop fever or 
chills should have a blood culture drawn).
x
Acetaminophen for headache.
x
Diphenhydramine for nausea and vomiting.
x
Fluid administration for hypotension.
x
Supplemental oxygen for hypoxemia.
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16.D.2. More severe reactions 
Including symptoms such as:
x
Hypotension with systolic BP <90 mmHg and >20 mmHg below baseline. 
x
Tachycardia with HR >150 and >50 above baseline.
x
Tachypnea with RR >32 and >10 above baseline. 
x
Hypoxemia with O2 saturation of <88% and >5% fall from baseline.
Suggested Management may include modifying the infusion rate or terminating the infusion, and 
administering supportive medical care:
x
If patient responds to supportive care by normalization of vital signs or resolution of 
hypoxemia, and the PI or designee deems it safe to continue, the infusion will be restarted 
at slower rate.  
x
If the patient does not respond by normalization of vital signs or hypoxemia after 
supportive care alone, corticosteroids to ablate the infused T cells may be administered as 
per Section 16.F.
16.D.3.  Any unexpected severe toxicity (see Section 16.B) occurring in the first 24 hours due 
to the T cell infusion and not attributable to a non-infusion related cause.
Management will be by supportive medical care, and corticosteroids to ablate cells may be
administered after discussion with the PI or designee as per Section 16.F. 
16.E. Management of Severe Cytokine Release Syndromes (or Cytokine Storm) 
Blood samples for research tests are planned to be collected in all patients prior to the T cell
infusion, and on specified time points after the T cell infusion as indicated in Appendix B. Plasma 
will also be isolated from each blood sample and stored for future cytokine analysis. 
If a patient becomes febrile or develops clinical evidence of a cytokine storm syndrome, we may 
measure cytokine levels (including IFNJ, TNFD, IL-6, IL-2 concentrations), serum ferritin, and 
markers of tumor lysis syndrome (for example, electrolytes, uric acid, lactate dehydrogenase 
[LDH]), and evaluate the persistence and the phenotype of the TCRC4-expressing cells at 
additional time points as clinically indicated.
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Any patient who develops clinical evidence of a cytokine storm syndrome will have a workup to 
exclude infection or other causes. Initial treatment should consist of supportive measures as 
dictated by the clinical and laboratory findings, and may include fluid replacement, antipyretics, 
oxygen supplementation, and broad-spectrum antibiotics if infection cannot be excluded as a 
potential etiology for the signs and symptoms. Serious and/or progressive symptoms and signs 
may result in the administration of corticosteroids and/or tocilizumab as described in Section 
16.F.  
16.F. Management of Severe Treatment-related Toxicities 
1. If a new and unexpected CTCAE vJUDGHWR[LFLW\LVREVHUYHGIROORZLQJT cell infusion, 
the patient will receive medical treatment appropriate for the clinical abnormality. 
2. $QHZDQGXQH[SHFWHGJUDGHWR[LFLW\WKDWLVDWWULEXWDEOHWRWKHT cell infusion, is 
unresponsive to supportive measures, or persists >7 days may be treated with corticosteroids
following the suggested dose schedule below (this schedule is adaptable depending on the 
presence of TCRC4-transduced cells and/or the clinical picture), and/or tocilizumab (anti-
interleukin-6 receptor) after discussion with the PI. These are general guidelines for the 
glucocorticoid administration and taper and may be adjusted based on each patient’s clinical 
situation. 
3. Patients who require corticosteroids for treatment of severe toxicities will not receive additional 
T cell or IL-2 treatment on this protocol and will continue to be followed for toxicity until 
symptoms have resolved. Methylprednisolone (or an equivalent dose of an alternative 
corticosteroid) can be tapered more rapidly when symptoms are significantly improving or 
WT1-specific T cells are <0.05% of CD8+ cells in peripheral blood. 
Day 1
i.v. Methylprednisolone at 2mg/kg/day if severe
p.o. Prednisone at 1mg/kg/day if clinically stable
Day 2
i.v. Methylprednisolone at 2mg/kg/day if severe
p.o. Prednisone at 1mg/kg/day if clinically stable
Day 3-4
Prednisone at 0.5-1mg/kg/day p.o.
Day 5-12
Taper p.o prednisone by 20% q 2 days
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4. It is suggested that patients receiving corticosteroids have daily assessments of peripheral 
blood for the presence of WT1-specific CTL cells (vE17+ and multimer+) for 5 days and then 
at least weekly for four weeks. 20 mL of blood (or up to 1 mL/kg in patients 15-20 kg) will be 
sent in ACD yellow top tube to the Greenberg Lab (D3-335) for the evaluation of the presence 
of the infused T cells.
16.G. Concomitant Therapy
16.G.1 Active infections occurring after initiating the study should be treated per clinical 
judgment. 
16.G.2 Patients with extramedullary disease may receive radiation therapy at the discretion of 
their treating physician and consulting radiation oncologist.
16.H. Off-study Criteria
A patient’s participation on the protocol will be terminated for any of the following reasons and the 
reasons for premature discontinuation must be recorded on the case report form. 
x
If the sponsor decides to stop the study
x
The participant withdraws consent.
x
Patient death.
x
At the discretion of the PI
MANAGEMENT OF T CELL INFUSIONS IN PATIENTS WHO RELAPSE OR 
PROGRESS DURING T CELL THERAPY
Patients who started treatment with no evidence of disease and relapsed during T cell therapy, or 
patients who started treatment with evidence of disease and experience progression of their 
disease during T cell therapy will be evaluated for the characteristics of the leukemic cells and 
advised about treatment options by their treating physician.
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T cell therapy may be continued if relapse or disease progression occurs prior to the last dose of 
T cells infusions, or at the discretion of the PI. The T cell infusions may be interrupted to allow 
administration of cytoreductive therapy. Patients who receive cytoreductive chemotherapy may 
resume treatment with WT1-specific T cells after evidence of recovery of hematopoiesis 
(ANC >200/mm3 and plts >20,000/ mm3).
This decision to continue WT1-specific T cell therapy despite disease progression is based on the 
extremely poor prognosis for these patients, the fact that they might benefit from infusion of 
available WT1-specific T cells, and the possibility of acquiring insights into safety and potential 
anti-leukemic effects of WT1-specific T cells.
Patients whose T cell therapy is interrupted for cytoreductive therapy will not undergo protocol 
specific evaluations, AE assessments or sample collections until T cell therapy resumes.
OPTIONS FOR FURTHER TREATMENT AFTER COMPLETION OF STUDY 
THERAPY
If adoptively transferred WT1-specific T cell infusion demonstrated anti-leukemic activity in the 
patient, the option of additional T cell infusions may be discussed with the PI, the IND Sponsor, 
the patient and the treating physician.
TARGETED/PLANNED ENROLLMENT
This study plans to treat 35 individuals with AML over 3 years.  
Table 5. Ethnic and gender distribution chart
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GUIDELINES FOR ADVERSE EVENTS REPORTING
20.A. Reporting of Adverse Events (AEs)
All unexpected and serious adverse events attributed to study treatment or intervention must be 
reported to the FHCRC Institutional Review Office (IRO) per the current reporting requirements. 
All JUDGH&7&$(YAEs will be recorded from the time of the first T cell infusion through 4 
weeks after the last T cell infusion. Beginning 4 weeks after the last T cell infusion, only study-
related SAEs may be recorded. For those patients with an interval of > 1 month between T cell 
LQIXVLRQVFROOHFWLRQRIDOOJUDGH$(VZLOObe temporarily put on hold, and then will resume at 
the onset of any additional study treatments. 
20.B. Reporting to the FDA
As a study conducted under IND (Investigational New Drug) regulations we will comply with the 
FDA regulations regarding safety reporting 21CFR312.32 including the following requirements:
1.
A sponsor must promptly review all information relevant to the safety of the drug 
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21CFR312.32 (b).
2.
A sponsor must notify FDA in an IND safety report of potential serious risks, as soon as 
possible but in no case later than 15 calendar days after the sponsor determines that the 
information qualifies for reporting under 21 CFR312.32 (c)(1). Information that is required to be 
reported includes, but is not limited to, a. Serious and unexpected adverse reactions and b. An 
increased rate of occurrence of serious suspected adverse reactions. 
3.
The IND safety report must be completed and sent to the FDA in a narrative format, on 
FDA Form 3500A, or an electronic format.
4.
A sponsor must also notify FDA of any unexpected fatal or life-threatening suspected 
adverse reaction as soon as possible but in no case later than 7 calendar days after the 
sponsor’s initial receipt of the information 21CFR312.32 (c)(2).
20.C. Definitions 
Definitions associated with reportable events can be found on the FHCRC IRO extranet website. 
(Relevant FHCRC policies include, but are not limited to the documents listed in Table 6. Please 
also refer to the FHCRC IRO website.)
Table 6. FHCRC IRB policies for reportable events
IRB Policy 2.6
Adverse Events and Other 
Unanticipated Problems 
Involving Risks to Subjects or
Others
http://extranet.fhcrc.org/EN/sections/iro/
irb/ae.html
IRB Policy 1.9
Noncompliance with the Office 
of the Director’s Human 
Research Protection Program 
Policy
http://extranet.fhcrc.org/EN/sections/iro/
irb/ae.html
IRB Policy 1.1
Reporting Obligations for 
Principal Investigators
http://extranet.fhcrc.org/EN/sections/iro/
irb/policy/index.html
IRB Policy 2.2
Continuing Review
http://extranet.fhcrc.org/EN/sections/iro/
irb/policy/index.html
IRB Policy 1.13
Investigational New Drugs 
(IND), Biologics and 
Investigational Device 
Exemptions (IDE)
http://extranet.fhcrc.org/EN/sections/iro/
irb/policy/index.html
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DATA AND SAFETY MONITORING PLAN
21.A. Primary Monitoring
The PI of the study and the IND Sponsor will have primary responsibility for ensuring that the 
protocol is conducted as approved by the Scientific Review Committee and IRB. The PI and the 
IND Sponsor will ensure that the monitoring plan is followed, that all data required for oversight of 
monitoring are accurately reported to the Protocol Office and Data Safety Monitoring Board 
(DSMB), that all adverse events are reported according to protocol guidelines, and that any 
adverse reactions reflecting patient safety concerns are appropriately reported.
21.B. Monitoring Plan
The PI, or a co-investigator on the study designated by the PI, will personally review with the 
Study Nurse the clinical course of all WT1-specific T cell infusions. The PI or his/her designee will 
review with the Study Nurse the progress of each patient undergoing therapy as well as the 
clinical course of all patients who have completed a course of T cell therapy. 
21.C. Monitoring the Progress of the Trial and the Safety of Participants
The study will be monitored by the Immunotherapy Integrated Research Center (IIRC) DSMB. 
The DSMB will be responsible for safeguarding the interests of trial participants and assessing the 
safety and efficacy of the interventions during the trial.  This responsibility will be exercised by 
providing recommendations about stopping or continuing the trial.  To contribute to enhancing the 
integrity of the trial, the DSMB may also formulate recommendations relating to the selection, 
recruitment and retention of participants and their management; adherence to protocol-specified 
regimens; and the procedures for data management and quality control. 
The DSMB will be advisory to the study Sponsor and the PI, who will be responsible for prompt 
review of the DSMB recommendations to guide decisions regarding continuation or termination of 
the trial and whether amendments to the protocol or changes in study conduct are required.
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The DSMB is an independent, multidisciplinary group consisting of clinical experts and a 
statistician who collectively have experience in leukemia, lymphoma, hematology, biostatistics, 
and the conduct and monitoring of clinical trials. The DSMB will meet approximately every 6 
months to review data. The current members are listed in the IIRC DSMB charter.
Institutional support of trial monitoring will be in accordance with the FHCRC/University of 
Washington Cancer Consortium Institutional Data and Safety Monitoring Plan.  Under the 
provisions of this plan, FHCRC Clinical Research Support coordinates data and compliance 
monitoring conducted by consultants, contract research organizations, or FHCRC employees 
unaffiliated with the conduct of the study.  Independent monitoring visits occur at specified 
intervals determined by the assessed risk level of the study and the findings of previous visits 
per the institutional DSMP. 
In addition, protocols are reviewed at least annually and as needed by the Consortium Data and 
Safety Monitoring Committee (DSMC), the FHCRC Scientific Review Committee (SRC), and the 
FHCRC/University of Washington Cancer Consortium Institutional Review Board (IRB).  The 
review committees evaluate accrual, adverse events, stopping rules, and adherence to the 
applicable data and safety monitoring plan for studies actively enrolling or treating patients.  The 
IRB reviews the study progress and safety information to assess continued acceptability of the 
risk-benefit ratio for human subjects.  Approval of committees as applicable is necessary to 
continue the study.
The trial will comply with the standard guidelines set forth by these regulatory committees and 
other institutional, state and federal guidelines. 
21.C.3  Statistical Monitoring Guidelines  
The DSMB will review all grade III or greater toxicities as defined by version 4.0 of NCI CTCAE,
and as further described in Section 16 of this protocol and determine whether the study should be 
prematurely discontinued due to toxicity. The clinical investigators assessing patients will be 
responsible for evaluations to grade toxicity. Criteria for discontinuing therapy in an individual 
patient are described in protocol section titled “Management of Toxicities and Complications”. 
Criteria and procedures for discontinuing the trial are described in the sections titled “Data and 
Safety Monitoring” and “Statistical Considerations”.
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The type and grade of toxicities noted during therapy will be summarized for each dose 
level/infusion. All AEs noted by the investigator will be tabulated according to the affected body 
system. Changes from baseline in clinical and laboratory parameters will be summarized in a 
table. Tumor responses will be determined as specified above.
RECORDS
The Clinical Research Division at the FHCRC maintains a patient database that allows for the 
storage and retrieval of specific types of patient data including demographic information, protocol 
registration information, and data from the treatment course. These data are collected from a wide 
variety of sources and conform to institutionally established guidelines for coding, collection, key 
entry, and verification. Each patient will be assigned a unique patient number (UPN) to assure 
patient confidentiality. Any publication or presentation will refer to patients by this number and not 
by name. Information about patients enrolled on this protocol that is forwarded to agencies such 
as the FHCRC IRB, NIH, and FDA will refer to the patients only by their UPN. 
Original inpatient and outpatient medical records will be maintained by the medical records 
departments at the institutions where the patients receive their care. The majority of the care 
related to this protocol will be received at the SCCA and UW Medical Center. The study nurse 
and/or data coordinator will maintain a Case Report Form (CRF) for each patient treated on this 
protocol. The CRFs and their contents will be identified by the patient’s initials and UPN only. All 
supporting documents used to verify the accuracy of the data in the case report forms will be kept 
separately. Patient research files will be kept in a locked, controlled-access building and/or 
secured computers. At least monthly, the PI or a designated co-investigator will review and cross 
check the data entered on the case report forms with the source documents.
STATISTICAL CONSIDERATIONS 
 
23.A. Analysis of Safety/Toxicity (Primary Endpoint)
The primary objective of this trial is to examine the safety of adoptive T cell therapy using 
autologous CD8+ T cells genetically modified to express a high affinity WT1-specific TCR for 
patients with high-risk AML. There is already an extremely high likelihood of toxicity with 
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chemotherapy in this high-risk leukemic population. In published trials of patients receiving 
cytarabine-based consolidation therapy, up to 57% of enrolled patients have been shown to 
develop grade 3 or 4 non-hematologic organ (e.g. lung, liver, kidney) toxicities and/or infectious
complications. 167-169 Considering the toxicity associated with undergoing re-induction 
chemotherapy for relapse, roughly 70% of older patients, based on published toxicity data from 
induction chemotherapy regimens in this population, would be expected to develop grade 3 or 4
non-hematologic organ toxicity and/or infectious complications.83 It is expected that nearly all 
patients receiving induction or consolidation chemotherapy will exhibit grade 3 or 4 hematologic 
toxicity following treatment. 
Given this extremely high degree of expected toxicity with chemotherapy, and in light of the 
overall very poor prognosis of patients with disease relapse, we will consider treatment with WT1-
specific T cells to have an acceptable safety profile if the true rate of grade 3 or 4 non-
hematologic toxicity (as defined in Section 16.C.2) and grade 4 hematologic toxicity (as defined in 
Section 16.C.4) following WT1-specific T cells is less than 50%. Therefore, if there is sufficient 
evidence to suggest that the true toxicity rate exceeds 50%, the study will be suspended pending 
review by the DSMB. Sufficient evidence will be taken to mean any observed proportion of 
toxicities for which the associated one-sided lower 90% confidence limit exceeds 50%. 
23.A.1. Safety analysis for sequential cohorts in Stage 1
A total of 5 patients are planned to be treated, sequentially, in each cohort. The cohort size of 5 
patients is based on feasibility rather than statistical considerations. Before allowing enrollment to 
a subsequent cohort, a full assessment of safety and other elements (such as T cell persistence) 
will be performed.  The study team will make this assessment.  Because of the length of time 
required to generate transduced T cells, we will continue to screen and enroll, and generate T cell 
product at the time of inter-cohort safety analysis, but will not begin treatment on the next cohort 
prior to reviewing safety data.   
If toxicity as defined above develops in 3 of 5 patients at any cohort specified, the study will be put 
on hold until such time as the DSMB can review the toxicity data. If 2 or fewer patients develop 
dose-limiting toxicity, as defined above, the next group of 5 patients will be treated on the next 
cohort, according to the respective treatment plan, if deemed suitable by the study team. 
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23.A.2. Safety, persistence and efficacy analysis for determination of treatment regimen 
used in Stage 2
At the completion of stage 1, the study team and DSMB will convene for a review of available 
toxicity data, as well as a review of T cell persistence data and any available efficacy data as 
noted above. Completion of stage 1 will be defined as the treatment and toxicity assessment 
(defined as administration of at least one T cell infusion, followed by safety monitoring for >28 
days) for 15 patients across 3 cohorts.
Comparative toxicities, both expected and unexpected, as detailed in Sections 16.B and 16.C
will be reviewed for all 3 cohorts. In terms of comparing percentage of CD8+ T cells at 30 days, if 
the true mean percentage in two groups is separated by 1.5 standard-deviation units, 5 patients 
per group will yield 83% power to observe a statistically significant difference in percentage of
persisting infused T cells present at 30 days (at the one-sided significance level of 0.10).  We will 
not, however, require a statistically significant difference in persistence to take one dose forward, 
rather we shall take forward the dose that yields the highest average percentage of persisting 
infused CD8+ cells at day 30, provided this dose has an acceptable safety profile.  The study team 
and the DSMB will meet to review these data and will make a recommendation on which dose to 
move forward to the second stage of the trial.
The group will make an informed decision as to which treatment plan should be advanced for 
Stage 2 testing (which T cell components will be used and if lymphodepletion chemotherapy will 
be modified or discontinued.) It is planned to treat 20 additional patients in stage 2 with the 
selected plan.  
23.A.3. Safety analysis in Stage 2
A total of 20 patients are planned to be treated in stage 2. We will include the initial 5 patients 
from the selected cohort in the overall toxicity analysis for a total of 25 patients considered for the 
respective therapy. If there is sufficient evidence to suggest that the true toxicity rate exceeds 
50%, the study will be suspended pending review by the DSMB. Sufficient evidence will be taken 
to mean an observed proportion of toxicities for which the associated one-sided lower 90% 
confidence limit exceeds 50%, which will be determined after every 5th patient becomes 
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evaluable. Operationally, any of the following observed ratios of toxicities/treated patients would 
yield such a confidence limit and would warrant suspension of the study and DSMB review:  4/5, 
7/10, 10/15, or 13/20.  If the true probability of toxicity is 40%, then the probability of reaching one 
of the above ratios after 20 patients is approximately 0.12. For a true probability of toxicity of 70%, 
the probabilities of one of these ratios occurring is approximately 0.86 (estimated from 5,000 
simulations).
23.B. Analysis of the Feasibility of infusing TCRC4 Transduced T Cells (Primary Endpoint)
Feasibility is defined as the ability to reproducibly generate and infuse the T cells for eligible 
patients.  We anticipate a 4-6 week period required for T cell generation. We will enroll patients 
until we are able to isolate, grow, and infuse T cells to at least 35 patients, presuming that the trial 
has not been suspended due to safety reasons. The proportion of patients for which this is 
possible will be estimated, and our goal is that at least 80% of patients will be treated, excluding 
patients who could not receive products because of frank relapse, uncontrolled infections or other 
patient-related factors. No formal statistical considerations will be used to evaluate this endpoint 
beyond a simple estimate of the proportion along with its 95% confidence interval.
23.C. Analysis of persistence in blood or bone marrow of TCRC4-transduced cells 
Transferred TCRC4 CTL will be enumerated in real time by staining with TCRC4-binding WT1 
multimer in PBMC obtained from patients prior to T cell infusions (baseline) and on days 1, 7, 14,
21 and 28, at 2 months and 3 months, and then every 3 months during the interval between 
infusions. Following the second infusion, PBMC will be analyzed for persistence on days 1, 7, 14, 
21 and 28, monthly x 2, and then every 3 months until WT1-specific T cells are no longer 
detectable by analysis (limit of detection 0.03% of total CD8+ T cells). Persistence will be 
measured and presented descriptively. Migration of T cells to bone marrow will be compared to 
that that in peripheral blood using a paired t-test. If the true distribution of differences (bone
marrow – peripheral blood) is 0.5 standard-deviation units from zero, 25 patients will provide 87% 
power to observe a statistically significant difference between bone marrow and peripheral blood. 
HTTCS (limit of detection 0.01% of CD3+ T cells) will be used to distinguish the frequency of 
individual T cell clonotypes from the TN, TCM, or TEBV subsets at day 7 (the peak of the response), 
at day +28 (establishment of short-term memory), at 3 months (long-term memory), and every 3 
months thereafter (at time points blood or marrow samples are obtained) as long as persistence 
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by multimer is observed. Clonotype frequencies >0.01% of CD3+ T cells will be summated to 
determine relative frequencies of TN and TCM at different time points. Because the sum of 
frequencies for each subset will likely still be >0.01% at 3-6 months, the population persisting less 
well will be identified when the sum of its clonotype frequencies reaches <25% of detectable 
transferred clonotypes. At each time point, we will assess the proportion of T cell subsets and test 
the null hypothesis that the proportions are equal at each time.  We’ll compare each subset to a 
fixed hypothesized frequency of 33%.  With 25 patients, a true effect size (frequency of cells 
divided by standard deviation of the frequency) of 0.5 standard-deviation units leads to 87% 
power to be able to conclude that the proportion of a specific T cell subset is statistically 
significantly different (at the one-sided significance level of .10) from 33%.
23.D.  Assessment of efficacy (Exploratory Endpoint)
We shall also summarize the probability of relapse/progression using cumulative incidence 
estimates, where death without relapse/progression will be regarded as a competing risk. In 
addition, Kaplan-Meier estimates will be used to estimate overall and disease-free survival. 
Efficacy assessment will be performed without distinction between TN vs TCM vs TEBV, as this trial 
is not designed to differentiate between relative efficacy of these three populations, but rather to 
assess T cell therapy targeting WT1 overall, and to gain insights into the subpopulation of cells 
that appears to provide the most functional and persistent response.
In order to gain a preliminary impression of potential efficacy, we will also follow patients who 
signed the study consent, but did not receive treatment, because they were not HLA-A*02:01, , 
they did not meet treatment criteria, or due to their preference (anticipated “observation” cohort 
size is 35). The populations being treated are at high risk of relapse. Patients with adverse-risk 
disease, MRD or poor count recovery after induction or with relapsed disease have a 1-year 
relapse-free survival of ~10-25% without HCT, 21-23,47 suggesting impact on relapse should be 
evident within 1-2 years of T cell infusions. Although the non-randomized nature of treated and 
untreated groups precludes definitive efficacy conclusions, this cohort of non-HCT patients (who 
will be receiving current standard or care) is considered a better benchmark by which potential 
efficacy can be assessed than an historical control group. Keeping in mind these limitations, if true 
1-year relapse rates are 50% (treated) and 75% (control), 25 treated and 35 control patients,
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respectively, provide 83% power to observe a statistically significant difference (one-sided 
significance level of .10).  
We will also consider clearance of MRD in determining efficacy of the T cells. We will estimate the 
percent of MRD positivity at 6 months for patients who were treated with MRD, which is defined 
as any detectable blast count by flow cytometry, or by persistently abnormal cytogenetics/FISH or 
PCR testing in patients with relevant disease markers. Corresponding confidence intervals will 
accompany each of these estimates.  While sample size is not contingent upon a specific 
benchmark for elimination of MRD, 20% of MRD+ treated patients who have no detectable MRD 
at 6 months would be considered a sufficiently encouraging result to warrant further study.
ADMINISTRATIVE CONSIDERATIONS
24.A.  Institutional Review Board
In accordance with federal regulations (21 CFR 312.66), an IRB that complies with regulations in 
21 CFR 56 must review and approve this protocol and the informed consent form prior to initiation 
of the study.
24.B.  Termination of Study
The study will be stopped if any of the following events occur:
x
All 35 patients have completed treatment.
x
Stopping rules for toxicity have been met. Accrual will be put on hold for discussion with 
the DSMB regarding a possible change in study design.
x
The PI and the IND Sponsor reserves the right to terminate the study at any time. The 
FDA may also terminate the study.
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APPENDICES
APPENDIX A
Karnofsky and Lansksy Scales
.DUQRIVN\6FDOHDJH\HDUV
Lansky Scale (age <16 years)
Able to carry on normal activity; no 
special care is needed.
Able to carry on normal activity; no 
special care is needed.
100
Normal, no complaints, no evidence of 
disease
100
Fully active
90
Able to carry on normal activity
90
Minor restriction in physically strenuous  
play
80
Normal activity with effort
80
Restricted in physically strenuous  play, tires
more easily, otherwise active
Unable to work, able to live at home and 
care for most personal needs; a varying 
amount of assistance is needed
Mild to moderate restriction
70
Cares for self, unable to carry on normal 
activity or to do active work
70
Both greater restrictions of and less time 
spent in active play
60
Requires occasional assistance but is able 
to care for most needs
60
Ambulatory up to 50% of time, limited active
play with assistance/supervision
50
Requires considerable assistance and 
frequent medical care
50
Considerable assistance required for any 
active play; full able to engage in quiet play
Unable to care for self, requires 
equivalent of institutional or hospital 
care, disease may be progressing rapidly
Moderate to severe restriction
40
Disabled, requires special care and 
assistance
40
Able to initiate quiet activities
30
Severely disabled, hospitalization indicated, 
although death not imminent
30
Needs considerable assistance for quiet 
activity
20
Very sick, hospitalization necessary
20
Limited to very passive activity initiated by 
others (e.g., TV)
10
Moribund, fatal process progressing rapidly
10
Completely disabled, not even passive play
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APPENDIX B
Monitoring Schedule*testing
Event
History and 
PE
Chemistry 
Panel
CBC
UA
Blood 
Draw
for 
research&,#
Other^ 
Bone Marrow
Evaluation@
Enrollment
X 
X 
X 
X 
Leukapheresis
X 
X 
X 
Pre-treatment
X 
X 
X 
X 
X 
X 
X
(within 6 weeks
of first infusion)
Infusion #1
Day 0
X 
X 
X 
X 
Day +1-+4
X 
X 
X 
X 
Day +7, +14, 
+21
X 
X 
X 
X (D14) 
X 
Day +28
X 
X 
X 
X 
X 
X
(only if delaying 2nd LQIXVLRQ
4 weeks)
After first 
infusion
Monthly for 2 
additional 
months then q 
3 months
X 
X 
X 
X 
Pre-infusion
#2
X
;RQO\LIZHHNVHODSVHG
since first infusion)
Infusion #2
Day 0
X 
X 
X 
X 
Day +1-+4
X 
X 
X 
X 
Day +7, +14, 
+21
X 
X 
X 
X (D14)
X 
Day +28
X 
X 
X 
X 
X 
X
(4 weeks after infusion)
At completion
of study 
treatment%
At 2, 3, 6 and 
12 months
X 
X 
X 
X 
12 months to 
15 years%$
X$
X$
* The dates listed on the study calendar above are approximate as patients may not always be able to follow the time points as 
dictated by the protocol.
& Peripheral blood in green-top (heparin) tubes: 60 ml for adults and children > 50kg; 30 ml for children 15-30kg; and 1ml/kg in 
children 15-30kg.
# Peripheral blood in serum separator (gold top) tubes: 10ml will be collected for adults and children > 50kg; 5 ml for children 15-
30kg; and 3 ml in children 15-30kg.
@ Bone marrow specimens to be collected: 7 ml in heparin for adults and children > 30kg; 5 ml for children 15-30kg; 
%: The following time points are recommendations for patient follow-up and may be completed by local providers.
^: “Other” testing includes determination of WT1 expression of leukemic blasts in blood or marrow, and HLA-A*0201 expression in 
patient cells from blood at the time of screening. Additionally, this includes HIV viral load testing, both at the time of enrollment, and 
also within 3 months of treatment, as needed.
$ Long Term Follow Up monitoring will begin at 12 months following the 2nd T cell infusion. During this period, patients should be 
monitored clinically as per FDA guidelines for up to 15 years. Research blood draws may be requested to detect persistence of 
WT1-specific T cells up to every 3 months until multimers are <0.03% CD8+ cells.
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APPENDIX C 
AML Response Criteriaa,b
Criteria
Complete Response (CR)
Partial Response (PR)
Stable Disease (SD)
Morphologic
Bone Marrow blasts <5%; 
no blasts with Auer rods; no 
extramedullary disease
Transfusion independent
Platelets  100,000/Pl
Absolute neutrophil count 
>1000/Pl
(CRi: incomplete 
hematologic recovery CRp: 
incomplete platelet 
recovery) 
Decrease of at least 50% in 
the percentage of blasts to 5-
25% in the bone marrow and 
the normalization of blood 
counts as for CR. 
(PRi and PRp if incomplete 
hematologic or platelet 
recovery)
Failure to achieve at least 
PR, but no evidence of 
progression for >4 weeks.
Cytogenetic
Disappearance of previous 
cytogenetic abnormality 
 50% reduction of abnormal 
metaphases
See above
FISH
Disappearance of previous 
FISH abnormality
As defined by individual 
assay.
See above
Molecular
Disappearance of molecular 
mutation/marker
As defined by individual 
assay.
See above
Flow cytometric Disappearance of cells with 
aberrant phenotype.
 10 fold reduction in 
percentage of leukemic cells 
if initial percentage of BM 
EODVWVLV 5%. Otherwise 
follow morphologic criteria. 
See above
a Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the International Working Group for 
Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic 
Trials in Acute Myeloid Leukemia. J Clin Oncol. Dec 15 2003;21(24):4642-4649.
b Cheson BD, Greenberg PL, Bennett JM, et al. Clinical application and proposal for modification of the International 
Working Group (IWG) response criteria in myelodysplasia. Blood. Jul 15 2006;108(2):419-425.
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APPENDIX D
Disease risk stratification based on leukemia-associated cytogenetic and molecular abnormalities, 
as per 2017 European LeukemiaNet (ELN) guidelines
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