Clinical trial NCT01640301
Conditions: Recurrent Adult Acute Myeloid Leukemia Recurrent Childhood Acute Myeloid Leukemia Secondary Acute Myeloid Leukemia Therapy-Related Acute Myeloid Leukemia Donor Hematopoietic Cell Transplant Recipient HLA-A*0201 Positive Cells Present Recurrent Acute Myeloid Leukemia
Sponsor: Fred Hutchinson Cancer Center
Start date: 2012-12-06; Completion date: 2020-03-20
Enrollment: 47 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This phase I/II trial studies the side effects of laboratory-treated T cells and to see how well they work in treating patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or chronic myelogenous leukemia (CML) that has returned after a period of improvement (relapsed), previously treated with donor stem cell transplant. Biological therapies, such as cellular adoptive immunotherapy, may stimulate the immune system in different ways and stop cancer cells from growing. Placing a gene that has been created in the laboratory into a person's T cells may make the body build an immune response to kill cancer cells.

Detailed description:
PRIMARY OBJECTIVES:

I. Determine the safety and potential toxicities associated with treating patients with high risk or relapsed AML, MDS, and CML after allogeneic hematopoietic cell transplantation (HCT) by adoptive transfer of virus-specific cluster of differentiation (CD)8 T cells genetically-modified to express a high affinity Wilms tumor 1 (WT1)-specific T cell receptor (TCR).

II. Determine the anti-leukemic activity associated with treating patients with relapsed AML, MDS and CML after allogeneic HCT by adoptive transfer of virus-specific CD8 T cells genetically-modified to express a high affinity WT1-specific T cell receptor (TCR).

SECONDARY OBJECTIVES:

I. Determine the in vivo persistence of transferred T cells and ability to migrate to and accumulate in bone marrow.

II. Determine the maintenance of TCR expression and function of transduced T cells.

OUTLINE: Patients are assigned to 1 of 2 treatment arms.

ARM I: Patients with no evidence of leukemia post-HCT receive WT1-sensitized T cells intravenously (IV) over 45 minutes (or longer for patients who are 15-30 kg) on days 0 and 14 and aldesleukin subcutaneously (SC) twice daily (BID) on days 14-28.

ARM II: Patients with evidence of AML (minimal residual disease or overt relapse) post-HCT receive cyclophosphamide IV and fludarabine phosphate IV daily on days -4 to -2. Patients also receive WT1-sensitized T cells IV over 45 minutes (or longer for patients who are 15-30 kg) on days 0 and 21 and aldesleukin SC BID on days 14-28.

After completion of study treatment, patients are followed up weekly for 4 weeks, at weeks 6 and 8, at 3, 6, 12 months, and then annually for up to 15 years.

Arms: arm 1: Patients with no evidence of leukemia or MDS post-HCT receive WT1-sensitized T cells IV over 45 minutes (or longer for patients who are 15-30 kg) on days 0 and 14 and aldesleukin SC BID on days 14-28. arm 2: Patients with evidence of AML (minimal residual disease or overt relapse) post-HCT receive cyclophosphamide IV and fludarabine phosphate IV daily on days -4 to -2. Patients also receive WT1-sensitized T cells IV over 45 minutes (or longer for patients who are 15-30 kg) on days 0 and 21 and aldesleukin SC BID on days 14-28.
Interventions: intervention 1: Aldesleukin intervention 2: Cyclophosphamide intervention 3: Fludarabine Phosphate intervention 4: Laboratory Biomarker Analysis intervention 5: WT1-Sensitized Allogeneic T-Lymphocytes
Intervention descriptions: intervention 1: Given SC intervention 2: Given IV intervention 3: Given IV intervention 4: Correlative studies intervention 5: Given IV

Protocol documents:
Prot_SAP_000.pdf: 
2498.00 
  Page 1 
  FRED HUTCHINSON CANCER RESEARCH CENTER (FHCRC) 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE (UW) 
DEPARTMENT OF MEDICINE, DIVISION OF ONCOLOGY 
SEATTLE CHILDRENS 
Current Version: 05/15/2019 
Previous Version: 12/28/2018 
PROTOCOL 2498.00 
Title of Protocol: Phase I/II study of adoptive immunotherapy after allogeneic HCT with virus 
specific CD8+ T cells that have been transduced to express a WT1-specific T cell receptor for 
patients with relapsed AML 
Investigator 
Professional Title 
Telephone Number 
Aude Chapuis, MD 
Assistant Member, FHCRC 
Assistant Professor, UW 
(206) 667-4369
Daniel Egan, MD (PI) 
Affiliate Investigator 
Merav Bar, MD 
Affiliate Investigator, FHCRC 
Affiliate Assistant Professor, UW 
Pager: (206) 540-0168 
Philip D. Greenberg, MD 
Member and Head, Program in 
Immunology, FHCRC, Professor, UW 
(206) 667-4462
Tom Schmitt, PhD. 
Postdoctoral Fellow, FHCRC 
(206) 667-2787
Jerald P. Radich, MD 
Member, FHCRC, Professor, UW 
(206) 667-4118
Ann E. Woolfrey, MD 
Member, FHCRC, Professor, UW 
(206) 667-4453
Ann Dahlberg, MD 
Assistant Member, FHCRC 
(206) 667-1959
Cecilia Yeung, MD 
Assistant Member, FHCRC 
(206) 288-7104
Frederick R. Appelbaum, 
M.D.
Member and Executive VP and Deputy 
Dir., Clinical Research Division, FHCRC 
Professor and Head, Division of  
Medical Oncology, UW 
(206) 667-4412
Biostatistician  
Ted A. Gooley, PhD 
Member, FHCRC,  
Affiliate Professor, UW 
(206) 667-6533
Research Staff 
Elizabeth Barnum, RN 
Research Nurse 
(206) 667-3792
Emergency (24 hours) Phone: (206) 598-8902; Fax: (206)-598-4034 
IRO REC'D OCT 28  2021
FHCRC IRB Approval  
11 18 2021 
Document Released Date


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              Page 2 
Table of Contents 
 
2. INTRODUCTION ........................................................................................................................ 5 
3. BACKGROUND .......................................................................................................................... 5 
3.A. HCT and Donor Lymphocyte Infusion as a Treatment for High Risk AML, MDS, and CML
 ........................................................................................................................................... 5 
3.B. Rationale for Treating AML, MDS, and CML with WT1-specific CD8+ T Cells ................... 6 
3.C. Rationale for Using Donor-derived CD8+ T Cells Transduced with Lentivirus to Express 
High Affinity WT1-specific TCR for Patients with High Risk or Relapsed AML, MDS, and 
CML ................................................................................................................................... 7 
3.D. Rationale for Transducing Donor derived EBV- or CMV-specific CD8+ T Cells to Express 
High Affinity WT1-specific TCR ......................................................................................... 8 
3.E. Rationale for Treating Patients with High Risk AML, MDS, or CML with Prophylactic 
Infusions of WT1-specific T cells (Arm 1 of the Protocol)................................................. 9 
3.F. Rationale for Treating Patients with Evidence of Persistent or Relapsed Disease Post-
transplant (Arm 2 of the Protocol) ................................................................................... 10 
3.G. Rationale for the Proposed WT1-specific T Cell Doses to be Infused in this Study......... 10 
3.H. IL-2 Administration to Augment Persistence of Adoptively Transferred T Cells ............... 12 
3.I. Safety Concerns Regarding Using Donor-derived EBV- or CMV-specific CD8+ T cells 
Lentivirally Transduced to Express a High Affinity WT1-specific TCR........................... 13 
3.J. Rationale for Allowable Doses of Immunosuppressive Therapy During the Study ........... 15 
3.K. Previous Human Experience with TCRC4: BB-IND 12046 – FHCRC Protocol 1655: Phase 
I/II Study of Adoptive Immunotherapy with CD8+ WT1-specific CTL Clones for Patients 
with Advanced MDS, CML, AML, or ALL after Allogeneic Hematopoietic Stem Cell 
Transplant. ....................................................................................................................... 18 
3.L. Ongoing Human Experience with TCRC4 in the current study (FHCRC protocol 2498 
Study) .............................................................................................................................. 18 
3.L.1. Patient characteristics on Protocol 2498 .................................................................... 20 
3.L.2. Persistence of TCRC4-transduced cells on Protocol 2498 .......................................... 22 
3.L.3. Toxicities observed on protocol #2498 ....................................................................... 24 
3.L.4. Efficacy observed on protocol #2498, Arm 1 .............................................................. 27 
3.L.5. Efficacy observed on protocol #2498, Arm 2 .............................................................. 28 
3.L.6. Protocol #2498: Conclusions to date .......................................................................... 29 
3.M.  Rationale for Lymphodepleting Chemotherapy Prior to T Cell Therapy .......................... 29 
3.N.  Rationale for Adjusting Eligibility Criteria for Ongoing Treatment on Protocol 2498 ....... 30 
4. OBJECTIVES ........................................................................................................................... 30 
4.A. Primary Objectives ............................................................................................................. 30 


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4.B. Secondary Objectives ........................................................................................................ 30 
5. STUDY ENDPOINTS ................................................................................................................ 31 
5.A. Primary Endpoints .............................................................................................................. 31 
5.B. Secondary Endpoints ......................................................................................................... 31 
6. STUDY SCHEMA ..................................................................................................................... 31 
7. PATIENT SELECTION ............................................................................................................. 32 
7.A. Eligibility for Enrollment ...................................................................................................... 32 
7.B. Exclusions for Enrollment ................................................................................................... 32 
7.C. Definition of Relapse Status or MRD ................................................................................. 33 
8. DONOR SELECTION ............................................................................................................... 33 
8.A. Inclusions ............................................................................................................................ 33 
8.B. Exclusions .......................................................................................................................... 33 
9. CONSENTING .......................................................................................................................... 34 
10. PROTOCOL REGISTRATION ............................................................................................... 34 
11. PROCEDURE TO OBTAIN PBMC FOR GENERATION OF WT1-SPECIFIC T CELLS .... 34 
12. GENERATION OF WT1-SPECIFIC T CELLS ....................................................................... 35 
13. T CELL PRODUCT HANDLING PRIOR TO INFUSION ....................................................... 35 
14. PLAN OF TREATMENT ......................................................................................................... 35 
14.A. Plan of Treatment – Arm 1 ............................................................................................... 35 
14.B. Plan of Treatment – Arm 2 ............................................................................................... 39 
14.C. Other Study Agents .......................................................................................................... 43 
14.C.1. Interleukin-2 .............................................................................................................. 43 
14.C.2. Cyclophosphamide ................................................................................................... 43 
14.C.3. Fludarabine ............................................................................................................... 43 
15. EVALUATION ......................................................................................................................... 43 
15.A. Patient and Donor Screening Prior to Enrollment ........................................................... 44 
15.B. Patient Evaluation after Transplant and Prior to Planned Initiation of CTL Infusions ..... 44 
15.C. Patient Evaluation at Time of Planned Initiation of T Cell Infusions (within 30 days of T 
cell infusion, unless otherwise indicated) ........................................................................ 45 
15.D. Evaluation Immediately Prior to each T Cell Infusion (Day 0)......................................... 45 
15.E. Evaluation During Each T Cell Infusion (Day 0) .............................................................. 46 
15.F. Evaluation After Each T Cell Infusion .............................................................................. 46 
15.G. Clinical and Laboratory Evaluation for Toxicity Following T Cell Infusions .................... 47 
15.H. Evaluation of Persistence and Function of Adoptively Transferred T Cells and for 
Evidence of Leukemia/MDS ............................................................................................ 49 
15. I. Evaluation for long term effects of using lentivirally transduced T cells .......................... 50 
16. MANAGEMENT OF T CELL INFUSION IN PATIENTS WHO RELAPSE OR PROGRESS 
DURING T CELL THERAPY ........................................................................................................ 51 


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17. OPTIONS FOR FURTHER TREATMENT AFTER COMPLETION OF STUDY THERAPY 51 
18. TOXICITIES ............................................................................................................................ 51 
18.A. Toxicity Grading ............................................................................................................... 51 
18.B. Immediate Toxicities:........................................................................................................ 51 
18.C.  Delayed Toxicities ........................................................................................................... 53 
18.D. Management of Severe Treatment-related Toxicities with Methylprednisolone ............. 53 
18.E. Definition and Management/Evaluation of Non-hematologic and Hematologic Toxicities 
Requiring Treatment Discontinuation ............................................................................. 53 
18.F. Chronic GVHD ................................................................................................................. 55 
18.G. Detection of Replication Competent Lentivirus (RCL) .................................................... 55 
20. PROTOCOL REGISTRATION AND SPECIAL CONSIDERATIONS ................................... 56 
20.A. Projected Target Accrual - Arm 1..................................................................................... 56 
20.B. Projected Target Accrual - Arm 2..................................................................................... 56 
21. GUIDELINES FOR ADVERSE EVENT REPORTING .......................................................... 57 
21.A. Reporting of Adverse Events (AEs) ................................................................................ 57 
21.B. Definitions ........................................................................................................................ 57 
22. DATA AND SAFETY MONITORING PLAN .......................................................................... 58 
22.A. Primary Monitoring .......................................................................................................... 58 
22.B. Monitoring Plan................................................................................................................ 58 
22.C. Monitoring the Progress of Trials and the Safety of Participants ................................... 58 
23. RECORDS .............................................................................................................................. 59 
24. STATISTICAL CONSIDERATIONS ...................................................................................... 60 
24.A. Analysis of Toxicity (Arm 1 and Arm 2) ........................................................................... 61 
24.B. Analysis of Efficacy in Preventing Relapse (Arm 1) ....................................................... 64 
24.C. Analysis of Anti-leukemic Potential Efficacy (Arm 2) ....................................................... 65 
25. TERMINATION OF THE STUDY ........................................................................................... 66 
26. REFERENCES ....................................................................................................................... 67 
27. APPENDICES ......................................................................................................................... 74 
APPENDIX A.............................................................................................................................. 74 
APPENDIX B.............................................................................................................................. 75 
APPENDIX C ............................................................................................................................. 78 
APPENDIX D ............................................................................................................................. 79 
APPENDIX E.............................................................................................................................. 81 
APPENDIX F .............................................................................................................................. 82 
 
 
 


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2. INTRODUCTION 
 
Allogeneic hematopoietic cell transplantation (HCT) has been shown to be an effective 
treatment for many otherwise incurable hematological malignancies, which at least partially 
reflects a therapeutic “graft-versus-leukemia” (GVL) effect mediated by transferred T cells.1 The 
benefit of GVL is often accompanied by harmful side effects, including graft-versus-host disease 
(GVHD).2,3 One way to harness the GVL activity of T cells and reduce toxicity might be to target 
the leukemia cells with CD8+ cytotoxic T cells (CTL) specific for antigens expressed by the 
malignant cells. Wilms’ tumor antigen 1 (WT1), a protein involved in regulation of gene 
expression that can promote the malignant phenotype, is over-expressed in Acute Myeloid 
Leukemia (AML), Myelodysplastic Syndrome (MDS), and Chronic Myeloid Leukemia (CML),4-7 
as well as many other malignancies. WT1-specific CTL can recognize and kill both in vitro and 
in vivo WT1-expressing leukemia cells, which express abnormally high levels of WT1, without 
affecting normal cells that express much lower levels.8-14 
 
Our group has treated patients with relapsed leukemia after allogeneic HCT on a clinical trial 
using WT1-specific CD8+ T cell clones generated and expanded from each patient’s normal 
human leukocyte antigen (HLA)-matched donor. Results indicate that this treatment has a very 
acceptable safety profile, but anti-leukemic efficacy has been limited. Analysis of the reasons for 
failure to reproducibly provide therapeutic benefit suggests this reflects in some patients’ poor 
persistence in vivo of the infused T cells and/or low avidity of the T cells for the leukemic target 
cells. In this phase I/II clinical trial, Epstein Barr virus (EBV)- or cytomegalovirus (CMV)-specific 
memory T cells that have the potential for longer in vivo persistence following transfer because 
they have already been programmed with the traits of memory T cells will be obtained from 
each patient’s HLA-matched donor and transduced with a lentiviral vector to express a 
characterized high affinity WT1-specific T cell receptor (TCR). The transduced cells will be 
expanded and infused into AML, MDS, or CML patients who are at high risk of relapse after 
HCT. The objectives of the study are to assess if the transduced cells can be safely 
administered to patients, maintain their function and persist in vivo, localize to sites of disease, 
and potentially prevent or treat relapse after HCT. 
 
3. BACKGROUND 
 
3.A. HCT and Donor Lymphocyte Infusion as a Treatment for High Risk AML, MDS, and 
CML 
 
Every year about 13,000 people are diagnosed with AML, 11,000 with MDS, and 5,000 with 
CML in the United States, with the incidence increasing in the advanced age group.15 AML is 
often incurable with standard systemic therapy, as evidenced by a one year relapse rate of  
~50% in the ~80% of AML patients with unfavorable cytogenetics16,17 and ~ 90% relapse rate in 
AML patients who are induced into second remission with chemotherapy only.18-20 High risk 
MDS patients, as defined by an International Prognostic Scoring System (IPSS) score of 1.5-2 
without therapy, have a median survival of 1.1 year, and those with an IPSS score >2.5 a 
median survival of 0.4 year.21 Patients with CML beyond chronic phase (CP) have a poor 
prognosis, especially if disease has progressed into blast phase (BP) (3% three-year survival).22  
Allogeneic HCT has been shown to have curative potential for patients with high risk AML, high 
risk MDS or CML in accelerated or blast phase, partially as a result of a GVL effect mediated by 
alloreactive T cells.1 
 


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However, despite the many advances in the field of HCT,23 relapse after transplantation 
continues to be a major problem, particularly in patients entering HCT with high risk/poor 
prognosis diseases. Patients with MDS or AML with poor prognostic factors, who receive 
myeloablative transplants from HLA-identical siblings, have a relapse rate of ~25% at 
three months and ~40% at one year post-transplant.24-28 Approximately 32% of patients 
undergoing non-myeloablative transplant for high risk AML have clinically relapsed or have 
detectable minimal residual disease (MRD) three months after transplant, and there is a three 
year relapse/progression rate after HCT of 55%.29 Relapse of AML or MDS after allogeneic HCT 
is typically characterized by chemo-refractory and rapidly progressive disease, with >75% of 
patients dying within a year.30,31 
  
Current options for patients who relapse after HCT include salvage chemotherapy, withdrawal of 
immunosuppressive therapy (WIST), donor lymphocyte infusion (DLI) and a second HCT.  
Although WIST followed by DLI has shown some long term benefit in the small number of 
patients who both relapse >6 months after HCT and can be successfully induced into remission 
by chemotherapy before DLI, in general efficacy is limited and the desired GVL effect is often 
accompanied or overshadowed by harmful side effects, including ~10% of patients developing 
grade III-IV acute GVHD within 30 days of DLI and an overall rate of grade III-IV acute GVHD of 
20-30%.2,3,32-34  A second HCT has some curative potential, but is associated with high morbidity 
and ~50% mortality rates.35 
 
Patients with chronic myeloid leukemia (CML) in either accelerated phase (AP) or blast crisis 
(BC) who receive transplants from an HLA-identical allogeneic donor have a probability of 
leukemic relapse post-transplant of approximately 50%, with most occurring within the first two 
years.36-39 Similar to relapsed AML after HCT, treatment of patients who relapse with CML in AP 
or BC with salvage chemotherapy, interferon, imatinib mesylate, or DLI has yielded few durable 
remissions.2,3,40,41 This markedly contrasts to results with DLI given to patients who have 
relapsed after HCT for CML in chronic phase, where ~75% of patients attain remission,2,3 
suggesting the leukemia in this setting is more susceptible to immunologic effector mechanisms 
not specifically targeted to the leukemia. However, as described above, DLI is also associated 
with significant toxicity from GVHD. Ultimately, alternative more-targeted options for the 
treatment of relapsed leukemia post-transplant are required. 
 
3.B. Rationale for Treating AML, MDS, and CML with WT1-specific CD8+ T Cells 
 
One way to maximize the GVL effects with less toxicity would be to selectively eliminate 
leukemia cells using CD8+ CTL specific for antigens expressed by the leukemia cells, but 
absent or expressed at only low levels by normal tissue. WT1 is a zinc finger, a protein involved 
in regulation of gene expression that has multiple functions, including gene transactivation, gene 
repression, and RNA binding.42 WT1 is an intracellular protein involved in the activation and 
repression of gene expression and regulation of posttranscriptional RNA processing.43 Up to 24 
different isoforms of WT1 can be generated by alternative splicing, alternative translation 
initiation, and RNA editing.43,44 All WT1 isoforms have four zinc finger domains at the carboxyl 
terminus of the protein, which are involved in binding to DNA motifs in the promoter region of 
WT1-regulated genes.45 In one isoform, insertion of three amino acids, lysine, threonine, serine 
(KTS) in the zinc finger domains by alternative splicing produces a WT1 isoform that is primarily 
involved in RNA processing. WT1 can be detected at low levels in adult podocytes of the 
kidney, sertoli cells of the testes, granulosa cells of the ovary, mesothelial cells of the lung, the 
uterus, and CD34+ hematopoietic progenitor cells.46 Over-expression of WT1 has been reported 
in various solid tumors, as well as hematologic malignancies, including ~90-100% of AML, 


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MDS, and CML.5 WT1 has been shown to be expressed in blast cells of the vast majority of 
patients with AML.5,47 
 
WT1 appears to have a role in oncogenesis, as suggested by studies showing inhibition of 
proliferation and induction of apoptosis of leukemia cells and other tumors following specific 
down-regulation of WT1 expression by antisense oligonucleotides.7 Additionally, WT1 increases 
expression of the anti-apoptotic molecule bcl-2, and constitutive expression of WT1 in 
hematopoietic progenitor cells increases proliferation and inhibits differentiation of myeloid cells 
in response to granulocyte colony-stimulating factor (G-CSF).6 Unfractionated leukemic cells 
express approximately 10 to >100-fold the level of WT1 protein found in normal CD34+ cells46 
with even higher levels detected in CD34+/CD38– leukemic stem cells compared to normal 
hematopoietic stem cells.48 This difference has made it possible to monitor WT1 expression as 
a sensitive molecular marker for relapse of leukemia and for progression of MDS from refractory 
anemia to more malignant forms.49-52 
 
Immunologic studies have demonstrated CD8+ T cells can also distinguish cells based on this 
difference in protein expression, suggesting it may be safe to therapeutically target the WT1 
protein. Using peptides representing epitopes predicted to bind the A2 and A24 human Class I 
molecules, CD8+ CTL have been generated that lyse leukemia cells but not normal CD34+ cells, 
and inhibit growth of leukemia colonies but not myeloid or erythroid colonies from normal CD34+ 
cells.8-10 
 
In vivo studies in mice demonstrated that WT1-specific CD8+ T cells inhibit engraftment of 
leukemia-initiating stem cells but not normal CD34+ hematopoietic cells in non-obese 
diabetic/severe combined immunodeficiency (NOD/SCID) mice9 and eliminate WT1+ leukemia 
cells in vivo without any evidence of injury to normal tissues, including kidney, lung, and bone 
marrow.14 Finally, detection of WT1-specific CD8+ T cells in patients with myeloid and 
lymphoblastic leukemias has been associated with beneficial GVL effects,53-56 and clinical 
vaccine trials with WT1 peptide have shown not only that CD8+ T cells can be induced from 
patients’ T cells but that induction of a WT1-specific CTL response is associated with anti-
leukemic activity in some patients including complete responses (CRs) in a few patients with 
advanced disease.57-62 
 
The WT1-specific TCR being used in this protocol (C4 TCR) (described in Section 3.C) targets 
the WT1126-134 epitope (RMFPNAPYL), which is highly conserved among species, expressed in 
all WT1 isoforms, and has not been reported as hotspot for mutation in AML. In fact, no 
mutations of this epitope have been reported. This epitope is presented by the class I major 
histocompatibility complex (MHC) HLA-A*0201, and is considered a promising lead for 
immunotherapeutic targeting.63 
 
3.C. Rationale for Using Donor-derived CD8+ T Cells Transduced with Lentivirus to 
Express High Affinity WT1-specific TCR for Patients with High Risk or Relapsed AML, 
MDS, and CML 
 
A review of cancer vaccine trials involving 440 patients 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 and, similarly, the majority of patients that have been 
vaccinated to WT1 have failed to achieve clinical benefit. Adoptive T cell therapy, in which the 
tumor-reactive T cells are generated and expanded in the laboratory and then infused into the 
patient, can overcome some of the obstacles to establishing an effective response. Our lab has 


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developed methods to generate high avidity CD8+ T cell responses specific for WT1 by primary 
in vitro sensitization of naïve T cells (TN) from healthy donors,66 and we have been performing a 
clinical trial in allogeneic HCT patients with relapsed leukemia, in which WT1-specific CD8+ T 
cell clones are generated and expanded from each patient’s normal matched donor for use in 
therapy BB-IND 12046 – FHCRC Protocol 1655 – Phase I/II Study of Adoptive 
Immunotherapy with CD8+ WT1-specific CTL Clones for Patients with Advanced MDS, 
CML, AML, or ALL after Allogeneic Hematopoietic Stem Cell Transplant [BB-IND 12046 - 
FHCRC Protocol 1655). Although we have been successful in generating WT1-specific CD8+ 
T cell clones from >85% of healthy donors, the avidity of the T cells generated from each donor 
for WT1 is variable and the lower avidity responses may have had limited anti-leukemic activity. 
 
Transfer of high affinity TCR genes into primary T cells as a strategy to impart specificity and 
high avidity for a cell expressing the desired target antigen can circumvent this problem. Genes 
encoding the α and β chains of a TCR have already been introduced into T cells and clinically 
evaluated in patients,67 and T cells transduced with a MART-1 or gp100 specific TCR were 
shown in patients with metastatic melanoma to migrate to the site of disease and mediate 
cancer regression.68,69 
 
One potential limitation to use of TCR-transduced T cells is that such T cells can exhibit 
decreased avidity compared to the original “donor” T cell due to reduced expression of the 
introduced TCR. However, our lab described the introduction of a point mutation that inserts a 
cysteine (cys) into the Cα and the Cβ domains of a WT1-specific A*0201-restricted TCR 
isolated from a high avidity CD8+ T cell clone to create a disulfide-bond. 
 
The cys-modification promoted preferential pairing of the introduced α and β chains with each 
other, resulting in more efficient expression of the introduced TCR compared to the unmodified 
TCR, and reduced mismatching with endogenous TCR chains, resulting in the transduced 
T cells demonstrating higher avidity for WT1-expressing leukemia cells compared to cells 
expressing chains without the mutation.70 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’ than others due to the relative availability of matching tRNAs as well as other factors.71 
Modifying the TCRα and β gene sequences to encode each amino acid by the optimal codon for 
mammalian gene expression, and to eliminate mRNA instability motifs and cryptic splice sites, 
has been shown to significantly enhance TCRα and β gene expression.72 
 
The WT1-specific TCR being used in this protocol (C4 TCR) was selected after screening 
>1000 T cell clones isolated from the repertoires of normal healthy individuals for avidity as 
reflected by WT1-specific cytolytic activity for targets expressing decreasing levels of WT1 and 
affinity for peptide/MHC tetramers. Thus, patients on this protocol will have their donor CD8+ 
T cells transduced with a lentiviral vector containing the C4 WT1-specific TCR chains that have 
been cys-modified and codon optimized. 
 
3.D. Rationale for Transducing Donor derived EBV- or CMV-specific CD8+ T Cells to 
Express High Affinity WT1-specific TCR 
 
T cell therapy is often limited by the ability of transferred T cells to expand and persist in vivo 
after transfer. The in vivo fate of transferred T cells is dependent in part on the intrinsic 
properties of the T cells from which infused cells were derived, and TCR gene therapy provides 
the opportunity to choose the nature and origin of the T cell type used for adoptive cell therapy. 


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Conventional CD8+ T cells can be divided into naïve T cells (TN), and antigen experienced 
memory T cells (TM). Memory T cells can be further divided into central memory T cell (TCM) and 
effector memory T cell (TEM) subsets, which have distinct transcriptional programs that dictate 
many characteristics, including homing, phenotype, and function.73 When TN and TEM cells are 
stimulated in vitro, they expand and differentiate largely into short-lived effector cells, which 
effectively kill targets, but generally have limited proliferative capacity and fail to persist for long 
periods after transfer in vivo.73-76 Although TCM cells also expand and differentiate into effector 
cells in response to in vitro stimulation, studies in non-human primates indicate that these 
effector cells have been imprinted and retain some of the beneficial properties of the parent 
TCM cell from which they were derived, and in particular the capacity for self-renewal, which 
translates into improved in vivo persistence and response to antigen challenge in vivo.74 
 
Over 90% of the human population is infected with EBV,77 and the sero-prevalence of CMV is 
~60% in the total US population (40-49 year old: ~65%; 50-59 year old: ~74%; 60-69 year old: 
83%),78 with almost the entire population being either EBV or CMV seropositive. The CD8+ 
T cells that are reactive with these viruses contain cells of central memory origin, with the 
proportion varying between donors, but in general is represented in a higher fraction among 
EBV-specific T cells.79-83 Therefore, introducing the WT1-specific high affinity TCR into EBV-
specific CD8+ T cells (or CMV-specific if the donor is EBV negative) may result in a more 
persistent and potent anti-leukemic response by the transduced T cells. 
 
In addition to ensuring 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, the use of 
virus-specific cells serves two additional 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; and 2) it 
removes the possibility of transducing TN cells that contribute disproportionally to GVHD 
following HCT. 84,85  
 
 
3.E. Rationale for Treating Patients with High Risk AML, MDS, or CML with Prophylactic 
Infusions of WT1-specific T cells (Arm 1 of the Protocol) 
 
Patients with high risk AML, CML, or MDS, but with no evidence of disease post HCT are 
eligible for treatment on Arm 1 of this study. The relapse rate for high risk AML, MDS, and CML 
after HCT remains high, with ~40-55% of patients eventually relapsing,24-29 and 85% of the 
relapses occurring within two years post HCT (FHCRC data). Treatment options available for 
relapsed leukemia after transplant generally have limited efficacy and/or are very toxic. 
Palliative therapy or enrollment in experimental clinical trials is therefore often considered in this 
patient group. The high risk of relapse and poor prognosis of relapsed patients highlights the 
importance of developing and testing strategies in patients with high risk AML, MDS, or CML 
that might prevent relapse, such as prophylactic infusion of WT1-specific T cells. Grade II-IV 
acute GVHD after allogeneic HCT requiring high dose glucocorticoids occurs in about 70% of 
patients (with grade III-IV 15-20%), with the cumulative incidence reaching a plateau by day +60 
after myeloablative transplant and day +90 after non-myeloablative transplant.86 Patients 
diagnosed with grade III-IV acute GVHD will be excluded from this protocol, as the use of drugs 
such as high doses of glucocorticoids would ablate the infused T cells. Patients with high risk 
AML, MDS, or CML, with no evidence of disease post HCT, and with no grade III-IV acute 
GVHD will be eligible to enroll in Arm 1 of this protocol.  
 


2498.00 
 
 
 
              Page 10 
3.F. Rationale for Treating Patients with Evidence of Persistent or Relapsed Disease 
Post-transplant (Arm 2 of the Protocol) 
 
Patients with evidence of residual or relapsed AML post-HCT are eligible for treatment on Arm 2 
of this study. Immune-based therapies are generally ineffective in the setting of large 
established tumor burdens, and such patients frequently have confounding toxicities from tumor 
progression. In murine leukemia models, the transfer of leukemia-specific T cells has been 
shown to be effective in mice with established disease only when the leukemia burden has been 
reduced with chemotherapy prior to T cell infusions.87 The lack of efficacy observed with 
advanced disease likely reflects multiple factors, including faster growth kinetics of the leukemia 
cells as compared to the infused CTL, alterations in T cell signal transduction (particularly of 
tumor-reactive T cells) in the tumor-bearing host, induction of T cell apoptosis, the presence of 
non-specific and/or specific regulatory cells that suppress the anti-tumor response, and the 
induction of antigen-specific T cell anergy during the course of tumor progression.88,89 
Therefore, it would likely be difficult to detect anti-leukemic effects of infused CTL if only patients 
with advanced disease were treated. 
 
Patients on this study who have evidence of residual disease post-HCT (Arm 2) will 
preferentially receive WT1-specific CTL infusions after detection of MRD – for example, 
immediately following detection of molecular relapse/progression by polymerase chain reaction 
(PCR) for leukemia-specific gene markers or detection of cytogenetic relapse/progression by 
analysis of bone marrow. 
 
3.G. Rationale for the Proposed WT1-specific T Cell Doses to be Infused in this Study  
 
In a prior Phase I/II BB-IND 12046 – FHCRC Protocol 1655 study, the most avid WT1-specific 
T cell clones that could be derived from sibling donors were used to treat 11 patients with high 
risk or relapsed leukemia post HCT. The first four patients on the study received a starting dose 
of 3.3 x 108 cells/m2 followed by a dose of 1 x 109 cells/m2 WT1-specific T cells. No unexpected 
adverse events (AEs) were observed among those patients. However, infused multimer+ CTL 
frequencies did not exceed 0.05% of total CD8+ T cells (limit of detection) after 3-7 days, 
suggesting that doses up to 1 x 109 cells/ m2 were insufficient to establish persistent responses. 
Therefore, the initial dose for the last seven patients was increased to 3.3 x 109 cells/m2, 
followed by two doses of 1 x 1010cells/m2. The last T cell infusion was followed by 14 days of 
low dose interleukin-2 (IL-2) administered subcutaneously (SC). With the exception of transient 
lymphopenia, which is consistently observed after antigen-specific CTL infusions,90 only two 
cases of flu-like symptoms and one case of grade ≥ 3 fatigue were observed (Table 1). No 
grade II-IV GVHD was attributed to the T cell therapy. 
 
Table 1. AEs on BB-IND 12046 – FHCRC Protocol 1655 
Grade ≥ 3 AE related to CTL infusion 
19 
Lymphopenia 
16 
Flu-like syndrome 
2 
Fatigue 
1 


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              Page 11 
Based on the observation that the frequencies of multimer+ CTL reached baseline undetectable 
levels within 3-7 days with infusions up to 1 x 109 cells/m2 donor-derived WT1-specific T cells, and 
that no toxicities were observed with doses up to 1 x 1010 cells/m2, the proposed starting WT1-
specific T cell dose for the current study was one log lower than the maximum dose administered 
on Protocol 1655 or 1 x 1010 cells/m2. The maximum WT1-specific T cell dose to be infused in this 
study, in both arms, is 1 x 1010 cells/m2, which was safely administered in our previous study. 
 
As of February 1, 2014 seven patients have been treated on the current study, and collectively 
received 17 T cell infusions represented the maximum target dose of 1 x 1010 WT1-specific 
CTL/m2.  
 
All adverse events 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, and AEs that were deemed possibly, probably or 
likely related were collected and graded according to NCI CTCAE v4.0 (Table 2).  
 
Categories 
NCI CTCAE v4.01 
Grade 1 Grade 2 Grade 3 Grade 4 
Cytokine 
Release 
Syndrome 
Fever 
5 
3 
1 
  
Chills 
2 
1 
1 
  
Generalized aches/pain/headache 
10 
2 
  
  
Fatigue 
1 
1 
  
  
Tachypnea 
4 
  
  
  
Hypotension 
5 
3 
1 
  
Sinus tachycardia 
4 
2 
  
  
Sinus bradycardia 
1 
  
  
  
Nausea 
3 
3 
  
  
Vomiting 
1 
  
  
  
Diarrhea 
2 
  
  
  
Hematological 
Abnormalities 
Lymphopenia 
3 
6 
4 
1 
Lymphocyte count increased  
 
 2 
  
  
Anemia 
1  
1 
  
  
Thrombocytopenia 
1 
2 
2 
 
Chemistry 
Abnormalities 
ALT increased 
6 
2 
  
  
AST increased 
8 
  
  
  
Alkaline phosphatase increased 
4 
  
  
  
Creatinine increased 
3 
  
  
  
Hypoalbuminemia 
4 
  
  
  
Hypocalcemia 
7 
  
  
  
Hypomagnesemia 
1 
  
  
  
Hyponatremia 
5 
  
  
  


2498.00 
 
 
 
              Page 12 
Hypokalemia 
3 
  
  
  
Miscellaneous  Dry eyes 
  
2 
  
  
Decreased respiratory rate 
4 
  
  
  
1 National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. 
 
Table 2. Protocol 2498 Adverse events 
 
Adverse events experienced by patients treated on protocol 2498 have been reviewed by the 
FDA, and in March 2014 an approval from the FDA was received to decrease the number of T cell 
infusion to two, to include the original last two infusions at a dose of 1 x 1010 WT1-specific T 
cell/m2.  
 
In our previous experience with BB-IND 12046 – FHCRC Protocol 1655, in vivo activity was 
only observed in patients with detectable transferred cells in the peripheral blood and/or bone 
marrow and no activity was observed in the absence of detectable WT1-specific CTL. 
Furthermore, no end-organ toxicities were observed with frequencies of transferred donor-
derived WT1-specific CTL >6% for >160 days. Therefore, to limit the potential risk of toxicity 
until safety has been demonstrated subsequent infusions of C4 CTL will be administered only if 
the frequency of WT1-specific T cells is ≤ 3% of total CD8+ T cells in the peripheral blood (50% 
of the long-term tolerated dose on BB-IND 12046 – FHCRC Protocol 1655). 
 
For BB-IND 12046 – FHCRC Protocol 1655, the most avid WT1-specific T cell clones that 
could be derived from sibling donors were used to treat eleven patients with high risk or 
relapsed leukemia post HCT. CTL clones with similar/higher avidities for peptide-pulsed T2 
B-LCL, and an increased capacity to lyse established leukemia cells lines compared to C4 TCR 
transduced EBV+ CD8+ cell products (C4 CTL) have been infused into high-risk post-transplant 
patients and were safe with no CTL-related end-organ toxicities. 
 
Additional safety data have been generated in pre-clinical studies in our lab (unpublished data). 
In these studies, splenocytes from P14 mice were transduced to express unmodified TCR-α and 
-β genes isolated from a WT1-specific murine T cell clone, or enhanced affinity TCR-α and -β 
genes generated by saturation mutagenesis of the CDR3α region of the wild-type TCR-α chain 
to yield a TCR with a > 100-fold higher affinity. Recipient B6 mice were infused with 
1 x 107 CD8+ gene-modified T cells. High frequencies (10-15%) of infused cells were achieved 
and detectable approximately one week after infusion, and there was no evidence of end 
organ toxicity (including bone marrow, kidney, and lung) in mice that received T cells expressing 
either the wild-type or enhanced affinity TCR up to six weeks after infusion. 
To limit the potential risk of toxicity until safety has been demonstrated, subsequent planned 
infusion of WT1-specific T cells will be delayed until the frequency of WT1-specific T cells is 
≤ 3% of CD8+ T cells (see Sections 14 and 24). 
 
3.H. IL-2 Administration to Augment Persistence of Adoptively Transferred T Cells 
 
IL-2 is a cytokine secreted by activated T cells with 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-chain (αβγ) IL-2 receptor, which is induced following TCR 
triggering, and at high concentrations can also activate resting natural killer (NK) cells and 
T cells via the intermediate affinity two-chain (βγ) IL-2 receptor. Studies in cancer patients have 


2498.00 
 
 
 
              Page 13 
evaluated IL-2 alone, with adoptively transferred in vitro-cultured lymphokine-activated killer 
(LAK) cells generated by culture in high dose IL-2, or with tumor infiltrating lymphocytes (TIL).91 
 
These studies used high doses (up to 50 x 106 units every 8 hours) of intravenous or 
subcutaneous 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 plasma levels 
obtained with high dose IL-2 exceed the concentrations needed to saturate intermediate affinity 
receptors, and can therefore induce non-specific widespread activation of NK cells and T cells 
that can mediate the observed toxicities via cytokine release and lysis of normal cells. However, 
CD8+ T cells activated by target recognition in vivo express high affinity IL-2 receptors and are 
responsive to very low concentrations of IL-2, suggesting that the doses necessary to augment 
survival and in vivo persistence of transferred T cells may be much lower than the doses that 
induce toxicity. 
 
Studies in cancer patients and HIV seropositive individuals have investigated the 
immunomodulatory effects of administering lower doses of IL-2. Doses of 1.25 x 105 to 
5 x 105 U/m2/day administered subcutaneously have been shown to be well tolerated for up to 
84 days.92 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 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 SC administration of low doses of 
IL-2 for 14 days following T cell transfer.93 
 
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 to sustain CTL activity/survival with minimal toxicity.  
Patients who have been treated on BB-IND 12046 - FHCRC Protocol 1655 received the same 
dosing of IL-2 planned for this trial and it has been well tolerated, with no evidence of induction of 
GVHD from non-specific T cell activation. 
 
3.I. Safety Concerns Regarding Using Donor-derived EBV- or CMV-specific CD8+ T cells 
Lentivirally Transduced to Express a High Affinity WT1-specific TCR 
 
Expression of WT1 can be detected at low levels in normal kidneys, testes, ovary, pleura, 
pericardium, the uterus and in CD34+ hematopoietic progenitor cells.46 Patients who were 
treated on BB-IND 12046 – FHCRC Protocol 1655 did not experience toxicities in any of these 
organs expressing physiologic levels of WT1, such as hematopoietic suppression or graft failure, 
renal failure, pleuritis or pericarditis, abdominal pain (from splenic capsule or ovaries), and/or 
testicular or ovarian pain. 
 
The “parental” WT1 specific CD8+ T cell clone C4 that provided the TCR to be used in this trial 
was isolated from a healthy donor. Of all the clones we screened, the TCR from this clone had the 
highest affinity TCR found in any tested clone (lowest KD value) as determined by tetramer 
titration. Even though this TCR was isolated from the repertoire of a healthy individual, it remains 
possible that patients who receive an infusion of virus-specific T cells transduced with the C4 TCR 
gene may potentially experience on-target organ toxicities, justifying the dose-escalation plan 
and the stopping rules described in the protocol. 
 
WT1 expression in normal and malignant tissues is comparable between mice and humans. We 
have developed a mouse model in which on-target toxicities can be assessed using a murine 
WT1-specific TCR that has been mutated to have an affinity that is ~100-250 fold higher than 


2498.00 
 
 
 
              Page 14 
can be isolated from the normal repertoire. Mice injected with CD8+ T cells transduced with this 
mutated high affinity TCR did not demonstrate any toxicity in WT1 expressing organs, and the 
transduced T cells functioned normally in vivo, responded to immunization with WT1, and 
recognizing WT1+ tumor cells with no evidence of activation from cells. These results suggest 
that the risk of toxicities to WT1-expressing organs after infusion of T cells transduced with a 
WT1-specific TCR derived from a naturally occurring T cell clone may be low. 
 
Acute GVHD can potentially be induced by alloreactive T cells or from the creation of new TCR 
specificities for host proteins in transduced T cells that result from mispairing of the endogenous 
α and β TCR chains with the introduced β or α chains. Laboratory studies have indicated that 
donor T cells that mediate acute GVHD are mainly found to have arisen from the TN population 
(TN) and not from the memory T cell pool, and removing TN from the stem cell graft has been 
effective in preventing GVHD in rodent models.85,94 This principle is now being tested in a 
clinical trial utilizing TN depleted allogeneic peripheral blood cells in myeloablative HCT (FHCRC 
Protocol 2222). 
 
By first enriching for EBV- or CMV-specific memory T cells for transduction with the 
WT1-specific TCR, most donor T cells that have either an alloreactive specificity or are TN 
should be excluded, therefore reducing the probability of inducing acute GVHD from the T cell 
infusions. We have also demonstrated that, if the inserted TCR genes have been cys-modified, 
mismatching of the inserted modified TCR chains with the endogenous chains is minimal, with 
<5% of the introduced chains forming mismatched pairs.66 The use of virus-specific T cells for 
transduction with WT1-specific TCR genes also limits the repertoire of endogenous TCR chains 
available to form mismatched pairs, further reducing the potential risk of the introduced α and β 
chains causing alloreactivity. 
 
It is possible that toxicity from acquired alloreactivity, if it occurred, could present not only in 
organs usually affected in acute GVHD (e.g. skin, gut, and liver) but also potentially at unusual 
sites for GVHD due to alloreactive cells that have a unique specificity. However, of note, no 
toxicity from mismatched pairing has been described in human trials utilizing TCR transduced 
T cells, even though in the trials that have been performed the combined strategies discussed 
above that will be employed in our trial to reduce the risk were not employed, suggesting the 
risk from this mechanism should be very low. 
 
IL-2 may also potentially increase the risk of acute GVHD. However, in BB-IND 12046 - FHCRC 
Protocol 1655, patients who received the same dosing of IL-2 as in this protocol have not 
experienced acute or worsening GVHD as a result of IL-2 therapy. Furthermore, IL-2, when given 
in 2-6 times higher doses than in the current protocol in association with DLI, did not increase the 
rate of acute GVHD compared to DLI without IL-2.33 A recent study demonstrated that IL-2 
administration to patients with steroid-refractory chronic GVHD was associated with preferential, 
sustained regulatory T cell expansion in vivo and amelioration of the manifestations of chronic 
GVHD in a substantial proportion of patients.95 Therefore, based on our experience and the 
reported observations of others,33,95 we do not expect an increased risk of GVHD due to the 
administration of low dose SC IL-2 in this study. 
 
Antigen-specific CD8+ T cells targeting CMV pp65 epitopes generated using antigen-presenting 
cells have been isolated either from bone marrow donors and adoptively transferred to 
immunodeficient bone marrow transplant recipients, or isolated from and re-infused in HIV+ 
patients receiving antiviral therapy. No toxicities were observed in either setting and in a subset 
of patients, in whom the clones provided persistent reconstitution of CD8+ CMV-specific CTL 


2498.00 
 
 
 
              Page 15 
responses.96,97 Donor-derived or autologous ex vivo expanded CD8+ T cells targeting EBV have 
also been infused into patients with EBV-related lymphoproliferative disorders and similarly to 
CMV-specific CD8+ T cells, no organ toxicities occurred in patients who experienced clinical 
benefit or with prolonged persistence of the infused cells.98 
Another safety concern is the potential for transformation from insertional mutagenesis, which 
has been documented following integration of retroviral vectors into the host genome. This is 
well-documented in two X-linked SCID gene therapy trials in which five patients developed 
Acute Lymphoblastic Leukemia (ALL), which was shown to reflect transactivation of either the 
LMO2 or CCND2 gene by the retroviral insert in transduced CD34+ hematopoietic stem cells 
(HSCs). However, retroviral transduction of HSCs represents a unique situation in which the 
retroviral vector preferentially inserts near genes that are actively expressed in stem cells, 
including genes that may confer a proliferative/survival advantage. These HSC and their 
lymphoid progeny then proliferate extensively, particularly in the context of having been 
transferred into an empty lymphoid compartment, allowing for rare pro-oncogenic integrations to 
yield very large numbers of progeny that are more susceptible to additional oncogenic events. 
 
It has also been suggested that the development of leukemia in these patients may reflect in 
part the nature of the gene inserted and severity of disease, since none of >30 patients with 
SCID from adenosine deaminase (ADA) deficiency who received therapy with gene modified 
CD34+ hematopoietic stem cells has experienced vector related toxicities.99 
 
By contrast to these treatment settings, transferred T cells are more differentiated with less 
proliferative potential, and a review of 31 patients treated with more than 1011 retrovirally-
transduced lymphocytes detected no adverse events from insertional mutagenesis.100 
Importantly, self-inactivating lentiviral vectors, in distinction to gamma-retroviral vectors, do not 
preferentially integrate near active promoters and have been shown to have a much lower risk 
of insertional mutagenesis.101 However, these concerns further justify initially pursuing therapy 
with transduced T cells in patients with high risk or relapsed AML, MDS, or CML, as described 
in our plan, rather than patients with better prognosis disease. 
 
The potential toxicities are listed in the protocol consent form, and will be discussed with 
patients as a part of consenting. If eligible patients elect to enroll on the protocol, they will be 
monitored and managed for potential toxicities as outlined in Section 15 and Section 19 and 
stopping rules applied as described in Section 24. 
 
3.J. Rationale for Allowable Doses of Immunosuppressive Therapy During the Study 
 
There are limited data examining the effects of pharmacological immunosuppression on the in 
vivo fate of donor-derived T cells, their function, and their antitumor efficacy.102 Among the 
eleven patients who received WT1-specific T cell infusions on BB-IND 12046 - FHCRC 
Protocol 1655, four patients received corticosteroids (at doses up to prednisone 0.5 mg/kg/day) 
during the time of T cell infusions. Of those patients, two patients received tacrolimus, and one 
patient received Mycophenolate mofetil (MMF) at a dose of 1 gm twice daily. Prednisone and 
tacrolimus did not seem to affect the persistence of the WT1-specific T cells, as the patients 
who received these drugs demonstrated persistence of more than 14 days after administration 
of WT1-specific T cells at a dose of ≥ 3.3 x 109 cells/m2, which was not different form the 
persistence demonstrated by patients who did not receive immunosuppression at the time of 
T cell infusion (see Patients #1, #21, #27 in Table 3). One patient who received MMF 
(2 gm/day) during the time of T cell infusion demonstrated persistence up to 14 days with an 
infused cell dose of 3.3 x 109 cells/m2 (Patient #6 in Table 3). Based on the effects of 


2498.00 
 
 
 
              Page 16 
immunosuppressive medications on T cell persistence observed in our previous study, for the 
current study we will allow continuation of calcineurin inhibitors, corticosteroids (prednisone 
≤ 0.5 mg/kg/day, or an equivalent dose of an alternative glucocorticoid), and low doses of MMF 
(≤ 1 gm/day). 
 
As we expect the toxicities related to the WT1-specific T cell infusion to occur within 14 days, 
the low dose immunosuppressive drugs will not affect our ability to evaluate toxicity related to 
the infused cells. 


 
 
 
Table 3. WT1 T cell persistence 
 
 
INFUSION #1 
INFUSION #2 
INFUSION #3 
Patient 
Immuno-
suppression 
WT1 Cell 
Dose 
Persistence 
(Days After 
Infusion) 
Immuno-
suppression 
WT1 Cell 
Dose 
Persistence 
(Days After 
Infusion) 
Immuno-
suppression 
WT1 Cell 
Dose 
Persistence 
(Days After 
Infusion) 
No IL-21 in Cell Culture 
#1 
Pred 45 mg QDAY 
Tacro 1.5 mg BID 
3.3x108/m2 
3 
Pred taper 
Tacro 1.5 mg BID 
1x109/m2 
7 
Pred 10 mg QDAY 
Tacro 1.5 mg BID 
3.3x109/m2 
14 
#2 
None 
3.3x108/m2 
7 
None 
1x109/m2 
7 
None 
3.3x109/m2 
1 
#6 
MMF 1 gm BID 
HC 30 mg/day 
3.3x108/m2 
0 
MMF 1 gm BID 
HC 30 mg/day 
1x109/m2 
1 
MMF 1 gm BID 
HC 30 mg/day 
3.3x109/m2 
14 
#10 
None 
3.3x108/m2 
0 
None 
1x109/m2 
1 
None 
3.3x109/m2 
1 
#15 
None 
3.3x109/m2 
7 
N/A 
 
 
N/A 
 
 
#17 
None 
3.3x109/m2 
7 
None 
1x1010/m2 
14 
None 
1x1010/m2 
(+IL-2) 
1 
#20 
None 
3.3x109/m2 
1 
None 
1x1010/m2 
1 
N/A 
 
 
IL-21 in Cell Culture 
#21 
Pred 15 mg QOD 
3.3x109/m2 
14+ 
Pred 15 mg QOD 
1x1010/m2 
14+ 
Pred 15 mg QOD 
1x1010/m2 
(+IL-2) 
427+ 
#24 
None 
3.3x109/m2 
14+ 
None 
1x1010/m2 
11 
None 
1x1010/m2 
(+IL-2) 
220+ 
#27 
Pred 60 mg QDAY 
Tacro 4 mg BID 
3.3x109/m2 
14+ 
Off Pred 
Tacro 4 mg BID 
1x1010/m2 
14+ 
Off Pred 
Tacro 2 mg BID 
1x1010/m2 
(+IL-2) 
160+ 
#28 
None 
3.3x109/m2 
14+ 
None 
1x1010/m2 
14+ 
None 
1x1010/m2 
(+IL-2) 
84+ 
Acronyms: Pred = Prednisone, Tacro = tacrolimus, MMF = Mycophenolate mofetil, HC = hydrocortisone 
 
 


FHCRC 2498.00 
 
 
 
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3.K. Previous Human Experience with TCRC4: BB-IND 12046 – FHCRC Protocol 1655: 
Phase I/II Study of Adoptive Immunotherapy with CD8+ WT1-specific CTL Clones for Patients 
with Advanced MDS, CML, AML, or ALL after Allogeneic Hematopoietic Stem Cell Transplant. 
 
Initially, TCRC4 was evaluated in a prospective, single-arm clinical trial (FHCRC protocol 1655) 
to determine the safety and potential toxicities associated with infusing donor CD8+ T cell clones 
specific for WT1 in patients. The secondary objectives were to assess in vivo persistence of 
transferred CTL, localization of transferred cells to bone marrow, and potential anti-leukemic 
activity. HLA-A*0201+ patients at high risk for relapse and undergoing HLA-matched allogeneic 
HCT at the FHCRC for AML, ALL, MDS, and CML were eligible for enrollment and treatment. 
CD8+ T cells from an EBV-seropositive healthy donor were stimulated with the HLA A*0201-
restricted EBVBMFL1 peptide (GLCTLVAML) and lentivirally transduced to express the C4 TCR. 
The transduced cells were then selected based on binding both EBV and WT1 multimers and 
expanded using the rapid expansion method (REP) described in the accompanying IND 
Section 7.  
 
Eleven patients received therapy on this protocol with three to five infusions of WT1-specific T 
cells (at doses of 3.3 x 109 to 1.0 x 1010 cells/m2) generated and expanded from the patient’s 
matched donor. The last infusion is followed by 5 x 105 U/m2/day IL-2 for two weeks.No grade II-
IV GVHD was attributed to the T cell therapy. With the exception of transient lymphopenia, 
which is consistently observed after antigen-specific CTL infusions,90 only two cases of flu-like 
symptoms and one case of grade ≥ 3 fatigue were observed. The infused T cells were 
detectable in the blood and localized to the bone marrow without observed toxicities to WT1-
expressing organs. A patient exhibited reduction in leukemic blasts after infusion of CTL (11.8% 
to 1.8% of PBMC). One patient, who entered HCT with >40% blasts in the bone marrow was 
treated prophylactically after HCT, and remained in CR at >22 months despite >80% risk of 
relapse. Three patients with relapsed leukemia after HCT were treated with salvage therapy and 
achieved remission, and then immediately received T cells, and remained in CR at >11, >13, 
and >21 months post therapy. 
 
 
3.L. Ongoing Human Experience with TCRC4 in the current study (FHCRC protocol 2498 
Study) 
 
For the current trial, TCRC4 is being introduced into HLA-A*0201-restricted Epstein–Barr virus 
(EBV)- or cytomegalovirus (CMV)-specific donor cells. 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.103 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 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. The initial treatment plan is shown in Figure 
1, below. 
 


FHCRC 2498.00 
 
 
 
              Page 19 
Figure 1. 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 is >3% of total CD8+ cells. The second infusion is followed by 14 days of s.c. 
low-dose IL-2 (Figure 2, below).  
 
 
 


FHCRC 2498.00 
 
 
 
              Page 20 
Figure 2. Revised treatment plan of protocol 2498, Arms 1 and 2 
 
3.L.1. Patient characteristics on Protocol 2498 
As of January 1, 2016, 25 patients with high-risk AML received a total of 48 doses of donor-
derived virus-specific cells (Tables 4A and 4B). 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 4A. 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 
CR1, 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 
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 
months after transplant; did not receive 
planned T cell doses 
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 
cytogenetics 
EBV 
1 
Persistent T cells, remains in remission 
 5 months after transplant 


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Table 4B. 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/ 
Gend
er 
Disease 
EB
V/ 
C
MV 
Ar
m 
Comments 
1 
56 
M 
AML, relapse with para-spinal 
chloroma 5 years after 1st 
myeloablative HC 
EB
V 
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 
CM
V 
2 
Normalization of counts and 
decreased abnormal blast population 
to 0.006% 
after T cell infusions. Remains in 
remission. 
4 
25 
M 
AML, relapse with medullary and 
extra medullary disease after 2nd 
HCT 
EB
V 
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) 
EB
V 
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 
CM
V 
2 
Poor T cell persistence (0.04% at 
day 173), in CR 24 months after 
HCT. 
7 
33 
F 
AML, MRD after myeloablative 
HCT 
EB
V 
2 
Progressive disease. Received 
Azacitidine after 1 T cell infusion. 
8 
63 
F 
AML, received HCT in CR2. MRD 
early after transplant 
EB
V 
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 
CM
V 
2 
Poor T cell persistence, progressive 
disease. 
14 
17 
M 
AML, 2nd HCT for relapse 5 years 
after first HCT. 
EB
V 
2 
Disease progression despite 
persistent T cells; received 
azacitidine. 
15 
69 
F 
MDS -> AML, second HCT for 
relapse 1 year after 1st HCT 
CM
V 
2 
Disease progression despite 
persistent T cells. 
20 
5 
F 
Relapsed AML after transplant; 
active disease (chloromas); MLL 
gene-rearrangement. 
EB
V 
2 
Disease progression despite 
persistent T cells; receiving re-
induction chemotherapy 
23 
75 
M 
Relapsed AML after transplant 
EB
V 
2 
CR at time of T cell infusion; 
continued complete remission 
 
 


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3.L.2. 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 16 patients who received transduced EBV-specific cells and could 
be followed beyond 4 weeks after their latest infusion, 12 had persistent frequencies >3% 
(range 3-60% of CD8 T cells) for an average duration of 44 weeks (range 3-85 weeks) (Figures 
3A and 3B, below) representing an average maximum of 215 cells/microliter (range 10-989 
cells/microliter). The remaining 5 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 is ≥3% of total CD8+ T cells. Therefore, at this 
time, Patients 10, 11, 12, 18, 19, 24 and 15 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. 
 
Figure 3A. Observed frequencies of infused WT1-specific T cells (% CD8+ cells) in Arm 1 
patients.    


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Figure 3B. Observed frequencies of infused WT1-specific T cells (% CD8+ cells) in Arm 2 
patients.    
 
 
 


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3.L.3. 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 5).  
 
Table 5. 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 experienced fevers (≥ 38.3°C) with or without 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 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.  


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Importantly, we have observed only a single incident of possible on-target/off-tissue toxicity in all 
of the 25 patients who received WT1-specific T cell infusions. Specifically, patient 22 on Arm 1 
developed left pleuritic chest pain in October 2016 that was attributed to pleuritis, but with no 
definitive underlying etiology. He did not have any detectable WT1-specific T cells in peripheral 
blood, making WT1-specific T cells unlikely to be the cause. However, because WT1 is 
expressed in the pleural tissue and without another identifiable cause, this SAE was reported. 
Notably, in the interim, the patient has developed frank leukemic recurrence with leukemic 
pleural effusion, making that the likely etiology in retrospect. Otherwise, there has not been any 
observed graft failure, renal failure, nephrotic-range proteinuria, 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.90,104 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, 105 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 4, 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.  
 
Figure 4. 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 
Platelet count 
3


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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 
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.  
 
 
 


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3.L.4. Efficacy observed on protocol #2498, Arm 1 
Thirteen patients with high-risk leukemia entering HCT (Table 6, below), and who had no 
detectable disease after HCT, received WT1-specific cells within a median of 90 days post-
HCT. With a maximum follow-up of 1,286 days and a median of 739 days after HCT, 12 of 13 
patients are alive in CR and have not relapsed to date. A single patient (Pt 22) who did not 
receive the intended dose of T-cells due to logistical difficulty with generating his T cell product, 
subsequently relapsed with extramedullary disease in the mediastinum and also with evidence 
of cytogenetic relapse in the marrow. Progression-free survival is shown in Figure 5. For 
comparison, patients undergoing HCT with high-risk disease have an ~30% chance of relapse 
at 1 year. 25,27,28,106,107 This suggests the infusion of WT1-specific cells in the post-HCT setting 
may prevent leukemia recurrence.  
 
 
Table 6. Cumulative risk factors for patients on Arm 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 5: 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 
 
 
X 
24 
 
X 
 
 
X 
 
25 
X 
 
 
X 
 
 


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3.L.5. Efficacy observed on protocol #2498, Arm 2 
Twelve patients with high-risk leukemia entering HCT with detectable disease after HCT had a 
higher cumulative risk based on adverse factors compared to patients treated on Arm 1 (Table 
7, below). Patients received T cell infusions at a median of 121 days after HCT. Although 3 of 
12 patients remain free of disease, with a median follow-up of 193 days after the first T cell 
infusion, 25% are still alive (Figure 6, below). For comparison, patients who relapse within 6 
months after HCT have <10% chances of being alive at 1 year.108 This suggests WT1-specific 
T-cells may confer a survival advantage despite the absence of complete clearance of relapsed 
AML post HCT.  
 
 
Table 7. 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 6: Overall survival of Arm 2 since 1st CTL infusion 
 
 


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3.L.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/μl. No durable toxicities to 
tissues expressing physiological levels of WT1 (including cells of the hematopoietic, urogenital 
and renal systems, pleura, or pericardium) were detected during the monitoring period. There 
was a single cases of pleuritis (Pt 22) that in occurred in the absence of detectable WT1-specific 
T cells, and in retrospect was likely due to leukemic disease itself involving in the pleural space. 
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.  
 
There is increasing evidence that WT1-specific T cells may protect against post-transplant 
leukemic relapse, as evidenced by the observed progression-free survival in Arm 1 of the study. 
Outcomes in Arm 2 of the study remain suboptimal, in terms of progression-free survival, 
survival and T cell persistence. Furthermore, most patients on the study have received 
engineered T cells while in clinical remission without detectable disease, limiting the study 
team’s ability to directly observe anti-leukemic activity.  
 
Thus, the study plan has been modified in an attempt to:  1) potentially improve outcomes in 
Arm 2 patients by the addition of lymphodepleting chemotherapy; and 2) increase the number of 
patients with AML and detectable disease at the time of T cell therapy.  
 
 
3.M.  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.109,110 The favorable impact of 
lymphodepleting conditioning on survival and efficacy of transferred T cells has been observed 
in animal models,111-114 donor lymphocyte infusions post-stem cell transplant,115 and in 
numerous clinical trials with adoptive T cell transfer in melanoma and other cancers.116-118 There 
are several proposed mechanisms by which lymphodepletion augments T cell expansion and 
function. One such explanation is that depletion of endogenous T cells minimizes T cell 
competition 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.119 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.120,121 
 
This study protocol initially did not include a component of lymphodepleting chemotherapy, 
given the potential adverse effects of chemotherapy on engraftment in the early post-transplant 
period (particularly for patients receiving the intervention prophylactically). However, due to the 
lack of observed efficacy in Arm 2 subjects treated thus far, the very poor prognosis of post-
transplant relapse, and the demonstrated safety with T-cells transduced to express TCRC4, we 
believe the potential benefit of adding lymphodepleting chemotherapy warrants further study in 
this population with established post-transplant relapse (Arm 2). We anticipate that in vivo 
expansion and persistence may be improved following lymphodepletion, and that hopefully this 
will be accompanied by an improved anti-leukemic effect. Notably, for the proposed 
modification, 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 


FHCRC 2498.00 
 
 
 
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current CD19 CAR T-cell trial FHCRC protocol 2639, ongoing pediatric studies 
(FHCRC/Seattle Children’s PLAT-02 protocol), and FHCRC protocol 9296, in which patients 
with AML who are at high risk for relapse but who are not transplant candidates will receive 
autologous T-cells transduced to express TCRC4.115,116,122-124 
 
3.N.  Rationale for Adjusting Eligibility Criteria for Ongoing Treatment on Protocol 2498 
As of January 1, 2017, a total of 42 patients were enrolled on the current protocol.  Of these, 25 
patients were treated.  Of the 42 enrolled patients, 40 patients had an underlying diagnosis of 
AML.  Only 2 patients were enrolled with a diagnosis of MDS; of these, 1 opted out of treatment 
because of disease remission and 1 expired prior to treatment with transduced T-cells. The 
availability of competing study protocols has been identified as the major barrier of enrollment 
for MDS patients. No CML patients have been enrolled on the protocol, with poor accrual 
attributed to the very small number of patients with CML who undergo transplant in the present 
era of TKI therapy, compounded by the additional requirement of HLA*0201 expression in this 
already small target population. Thus, only patients with AML have been treated to date. Moving 
forward, given the poor accrual of MDS and CML patients thus far, it is not expected that 
continued efforts at enrolling and treating patients with MDS or CML will yield sufficient 
information as to justify the risk of added lymphodepleting chemotherapy.  
 
On Arm 2 of the study, of 12 patients with post-transplant MRD or relapse who were treated on 
the study, an objective response was observed in one patient with MRD at the time of T-cell 
infusion, with subsequent clearance of disease; she remains in remission at 43 months after T 
cell infusion. Five patients with detectable disease at the time of T cell infusion demonstrated 
disease progression. However, 6 patients (50% of the Arm 2 cohort) were in CR at the time of 
initial T cell infusion – of which 2 remain in CR – while 4 patients relapsed/progressed. By 
definition, all Arm 1 patients were in CR at the time of treatment. Thus, most patients on the 
study have received engineered T cells while in clinical remission and without detectable 
disease, significantly limiting the study team’s ability to measure anti-leukemic activity.  
 
Thus, this protocol has been modified to limit prospective enrollment to patients with relapsed 
AML on Arm 2 of the study protocol, all of whom will be treated with lymphodepleting 
chemotherapy prior to WT1-specific T cells. Any patients previously enrolled on the protocol, but 
who have not completed the study intervention, will remain eligible to receive T-cells, as outlined 
in the originally approved plan of treatment.  
 
4. OBJECTIVES 
 
4.A. Primary Objectives 
 
a) Determine the safety and potential toxicities associated with treating patients with high risk 
or relapsed AML, MDS, and CML after allogeneic HCT by adoptive transfer of virus-specific 
CD8 T cells genetically-modified to express a high affinity WT1-specific TCR. 
b) Determine the anti-leukemic activity associated with treating patients with relapsed AML, 
MDS and CML after allogeneic HCT by adoptive transfer of virus-specific CD8 T cells 
genetically-modified to express a high affinity WT1-specific T cell receptor (TCR). 
 
4.B. Secondary Objectives 
 
a) Determine the in vivo persistence of transferred T cells and ability to migrate to and 
accumulate in bone marrow. 


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b) Determine the maintenance of TCR expression and function of transduced T cells. 
 
5. STUDY ENDPOINTS 
 
5.A. Primary Endpoints 
 
a) Evidence and nature of toxicity related to study treatment. 
b) Disease response when treating patients with active disease (MRD or overt disease). 
 
5.B. Secondary Endpoints 
 
a) Persistence and migration of transferred T cells to bone marrow. 
b) Maintenance of TCR expression and function of transduced T cells. 
c) Disease-free survival, relapse and time to progression after T cell therapy. 
 
6. STUDY SCHEMA 
 
The study schema is described below in Figure 7, representing the initial eligibility criteria and 
treatment plan. Note that prospective enrollment is now limited only to patients with post-
transplant relapse of AML on Arm 2 of the study.  
 
Figure 7. Study schema 
 
 
 
 
 
 
 
Initial evaluation to confirm eligibility 
High risk AML 
Post-HCT evaluation for disease status  
ARM 1 -  No evidence of disease post-HCT 
Patients who entered transplant as high risk 
and have no detectable leukemia/MDS after 
HCT will proceed on this Arm 
ARM 2 -  Evidence of disease post-HCT  
(minimal residual disease or overt 
disease) 
Patients who have relapsed post-HCT (even 
if not previously enrolled as high risk disease 
before HCT) may be enrolled on Arm 2 post-
HCT 
 


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7. PATIENT SELECTION 
 
7.A. Eligibility for Enrollment 
 
1. Patients must express HLA-A*0201. 
 
2. Patients who are currently undergoing or who previously underwent matched 
allogeneic HCT for: 
a. AML: Prospective enrollment will now be limited to patients with relapsed disease 
(overt relapse or minimal residual disease) at any time post allogeneic HCT. 
b. MDS will no longer be a criterion for eligibility. 
c. CML will no longer be a criterion for eligibility.  
 
 
3. Patients must have an HLA-matched donor of hematopoietic stem cells (related or 
unrelated). 
 
4. Patients must be able to provide blood and bone marrow samples and undergo the 
procedures required for this protocol. 
 
5. Patients must be ≥15kg, as patients with lower weight would be incapable of providing 
high volume and frequent blood samples for monitoring and analysis. 
 
6. Patients must be able to give informed consent. Parent or legal representative will be 
asked to consent for patients younger than 18 year old. 
 
7.B. Exclusions for Enrollment 
 
1. Central nervous system (CNS) tumor refractory to intrathecal chemotherapy and/or 
cranio-spinal radiation. 
 
2. In patients whose leukemic cells are available for evaluation, the expression of WT1 in 
the patient’s bone marrow will be determined. If WT1 expression in the patient’s bone 
marrow is not highly expressed by PCR, the patient will be excluded from the study. 
Patients with no evaluable leukemia will be eligible for enrollment based on the high 
frequency of positive leukemias (>90%),5,47 and leukemia will be evaluated for WT1 
expression if recurrence is detected. 
 
3. HIV seropositive. Testing for HIV should be within 6 months of enrollment.  
 
4. Medical or psychological conditions that would make the patient unsuitable candidate 
for cell therapy at the discretion of the PI. 
 
5. Pregnancy or breast-feeding. Women of childbearing potential must have a negative 
serum or urine β-hCG pregnancy test result within 14 days before the first dose of 
WT1-specific T cell infusion. 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. 


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              Page 33 
 
7.C. Definition of Relapse Status or MRD 
 
1.  Morphologic relapse defined as one or more of the following: 
a. Abnormal blasts in peripheral blood in absence of growth factor therapy. 
b. Abnormal blasts in bone marrow that represent >5% of nucleated cells. 
c.  Extramedullary chloroma or granulocytic sarcoma. 
 
2.  Flow cytometric relapse defined as: 
a. The re-appearance in the peripheral blood or bone marrow of cells with an abnormal 
immunophenotype detectable by flow cytometry that is characteristic of the patient’s 
leukemia. 
 
3.  Cytogenetic relapse defined as: 
a. The appearance in one or more metaphases from bone marrow or peripheral blood cells 
of either a non-constitutional cytogenetic abnormality identified in at least one 
cytogenetic study performed prior to transplant or a new abnormality known to be 
associated with leukemia. 
 
4.  Molecular relapse defined as: 
a. Positive PCR assay for the expression of genes associated with leukemia. 
 
5.  MRD defined as: 
a. Flow cytometric, cytogenetic or molecular relapse without fulfilling the criteria for 
morphologic relapse. 
 
8. DONOR SELECTION 
 
8.A. Inclusions 
 
1.   Patient and donor (related or unrelated) must be HLA-matched and express HLA-
A*0201. 
 
a) Donor must be EBV or CMV seropositive. 
 
b) Donor must be age 18 or older.  
 
c) In good general health. 
 
d) Able to give informed consent. 
 
8.B. Exclusions 
 
1. Less than 18 years old. 
 
2. Active infectious hepatitis. 
 
3. HIV or HTLV seropositive. 
 
4. Pregnancy or nursing. 
 


FHCRC 2498.00 
 
 
 
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5. Significant medical conditions (e.g. immunosuppressive therapy) that would make the donor 
an unsuitable T cell donor. 
 
6. Unable to give informed consent. 
 
9. CONSENTING 
 
An enrollment conference will be held with the patient, and the related donor (unrelated donors 
are consented through the National Marrow Donor Program). The PI or a delegated 
representative will discuss this study and alternative treatments available for high risk and 
relapsed AML. All known risks and potential hazards of treatment with virus-specific CD8+ T 
cells transduced to express a WT1-specific TCR will be discussed. Informed consent will be 
obtained from the patient using forms approved by the Institutional Review Board (IRB) of the 
FHCRC. 
 
Patients with high risk disease may be consented and enrolled on the study prior to 
transplant. Patients with high risk disease who met study criteria before transplant but 
had not been enrolled pre-transplant, may be enrolled and consented after transplant. In 
addition, any patient who experiences relapse post-HLA matched HCT (MRD or overt 
relapse) will be eligible to enroll in the study post-HCT. 
 
10. PROTOCOL REGISTRATION 
 
Patients will be assigned to the protocol by the Clinical Coordinator who will register the patient 
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. 
 
11. PROCEDURE TO OBTAIN PBMC FOR GENERATION OF WT1-SPECIFIC T CELLS 
 
The preferred source for EBV/CMV-specific CD8+ T cells isolation is the GCSF-mobilized PBSC 
product. If insufficient peripheral blood mononuclear cell (PBMC) product is obtained from the 
PBSC product, or if the patient is enrolled on study after PBSC collection and no cryopreserved 
PBSC product can be used, a separate leukapheresis or high volume peripheral blood draw 
should be performed as described below. 
 
1. The G-CSF-mobilized PBSC product will be the preferred source for EBV/CMV-specific 
CD8+ T cells isolation, since it will not require the donor to undergo an additional 
procedure. 5% of the PBSC product will be removed for the purpose of generating WT1-
specific T cells.  If insufficient PBMC product is obtained from the PBSC product, an 
additional day of 6 or 12 liter leukapheresis may be added, at the discretion of the PI and 
the attending physician. No additional G-CSF will be given after completion of stem cell 
collection. 
 
2. If the patient is enrolled on study after PBSC collection, an attempt will be made to use a 
cryopreserved PBSC product, if available. 
 
3. If fresh or cryopreserved PBSC product cannot be used for T cell isolation or inadequate 
EBV/CMV-specific CD8+ T cells were isolated from the PBSC product, a separate 6-12 
liter leukapheresis via intravenous access will be scheduled. 


FHCRC 2498.00 
 
 
 
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2. Donors must be age 18 or older and meet all the donor criteria for leukapheresis as 
described in Protocol Appendix C in order to undergo leukapheresis. 
3. Donors 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 include: 
i. Infection, with or without antibiotic treatment. 
ii. Recent hepatitis exposure, hepatitis A or B antigenemia, or hepatitis C 
antibody positivity. 
iii. Pregnancy or nursing. 
iv. HIV or HTLV infection. 
 
4. If a portion of the PBSC product cannot be used for T cell selection, and the donor is 
unable or unwilling to undergo separate leukapheresis, 400 mL of peripheral blood can 
alternatively be drawn for generation of the T cells. This is a non-preferred option as the 
total number of CD8+ T cells is significantly lower than from leukapheresis or stem cell 
collection, and will require additional expansion of the transduced cells to achieve the 
therapeutic dose. 
 
12. GENERATION OF WT1-SPECIFIC T CELLS 
 
Donor peripheral blood lymphocytes will be enriched for virus-specific CD8+ T cells (preferably 
EBV because of the high frequency of TCM, but alternatively CMV) using clinical grade reagents 
according to Standard Operating Procedures (SOPs) in the Cell Processing Facility (CPF). The 
methods employed to enrich for virus-specific CD8+ T cells and generate WT1-specific T cells 
are described in details in IND Section 7 – Chemistry, Manufacturing and Controls. 
 
13. T CELL PRODUCT HANDLING PRIOR TO INFUSION 
 
For each infusion dose, the gene-modified cell product is formulated at the desired cell dose in a 
final volume of 250ml (final volume may be less for patients who are 15-30kg). The final product 
will be prepared and labeled according to SOPs in the CPF. After releasing from the Cell 
Processing Facility (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 nurse will then administer the cells to the patient over approximately 
45 minutes (+/- 15minutes) (or longer for patients who are 15-30kg and if clinically indicated, as 
described in Section 14 below). 
 
14. PLAN OF TREATMENT 
 
14.A. Plan of Treatment – Arm 1 
  
Two T cell infusions at a dose of 1x1010 cells/m2 will be infused at an interval of at least 14 days 
(Figure 8). First infusion may be given as soon as CD8+ WT1-specific transduced T cells have 
been generated. The second infusion will be followed by IL2 injections as discussed below 
(Section 14A.c.4). To limit the potential risk of toxicity second infusions of WT1-specific T cells 
will be delayed beyond 14 days until the frequency of WT1-specific T cells is ≤ 3% of 
CD8+ T cells. Second infusion may also be delayed due to the patient’s clinical status per the PI 
and the attending physician discretion.  
 
Figure 8. Treatment schema – Arm 1 


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A) Eligibility for Treatment on Arm 1 
1. Disease status: Patients must have no evidence of leukemia after transplant. Patients 
who have evidence of leukemia (MRD or overt relapse) prior to initiation of WT1-specific 
T cell therapy will be eligible for treatment on Arm 2. See Section 7.C for definition of 
relapse or MRD. 
 
2. Timing: Patients can be treated post-HCT as soon as CD8+ WT1-specific transduced T 
cells have been generated, expanded, and completed Quality Control (QC) testing. 
 
3. Patients should have evidence of post-transplant recovery of hematopoiesis (absolute 
neutrophil count [ANC] ≥ 1000/µl and platelets ≥ 50,000/µl for at least one week) prior to 
the initiation of T cell infusions. 
 
4. Patients on immunosuppressive therapy for GVHD at the time of therapy are eligible for 
treatment if:  (i) the patient has not developed grade III-IV acute GVHD prior to T cell 
therapy (Protocol Appendix A), (ii) the patient is not receiving corticosteroids, or if the 
dose of corticosteroids has been tapered to ≤ 0.5 mg/kg/day of prednisone (or an 
equivalent dose of an alternative glucocorticoid), and patients symptoms have been 
stable or improving for at least 14 days on tapering of immunosuppressive therapy, (iii) if 
patient is receiving MMF; MMF should be tapered to ≤ 1 gm/day prior to starting a T cell 
infusion or preferably be discontinued, particularly prior to the last infusion, which is 
followed by IL-2 administration, as MMF can lead rapid cell death of activated 
proliferating T cells. 
 
B) Exclusions for Treatment on Arm 1 
 
1. Patients who have evidence of leukemia (MRD or overt relapse) prior to initiation of 
WT1-specific T cell therapy (these patients will be eligible for treatment on Arm 2). 
 
2. Patients with grade III or IV acute GVHD at any time prior to study therapy (Protocol 
Appendix A). 
 
3. Anti-thymocyte globulin or other T cell suppressive treatment (such as anti-CD3 mAb) 
within one month prior to scheduled T cell infusion. 
 
4. Prednisone >0.5 mg/kg/day (or an equivalent dose of an alternative glucocorticoid). 


FHCRC 2498.00 
 
 
 
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5. MMF should be tapered to ≤ 1 gm/day prior to starting T cell infusion or preferably be 
discontinued, particularly prior to the last infusion, which is followed by IL-2 
administration, as MMF can lead to rapid cell death of activated proliferating T cells. 
Concurrent use of calcineurin inhibitors (cyclosporine, FK506) is not strictly an exclusion 
criterion, although attempts should be made to discontinue or reduce dosages of such 
drugs if possible. 
 
6. Graft rejection or graft failure. 
 
7. Karnofsky performance status score (age ≥ 16 years) or Lansky play score (age 
<16 years) ≤ 40%. 
 
8. Unable to generate adequate numbers of CD8+ WT1-specific transduced T cells from 
virus-specific T cells that were derived from the patient’s matched donor. 
 
9. Ongoing ≥ grade 3 cardiac, pulmonary, renal, gastrointestinal or hepatic toxicities 
according to NCI CTCAE version 4 toxicity criteria. 
 
10. Neurologic: Encephalopathy.  
 
11. Any condition or organ toxicity that is deemed by the principle investigator or the 
attending physician to place the patient at unacceptable risk for treatment on the 
protocol. 
 
12. Pregnancy or breast-feeding. 
 
C) Administration of Therapy on Arm 1  
Patients will receive two intravenous infusions of CD8+ WT1-specific transduced T cells if no 
serious infusion related toxicities are observed (see Section 18). Cell doses for infusions will be 
based on patient’s height and weight prior to first infusion (baseline); if time interval between 
infusions is greater than 60 days, or if the weight prior to subsequent infusion has changed by 
>20% compared to baseline weight, the subsequent cell dose will be calculated on the new 
weight. Actual cell doses administered may be less than the planned cell doses if insufficient 
numbers of transduced cells are generated. The interval between T cell infusions may be 
prolonged if deemed clinically to be in the best interest of the patient due to ongoing events. 
 
 
 


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Table 8. Schedule of WT1-specific T cell infusions on Arm 1 
 
 
Infusion #1 
Infusion #2 
Treatment day*  
0 
14 
Cell Dose 
(WT1-specific 
transduced T cells) 
1.0 x 1010/m2 
1.0 x 1010/m2 
IL-2 
 
2.5 x 105 U/m2 
every 12 hours 
for 14 days 
* If indicated, second infusions will be delayed (see Section 14.A.c.3).  
 
1.  First infusion of CD8+ WT1-specific transduced T cells; Day 0 
Patients will receive the infusion of T cells at the Seattle Cancer Care Alliance, either on the 
Immunotherapy service or alternatively in the infusion room. Patients admitted at the University 
of Washington Medical Center at the time of planned infusion will receive the T cells as an 
inpatient. The infusion will consist of CD8+ WT1-specific transduced T cells at a planned cell 
dose of 1.0 x 1010 cells/m2 administered by intravenous (IV) infusion over approximately 45 
minutes (+/- 15minutes) or longer for patients who are 15-30kg. Infusion time may be longer if 
required based on CPF endotoxin results or patient reaction to infusion. Vital signs and O2 
saturation should be monitored and recorded at around the following time points time 0, every 
15 minutes during T cell infusion, and then hourly for 2 hours following the T cell infusion (see 
Section 15.E). Infusion-related toxicities, if observed, will be treated as outlined in Section 18. 
 
2.  Second infusion of CD8+ WT1-specific T cells; Day +14  
Patients may receive a second infusion of CD8+ WT1-specific transduced T cells at a dose of 
1 x 1010 cell/m2 at least 14 days after the first infusion if the patient meets persistence criteria 
and did not experience a toxicity requiring delaying/discontinuation of study treatment (see 
Section 19).  Monitoring, and management of toxicity will be performed as described for the first 
infusion. 
 
Several study participants have received T cells at doses lower than the current dose of 1.0 x 
1010 cells/m2, prior to the protocol being modified as such, and are awaiting subsequent infusion 
of T cells at the current (higher) dose. Such patients who go on to receive a subsequent dose of 
1.0 x 1010 cells/m2 shall be considered to have completed the study treatment, and are therefore 
eligible to receive IL-2, as outlined below.   
 
3.  Criteria for delaying subsequent T cell infusion  
Subsequent T cell infusion will be delayed if clinically indicated, or if WT1-specific T cell 
frequency in the peripheral blood is >3% of total CD8+ T cells. Up to 60mL (45mL for patients 
who are 30-50kg, 1ml/kg for patients who are 15-30kg) of blood should be drawn within 21 days 
prior to the scheduled date for the subsequent T cell infusion for evaluation of persistence of 
adoptively transferred T cells. If the WT1-specific T cell frequency is >3%, the following T cell 
infusion will be held. Repeat evaluation of persistence of WT1-specific T cells should be 
performed up to twice weekly for one month and then at the PI discretion until the WT1-specific 
cell frequency is ≤ 3%. Subsequent T cell infusion may then be administered as soon as T cell 
product and an infusion appointment are available. 
 
4.  IL-2 administration 


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If the second T cell infusion is tolerated without serious toxicity as detailed in Section 18, 
patients should then receive twice daily SC injections of recombinant IL-2 at a dose of 2.5 x 105 
U/m2 approximately every 12 hours for 14 days, starting between 2-4 hours after completing the 
T cell infusion. Patients or caregivers will be trained on IL-2 administration. IL-2 therapy may be 
omitted in any patient with a contraindication to receiving the drug (e.g. graft versus host 
disease), as determined by the PI and/or treating physician. IL-2 therapy may be discontinued in 
any patient developing grade 3 or greater treatment-related toxicities while receiving daily SC 
IL-2 injections.  
 
14.B. Plan of Treatment – Arm 2 
 
Patients with history of AML and evidence of disease relapse or MRD post-transplant will be 
eligible for treatment on Arm 2 of the study. While T cells are being generated, it is anticipated 
that debulking systemic therapy will be administered for patients with overt relapse. Morphologic 
remission will not be required to receive the study treatment (lymphodepleting chemotherapy 
and WT1-specific T cells), although additional systemic therapy may be administered prior to T 
cells depending on patient-specific parameters (e.g. tempo of disease relapse and/or disease 
burden) and the preference of the patient’s treating physician. For patients with MRD only post-
transplant, it is preferred that systemic therapy not be administered prior to the study 
intervention, in order to maximize the number of patients with detectable disease at the time of 
T cell therapy. 
 
Two T cell infusions at a dose of 1x1010 cells/m2 will be infused at an interval of at least 21 days 
(Figure 9). The interval was extended from 14 to 21+ days due to the addition of 
lymphodepleting chemotherapy and allowance for recovery of blood counts between infusions. 
The first infusion may be given on or after day +60 post-transplant when CD8+ WT1-specific 
transduced T cells have been generated. The second infusion will be followed by IL2 injections, 
as discussed further below (Section 14B.d.4). To limit the potential risk of toxicity second 
infusions of WT1-specific T cells will be delayed beyond 21 days until the frequency of WT1-
specific T cells is ≤ 3% of CD8+ T cells. Second infusion may also be delayed due to the 
patient’s clinical status per the PI and the attending physician discretion.  
 
Patients previously enrolled on Arm 2 who have not yet received both T cell infusions will 
complete the planned study treatment by proceeding with the 2nd T cell infusion but without 
lymphodepleting chemotherapy. 
 


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Figure 9. Revised treatment schema – Arm 2 
 
 
  
A) Eligibility for Treatment on Arm 2 
 
1.  Disease status: Patients must have evidence of AML (MRD or overt relapse) post HCT. See 
Section 7.C for definition of relapse or MRD. 
 
2.  Timing: Patients can be treated post-HCT when CD8+ WT1-specific transduced T cells have 
been generated, expanded, and completed QC testing. In addition, because of the potential 
negative impact of lymphodepleting chemotherapy on engraftment, patients must also be at 
least 60 days post-transplant to begin treatment.  
 
3.  Patients should have evidence of post-transplant and/or post-chemotherapy recovery of 
hematopoiesis (ANC ≥ 500/µl and platelets ≥ 30,000/µl for at least one week) prior to the 
initiation of T cell infusions. However, patients who have ANC <500/µl and/or platelets 
<30,000/µl due to underlying leukemia or lasting more than 6 weeks following systemic 
chemotherapy will be allowed to undergo therapy, but marrow suppression will not be evaluated 
as a stopping criteria in these patients. 
 
4.  Patients will receive two intravenous infusions of CD8+ WT1-specific transduced T cells if no 
serious infusion related toxicities are observed (see Section 18). Cell doses for infusions will be 
based on patient’s height and weight prior to first infusion (baseline); if time interval between 
infusions is greater than 60 days, or if the weight prior to subsequent infusion has changed by 
>20% compared to baseline weight, the subsequent cell dose will be calculated on the new 
weight. Actual cell doses administered may be less than the planned cell doses if insufficient 
numbers of transduced cells are generated. The interval between T cell infusions may be 
prolonged if deemed clinically to be in the best interest of the patient due to ongoing events. 
 
5.  Patients on immunosuppressive therapy for GVHD at the time of therapy are eligible for 
treatment if: (i) the patient has not developed grade III-IV acute GVHD prior to T cell therapy 
(Appendix A), (ii) the patient is not receiving corticosteroids, or if the dose of corticosteroids 
has been tapered to <0.5 mg/kg/day of prednisone (or an equivalent dose of an alternative 
glucocorticoid), and patients symptoms have been stable or improving for at least 14 days on 


FHCRC 2498.00 
 
 
 
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tapering of immunosuppressive therapy, (iii) if patient is receiving MMF; MMF should be tapered 
to ≤ 1 gm/day prior to starting T cell infusion or preferably be discontinued, particularly prior to 
the last infusion, which is followed by IL-2 administration, as MMF can lead rapid cell death of 
activated proliferating T cells. 
 
B) Exclusions for Treatment on Arm 2 
 
1.  Patients with grade III or IV acute GVHD at any time prior to study therapy (Appendix A). 
 
2.  Anti-thymocyte globulin or other T cell suppressive treatment (such as anti-CD3 mAb) within 
one month prior to scheduled T cell infusion. 
 
3.  Prednisone >0.5 mg/kg/day (or an equivalent dose of an alternative glucocorticoid). 
 
4.  MMF should be tapered to ≤ 1 gm/day prior to starting T cell infusions or preferably be 
discontinued, particularly prior to the last infusion, which is followed by IL-2 administration, as 
MMF can lead to rapid cell death of activated proliferating T cells.  Concurrent use of calcineurin 
inhibitors (cyclosporine, FK506) is not strictly an exclusion criterion, although attempts should 
be made to discontinue or reduce dosages of such drugs if possible. 
 
5.  Graft rejection or graft failure. 
 
6.  Patients with Karnofsky performance status score (age ≥ 16 years) or Lansky play score 
(age <16 years) ≤ 30%. 
 
7.  Unable to generate adequate numbers of CD8+ WT1-specific transduced T cells from virus-
specific T cells that were derived from the patient’s matched donor. 
 
8.  Ongoing ≥ grade 3 cardiac, pulmonary, renal, gastrointestinal, or hepatic toxicities according 
to NCI CTCAE version 4 toxicity criteria. 
 
9.  Neurologic: Encephalopathy. 
 
11.  Any condition or organ toxicity that is deemed by the principle investigator or the attending 
physician to place the patient at unacceptable risk for treatment on the protocol.  
 
12.  Pregnancy or breast-feeding. 
 
 
C) Cytoreductive Therapy Prior to T Cell Infusion on Arm 2 
 
It is expected that some patients (e.g. those with >5% bone marrow leukemic blasts at 
diagnosis of relapse) will receive cytoreductive therapy prior to T cell infusion, with such 
treatment decisions at the discretion of the treating physician. These patients will require a 
repeat bone marrow sampling prior to T cell therapy to document disease and marrow status. 
Patients with disease relapse who receive cytoreductive chemotherapy will begin treatment with 
CD8+ WT1-specific transduced T cells after evidence of recovery of hematopoiesis (ANC ≥ 
500/µl and platelets ≥ 30,000/µl), or at least 6 weeks after chemotherapy if neutrophil and/or 
platelet counts remain below threshold, given the risk of further increase in disease burden with 
prolonged delay in treatment. For patients with MRD only post-transplant, it is preferred that 


FHCRC 2498.00 
 
 
 
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systemic therapy not be administered prior to the study intervention, in order to maximize the 
number of patients with detectable disease at the time of T cell therapy. 
 
  
D) Administration of Study Treatment on Arm 2 
Patients will receive two intravenous infusions of CD8+ WT1-specific transduced T cells if no 
serious infusion related toxicities are observed (see Section 18). Cell doses for infusions will be 
based on patient’s height and weight prior to first infusion (baseline); if time interval between 
infusions is greater than 60 days, or if the weight prior to subsequent infusion has changed by 
>20% compared to baseline weight, the subsequent cell dose will be calculated on the new 
weight. Actual cell doses administered may be less than the planned cell doses if insufficient 
numbers of transduced cells are generated. The interval between T cell infusions may be 
prolonged if deemed clinically to be in the best interest of the patient due to ongoing events. 
 
 
Table 9. Schedule of WT1-specific T cell infusions on Arm 1 
 
 
Infusion #1 
Infusion #2 
Treatment day*  
0 
21+* 
cyclophosphamide 
/ fludarabine 
Yes 
Yes 
Cell Dose 
(WT1-specific 
transduced T cells) 
1.0 x 1010/m2 
1.0 x 1010/m2 
IL-2 
 
2.5 x 105 U/m2 
every 12 hours 
for 14 days 
* Timing of second infusions may be delayed beyond 21 days (see Section 
14B.d.3).  
 
1.  First infusion of CD8+ WT1-specific transduced T cells; Day 0 
 
Patients will receive lymphodepleting chemotherapy prior to receiving donor-derived CD8+ 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). 
Pediatric patients meeting treatment eligibility will receive chemotherapy and T cells 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 and T cells in the outpatient setting at the Seattle Cancer Care Alliance under the 
care of the Immunotherapy service.  Patients admitted at the University of Washington Medical 
Center or Seattle Children’s Hospital at the time of planned infusion may receive the T cells as 
an inpatient. The infusion will consist of CD8+ WT1-specific transduced T cells at a planned cell 
dose of 1.0 x 1010 cells/m2 administered by intravenous (IV) infusion over approximately 45 
minutes (+/- 15minutes) or longer for patients who are 15-30kg. Infusion time may be longer if 
required based on CPF endotoxin results or patient reaction to infusion. Vital signs and O2 
saturation should be monitored and recorded at around the following time points time 0, every 
15 minutes during T cell infusion, and then hourly for 2 hours following the T cell infusion (see 
Section 15.E). Infusion-related toxicities, if observed, will be treated as outlined in Section 18. 
 
2.  Second infusion of CD8+ WT1-specific T cells; Day ≥21  


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Patients may receive a second infusion of CD8+ WT1-specific transduced T cells at a dose of 
1 x 1010 cell/m2 at least 21 days after the first infusion if the patient meets persistence criteria 
and did not experience a toxicity requiring delaying/discontinuation of study treatment (see 
Section 19).  Administration of lymphodepleting chemotherapy, provision of subsequent 
infusion of T cells, and monitoring will be performed as described above for infusion #1.  
 
3.  Criteria for delaying subsequent T cell infusion  
Subsequent T cell infusion will be delayed if clinically indicated, or if WT1-specific T cell 
frequency in the peripheral blood is >3% of total CD8+ T cells. Up to 60mL (45mL for patients 
who are 30-50kg, 1ml/kg for patients who are 15-30kg) of blood should be drawn within 21 days 
prior to the scheduled date for the subsequent T cell infusion for evaluation of persistence of 
adoptively transferred T cells. If the WT1-specific T cell frequency is >3%, the following T cell 
infusion will be held. Repeat evaluation of persistence of WT1-specific T cells should be 
performed up to twice weekly for one month and then at the PI discretion until the WT1-specific 
cell frequency is ≤ 3%. Subsequent T cell infusion may then be administered as soon as T cell 
product and an infusion appointment are available. 
 
4.  IL-2 administration 
If the second T cell infusion is tolerated without serious toxicity as detailed in Section 18, 
patients should then receive twice daily SC injections of recombinant IL-2 at a dose of 2.5 x 105 
U/m2 approximately every 12 hours for 14 days, starting between 2-4 hours after completing the 
T cell infusion. Patients or caregivers will be trained on IL-2 administration. IL-2 therapy may be 
omitted in any patient with a contraindication to receiving the drug (e.g. graft versus host 
disease), as determined by the PI and/or treating physician. IL-2 therapy may be discontinued in 
any patient developing grade 3 or greater treatment-related toxicities while receiving daily SC 
IL-2 injections.  
 
14.C. Other Study Agents 
14.C.1. 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. 
14.C.2. 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 24 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. 
14.C.3. 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. Standard practice policy guidelines will be followed, and its 
administration will be completed at least 24 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. 
 
 
15. EVALUATION 
 


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To note: The time points for evaluation, indicated in this section, may be varied due to 
clinical or logistic factors. 
 
15.A. Patient and Donor Screening Prior to Enrollment 
 
See Standard Practice Guidelines for standard Evaluation. In addition, the following samples will 
be obtained: 
 
1. 5 mL of blood in EDTA (purple top) tube may be collected from the donor, if needed, for 
high resolution HLA typing. 
 
2. 10 mL of donor’s blood may be sent for EBV serology. 
 
3. 2 mL of bone marrow should be sent in EDTA (purple top tubes) to the FHCRC 
Research Cell Bank (E1-305) for screening for WT1 expression, and for potential genetic 
markers (e.g. translocations, deletions, mutations) associated with the disease. If a 
patient does not have a sample containing leukemia cells at the FHCRC, we may 
request samples containing leukemia cells if they exist at outside facilities to assess for 
expression of WT1. 
 
4. 5mL of blood in EDTA (purple top) tube may be sent on the patient, if needed, for high 
resolution HLA typing. 
 
5. 10mL of blood in EDTA (purple top) tube may be sent on patient with circulating blasts 
for screening of WT1 expression and for potential genetic markers associated with 
disease. 
 
6. If leukemic cells are detected by flow cytometry in blood or bone marrow during the 
clinical work up, the hematopathology lab may be asked to sort the leukemic cells for 
expression analysis of WT1 and other potential genetic markers. 
 
7. Karnofsky performance status score (age ≥ 16 years) or Lansky play score (age 
<16 years) ≤ 40% for patient with no evidence of leukemia or MDS at time of screening 
(ARM1) 
 
8. Patients with Karnofsky performance status score (age ≥ 16 years) or Lansky play score 
(age <16 years) ≤ 30% for patient with evidence of leukemia or MDS at time of screening 
(ARM2) 
 
 
15.B. Patient Evaluation after Transplant and Prior to Planned Initiation of CTL 
Infusions 
 
See Standard Practice Guidelines and transplant protocol guidelines for evaluation after 
transplant. When bone marrow is sampled for clinical evaluation (routinely at ~day +28 and +80-
100 for myeloablative transplant and ~day+28, +56 and +80-100 for non-myeloablative 
transplant, as well as when clinically indicated), the following samples may also be requested: 
 


FHCRC 2498.00 
 
 
 
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1. 10 ml blood (3ml for patients who are 15-50kg) to be sent in EDTA tubes to the FHCRC 
Research Cell Bank (E1-305) for screening for WT1 expression, and for potential genetic 
markers (e.g. translocations, deletions, mutations) associated with the disease. 
2. 2 mL of bone marrow aspirate to be sent in EDTA tubes to the FHCRC Research Cell 
Bank (E1-305) for screening for WT1 expression, and for potential genetic markers (e.g. 
translocations, deletions, mutations) associated with the disease. 
3. 5 ml of bone marrow aspirate to be sent in ACD tube to the FHCRC Research Cell Bank 
(E1-305). 
 
15.C. Patient Evaluation at Time of Planned Initiation of T Cell Infusions (within 30 days 
of T cell infusion, unless otherwise indicated) 
 
A. History: An updated history. 
 
B. Physical exam with attention for signs of pre-existing GVHD or toxicity to 
WT1-expressing tissues [e.g. pleuritis or pericarditis, pneumonitis, abdominal pain 
(particularly in region of splenic capsule or ovaries), and testicular pain].  
 
C. Assessment of Karnofsky or Lansky performance status. 
 
D. Laboratory studies including CBC, chemistry battery, and urinalysis, with special 
attention for evidence of pre-existing toxicity to WT1-expressing tissues as manifested 
by graft failure, renal dysfunction, or hematopoietic suppression. 
 
1. Chest X-ray (CXR) and pulmonary function tests, if clinically indicated. 
 
2. Assessment of chimerism by FISH or Variable Number Tandem Repeat (VNTR) analysis 
if clinically indicated. 
 
3. Bone marrow aspirate and if clinically indicated, a biopsy should be performed within 
three weeks prior to initiation of T cell therapy. Bone marrow evaluation should include 
morphology, cytogenetics, flow cytometry, FISH, and PCR studies for MRD (if 
applicable). Research marrow aspirate samples also should be sent (5 ml in ACD) (2 ml 
in EDTA) to FHCRC Research Cell Bank (E1-305). 
 
15.D. Evaluation Immediately Prior to each T Cell Infusion (Day 0)  
1) Interval history and review of systems. 
 
2) Physical exam, vital signs. 
 
3) CBC, chemistry battery, urinalysis. 
 
4) Serum or urine β-hCG pregnancy test within 14 days of planned T cells infusion. 
 
5) 10 mL of blood in EDTA tubes should be sent to FHCRC Research Cell Bank (E1-
305) (3mL for patients who are 30-50kg, not required for patients who are 15-30kg).  
 
6) 60 mL of blood in ACD tubes should be sent to FHCRC Research Cell Bank (E1-
305) (40 mL for patients who are 30-50kg, 1mL/kg for patients who are 15-30kg).  
 


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15.E. Evaluation During Each T Cell Infusion (Day 0) 
 
1. Blood pressure, heart rate, temperature, respiratory rate and O2 saturation (by pulse 
oximetry) will be recorded at around the following time points: time 0, every 15 minutes 
during the T cell infusion, then hourly for 2 hours following the T cell infusion. 
 
2. Events will be managed by standard medical practice (see also Section 18 for 
management of toxicities). 
 
15.F. Evaluation After Each T Cell Infusion  
All post-treatment monitoring shall start from the T cell infusion for both first and second 
infusion (to avoid any confusion that such monitoring should begin after 2nd T cell infusion or 
completion of IL-2). 
 
1. For patients > 50kg 
a. Interval history and review of systems on day +1, and weekly (starting on day +7) 
for four weeks after infusion #1; and day +1, and weekly for six weeks after 
infusion #2 (to correspond with four weeks of monitoring post-SC IL-2). 
 
b. Physical exam, vital signs on day +1, and weekly (starting on day +7) for four 
weeks after infusion #1; and day +1, and weekly for six weeks after infusion #2 
(to correspond with four weeks of monitoring post-SC IL-2). 
 
c. CBC with differential and platelet count, serum electrolytes, BUN, creatinine, 
glucose, and urinalysis on day +1, +4, and weekly (starting on day +7) for four 
weeks after infusion #1; and day +1, +4, and weekly for six weeks after infusion 
#2 (to correspond with four weeks of monitoring post-SC IL-2). 
 
d. 10 mL in EDTA purple top tubes should be sent to FHCRC Research Cell Bank 
(E1-305) on day +1, +4, +7, +14, +21 and +28 following each infusion.   
 
e. 60mL of blood in ACD yellow top tubes should be sent to FHCRC Research Cell 
Bank (E1-305) on day +1, +4, +7, +14, +21 and +28 following each infusion. 
 
2. For patients 30-50kg 
a. Interval history and review of systems on day +1, and weekly (starting on day +7) 
for four weeks after infusion #1; and day +1, and weekly for six weeks after 
infusion #2 (to correspond with four weeks of monitoring post-SC IL-2). 
 
b. Physical exam, vital signs on day +1, and weekly (starting on day +7) for four 
weeks after infusion #1; and day +1, and weekly for six weeks after infusion #2 
(to correspond with four weeks of monitoring post-SC IL-2). 
 
c. CBC with differential and platelet count, serum electrolytes, BUN, creatinine, 
glucose, and urinalysis on day +1, +4, and weekly (starting on day +7) for four 
weeks after infusion #1; and day +1, +4, and weekly for six weeks after infusion 
#2 (to correspond with four weeks of monitoring post-SC IL-2). 
 
d. 3 mL in EDTA purple top tubes should be sent to FHCRC Research Cell Bank 
(E1-305) on day +1, +4, +7, +14, +21 and +28 following each infusion. 


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e. 40mL of blood in ACD yellow top tubes should be sent to FHCRC Research Cell 
Bank (E1-305) on day +1, +4, +7, +14, +21 and +28 following each infusion. 
 
3. For patients 15-30kg 
a. Interval history and review of systems on day +1,+4, and weekly for four weeks 
after infusion #1; and on day +1, +4, and weekly for six weeks after infusion #2 
(to correspond with four weeks of monitoring post-SC IL-2). 
 
b. Physical exam, vital signs on day +1, +4, and weekly for four weeks after infusion 
#1; and on day +1, +4, and weekly for six weeks after infusion #2 (to correspond 
with four weeks of monitoring post-SC IL-2). 
 
c. CBC with differential and platelet count, serum electrolytes, BUN, creatinine, 
glucose, and urinalysis on days +4, +14, +28 following infusion #1, and on days 
+4, +14, +28 and +42 following infusion #2. Note that additional laboratory 
monitoring may be performed more frequently outside of these specific dates, as 
per the primary treating pediatric team.  
 
d. 1mL/kg of blood in ACD yellow top tubes should be sent to FHCRC Research 
Cell Bank (E1-305) on day +4, +14 and +28 following infusion #1, and on days 
+4, +14, +28 and +42 following infusion #2. Note that additional laboratory 
monitoring may be performed more frequently outside of these specific dates, as 
per the primary treating pediatric team.  
 
Additional evaluation and laboratory as indicated in section 15.G.1.b 
 
15.G. Clinical and Laboratory Evaluation for Toxicity Following T Cell Infusions 
 
The period of monitoring for treatment-related toxicity will start with the first T cell infusion. 
Patients will be followed during and after therapy as described below. To note: the evaluation 
days listed below are approximate, as many of the patients treated in our center reside out of 
the area, and it is anticipated that at four weeks after completing T cell infusions many patients 
will have returned home to the care of their primary physician, and not always be able to follow 
the exact time points as dictated by the protocol. In addition, frequency of monitoring may 
potentially be increased if indicated based on the patient’s clinical condition. 
 
1. Time points of evaluation for toxicities: 
a. During therapy: 
i. Interval history and physical exam: Day 0 prior to infusion, day +1 after each 
infusion and then weekly for 4 weeks following any T cell infusion, or for 6 weeks 
if IL-2 is administered. If the second infusion is delayed beyond 8 weeks, efforts 
will be made to obtain an interim H and P at 8 weeks, 3 months, and then every 3 
months until the second infusion is administered or patient is taken off study.  
ii. Laboratory evaluation (as described in Section 15.G.2.b): Day 0 prior to each T 
cell infusion, days +1, +4, and then weekly after each infusion. For patients 15-
30kg: laboratory evaluation on Day 0 prior to each T cell infusion, days +4, +14 
and +28. If the second infusion is delayed beyond 8 weeks, clinical and research 
samples will be requested at 8 weeks, 3 months, and then every 3 months until 
the second infusion is administered, or more frequently as the P.I. or treating 


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physician recommends, depending on clinical factors and T cell persistence 
level.  
iii. Bone marrow: Before any T cell infusion (if marrow sampling has not been 
performed within the prior three weeks), 3-5 days after IL-2 has been completed 
and then as clinically indicated (see also Sections 15.H). An additional “interim” 
restaging bone marrow sample may be requested following treatment with the 
first T cell infusion if the second T cell infusion is delayed by > 4 weeks.  
b. After study therapy has been completed: 
i. In the first 6 weeks after completion of the second T cell infusion if IL-2 is 
administered (or in the first 4 weeks after completion of the second T cell infusion 
if there is no inclusion of IL-2): weekly interval history and physical exam, and 
laboratory evaluation. For patients 15-30kg, interval history and physical exam, 
and laboratory evaluation as per 15.G.1.a.i and ii. 
ii. ≥6 weeks after infusion of T cells, efforts will be made to obtain:  
Interval history and physical and laboratory evaluation at 6 weeks and 8 weeks; 
3, 6, and 12 months for the first year and then annually for a total of 15 years 
depending on persistence of transferred cells and in compliance with FDA 
guidelines for patients receiving lentivirally-transduced cells (see also Section 
15.I). 
 
2. General Toxicity Assessment 
a. Interval history and physical exam with vital signs, with particular attention for signs of 
GVHD or toxicity to WT1-expressing tissues [e.g. pleuritis or pericarditis, pneumonitis, 
abdominal pain (from splenic capsule or ovaries), and testicular pain]. 
b.   Laboratory evaluation: CBC with differential and platelet count (with attention for signs 
of graft failure or hematopoietic toxicity), serum electrolytes, BUN, creatinine, glucose, and 
urinalysis (for evidence of renal toxicity). 
c. Bone marrow: Bone marrow aspirate and biopsy, if needed, to evaluate for graft 
failure or hematopoietic toxicity (aplasia). 
d. Weekly exam during SC IL-2 administration. 
e. Daily record of temperature and symptoms by the patient or medical staff during 
SC IL-2 administration. 
f. CXR day +1 after each T cell infusion, if clinically indicated. 
 
3. GVHD Assessment 
a. Skin involvement will be assessed by clinical exam, and, if abnormalities suggestive of 
GVHD are detected, the diagnosis should be established by skin biopsy. The 
percentage of body surface area involved will be recorded. 
b. Gastrointestinal symptoms suspicious for GHVD may be evaluated by endoscopy and 
biopsy if indicated. 
c.  Liver function abnormalities should be evaluated in the context of the clinical status and 
a liver biopsy may be performed if indicated to establish a diagnosis. 
d. GVHD assessment should be performed, if the patient is in Seattle, by a trained 
member of the medical staff, and staged and graded according to established criteria 
(Protocol Appendices A and B). 
 
4. Toxicity 
The occurrence of infusion related toxicity requiring treatment discontinuation will result in 
no further infusions being given and also in the administration of glucocorticoids to ablate 
the transferred T cells (see Section 19 for details). 


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15.H. Evaluation of Persistence and Function of Adoptively Transferred T Cells and for 
Evidence of Leukemia/MDS 
 
1. Blood samples: 
a. To evaluate persistence and function of adoptively transferred T cells: up to 60mL for 
patients > 50kg (40mL for patients who are 30-50kg, 1mL/kg for patients who are 15-
30kg) of blood in ACD yellow top tubes to FHCRC Research Cell Bank (E1-305) should 
be obtained immediately prior to each infusion and as close as possible to the time 
points: days +1, +4, +7 and +14 after each infusion of T cells (days +4, +14, +28 for 
patients who are 15-30kg).  
 
b. If second T cell infusion is delayed, up to 60 mL (40mL for patients who are 30-50kg, 
1mL/kg for patients who are 15-30kg) of blood in yellow top tubes may be obtained up to 
twice weekly for a month and then at the PI’s discretion and sent to the FHCRC 
Research Cell Bank (E1-305) to evaluate for WT1 specific T cell persistence. All other 
research samples may be discontinued until T cell therapy resumes. 
 
c. 60 mL (40mL for patients who are 30-50kg) blood samples in ACD yellow top tubes 
to FHCRC Research Cell Bank (E1-305) will be obtained at days +1, +4, +7 and +14 
after each infusion of T cells and approximately 3-5 days after the last T cell infusion and 
completion of IL-2 injections, weekly in the first four weeks, week 6, week 8, three 
months, six months, and 12 months.  
 
For patients who are 15-30kg, 1mL/kg of peripheral blood in ACD yellow top tubes 
will be obtained at days +4, +14, +28 after each T cells and approximately 3-5 days 
after the last T cell infusion and completion of IL-2 injections.  At week 8, three 
months, six months, and 12 months up to 2mL/kg of blood samples will be obtained. 
 
If adoptively transferred T cells are <0.1% of CD8+ T cells, persistence evaluation 
may be discontinued or evaluation frequency decreased, per the PI’s discretion. If 
persistent adoptively transferred T cells are detected additional samples may be 
requested. 
 
 
d. 10 mL (3mL for patients who are 30-50kg) blood sample in EDTA purple top tubes 
should be sent to FHCRC Research Cell Bank (E1-305) to be evaluated for expression 
of WT1 at days +1, +4, +7 and +14 after each infusion of T cells and approximately 3-5 
days after the last T cell infusion and completion of IL-2 injections, weekly in the first four 
weeks, week 6, week 8, three months, six months, and 12 months. 
 
For patients who are 15-30kg, up to 3mL blood sample in EDTA purple top tubes 
should be obtained and sent to FHCRC Research Cell Bank (E1-305) for evaluation 
of WT1 expression at week 8, three months, 6 months and 12 months. 
 
2. Bone marrow samples: 
a. Patients will be assessed for evidence of disease by morphology, flow cytometry, 
PCR or cytogenetic analysis of bone marrow samples obtained as close as possible to 
days +0 (if marrow sampling has not been performed within the prior three weeks), 3-5 
days after IL2 has been completed, and then as clinically indicated. 
b. In addition to the samples sent for clinical purposes: 


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i.5 ml of bone marrow (3 ml for patients 15-30 kg) in ACD yellow top tubes to 
FHCRC Research Cell Bank (E1-305) should be sent of any bone marrow 
samples obtained during this study to be evaluated for the presence of adoptively 
transferred T cells by WT1 and EBV (or CMV) HLA-A*0201 double 
peptide/multimer analysis and for other immunological parameters. 
ii.2 ml of bone marrow in EDTA purple top tubes should be sent to FHCRC 
Research Cell Bank (E1-305) of any bone marrow samples obtained during this 
study to be evaluated for expression of WT1. 
 
3. Other samples: If a biopsy of an organ is performed for clinical reasons, an additional 
sample may be obtained for research tests. 
 
15. I. Evaluation for long term effects of using lentivirally transduced T cells 
 
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” (November 28, 2006, the same rules 
apply to lentiviruses), every effort will be made to obtain blood samples for testing for RCL at the 
following time points: Pretreatment, at 3, 6, and 12 months for the first year, and annually 
thereafter. If all post-treatment assays are negative during the first year, subsequent yearly 
samples will be archived for future analysis, if such analysis becomes clinically or scientifically 
indicated. Samples will be archived with appropriate safeguards to ensure long-term storage 
and an efficient system 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 Center for Biologics Evaluation & Research (CBER). 
 
2.  Long-Term Follow-Up 
Every effort 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 lentiviral disease, such as cancer, 
neurologic disorders, or other hematologic disorders. 
 
Additionally, efforts will be made to collect samples to determine levels of gene-modified cells in 
peripheral cells. Suspect clinical symptoms or findings will trigger 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 metabolic panel, and levels of gene-modified cells. In case histories, 
physicians will be asked to record details of all exposures to mutagenic agents and other 
medications. 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 illness and 


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hospitalization. Patients will generally be seen by their doctors in their local area. However, the 
FHCRC in collaboration with Seattle Cancer Care Alliance (SCCA) will be available to assist in 
the LTFU of participants in this clinical trial. If patients die or develop neoplasms, 
recommendation  will be made to assay for RCL in a biopsy sample of the neoplastic tissue or 
the pertinent autopsy tissue. 
 
16. MANAGEMENT OF T CELL INFUSION IN PATIENTS WHO RELAPSE OR PROGRESS 
DURING T CELL THERAPY 
 
Patients who started treatment with no evidence of disease (Arm 1) and relapsed during T cell 
therapy, or patients who started treatment with evidence of disease (Arm 2) 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. 
 
T cell therapy may be continued if relapse or disease progression occurs prior to the last dose 
of T cells infusion, 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 >500/mm3 and plts > 30K). 
 
This decision 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. 
 
17. OPTIONS FOR FURTHER TREATMENT AFTER COMPLETION OF STUDY THERAPY 
 
If adoptively transferred WT1-specific T cell infusion demonstrated anti-leukemic activity, the 
option of additional T cell infusions may be discussed with the patient and treating physician, at 
the discretion of the PI. 
 
18. TOXICITIES 
 
18.A. Toxicity Grading 
 
Toxicities will be graded according to the NCI Common Terminology Criteria for Adverse Events 
(CTCAE) Version 4. The full text of the NCI CTCAE is available online at:  
http://evs.nci.nih.gov/ftp1/CTCAE/About.html. 
 
18.B. Immediate Toxicities: 
 
Immediate reaction to infusion (i.e. defined as those occurring 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, due to direct cytotoxic effects of CTL on target tissues expressing WT1, from normal 
tissues expressing antigens recognized by mispaired TCR, or from allogeneic T cells not 
specific for EBV that were not completely excluded during the generation of the WT1-specific T 
cells. 
 


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a) Milder reactions (i.e. < grade 3 CTCAE v.4 or less severe than specified below) would 
include symptoms such as: 
 
• 
Fever, chills, fatigue. 
 
• 
Dyspnea, chest tightness, myalgia, or pain (chest, pleuritic, abdominal, back, 
testicular). 
 
• 
Alteration in vital signs such as: 
• 
Lowering of blood pressure, but with systolic BP ≥ 90 mm Hg, or ≤ 20 mm Hg below 
baseline. 
• 
Tachycardia, but with HR ≤ 130 or ≤ 30 above baseline. 
• 
Tachypnea, but with RR ≤ 32/min or ≤ 10 above baseline. 
• 
Hypoxemia, but O2 saturation ≥ 88% on room air, or ≤ 5% fall from baseline. 
• 
Skin changes such as erythema, urticaria, or other rash. 
 
Management will be at the discretion of the PI or designee, and may include appropriate 
supportive care such as: 
o Acetaminophen or Demerol for fever and chills (All subjects who develop fever or chills 
should have a blood culture drawn). 
o Acetaminophen for headache. 
o Diphenhydramine for nausea and vomiting. 
o Fluid administration for hypotension. 
o Supplemental oxygen for hypoxemia. 
o Decreasing the rate of the infusion 
 
b) More severe reactions would include symptoms such as, 
 
• 
Hypotension with systolic BP <90 mm Hg and >20 mm Hg below baseline. 
• 
Tachycardia with HR >130 and >30 above baseline. 
• 
Tachypnea with RR >32 and >10 above baseline. 
• 
Hypoxemia with O2 saturation of <88% and >5% fall from baseline. 
 
Management will be by modifying the infusion rate or terminating the infusion at the 
discretion of the PI or designee, and administering supportive medical care. 
o If patient responds to supportive care by normalization of vital signs or resolution of 
hypoxemia and PI deems it safe to continue, the infusion may be resumed/infusion rate 
increased. 
 
o If the patient does not respond by normalization of vital signs or hypoxemia after 
supportive care alone, methylprednisolone (MP) to ablate the infused T cells should 
be administered as per Section 18.D. 
 
c) Any unexpected severe toxicity (see Section 18.E) 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 MP will be administered, per the 
discretion of the PI or designee, as per Section 18.D. 
 


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18.C.  Delayed Toxicities 
 
It is possible that the infusion of CD8+ WT1-specific transduced T cells will result in delayed 
toxicities related to the recognition of tissues expressing WT1. It is anticipated that the 
symptoms and signs will occur within hours to four weeks after completion of either the T cell 
infusion or IL-2 administration. Any toxicities requiring treatment discontinuation (as 
described in Section 18.E) occurring within four weeks of study treatment will be 
managed by supportive care and MP will be administered, per the discretion of the PI or 
designee, as per Section 18.D. Evaluation and management of hematologic toxicities are 
described in Section 18.E.2. Acute GVHD will be managed as described in Section 18.E.3. 
 
Any patient who develops clinical evidence of a cytokine storm will have a workup to exclude 
infection or other causes. Initial treatment will 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 as described in Section 18.D addressing 
management of serious adverse events. 
 
Blood samples for research tests are collected in all patients prior to the T cell infusion and on 
days +1, +4, +7, and +14 (days +4, +14 and +28 for patients weighing 15-30kg) after the T cell 
infusion, as indicated in Section 15.G. Plasma will be isolated from each blood sample and 
stored for cytokine analysis. If patients have clinical evidence of a cytokine release storm, the 
samples will be analyzed for cytokine analysis (including IFN-γ, TNF-α, IL-2 concentrations). 
 
18.D. Management of Severe Treatment-related Toxicities with Methylprednisolone 
 
Patients who require glucocorticoids for treatment of severe toxicities will receive 
Methylprednisolone (MP) at 2 mg/kg/day IV if WT1-specific T cells are ≥ 0.05% of CD8+ cells 
in peripheral blood, and the patient will be taken off study. MP (or an equivalent dose of an 
alternative glucocorticoid) can be tapered when symptoms are significantly improving or WT1-
specific T cells are <0.05% of CD8+ cells in peripheral blood. These are general guidelines for 
the glucocorticoid taper and may have to be adjusted based on each patient’s clinical situation. 
 
Patients receiving glucocorticoids should have assessments of peripheral blood for the 
presence of WT1-specific CTL cells (vβ17+ and dual-multimer+) will be performed according 
to the PI or designee discretion. Twenty ml of blood will be sent in ACD yellow top tube to the 
FHCRC Research Cell Bank (E1-305) for the evaluation of the presence of the infused T cells. 
Modification of WT1-specific CTL persistence assessment may be made per the PI or designee 
discretion. 
 
18.E. Definition and Management/Evaluation of Non-hematologic and Hematologic 
Toxicities Requiring Treatment Discontinuation 
 
• 
Non-hematologic toxicity requiring treatment discontinuation is defined as any 
grade 3 or 4 non-hematologic toxicity (CTCAE 4) that is deemed to be caused by 
infusion of the study treatment (i.e. not attributable to infection, recurrent/progressive 
leukemia, toxicity from other therapies, or any identifiable cause other than T cell 
infusion or IL-2 administration; and that is not an expected cytokine-mediated 
symptom as further described below) that occurs at any time point after the first T 


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cell infusion. It is expected that the onset of non-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.G.  
Transient high fever may occur due to release of cytokines from T cells stimulated by the 
recognition of targets, and therefore grade 4 toxicity from fever (>40 °C) lasting ≤ 36 
hours will not require treatment discontinuation. Symptoms of cytokine release that are 
grade 3 or less (cytokine release syndrome, tachycardia, chills, rigors, fatigue, myalgias, 
arthralgias, dizziness, rash, urticaria, hypotension not requiring pressor support or > 3L 
fluid boluses in 24 hours, dyspnea or hypoxemia not requiring > 2L/min supplemental 
oxygen for longer than 48 hours) are similarly expected to possibly occur due to release 
of cytokines from T cells stimulated by the recognition of targets, and therefore will not 
require treatment discontinuation. 
 
If possible, an attempt will be made to obtain biopsies of affected tissues to determine if 
transduced T cells are accumulating at the site. 
 
Toxicities will be managed as described in Section 18.C.  
 
2. Hematologic toxicity requiring treatment discontinuation (blood/bone marrow CTCAE 4) is 
defined as any new or recurrent onset of grade 4 hematologic toxicity that occurs at any time 
point after the first T cell infusion, and last for two consecutive days, such as neutropenia (ANC 
<500/mm3) in the context of new bone marrow aplasia (<5% cellularity), which is attributed to 
the study treatment (i.e. not associated with prior chemotherapy, leukemic progression in the 
bone marrow, transplantation, infection, toxicity from other therapies, or any identifiable cause 
other than T cell infusion or IL-2 administration). Transient drop in lymphocyte count is expected 
after T cell infusion. Therefore, grade 4 lymphopenia, if returns to pre-infusion level within 14 
days, will not require treatment discontinuation. Thrombocytopenia threshold for treatment 
discontinuation is platelets <20,000/mm3. This differs from CTCAE 4 for guidelines for Grade 4 
hematologic toxicity for thrombocytopenia, but will be used in this trial, as thrombocytopenia is a 
common problem in this patient population 
 
Management: If a patient develops new onset hematologic toxicity such as ANC <500/mm3 or 
platelets <20,000/mm3 for two consecutive days, which cannot be explained by alternative 
cause, a bone marrow sample should be obtained. 
• 
If the bone marrow shows evidence of aplasia, MP will be started as described in 
Section 18.D and study treatment discontinued. G-CSF may be administered at the 
discretion of the medical team. 
• 
If the bone marrow shows no evidence of aplasia and no disease progression or other 
clear etiology for marrow suppression is found, patients will be observed for three more 
days. If by that time there is no improvement of bone marrow function and no clear 
etiology for marrow suppression (e.g. infection) is detected, MP will be started as 
described in Section 18.D and study treatment discontinued. G-CSF may be 
administered at the discretion of the medical team. 
• 
If bone marrow cannot be obtained and no alternative cause of hematologic toxicity is 
identified by the third consecutive day of neutropenia or thrombocytopenia, MP will be 
started as described in Section 18.D 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.G. 
 
3.  Acute GVHD: Unlike the unselected polyclonal population of T cells administered as part of 
DLI, this protocol utilizes selected populations of virus-specific CD8+ T cells that have been 
transduced to recognize WT1.  Therefore the incidence of classical GVHD due to CTL infusions 
is expected to be very low if at all detectable. The risk should be even further reduced compared 
to DLI since no naïve T cells will be included in the infusion. 
 
Patients will be assessed for status of acute and/or chronic GVHD within 3 weeks prior to 
receiving the study treatment, and again at 3 months, 6 months and 12 months following any T 
cell infusion, and then annually, as clinically indicated.  
 
Management: Patients developing grade III-IV acute GVHD will be treated for acute GVHD with 
glucocorticoids according to Standard Practice guidelines. If possible, biopsies of affected 
tissues will be obtained prior to glucocorticoids treatment to determine if transduced T cells are 
accumulating at the site. Patients receiving glucocorticoids will have assessments of peripheral 
blood for the presence of WT1-specific T cells. If GVHD is attributed to the study treatment, 
treatment will be discontinued. 
 
• 
A patient will no longer be eligible to receive additional T cell infusions if the PI or 
designee determines that additional T cell infusions are not in the best interest of the 
patient. 
 
 
18.F. Chronic GVHD 
 
Patients found to have chronic GVHD will be treated as directed by their primary team and/or 
per recommendations of the GVHD or LTFU attending physicians. If possible, biopsies of 
affected tissues will be obtained prior to treatment to determine if transduced T cells are 
accumulating at the site. 
 
18.G. Detection of Replication Competent Lentivirus (RCL) 
 
Finding evidence of RCL or evidence suggestive of lentiviral disease (see Section 15.I) will be 
considered a treatment-related toxicity. 
 
 
19. OFF STUDY CRITERIA 
 
A patient’s participation on the protocol will be terminated for any of the reasons listed below: 
 
1. 
The participant withdraws consent. 
 
2. 
Inability to generate transduced t cells that meet release criteria for t cell therapy. 
 
3.   
Patient death. 
 


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If a patient progresses on T cell therapy, pursues alternative systemic therapy, cannot receive 
further T cell therapy due to toxicity or clinical status, or if the treating physician or the protocol 
PI deems that it is not in a patient’s best interest to continue in this study, that patient will be 
designated as “off treatment”. To comply with FDA requirements for long-term monitoring for 
potential toxicities related to use of lentivirally-transduced T cells, such patients who do not 
otherwise withdraw consent will nevertheless continue to be followed by the study team for 
survival outcomes and for late toxicities, as per Section 15.I. 
 
 
 
 
20. PROTOCOL REGISTRATION AND SPECIAL CONSIDERATIONS 
 
HLA-A*0201 is expressed in all ethnic groups, but is most common in Caucasians, which 
represents the most common ethnic group treated at our center, and therefore it is expected 
that the majority of patients on this trial will be Caucasian. However, patients from all ethnic 
groups will be enrolled if they meet all eligibility criteria. If these studies demonstrate safety and 
potential benefit, it is anticipated that TCR genes specific for WT1 in the context of other HLA 
alleles, more predominant in other ethnic groups, will be isolated and afford the option of 
expanding the trial to be more inclusive of all patients. 
 
20.A. Projected Target Accrual - Arm 1 
Table 5. Ethnic and gender distribution chart – Arm 1 
TARGETED / PLANNED TREATMENT:  Number of Subjects 
 
ETHNIC CATEGORY 
Sex / Gender 
Females 
Males 
Total 
Hispanic or Latino 
1 
1 
2 
Not Hispanic or Latino 
17 
16 
33 
Ethnic Category Total of All Subjects 
18 
17 
35 
 
RACIAL CATEGORIES 
Sex / Gender 
Females 
Males 
Total 
American Indian / Alaska Native 
0 
0 
0 
Asian 
1 
1 
2 
Native Hawaiian or Other Pacific Islander 
0 
1 
1 
Black or African American 
1 
0 
1 
White 
16 
15 
31 
Racial Categories:  Total of All Subjects 
18 
17 
35 
 
20.B. Projected Target Accrual - Arm 2 
 
Table 6. Ethnic and gender distribution chart – Arm 2 


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TARGETED / PLANNED TREATMENT:  Number of Subjects 
 
ETHNIC CATEGORY 
Sex / Gender 
Females 
Males 
Total 
Hispanic or Latino 
1 
1 
2 
Not Hispanic or Latino 
9 
9 
18 
Ethnic Category Total of All Subjects 
10 
10 
20 
 
RACIAL CATEGORIES 
Sex / Gender 
Females 
Males 
Total 
American Indian / Alaska Native 
0 
0 
0 
Asian 
1 
1 
2 
Native Hawaiian or Other Pacific Islander 
0 
0 
0 
Black or African American 
0 
0 
0 
White 
9 
9 
18 
Racial Categories:  Total of All Subjects 
10 
10 
20 
 
Since not all patient’s enrolled on the study will receive treatment the enrollment numbers may 
be higher than those in the tables above. 
 
21. GUIDELINES FOR ADVERSE EVENT REPORTING 
 
21.A. Reporting of Adverse Events (AEs) 
 
All unexpected and serious adverse events which may be due to study treatment or intervention 
must be reported to the FHCRC Institutional Review Office per their current reporting 
requirements.  
 
All grade ≥ 3 CTCAE v.4 AEs will be collected from the time of first T cell infusion through 30 
days after the last study intervention (T cell infusions + IL2). For those patients with an interval 
of > 3 months between T cell infusions, collection of all grade ≥ 3 AEs will temporarily put on 
hold after 3 months, and then resume at the onset of any additional study treatments (e.g. 
lymphodepleting chemotherapy and 2nd T cell infusion.) Beginning 30 days after last study 
intervention, study related toxicities will be collected as discussed in Section 15 and Section 
18. 
 
All serious adverse events that are unexpected and related to study treatment will be reported to 
the FDA within 15 calendar days. The reports will include the date and time of onset, severity 
and duration of the event, the relationship to study treatment, the treatment given, and the 
eventual outcome. 
 
21.B. 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 following documents. 
Please also refer to the FHCRC IRO website.) 
 


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Table 7. 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 
 
Toxicity grading  
 
Toxicities will be graded using the NCI Common Terminology Criteria for Adverse Events 
(CTCAE) Version 4. Information on this scale can be found at 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html. 
 
22. DATA AND SAFETY MONITORING PLAN 
 
22.A. Primary Monitoring 
 
The PI of the study will have primary responsibility for ensuring that the protocol is conducted as 
approved by the Scientific Review Committee and Institutional Review Board. The PI 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. 
 
22.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. At least twice monthly the PI 
or his/her designee will also review with the Study Nurse the progress of each patient 
undergoing therapy with WT1-specific T cells.  At least monthly the PI will also meet with the 
Study Nurse to review the clinical course of all patients who have completed a course of T cell 
therapy. 
 
22.C. Monitoring the Progress of Trials and the Safety of Participants 
 


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The FHCRC PI is responsible for monitoring this clinical trial, with oversight by a DSMB, the 
Protocol and Data Monitoring Committee (PDMC) at the FHCRC, and the FHCRC IRB. This is a 
Phase I/II study and the assessment of risk is considered above minimal. 
 
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. 
 
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. 
 
An external monitor will be retained to monitor study progress. The scope of monitoring will be 
based on the FHCRC/UW Data and Safety Monitoring Plan (DSMP): 
http://www.cancerconsortium.org/rto/prr/DSMPlan.pdf. Per the DSMP, subjects will be randomly 
selected for verification. An initial monitoring visit is expected within six months of enrollment of 
the first subject and preferred prior to enrollment of > 4 subjects as 100% verification is 
expected during an initial visit.  
 
Flow of information concerning clinical trial participants originates with the clinicians and nurses 
in the clinic or referring clinicians at other institutions and will be transmitted to the FHCRC 
Research Nurse. At the FHCRC, health care providers and rotating attending physicians assess 
patients and will record their observations regarding toxicity and response outcomes in the 
medical record. Thus, multiple health care providers will be providing independent observations 
and participating in monitoring this trial. The PI may be a responsible clinician for some patients 
entered on this trial. However, assessments will be the sum total of the multiple clinicians 
involved with the patient, which should avert possible conflict of interest if the PI is the attending 
clinician for a protocol patient. If determination of adverse events is controversial, co-
investigators will convene on an ad hoc basis as necessary to review the primary data and 
render a decision. 
 
23. 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 transplant 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 


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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 their care 
related to this protocol will be received at the SCCA, UW Medical Center and Seattle Children’s. 
The study nurse and/or data coordinator will maintain a Case Report Form (CRF) Notebook for 
each patient treated on this protocol. The CRF notebooks 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. 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. 
 
24. STATISTICAL CONSIDERATIONS 
 
The primary objectives of this study are to estimate the toxicity rate associated with infusing 
donor CD8+ T cells that have been transduced to express a high affinity T cell receptor specific 
for WT1 in patients at high risk for post-transplant relapse of AML, MDS, or CML (Arm 1) and to 
examine the anti-leukemic activity and potential toxicities of these cells in patients with relapsed 
AML, MDS, or CML post transplant (Arm 2). The protocol will enroll eligible patients at or post 
transplant, and accrual will continue until either 35 patients have been enrolled and treated on 
Arm 1 and 20 patients have been enrolled and treated on Arm 2 or until five years have 
elapsed. 
 
Patients will be followed for 15 years, in compliance with the FDA guidance for patients that 
have received lentivirally-transduced cells. As with all transplant patients, individuals on this 
protocol will also be seen for a detailed follow-up at one year post HCT at the LTFU Clinic at the 
FHCRC. This should provide the opportunity to assess potential toxicities of the study therapy 
as well as anti-leukemic activity as reflected by prevention of relapse and/or prolongation of 
disease free survival. If the safety profile is acceptable (i.e. study is not stopped due to excess 
toxicity as detailed in stopping rules below) and the anti-leukemic efficacy in this study appears 
promising (as described below) then a subsequent study will be designed to more definitively 
address potential efficacy in preventing and treating relapse of leukemia or MDS after HCT. 
Such a subsequent study would also utilize additional insights obtained from this trial regarding 
limitations to efficacy and could involve exploring strategies to increase the observed anti-
leukemic efficacy, e.g. by transducing T cells with a higher affinity TCR, and/or by co-
administering a mAb that blocks signaling to the infused T cells through the PD-1 receptor. 
 
From 2005-2010, an average of approximately 50 patients with high-risk AML, 25 patients with 
high-risk MDS, and 7 patients with CML beyond chronic phase underwent allogeneic transplant 
each year at our center. It is expected that nearly all of these patients will have disease that 
expresses WT1 significantly above background levels (~90-100% of cases),7 and at the FHCRC 
~40-44% of treated patients are HLA-A*0201 positive, similar to the general US population. 
Given these numbers, the desired patient accrual would appear to be achievable even after 
accounting for patients who are likely to be excluded due to Grade III-IV acute GVHD 
(~15-20%), meeting other exclusion criteria, and/or refusal to consent. 
 
The sample size of 35 patients to be enrolled on Arm 1 was not chosen based on formal power 
calculations, but rather was chosen after consultation with our biostatistics team to represent a 
number that is achievable (as detailed above) and will be sufficient to provide important 


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descriptive information regarding the promise (see below) of this approach and determine if it is 
worthy of further study and to reveal if the toxicity is acceptable. Regarding the precision of 
estimates for the parameters that are of interest, with 35 patients we can be 80% confident that 
the estimate of toxicity (or any alternative binary outcome) will be within at least 0.11 of the true 
value. 
 
The sample size of 20 patients to be treated on Arm 2 of this trial was not chosen based on 
formal power calculations, but rather was chosen after consultation with our biostatistics team to 
represent a number that is achievable and will be sufficient to provide important descriptive 
information regarding the promise (see below) of this approach and determine if it is worthy of 
further study. It is expected that ~15-20% of the high risk eligible patients, who enrolled on the 
study at the time of transplant, will have evidence of MRD or relapse by day 60 after 
myeloablative HCT and ~30-35% will have evidence of disease after non-myeloablative HCT by 
day 90.125,126 These relapsed patients will be eligible for treatment on Arm 2 of this study. Based 
on the above anticipated relapse rates, we expect 6-8 patients to be eligible for treatment on 
Arm 2 per year. We also expect that 2-4 HLA-A*0201+ patients/year who received an HCT for 
lower risk AML, MDS, or CML will relapse post-HCT, with nearly all having disease that 
expresses WT1 significantly above background levels (~90-100%),5 and these patients will also 
be offered enrollment on Arm 2 of this study. Assuming some patients will not fulfill inclusion 
criteria and some will choose not to participate on the trial, with the numbers above we 
anticipate no difficulty enrolling/treating 20 patients on Arm 2 of the trial within five years. 
 
Regarding the precision of estimates for the parameters that are of interest, with 20 patients we 
can be 80% confident that the estimate of a binary outcome (such as durable response as 
defined below) will be within at least 0.14 of the true value. With 20 patients, we will not likely be 
able to say that outcomes such as response rate are statistically significantly better than 
historical rates, but if our observed rates are deemed encouraging as defined below, a larger 
trial could be designed to more definitively assess efficacy as stated above. Additionally, if 
evidence of anti-leukemic activity are demonstrated in Arm 2, in concert with data from Arm 1, 
and demonstrate the absence of serious toxicity, that would justify extending this therapy in 
normal risk leukemia patients undergoing HCT and patients not undergoing an HCT. 
 
24.A. Analysis of Toxicity (Arm 1 and Arm 2) 
 
Toxicity analysis will be performed for both study arms; however, each arm will be 
analyzed independently. 
 
The period of monitoring for treatment-related toxicity will start with the first T cell infusion. For 
details regarding toxicity evaluation during treatment see Section 15 and Protocol Appendix A 
and B. During the first month after completion of T cell therapy, patients will be followed with a 
weekly clinical exam and laboratory evaluation, and during the second month a clinical exam 
and laboratory evaluation will be performed every two weeks. For the rest of the first year, 
patients will be seen and laboratory tests performed at 3, 6, and 12 months after completion of 
therapy and annually thereafter for a total of 15 years in compliance with FDA guidelines for 
patients receiving lentivirally-transduced cells (see Section 15.I. for details). To note; the 
evaluation time points listed above are approximate, as many of the patients treated in our 
center reside out of the area, and it is anticipated that at four weeks after completing therapy 
many patients will have returned home to the care of their primary physician, and not always be 
able to follow the exact time points as dictated by the protocol. In addition, frequency of 
monitoring may potentially be increase if indicated based on the patient’s clinical condition. 


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a) Toxicities to organs expressing WT1 
As described earlier, potential toxicities of WT1-specific T cell infusions include damage to 
tissues that express low levels of WT1, possibly resulting in hematopoietic suppression or graft 
failure, renal dysfunction, pleuritis or pericarditis, abdominal pain (from splenic capsule or 
ovaries), and/or testicular or ovarian pain. 
 
Treatment on  both arms will be initiated as soon as the WT1-specific T cells have been 
generated.  This means that patients may receive therapy at a time point when they are still at 
risk to develop GVHD or other transplant-related toxicities. Thus, there may be situations when 
study treatment-related toxicity may be difficult to differentiate from transplant-related toxicities. 
However, due to the poor prognosis of the patients enrolled on this study, the potentially higher 
risk of indeterminate toxicities will be explained to the patient and has been considered in the 
design of stopping criteria. The toxicity analysis will be performed for Arm 1 and Arm 2 
independently. 
 
Arm 1: 
Patients after allogeneic HCT develop major organ (e.g. lung, liver, kidney) toxicity at a rate of 
15-20%.23 Based on this expected toxicity rate, the acceptable toxicity rate determined to be 
related to the study treatment on Arm 1 will be 30%. If there is sufficient evidence to suggest 
that the true toxicity rate exceeds 30%, Arm 1 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 the acceptable threshold. If the true 
significant (requiring treatment discontinuation) toxicity rate (as defined in Section 19) exceeds 
30% on Arm 1, infusion of T cells transduced to express a high affinity WT1-specific TCR will be 
considered excessively toxic for use for prophylactic therapy post-HCT. With 35 treated 
patients, the likelihood of having stopped the trial if the true toxicity is only 15% would be 0.08, 
and if the true toxicity is 40% it would be 0.91 (estimated from 5,000 simulations). 
 
1. Operationally, any of the following observed ratios of toxicities/treated patients would yield 
such a confidence limit and would warrant suspension of Arm 1 and DSMB review:  2/2, 3/3-
4, 4/5-6, 5/7-9, 6/10-11, 7/12-14, 8/15-16, 9/17-19, 10/20-22, 11/23-25, 12/26-27, 13/28-30, 
14/31-33, 15/34-35-. If the true probability of toxicity is 15%, then the probability of reaching 
one of the above ratios after 20 or 30 patients is approximately 0.08 and 0.08, respectively. 
For a true probability of toxicity of 40%, the probabilities of one of these ratios occurring is 
approximately 0.83 and 0.91, respectively (estimated from 5,000 simulations). 
 
Arm 2: 
 
1. As for arm 1, the acceptable toxicity rate on on arm 2 is 30%. If there is sufficient evidence 
to suggest that the true toxicity rate on this arm exceeds 30%, Arm 2 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 the acceptable threshold. If the true significant (requiring treatment discontinuation) 
toxicity rate (as defined in Section 19) exceeds 30% on Arm 2, infusion of T cells 
transduced to express a high affinity WT1-specific TCR will be considered excessively toxic 
for treating relapse post HCT. 
 
2. Operationally, any of the following observed ratios would yield such a confidence limit and 
would warrant suspension of Arm 2 and DSMB review:  3/3-4 (3 of the first 3 or 4 patients), 


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4/5-6, 5/7-9, 6/10-11, 7/12-14, 8/15-16, 9/17-19, 10/20. If the true probability of toxicity is 
15%, then the probability of reaching one of the above ratios after 20 patients is 
approximately 0.11. For a true probability of toxicity of 50%, the probability of one of these 
ratios occurring is approximately 0.79 (estimated from 5,000 simulations). 
 
Clinical and laboratory information will be gathered that may be linked to or predict toxicities, 
and will be used to help design future clinical trials. This may include the patient’s age, 
comorbidities, or transplant-related toxicities prior to T cell therapy, e.g. in WT1-expressing 
organs (such as renal insufficiency related to use of calcineurin inhibitors), as well as the peak 
frequency and the persistence of infused T cells after transfer. 
 
b) Acute GVHD 
Induction of acute GVHD is another potential toxicity caused by WT1-specific T cell infusion. At 
the FHCRC the incidence of grade III-IV acute GVHD in the first 30 days after DLI is ≤ 10%. 
Selecting virus-specific CD8+ T cells (EBV- or CMV-specific) for transduction with the 
WT1-specific TCR decreases the possibility of acute GVHD by excluding TN cells and 
eliminating most if not all alloreactive donor T cells. GVHD may also potentially result from the 
creation of new TCRs on transduced T cells as a result of mispairing of the endogenous α and β 
chains with the introduced β or α chains. However, several strategies are being employed to 
reduce/eliminate this risk, including introduction of a point mutation to create a cysteine in each 
of the introduced TCR chains that forms an interchain bond and has been shown to nearly 
completely eliminate any mispairing, and the use of virus-specific T cells for transduction with 
the TCR chains which greatly limits the repertoire of endogenous TCR chains available to form 
mismatched pairs.66 Even in the absence of using these combined strategies, no toxicity from 
mismatched pairing has ever been described in human trials utilizing TCR-transduced T cells. It 
is expected that such toxicities would present in organs usually affected in acute GVHD 
(e.g. skin, gut, and liver), but could result in unusual manifestations of acute GVHD due to 
alloreactive cells with a unique specificity. Theoretically, IL-2 may also potentially increase the 
risk of acute GVHD. However, in a recent study at our institute, DLI given for relapsed acute 
leukemia was followed by IL-2 (1-3 x 106 IU/m2 /day for nine days followed by 1 x 106 IU/m2 /day 
for five days, which is 2-6 times higher than the dose of IL-2 that will be used in this study), 
without evidence of higher rate of grade II-IV GVHD compared to DLI without IL-2.34 Also, in 
BB-IND 12046 - FHCRC Protocol 1655, patients have not experienced evidence of new or 
flared acute GVHD after receiving the same IL-2 treatment that will be used in this protocol. 
 
In the absence of DLI, the cumulative incidence of grade III-IV acute GVHD is ~15-20% at the 
FHCRC and has reached a plateau by day +60 after myeloablative transplant and day +90 after 
non-myeloablative transplant. At the FHCRC, ~27% of patients will reach these time points 
without a diagnosis of any grade of acute GVHD, and only ~5% of this 27% of patients 
subsequently develop grade III-IV GVHD. In patients who have less than grade III GVHD at 
these time points, it is estimated that ~10% of those patients will progress to develop grade 
III-IV GVHD. It is therefore expected that some patients may experience grade III-IV acute 
GVHD after these time points, even if they would never have received the study therapy. 
• 
Patients with grade III or IV acute GVHD prior to initiation of therapy will be considered 
ineligible for treatment on this trial. However, study treatment may start early post- 
transplant, potentially during the time period when acute GVHD may still develop due to the 
transplant itself, and not due to the infused T cells Based on the incidence of grade III-IV 
acute GVHD after HCT (15-20%)81 and after DLI (20-30%), 2,3,32-34 the acceptable rate of 
grade III-IV acute GVHD determined to be related to the study treatment on each arm of the 
study will be 30%, in the context of the poor prognosis of the patients enrolling in this study 


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with the potential early initiation of T cell infusion during the time period when GVHD may 
still develop due to the transplant itself.. If there is sufficient evidence to suggest that the 
true grade III-IV acute GVHD rate exceeds 30% on any arm of the study, this arm will be 
suspended pending review by the DSMB. Rules that would trigger such a suspension for 
each or the study arms are as described above.  
 
d) Chronic GVHD 
It is expected that symptoms of GVHD due to T cell infusion would be similar to what has been 
observed with DLI, and thus similar to classical acute GVHD. However, it is also possible that 
the T cell infusions could lead to the development of chronic GVHD. The probability of 
developing extensive chronic GVHD one year after HCT in patients currently treated on 
protocols at the FHCRC is ~70%, and this rate will be used as a guideline to retrospectively 
assess at the end of the study the risk of chronic GVHD due to T cell infusions. However, based 
on this high frequency and the delayed time of occurrence after transplant, no formal 
stopping/suspension rules will be put in place for chronic GVHD, and all chronic GVHD will be 
treated per standard therapy. 
 
As described above for toxicity, we will gather information in an attempt to determine if any 
increased risk of acute and/or chronic GVHD in patients treated with infusion of virus-specific 
T cells transduced to express a WT1-specific TCR is associated with either the infused T cells 
or other factors such as IL-2, and such information will be used to inform the potential design of 
subsequent trials. This will include correlating the timing of study therapy after transplant, 
previous GVHD, age, donor-patient gender mismatch, conditioning therapy for HCT, and GVHD 
prophylactic therapy, with the frequency of acute and chronic GVHD.  Also, in patients who 
develop GVHD, efforts will be made to obtain biopsies and assess the samples for infiltration of 
the transduced T cells and to determine if there is emergence of an oligoclonal population with a 
particular set of endogenous TCR chains that might be responsible for forming alloreactivity by 
mispairing with the transduced TCR. Additionally, especially in cases of chronic GVHD, the 
frequency and persistence of infused T cells with development of GVHD will be determined.  
These analyses need to be considered as hypothesis generating as the power to observe 
statistically significant associations is somewhat limited given the sample size. 
 
24.B. Analysis of Efficacy in Preventing Relapse (Arm 1) 
 
Even though examination of safety is the primary objective of Arm 1 of this protocol, we shall 
also look at potential efficacy as defined by prevention of relapse at one year after transplant.  
For the high risk population that will be eligible for this arm, we would expect a one-year relapse 
probability of ~40% after myeloablative HCT and ~45% after non-myeloablative HCT.28,29 The 
rate of 40% will therefore be used as a guideline for assessment of potential efficacy in the 
current trial, but no formal rules will be in place to deem this approach as potentially efficacious.  
Using 40% as the benchmark for the expected outcome in this trial, ≤ 9 relapses among 
35 patients (observed rate of 26% or less) would be a statistically significant improvement over 
the fixed benchmark of 40% (one-sided p-value of .04), and the likelihood of making such an 
observation is 82% (i.e., power) if the true one-year relapse with the proposed regimen is 20%. 
Additionally, if the observed one-year relapse probability in this trial is lower than 40% and the 
treatment is not toxic, we will be sufficiently encouraged to consider this approach worthy of 
further study in a subsequent trial to address potential efficacy in preventing relapse more 
definitively. 
 


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As described above, the relapse rate will be examined and assessed to acquire insights into 
potential efficacy, but it is unlikely that formal conclusions will be drawn. The results will also be 
used to set benchmarks for potential future clinical trials aimed at improving anti-leukemic 
efficacy of T cell therapy. In addition to assessing prevention of relapse, information will be 
gathered that may be linked to efficacy after adoptive therapy for leukemia. This will include 
disease characteristics such as cytogenetics, level of expression of WT1 (and potentially other 
genetic markers), and other concurrent therapies the patient has or is receiving that may affect 
response (e.g. immuno-suppression). Although the small sample size will most likely preclude 
detecting statistically significant differences for such parameters, this information will be 
hypothesis generating and may again serve as a platform for designing future clinical trials. 
 
24.C. Analysis of Anti-leukemic Potential Efficacy (Arm 2) 
 
Relapse of AML and MDS after allogeneic HCT is typically characterized by progressive 
disease, and >75% of patients will die within one year of the diagnosis of relapse.127,128 Patients 
with relapsed AML or MDS (see Section 7.C for definition of relapse), who have no evidence of 
disease after T cell therapy, will be considered complete responders (see Protocol Appendix D 
for response criteria). These patients will be followed to determine the duration of response.  
Treatment options for relapsed AML or MDS after HCT, e.g. chemotherapy or DLI, are limited 
by poor response rates and non-durable responses with less than 3% two-year overall survival 
for patients who relapse within six months after HCT. Durable response can be attained in a few 
patients who undergo a second HCT, but a second HCT is associated with a treatment-related 
mortality of ~50%. Using 3% as the benchmark, two or more two-year survivors among 
20 treated patients (observed rate of 10% or more) would represent a statistically significant 
improvement over 3% (one-sided p-value of .03), and, if the true two-year overall survival (OS) 
with the proposed regimen is 20%, the likelihood of such an observation is 88% (i.e., power). 
Alternatively, the probability of observing a durable response in >20% of 20 patients (four or 
more responses among 20) is 0.13 if the true response rate is 0.10, 0.77 if the true response 
rate is 0.25, and 0.89 if the true response rate is 0.30. 
 
Patients who do not attain a durable complete remission will be evaluated for the nature of their 
response. Samples will be requested from all patients with detectable disease after T cell 
therapy. If expression of WT1 and/or HLA-A2 has decreased in purified leukemia cells isolated 
following T cell therapy, it will be considered suggestive evidence that the T cells exerted 
immunologic pressure and that the leukemia may have escaped recognition by WT1-specific 
T cells by down-regulating presentation of antigen. The ability of the infused transduced T cells 
to recognize WT1+ leukemia cells pre- and post-therapy will be assessed. Limited direct 
recognition of pre-treatment leukemia cells and/or escape due to decreased expression of 
HLA-A2 or WT1 will be considered presumptive evidence that the avidity of the transduced 
T cells may be too low, and that strategies to increase the recognition of leukemic cells by the 
transferred T cells (e.g. by transducing donor T cells with a synthetic higher affinity TCR), 
should be explored in subsequent clinical trials if safety from the current clinical trials proves 
acceptable. By contrast, if the safety data suggests this affinity TCR has reached the limits of 
acceptable toxicity, then this will be considered the maximal achievable efficacy with this 
approach. Patients with partial responses (PR) or stable disease (SD) (see Protocol 
Appendix D) will be considered as evidence that adoptive T cell therapy targeting WT1 is 
potentially effective and that strategies to increase the anti-leukemic activity of the transferred 
T cells, such as by increasing the number of infusions, cell doses, or affinity may result in 
improved therapeutic efficacy. 
 


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As described above, the response rate in this trial will be examined and assessed for insights 
into therapeutic promise. In addition to assessing responses to therapy, information will be 
gathered that may be linked to disease response after adoptive therapy for leukemia. This will 
include characterizing disease burden and characteristics at time of therapy, time of relapse 
after HCT, timing of adoptive immunotherapy, level of expression of WT1 (and potentially other 
genetic markers) by the leukemia, and other concurrent therapies the patient has received or is 
receiving that may affect response (e.g. immunosuppression). Although the small sample size 
will most likely preclude detecting significant statistical differences for such parameters, this 
information will be hypothesis generating and may serve as a platform for designing future 
clinical trials. 
 
25. TERMINATION OF THE STUDY 
 
The study will be stopped if any of the following events occur: 
 
2. All cohorts have been filled and all patients have completed treatment. 
3. Evidence is detected of RCL or leukemia due to insertional mutagenesis. 
4. 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. 
5. The PI reserves the right to terminate the study at any time. The FDA may also terminate 
the study. 
 


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Wood, Lorinda Soma, Xueyan Chen, Shelly Heimfeld, Michael C Jensen, Stanley R. Riddell, David G 
Maloney. Anti-CD19 Chimeric Antigen Receptor-Modified T Cell Therapy for B Cell Non-Hodgkin 
Lymphoma and Chronic Lymphocytic Leukemia: Fludarabine and Cyclophosphamide Lymphodepletion 
Improves In Vivo Expansion and Persistence of CAR-T Cells and Clinical Outcomes. Blood cancer 
journal. 2015;126:184. 
119. 
Dummer W, Ernst B, LeRoy E, Lee D, Surh C. Autologous regulation of naive T cell homeostasis within 
the T cell compartment. Journal of immunology (Baltimore, Md : 1950). 2001;166(4):2460-2468. 
120. 
Woo EY, Chu CS, Goletz TJ, et al. Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-
stage non-small cell lung cancer and late-stage ovarian cancer. Cancer research. 2001;61(12):4766-4772. 
121. 
Woo EY, Yeh H, Chu CS, et al. Cutting edge: Regulatory T cells from lung cancer patients directly inhibit 
autologous T cell proliferation. Journal of immunology (Baltimore, Md : 1950). 2002;168(9):4272-4276. 
122. 
Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative 
but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. 
Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 
2005;23(10):2346-2357. 
123. 
Shaffer BC, Le Luduec JB, Forlenza C, et al. Phase II Study of Haploidentical Natural Killer Cell Infusion 
for Treatment of Relapsed or Persistent Myeloid Malignancies Following Allogeneic Hematopoietic Cell 
Transplantation. Biology of blood and marrow transplantation : journal of the American Society for Blood 
and Marrow Transplantation. 2016;22(4):705-709. 
124. 
Gunturu KS, Meehan KR, Mackenzie TA, et al. Cytokine working group study of lymphodepleting 
chemotherapy, interleukin-2, and granulocyte-macrophage colony-stimulating factor in patients with 
metastatic melanoma: clinical outcomes and peripheral-blood cell recovery. Journal of clinical oncology : 
official journal of the American Society of Clinical Oncology. 2010;28(7):1196-1202. 
125. 
Walter RB, Pagel JM, Gooley TA, et al. Comparison of matched unrelated and matched related donor 
myeloablative hematopoietic cell transplantation for adults with acute myeloid leukemia in first remission. 
Leukemia.24(7):1276-1282. 
126. 
Gyurkocza B, Storb R, Storer BE, et al. Nonmyeloablative allogeneic hematopoietic cell transplantation in 
patients with acute myeloid leukemia. J Clin Oncol.28(17):2859-2867. 
127. 
Frassoni F, Barrett AJ, Granena A, et al. Relapse after allogeneic bone marrow transplantation for acute 
leukaemia: a survey by the E.B.M.T. of 117 cases. Br J Haematol. 1988;70(3):317-320. 
128. 
Mortimer J, Blinder MA, Schulman S, et al. Relapse of acute leukemia after marrow transplantation: 
natural history and results of subsequent therapy. J Clin Oncol. 1989;7(1):50-57. 
129. 
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. 2003;21(24):4642-4649. 
130. 
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. 2006;108(2):419-425. 
131. 
Kröger N, Bacher U, Bader P, et al. NCI First International Workshop on the Biology, Prevention, and 
Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation: Report from the 
Committee on Disease-Specific Methods and Strategies for Monitoring Relapse following Allogeneic Stem 
Cell Transplantation. Part I: Methods, Acute Leukemias, and Myelodysplastic Syndromes. Biology of 
Blood and Marrow Transplantation.16(9):1187-1211. 


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132. 
Hughes T, Deininger M, Hochhaus A, et al. Monitoring CML patients responding to treatment with 
tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting 
BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood. 2006;108(1):28-37. 
133. 
Hughes TP, Hochhaus A, Branford S, et al. Long-term prognostic significance of early molecular response 
to imatinib in newly diagnosed chronic myeloid leukemia: an analysis from the International Randomized 
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134. 
Talpaz M, Shah NP, Kantarjian H, et al. Dasatinib in imatinib-resistant Philadelphia chromosome-positive 
leukemias. N Engl J Med. 2006;354(24):2531-2541. 
135. 
Radich JP. How I monitor residual disease in chronic myeloid leukemia. Blood. 2009;114(16):3376-3381. 
 
 


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27. APPENDICES 
 
APPENDIX A 
 
Staging and grading of acute GVHD* 
Staging 
 
Skin  1  
Rash on <25% of skin 
2  
Rash on 25-50% of skin  
3  
Rash on >50% of skin 
4  
Generalized erythroderma with bullous formation  
 
Liver  1  
Bilirubin 2.0-3.0 mg/dl a 
2  
Bilirubin 3.1-6.0 mg/dl  
3  
Bilirubin 6.1-15.0 mg/dl  
4  
Bilirubin > 15.0 mg/dl  
 
 
 
Gut 
1  
Diarrhea > 500 mL /day or >280 mL/m 2   b or persistent nausea c 
2  
Diarrhea >1000 mL /day or >555 mL/m 2    
3  
Diarrhea >1500 mL /day or >833 mL/m 2    
4  
Severe abdominal pain with or without ileus 
 
 
Grade d 
 
Skin 
 
 
Liver 
 
 
Gut 
I 
 
Stage 1-2 
 
None  
 
None 
II 
 
Stage 3 or 
 
Stage 1 or 
 
Stage 1 
III 
 
- 
 
 
Stage 2-3 or  
Stage 2-4 
IV e 
 
Stage 4 
 
Stage 4 
 
- 
 
 
 
 
_______________ 
* Przepiorka et al. Bone Marrow Transplantation 15:825-8, 1995 
a Range given as total Bilirubin. Downgrade one stage if an additional cause of elevated bilirubin 
has been documented   
b For pediatric patients, the volume of diarrhea should be based on body surface area.  
Downgrade one stage if an additional cause of diarrhea has been documented.   
c Persistent nausea with histologic evidence of GVHD in the stomach or duodenum 
d Criteria for grading given as minimum degree of organ involvement required to confer that 
grade 
e Grade IV may also include lesser organ involvement but with extreme decrease in 
performance status 
 
 


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              Page 75 
APPENDIX B 
Chronic GVHD-scoring 
Mild chronic GVHD involves only 1 or 2 organs or sites (except the lung: see below), with no 
clinically significant functional impairment (maximum of score 1 in all affected organs or sites). 
Moderate chronic GVHD involves (1) at least 1 organ or site with clinically significant but no 
major disability (maximum score of 2 in any affected organ or site) or (2) 3 or more organs or 
sites with no clinically significant functional impairment (maximum score of 1 in all affected 
organs or sites). A lung score of 1 will also be considered moderate chronic GVHD. Severe 
chronic GVHD indicates major disability caused by chronic GVHD (score of 3 in any organ or 
site). A lung score of 2 or greater will also be considered severe chronic GVHD. 


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Organ scoring of chronic GVHD. *AP may be elevated in growing children, and not reflective 
of liver dysfunction. †Pulmonary scoring should be performed using both the symptom and 
pulmonary function testing (PFT) scale whenever possible. When discrepancy exists between 
pulmonary symptom or PFT scores the higher value should be used for final scoring. Scoring 
using the Lung Function Score (LFS) is preferred, but if DLCO is not available, grading using 
FEV1 should be used. The LFS is a global assessment of lung function after the diagnosis of 
bronchiolitis obliterans has already been established [29]. The percent predicted FEV1 and 
DLCO (adjusted for hematocrit but not alveolar volume) should be converted to a numeric score 
as follows:>80% =1; 70-79% = 2; 60-69% = 3; 50-59% = 4; 40-49% = 5; <40% =6. The LFS _ 
FEV1 score _ DLCO score, with a possible range of 2-12. GVHD indicates graft versus host 
disease; ECOG, Eastern Cooperative Oncology Group; KPS, Karnofsky Performance Status; 
LPS, Lansky Performance Status; BSA, body surface area; ADL, activities of daily living; LFTs, 
liver function tests; AP, alkaline phosphatase; ALT, alanine  aminotransferase; AST, aspartate 
aminotransferase; ULN, upper limit of normal. 
 


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From: Filipovich AH, et. al:Biology of  Blood and Marrow Transplant. 2005 Dec;11(12):945-
56.National Institutes of Health consensus development project on criteria for clinical trials in 
chronic graft-versus-host disease: I. Diagnosis and staging working group report.   


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              Page 78 
APPENDIX C 
Donor Criteria for Leukapheresis 
 
Clinical parameter 
Criterion for healthy donors 
Donor Questionnaire for Allogeneic Apheresis 
Product intended for infusion in recipient  
Required—donors deemed suitable for 
apheresis for therapeutic purposes will be 
eligible for research apheresis under this 
protocol 
Virology Testing 
Required  
Blood Pressure 
Systolic: 90-180 
Diastolic: 50-100 
Pulse 
50-110 and Regular 
Weight 
≥ 50 Kg 
Temperature 
≤ 37.5C (99.5F) 
WBC 
≥ 3,000 
HCT 
Men: ≥ 35% 
Women: ≥ 34% 
Platelets 
>130,000 
Cardiovascular complications during 
therapeutic apheresis specifically, any chest 
pain, clinical evidence of cardiac ischemia, or 
significant arrhythmia 
No cardiovascular complications unless 
deemed safe by cardiovascular consult 
service after appropriate medical 
management. 
 
  
 


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APPENDIX D 
Response criteria: 
 
Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) 129-131 
 
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/µl  
Absolute neutrophil 
count >1000/µl  
(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 blasts is ≤ 5%. 
Otherwise follow 
morphologic criteria.  
See above 
 
 
Chronic Myeloid Leukemia (CML) 132-135 
  
1) Patients with CML beyond chronic phase at the beginning of T cell therapy: 
Hematologic response: Complete Hematologic Response (CHR): WBC <10,000/µl and platelet 
count <450,000/µl maintained for at least 4 weeks.  
  
2) Patients with CML in chronic phase. It is considered a response when patients reach a 
disease category indicating lower disease burden (example minimal response reaching the level 


FHCRC 2498.00 
 
 
 
              Page 80 
of minor response after T cell therapy). Complete response is considered attaining complete 
molecular response (CMR): 
  
Disease categories: 
1. >96% Ph+ cells (Full cytogenetic relapse)  
2. 66-95% Ph+ cells 
3. 36-65% Ph+ cells 
4. 1-35% Ph+ cells 
5. No Ph+ cells but > MMR (BCR-ABL>0.1% on the international scale)  
6. No BCR-ABL transcripts by quantitative PCR (CMR). 
  
3) Response in all CML patients receiving T cell therapy will also be assessed by quantitation of 
BCR-ABL transcripts by quantitative PCR in peripheral blood 
 
 
 


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APPENDIX E 
 
Potential Adverse Events Associated or Expected with Hematopoietic Cell Transplantation 
 
1. Opportunistic infections, including viral and fungal infections, can results in severe pulmonary, 
neurologic, hepatic and other organ dysfunction, and possible death. 
 
2. Gastrointestinal toxicity. Nausea and vomiting can be anticipated during the entire course of 
ablative therapy. Mucositis and diarrhea should be expected. Prednisone can cause GI 
bleeding. 
 
3. Cardiac toxicity. Cardiotoxicity (congestive heart failure, pericardial effusion, EKG changes) is 
uncommonly associated with chemotherapy agents and TBI and these sequelae may prove 
lethal. 
 
4. Pulmonary toxicity. Diffuse interstitial pneumonitis of unknown etiology occurs with some 
regularity after BMT and interstitial fibrosis occurs much more rarely. Both are well-described 
complications of intensive chemotherapy and TBI regimens and may prove lethal. 
 
5. Hepatic toxicity. Veno-occlusive disease of the liver is a common toxicity of high-dose 
chemoradiotherapy and may result in death. Tacrolimus may cause elevation of ALT/AST. 
 
6. Renal dysfunction. Chemoradiotherapy may uncommonly cause renal dysfunction. More 
commonly, nephrotoxicity results from tacrolimus and generally responds to dose reduction. 
Rarely, idiopathic or calcineurin inhibitor-associated hemolytic-uremic syndrome may occur and 
may be progressive and fatal. A syndrome of moderate renal insufficiency and hemolysis has 
been seen 5-7 months post HSCT after intensive conditioning plus TBI. 
 
7. Hemorrhagic cystitis, manifested either as gross or microscopic hematuria, is a common 
toxicity after high-dose chemoradiotherapy, but usually associated with regimens that include 
cyclophosphamide. Hemorrhagic cystitis may predispose to a long-term increased risk of 
bladder cancer. 
 
8. Central nervous system toxicity. Radiation and chemotherapy can cause CNS toxicity, 
including seizures, depressed mental status, or leukoencephalopathy. Calcineurin inhibitors can 
cause seizures or other CNS toxicity. 
 
9. Marrow aplasia. Severe neutropenia, thrombocytopenia, and anemia, is expected to occur for 
a period of 7 to 42 days following infusion of marrow. Transfusion of platelets and red blood 
cells is expected as supportive care. Transfusion of blood products may be associated with 
acquisition of HIV or a hepatitis virus. Neutropenia may increase the risk for acquiring serious 
infection. Thrombocytopenia may increase the risk of life-threatening hemorrhage. Hemorrhagic 
or infectious complications during the expected period of aplasia may result in death. 
 
10. Miscellaneous. Alopecia and sterility are expected complications of the program as a whole. 
Cataract development is possible after TBI and/or steroids. Deficiencies of growth hormone, 
thyroid hormone, and sex hormones are possible after TBI. Calcineurin inhibitors can cause 
transient gingival hyperplasia, tremor, seizure, hypertension, headache, dysesthesia and 
hirsutism. Steroid therapy can also contribute to fluid retention, easy bruising, hypertension, 


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              Page 82 
aseptic necrosis of bone and increased susceptibility to infection. Hospitalization during 
conditioning and recovery period is expected to be 5-9 weeks in duration. 
 
 


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              Page 83 
APPENDIX F 
 
Arm 2 
(new cohort) 
Event 
History  
and PE8 
Chemistry  
Battery/ 
Urinalysis 
CBC w/ 
differential 
Recipient 
Battery  
GVHD 
Assessment1 
HLA*A 0201  
WT1 expression 
EBV Serology3 
Research Blood9 
10mL EDTA Purple 
60mL ACD Yellow 
Bone Marrow Aspirate2,9 
2mL EDTA Purple 
5mL ACD Yellow 
Screening 
Evaluation for study 
participation 
 X 
X  
X  
X 
X  
X 
  
  
Leukapheresis 
Donor leukapheresis 
  
  
  
 X11 
  
  
  
  
Re-induction 
  
Day -76 
Evaluate count 
recovery 
  
  
X  
  
  
  
  
  
Lympho-
depletion16 
Day -4 
Cyclophosphamide 
300mg/m2 
Fludarabine 30mg/m2 
  
  
  
  
  
  
  
  
Day -3 
  
  
  
  
  
  
  
  
Day -2 
  
  
  
  
  
  
  
  
Pre-Treatment4,5  
Treatment Eligibility 
X 
X 
X 
 
X 
  
X 
X7 
Week 0 
 (Infusion 1) 
Day 0 
T cell infusion (1x1010 
cells/m2) 
 X 
X  
X  
 
  
  
X 
  
Day +1 
Evaluations post T cell 
infusion 
  
X  
X  
X  
  
  
  
X 
  
Day +4 
  
X  
X  
  
  
  
X  
  
Week 1 
Day +7 
 X 
X  
X  
  
  
  
X  
  
Week 2 
Day +14 
 X 
X  
X  
  
  
  
X  
  
Week 3 
Day +21 
 X 
X  
X  
  
  
  
X  
  
Week 4 
Day +28 
 X 
X  
X  
  
  
  
X  
  
Week 810 
Day +56 
 X 
X  
X  
  
  
  
X  
X7, 13  
If second T cell infusion is delayed beyond 8 weeks, an interim H&P is to be completed at 3 months and every 3 months until second infusion or taken off study 
Day -76 
Evaluate count 
sufficiency 
  
  
X  
  
  
  
  
  
Pre-Treatment4,5  
Treatment Eligibility 
X 
X 
X 
 
X 
  
X 
X7 
Week 0 
 (Infusion 2) 
Day 0 
 
T cell infusion (1x1010 
cells/m2) 
  
IL-2 Administration 
 X 
X  
X  
 
  
  
X 
 


FHCRC 2498.00 
 
 
 
              Page 84 
 
1) 
Provider to complete study-specific GVHD Assessment Form (15.G.3; Appendix B) 
2) 
To be collected at every clinical bone marrow procedure (15.H); 10mL EDTA purple top whole blood is to be collected along with bone marrow aspirate research samples 
3) 
EBV serology is to be assessed for the final 5 patients on Arm 2 (new cohort) 
4) 
Must be performed < 30 days of T cell infusion (15.C) 
5) 
Chest X-ray and pulmonary function tests are to be completed if clinically indicated 
6) 
Serum or urine pregnancy test to be performed < 14 days of T cell infusion 
7) 
Must be performed < 21 days of T cell infusion and as close as possible to day 0 (15.H.2.a) 
8) 
Karnofsky or Lansky performance status to be recorded at each clinic visit 
9) 
As defined in section 15.F.1.d and 15.F.1.e; refer to section 15.F.2 for patients 30-50 kg and 15.F.3 for patients 15-30kg 
10) If T cell infusion is delayed >8 weeks, efforts will be made to obtain an interim H&P at 8 weeks, 3 months, then every 3 months until second infusion is administered or 
patient is taken off study (15.G.1.a.i). 
11)  Must be performed < 30 days prior to leukapheresis 
12)  To be collected 3-5 days after last dose of IL-2 (15.H.2.a) 
13) An additional “interim” restaging bone marrow may be requested if second infusion is delayed >4 weeks 
14)  To be performed q1 year for 15 years (15.I.2) 
15) First dose of IL-2 is to be administered 2-4 hours after second T cell infusion (14.B) 
16) Standard administration guidelines are to be followed for cyclophosphamide and fludarabine (14.B.d.1) 
 
 250,000 U/m2 BID for 
14 days 
 
   
Arm 2 
(new cohort) 
Event 
History  
and PE8 
Chemistry  
Battery/ 
Urinalysis 
CBC w/ 
differential 
Recipient 
Battery  
GVHD 
Assessment1 
HLA*A 0201  
WT1 expression 
EBV Serology3 
Research Blood9 
10mL EDTA Purple 
60mL ACD Yellow 
Bone Marrow Aspirate2,9 
2mL EDTA Purple 
5mL ACD Yellow 
 
Day +1 
IL-2 Administration 
 250,000 U/m2 BID for 
14 days15 
 
X  
X  
X  
  
  
  
X 
  
Week 0 
(Infusion 2)  
Day +4 
  
  
  
  
  
  
X  
  
Week 1 
Day +7 
 X 
X  
X  
  
  
  
X  
  
Week 2 
Day +14 
X  
X  
X  
  
  
  
X  
 
Week 3 
Day +21 
Evaluations post-T cell 
infusion 
X  
X  
X  
  
  
  
X  
X  
Week 4 
Day +28 
X  
X  
X  
  
 X 
  
X  
X  
Week 6 
Day +42 
X  
X  
X  
  
  
  
X  
X  
Week 8  
(month 2) 
Day +56 
X  
X  
X  
  
  
  
X  
X  
Week 12 
(month 3) 
Day +84 
X  
X  
X  
  
  
  
X  
X  
Week 24 
(month 6) 
Day +168  
X  
X  
X  
  
  
  
X  
  
 Year 1 
Long-Term Follow-Up14 
 X 
  
  
  
  
  
X