Clinical trial NCT00562640
Conditions: Fallopian Tube Cancer Ovarian Cancer Primary Peritoneal Cavity Cancer
Keywords: recurrent ovarian epithelial cancer stage IV ovarian epithelial cancer recurrent primary peritoneal cavity cancer stage IIIA primary peritoneal cavity cancer stage IIIB primary peritoneal cavity cancer stage IIIC primary peritoneal cavity cancer stage IV primary peritoneal cavity cancer recurrent fallopian tube cancer stage IIIA fallopian tube cancer stage IIIB fallopian tube cancer stage IIIC fallopian tube cancer stage IV fallopian tube cancer stage IIIA ovarian epithelial cancer stage IIIB ovarian epithelial cancer stage IIIC ovarian epithelial cancer 06-155
Sponsor: Memorial Sloan Kettering Cancer Center
Start date: 2007-10-16; Completion date: 2021-08-03
Enrollment: 12 participants; Allocation: NA; Intervention model: SEQUENTIAL

Brief summary:
RATIONALE: Giving colony-stimulating factors, such as G-CSF, helps stem cells move from the bone marrow to the blood so they can be collected. Treating stem cells collected from the patient's blood in the laboratory may increase the number of immune cells that can mount an immune response against the tumor. The treated stem cells may help destroy any remaining tumor cells (graft-versus-tumor effect). Chemotherapy may also be given to the patient to prepare the bone marrow for the stem cell transplant.

PURPOSE: This phase I trial is studying the side effects and best dose of autologous T cells when given with or without cyclophosphamide and fludarabine in treating patients with recurrent or persistent advanced ovarian epithelial cancer, primary peritoneal cavity cancer, or fallopian tube cancer. (fludarabine treatment closed as of 12/012009)

Detailed description:
OBJECTIVES:

* To assess the safety and tolerability of in vitro expanded autologous WT1 specific T cells, when administered alone or in combination with non-myeloablative, immunosuppressive conditioning, in patients with recurrent or persistent, advanced, WT1-positive, ovarian epithelial cancer, primary peritoneal cavity cancer, or fallopian tube cancer.
* To determine the maximum tolerated dose of autologous WT1 specific T cells in these patients.
* To quantitate alterations in the concentration of WT1 specific T cells in the blood at defined intervals post infusion with or without non-myeloablative, immunosuppressive conditioning in order to gain estimates regarding their survival and proliferation.
* To assess the effects of the adoptively transferred T cells on the growth and progression of advanced ovarian epithelial cancer, primary peritoneal cavity cancer, or fallopian tube cancer.

OUTLINE: This is a dose-escalation study of WT1 peptide-specific T cells.

* T-cell generation and isolation: Patients undergo collection of peripheral blood stem cells (PBMC) from which T cells are purified, stimulated in vitro with WT1 peptide-pulsed autologous EBV BLCL, and expanded ex vivo.
* Stem cell mobilization and harvest: Patients receive filgrastim (G-CSF) subcutaneously daily for five days. PBMC are collected by leukapheresis on the fifth day and then cryopreserved for subsequent reinfusion into the patient, in the event of prolonged cytopenia.
* Autologous T-cell infusion with or without conditioning chemotherapy ( fludarabine treatment closed as of 12/01/2009): Approximately 4-6 weeks after T-cell sensitization, patients receive an infusion of autologous WT1-specific T cells over 5-10 minutes on day 0. Patients enrolled in dose levels II and III also undergo pre-infusion lymphodepletive conditioning comprising cyclophosphamide IV on day -2 and fludarabine phosphate IV over approximately 30 minutes on days -6 to -2. After a 48-hour rest period, patients receive autologous WT1-specific T cells. Treatment repeats every 14 days for up to 4 courses in the absence of disease progression or unacceptable toxicity. Patients with responsive or stable disease after completion of therapy, may receive additional courses of autologous WT1-specific T cells every 14 days.

Blood samples are obtained at baseline and periodically during study and assayed for alterations in circulating levels of WT1 peptide-specific T cells, for biochemical indices of tumor burden, and for radiologic evidence of tumor response. Serum CA125 levels are measured and the number of T cells generating interferon gamma in response to autologous EBV BLCL is quantitated.

After completion of study therapy, patients are followed for up to 12 weeks.

Arms: arm 1: This is a phase I dose escalating trial designed to identify tolerable, clinically active doses of Wilms' tumor gene (WT1) peptide sensitized T cells when administered alone or with nonmyelosuppressive chemotherapy in patients with recurrent or persistent, evaluable WT1+ ovarian, primary peritoneal, or fallopian tube carcinomas.
Interventions: intervention 1: filgrastim intervention 2: therapeutic autologous lymphocytes intervention 3: cyclophosphamide intervention 4: laboratory biomarker analysis
Intervention descriptions: intervention 1: Stem cell mobilization and harvest: Patients receive filgrastim (G-CSF) subcutaneously daily for five days. PBMC are collected by leukapheresis on the fifth day and then cryopreserved for subsequent reinfusion into the patient, in the event of prolonged cytopenia. intervention 2: Autologous T-cell infusion with or without conditioning chemotherapy ( fludarabine treatment closed as of 12/01/2009): Approximately 4-6 weeks after T-cell sensitization, patients receive an infusion of autologous WT1-specific T cells over 5-10 minutes on day 0. Patients enrolled in dose levels II and III also undergo pre-infusion lymphodepletive conditioning comprising cyclophosphamide IV on day -2 and fludarabine phosphate IV over approximately 30 minutes on days -6 to -2. After a 48-hour rest period, patients receive autologous WT1-specific T cells. Treatment repeats every 14 days for up to 4 courses in the absence of disease progression or unacceptable toxicity. Patients with responsive or stable disease after completion of therapy, may receive additional courses of autologous WT1-specific T cells every 14 days. intervention 3: Patients enrolled in dose levels II and III also undergo pre-infusion lymphodepletive conditioning comprising cyclophosphamide IV on day -2 intervention 4: Obtained prior to adoptive therapy to quantitate baseline levels of WT1 reactive T cells, by quantitation of WT1 specific CTLp by LDA, T cells secreting IFNγ in response to peptide and, in HLA A0201+ patients, T cell binding WT1 peptide HLA A2 tetramers.

Protocol documents:
Prot_SAP_000.pdf: 
Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
A Phase I Dose Escalation Safety and Feasibility Study of WT1-Specific T Cells for the 
Treatment of Patients with Advanced Ovarian, Primary Peritoneal, and Fallopian Tube 
Carcinomas 
MSKCC THERAPEUTIC/DIAGNOSTIC PROTOCOL 
 
Principal 
Investigator/Department: 
Roisin O’Cearbhaill, MB BCh 
Medicine/Gynecologic 
Medical Oncology 
Co-Principal 
Investigator(s)/Department: 
Richard O’Reilly, MD 
Pediatrics/Bone Marrow 
Transplant Service 
Investigator(s)/Department: 
Carol Aghajanian, MD 
Katherine Bell-McGuinn, MD, PhD 
Rachel N. Grisham, MD 
Martee Leigh Hensley, MD, MSc 
David Hyman, MD 
Jason A. Konner, MD 
Vicky Makker, MD 
Paul Sabbatini, MD 
David R.Spriggs, MD 
William P. Tew, MD 
Guenther Koehne, MD 
Howard Thaler, PhD 
Alexia Iasonos, PhD 
Svetlana Mironov, MD 
Eric Pamer, MD 
Robert Soslow, MD 
Ekaterina Doubrovina, MD, PhD 
Medicine/Gynecologic 
Medical Oncology 
Medicine/Bone Marrow 
Transplant Service 
Biostatistics 
Radiology 
Medicine/Infectious Diseases 
Services 
Pathology 
Pediatrics/Bone Marrow 
Transplant Service 
Consenting 
Professional(s)/Department: 
Roisin O’Cearbhaill, MB BCh 
Carol Aghajanian, MD 
Katherine Bell-McGuinn, MD, PhD 
Rachel N. Grisham, MD 
Martee Leigh Hensley, MD, MSc 
David Hyman, MD 
JasonA. Konner, MD 
Vicky Makker, MD 
Paul Sabbatini, MD 
Medicine/Gynecologic 
Medical Oncology 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
David R. Spriggs, MD 
William P. Tew, MD 
Guenther Koehne, MD 
Medicine/Bone Marrow 
Transplant Service 
 
 
Please Note: A Consenting Professional must have completed the mandatory Human Subjects 
Education and Certification Program. 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
 
 
IRB#: 06-155 A(10) 
 
Table of Contents 
 
THERAPEUTIC/DIAGNOSTIC PROTOCOL 
1.0 PROTOCOL SUMMARY AND/OR SCHEMA……………………………………… 2 
2.0 OBJECTIVES AND SCIENTIFIC AIMS .......................................................................7 
3.0 BACKGROUND AND RATIONALE ............................................................................7 
4.0 OVERVIEW OF STUDY DESIGN/INTERVENTION ..................................................30 
4.1 
DESIGN…………………………………………………………………………..  30 
4.2 
INTERVENTION………………………………………………………………… 30 
5.1 THERAPEUTIC/DIAGNOSTIC AGENTS ....................................................................31 
5.2 
WT1 SPECIFIC T LYMPHOCYTES…………………………………………….. 31 
5.2 
CYCLOPHOSPHAMIDE………………………………………………………..  31 
6.1 CRITERIA FOR SUBJECT ELIGIBILITY ....................................................................32 
6.2 
SUBJECT INCLUSION CRITERIA…………………………………………….. 32 
6.3 
SUBJECT EXCLUSION CRITERIA……………………………………………. 33 
7.0 RECRUITMENT PLAN ..................................................................................................34 
8.0 PRETREATMENT EVALUATION ...............................................................................34 
9.1 TREATMENT/INTERVENTION PLAN ........................................................................36 
9.2 
GENERATION/ISOLATION OF WT1 PEPTIDE SPECIFIC T CELLS……….  36 
9.3 
IMMUNOSUPPRESSION & TREATMENT W/ WT1 T CELLS………………. 38 
9.4 
DOSE ESCALATION & DOSE LIMITING TOXICITY……………………….. 38 
10.0 EVALUATION DURING TREATMENT/INTERVENTION ......................................39 
11.1 TOXICITIES/SIDE EFFECTS ......................................................................................43 
11.2 
RISKS OF WT1 T CELL INFUSIONS…………………………………… 43 
11.3 
RISKS OF CYCLOPHOSPHAMIDE……………………………………………. 44 
11.3 
RISKS OF LEUKAPHERESIS…………………………………………………..  45 
12.0 CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT ............45 
13.0 CRITERIA FOR REMOVAL FROM STUDY .............................................................48 
14.0 BIOSTATISTICS ...........................................................................................................49 
15.0 SUBJECT REGISTRATION AND RANDOMIZATION PROCEDURES ..................50 
15.1 
SUBJECT REGISTRATION………………………………………………… 50 
16.0 DATA MANAGEMENT ISSUES ....................................................................................50 
16.1 
QUALITY ASSURANCE…………………………………………………………51 
16.2 
DATA AND SAFETY MONITORING…………………………………………...51 
17.0 PROTECTION OF HUMAN SUBJECTS .................................................................... 52 
17.1 
PRIVACY………………………………………………………………………….52 
17.2 
SERIOUS ADVERSE EVENT REPORTING…………………………………….52 
18.0 INFORMED CONSENT PROCEDURES .................................................................... 54 
19.0 REFERENCES .............................................................................................................. 54 
20.0 APPENDICES 
APPENDIX A: …………………………………………………………………62 
APPENDIX B: …………………………………………………………………63 
APPENDIX C: ……………………………………………………………….  64 
APPENDIX D:  Generation of WT1-Specific Cytotoxic T-Lymphocytes …….65 
Amended: 10/09/12 


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Amended: 10/09/12 
 
 
APPENDIX E: Chemistry, Manufacturing and Control Information 
APPENDIX F: WT 1 Immunoreactive Score (IRS) of Archived Tumor Tissue 
APPENDIX G: Physical Examination and Toxicity Assessment Form 
APPENDIX H: Phone Assessment Form 


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IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
1.0       PROTOCOL SUMMARY AND/OR SCHEMA 
 
This is a phase I dose escalating trial designed to identify tolerable, clinically active doses of 
Wilms’ tumor gene (WT1) peptide sensitized T cells when administered alone or with non- 
myelosuppressive chemotherapy in patients with recurrent or persistent, evaluable WT1+ 
ovarian, primary peritoneal, or fallopian tube carcinomas. Patients eligible for this study have 
recurrent ovarian, primary peritoneal or fallopian tube cancer that expresses the WT1 antigen. In 
this phase I trial, eligible patients will be treated with autologous T cells sensitized with WT1 
peptides. Initially, one dose of the WT1 cells alone will be tested specifically for any toxicities 
as well as to determine the levels of WT1 specific T cells achieved in the blood and the 
persistence of these cells post infusion. In association with these studies, patients will be 
evaluated for any effects these T cells might have on their disease. Thereafter, certain patients 
will receive the T cells following lymphodepletion with cyclophosphamide. Previously, Dudley 
et al (1,54) described lymphoablative chemotherapy with cyclophosphamide and fludarabine 
which was then followed by T cell infusion. Childs et al also developed a similar lymphoablative 
regimen for conditioning renal carcinoma patients for allografts.(61) Many of the patients in our 
study will already have been heavily pretreated with chemotherapy and therefore have a reduced 
bone marrow reserve. We therefore, propose using lymphodepletive chemotherapy with single 
agent cyclophosphamide. The proposed treatment dose has been studied extensively at our 
institute in patients will CLL, and found to be well tolerated and safe.(104) Furthermore, 
cyclophosphamide is an active agent in ovarian cancer.(105) 
 
TREATMENT PLAN 
 
A. Initial Evaluation. Patients with ovarian, primary peritoneal, or fallopian tube carcinomas 
will be identified. Patients with these tumor types will be offered Informed Consent 1 that 
allows for immunohistochemical (IHC) testing of their banked tumor for WT1 expression. 
To be eligible, patients must have WT1 expressing tumors. Once WT1 expression has been 
verified, a leukapheresis will be performed on eligible patients to collect peripheral blood 
mononuclear cells to establish autologous Epstein Barr Virus (EBV) transformed B cell lines 
(BLCL) and for the generation of the autologous WT1-specific T cell lines. 
 
B. Pretreatment Evaluation. Patients will be offered Informed Consent 2 for treatment with 
autologous WT1-specific T cells if they meet the following criteria: (1) recurrent or 
persistent, evaluable ovarian, primary peritoneal or fallopian tube cancer, (2) autologous 
EBV BLCL established, (3) and WT1 tumor expression. Approximately eight to twelve 
weeks will elapse between Informed Consent 1 and 2, while patients’ banked tumor 
specimen will be tested for WT1 expression, as well as generation of EBV BLCL and 
autologous WT1-specific T cell lines. Patient EBV serology will be assessed at the time of 
leukapheresis. Autologous T cells will be sensitized in vitro to recognize WT1 with the 
patient’s autologous EBV B lymphoblastoid cell line (BLCL) or cytokine-activated 
monocytes (CAMs) pulsed with WT1 peptide(s). Autologous WT1-specific T cell 
populations will be generated in quantities sufficient for adoptive therapy. Blood samples 
will be obtained immediately prior to adoptive therapy to quantitate baseline levels of WT1 


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Amended: 10/09/12 
 
 
 
peptide reactive T cells, by quantitation of T cells secreting IFN in response to peptide, T 
cells binding WT1 peptide HLA tetramers, if applicable, and WT1 cytotoxic T lymphocyte 
precursors (CTLp) by limiting dilution analysis. 
 
C. Generation and Isolation of WT-1 Peptide-specific T cells for Adoptive Therapy. The 
patient’s EBV BLCL or CAMs, loaded with WT1 peptide(s), known to induce WT1-specific 
cytotoxic T cells in normal donors and in patients, will be used for sensitization of the 
patient’s T cells. Prior to use as sensitizers, the EBV BLCL are irradiated to 9,000 rads in a 
calibrated certified blood irradiator to eradicate the capacity of these cells to grow within co- 
cultures. In addition, EBV BLCL will be cultured in media containing acyclovir to eliminate 
the risk of transferring infectious EBV in the T cells transferred. After sensitization, T cells 
will be expanded with IL2, IL15 and OKT3 according to a modification of the technique of 
Dudley and Rosenberg using autologous WT-1 peptide-loaded EBV BLCL and autologous 
PBMC as irradiated feeders (1). The T cells will be assessed for WT-1 specific cytotoxic 
reactivity in standard Cr51 release assays. 
 
D. Treatment with WT1 Peptide-specific T cells. 
Once the requisite doses of T cells have been generated, the patients will undergo treatment 
with the T cells. The WT1 peptide-specific T cells will be administered intravenously. 
Patients will be treated in 4 sequential groups of 3-6 patients. In this phase I trial, dose 
escalation will be based on dose limiting toxicity (DLT). In cohort 1 patients will receive 
WT1 specific T cells dosed at 5 x 106/m2 (level I) without cyclophosphamide 
lymphodepletion. The first two patients in cohort 1 received only a single administration of 
WT1 peptide-specific T cells, whereas the third patient is to receive infusions every two 
weeks for four doses. The next three cohorts of patients (levels II, III, and IV) will undergo 
lymphodepletion first with cyclophosphamide (Cytoxan®, CTX) at 750mg/m2 administered 
intravenously, followed 48 hours later by treatment with an infusion of WT1 peptide 
sensitized autologous T cells. For patients receiving cyclophosphamide (levels II, III, and IV) 
only a single dose of cyclophosphamide will be administered 48 hours prior to the first 
infusion of T cells. No further doses of cyclophosphamide will be administered during the 
study. These patients will then receive additional infusions of T cells every 2 weeks for a 
total of 4 doses. These three dose levels represent administration of WT1 specific T cells at 
escalated doses (2 x 107/m2 [level II], 5 x 107/m2 [level III] and 1 x 108/m2 [level IV]). This 
is summarized in Table 1. Each cycle comprises of 28 days or 2 WT1 peptide-specific T cell 
infusions. 
 
All patients (apart from the first two patients on cohort I who have already been  treated) will 
receive WT1 peptide-specific T cell infusions once every two weeks for up to 4 doses (2 
cycles) as outlined in Table 7c. 
 
If 2 x 107/m2 T cells represents the DLT during treatment of patients on level II, the dose 
below the DLT, that i.e. 5 x 106/m2 T cells will be used for the next cohort of patients level 
III. If 5 x 107/m2 T cells represents the DLT during treatment of patients on level III, the dose 
below the DLT, that i.e. 2 x 107/m2  T cells will be used to complete protocol requirements 


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Amended: 10/09/12 
 
 
 
within the current cohort. It is therefore possible that the dose level: 1 x 108/m2 WT1 peptide 
sensitized autologous T cells may not be reached. 
 
One dose level of WT1–specific T cells alone and three dose levels of cyclophosphamide 
conditioning followed by WT1-specific T cells will be tested. Groups of three patients will 
be accrued sequentially as summarized in Table 1: 
 
Table 1: Dose Levels of T cells and Conditioning Chemotherapy 
Level 
Patients 
Cyclophosphamide 
WT1 Specific T cells 
Level I 
3-6 
NO 
5 x 106/m2 
Level II 
3-6 
Yes 
2 x 107/m2 
Level III 
3-6 
Yes 
5 x 107/m2 
Level IV 
3-6 
Yes 
1 x 108/m2 
In this phase I trial, dose escalation will be based on the DLT, defined as a grade 3-4 toxicity 
developing after infusion of the T cells as graded by the National Cancer Institute (NCI) 
Common Terminology Criteria for Adverse Events Scale (CTCAE) Version 4.0. Only 
toxicities that are possibly, probably, or definitely related to T cell infusions will be 
considered DLTs. Patients will be observed for DLTs for 2 weeks following each T-cell 
infusion and for 4 weeks following the last T-cell infusion. The first two patients treated on 
the study in cohort one received a single T cell infusion at Level I. All subsequent patients 
treated on study will receive up to four T cell infusions, each infusion being separated by 2 
weeks. Each patient will then be followed for four weeks after the last infusion. If no patients 
experience DLT during the 28-day period following the patient’s initial T cell infusion, we 
will proceed to the next dose level. If any patient at a given dose level is not evaluable 
through the first 28 days post the initial T cell infusion, the patient will be replaced such that 
three patients are evaluated prior to dose escalation to the next level. 
For patients receiving cyclophosphamide (Levels II,III, or IV), hematopoietic 
suppression will not be considered a DLT during the first 21 days post cyclophosphamide, 
since cytopenia is an expected toxicity of the pretreatment course of cyclophosphamide. 
However, severe grade 3 cytopenia extending beyond 21 days of the first 28-day cycle will 
be graded as a severe DLT. 
 
 
 
 
 
The dose escalation scheme for this study is as follows: 


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IRB Protocol 
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Amended: 10/09/12 
 
 
 
Dose Escalation and DLT 
 
1. If 0 of 3 patients experience DLT at a given dose level, then proceed to the next 
dose level. 
 
2. If 1 of 3 patients experience DLT at a given dose level, 3 additional patients will 
be treated at that level. If 0 of those 3 develop DLT, the dose will be further 
escalated. 
3. If 2 or more of the 6 patients experience DLT at a given dose level, the maximum 
tolerated dose (MTD) is defined as the previous dose level. 
 
4. If only 3 patients are treated at the MTD, an additional 3 patients will be treated at 
that level. If among these 6 patients, 2 or more experience DLT, the MTD will be 
lowered and the same criterion will be applied to the previous dose level. 
 
 
E. Additional WT1 specific T cell infusions 
Only a single infusion of WT1 peptide-specific T cells was administered to the first two 
patients at dose level I. Additional patients treated on dose level I (cohort 1) and patients 
treated on dose level II,III, or IV who have been observed for 2 weeks post infusion and who 
do not develop a grade 3 or greater toxicity (with the exception of grade 3 hematopoietic 
suppression during the first 21 days after the cyclophosphamide) may receive at least 4 
infusions of WT1 peptide sensitized T cells administered at two week intervals (i.e. on 
approximately days 1, 15, 29 and 43). If, after 4 infusions, the patient has a clinical response 
or at least stable disease, additional infusions of WT1 peptide sensitized T cells may continue 
to be administered once every 2 weeks until disease progression, DLT, withdrawal of consent 
or until the stock of the patient-specific WT1 peptide sensitized T cells has been exhausted. 
These additional infusions of WT1 peptide sensitized T cells will be administered at the same 
dose level as the patient’s initial infusion. 
 
F. Post-Treatment Evaluation. 
Patients will be evaluated clinically by vital signs pre-infusion and hourly for 6 hours 
post infusion of T cells. During cycle 1 patients will undergo physical examinations and 
toxicity assessments the day of and the day following each WT1 peptide-specific T cell 
infusion. A phone toxicity assessment will be performed on Day 3 post each T cell infusion. A 
+72 hour window to contact patient via phone is permissible. In addition, during cycle 1 
patients will also undergo physical examinations and toxicity assessments on the non- 
treatment weeks 2 and 4. 
 
Patients who undergo further WT1 peptide-specific T cell infusions will undergo physical 
examinations and toxicity assessments on the first day following each infusion and then at 
least once every two weeks thereafter. A phone toxicity assessment will be performed on Day 
3 post each T cell infusion. This schedule of assessment will continue until 8 weeks following 
the patient’s final T cell infusion. Patients will also have at least weekly complete blood 


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Amended: 10/09/12 
 
 
 
counts (CBCs) with differentials and comprehensive metabolic panels (CMPs) to monitor for 
renal or hepatic toxicity for the first four weeks and then every two weeks thereafter to 
complete 4 weeks following completion of the final T cell infusion. Finally, patients will 
undergo physical examinations, toxicity assessments and bloods as above at the 8-week safety 
follow-up visit (i.e. 8 weeks following the final T cell infusion). 
 
Subjects will have 2 safety follow-up visits approximately 4 to 8 weeks after final WT1 T 
cell infusion. In the case that a patient has an overall decline of health status, the follow-up 
safety visits will at the discretion of the investigator. 
 
Tumor responses will be monitored radiographically using RECIST criteria by computed 
tomography scans (CT scans) of the chest, abdomen and pelvis. A baseline CT scan will be 
performed. A second CT scan will be performed at approximately 8 weeks post the first 
infusion. Thereafter, CT scans will be performed approximately every 8 weeks if the patient 
continues on study. Patients requiring surgery for management of their disease any time after 
T cell infusion may have biopsies. If such biopsies are performed, samples will be obtained 
and tumors examined by immunohistology for T cell infiltration and for residual WT1+ tumor 
cells. Serum CA125 levels will be measured prior to the first infusion of WT1 specific T cells. 
For patients who continue to receive additional infusions of WT1 specific T cells, serum 
CA125 will be monitored prior to each WT1 specific T cell infusion, and if applicable at 4 
and 8 weeks after the final WT1 specific T cell infusion. CA125 levels will not be used to 
make treatment decisions, as progression will be assessed using RECIST criteria. 
 
In addition, patients will be tested for circulating levels and phenotype of T cells. All 
patients will have these levels assessed at baseline. The two patients treated with a single T 
cell infusion were assessed for T cells responding to WT1 peptide pool prior to the WT1 
specific T cell infusion, 24-hours post infusion and at 1, 2, 4 and 8 weeks post infusion, when 
applicable. For patients who will receive multiple T cell infusions, frequencies of WT1 
specific T cells will also be assessed prior to each WT1 specific T cell infusion and 24-hours 
post first and last infusion and at weeks 4 and 8 post the last infusion, when applicable. 
 
The frequencies of the WT1 and EBV specific CTLp will be assessed by limiting dilution 
assay (LDA). The proportion of T cells generating intracellular IFN in response to WT1 and 
autologous EBV BLCL (if applicable) will be assessed by FACS analysis. Concurrent 
quantitation of T cells binding tetramers presenting WT1 and EBV epitopes in the context of 
the HLA alleles expressed on the T cells will be performed whenever relevant tetramers are 
available, as a second marker of the persistence and expansion of adoptively transferred T 
cells as well as a marker of recovery of autologous immune function. 
 
Estimated time to completion: 48 months. 


Memorial Sloan-Kettering Cancer Center 
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IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
2.1 
OBJECTIVES AND SCIENTIFIC AIMS 
 
1) To assess the safety and tolerability of in vitro expanded autologous WT1 specific T cells, 
when administered alone or with non-myeloablative, immunosuppressive conditioning to 
patients with recurrent ovarian, primary peritoneal or fallopian tube cancer. 
 
2) To determine the maximum tolerated dose (MTD) of autologous WT1 specific T cells, 
when administered alone or with non-myeloablative, immunosuppressive conditioning. 
 
3) To quantitate alterations in the concentration of WT1 specific T cells in the blood at 
defined intervals post infusion with or without non-myeloablative, immunosuppressive 
conditioning in order to gain estimates regarding their survival and proliferation in these 
patients. 
 
4) To assess the effects of the adoptively transferred T cells on the growth and progression of 
advanced ovarian, primary peritoneal or fallopian tube cancer. 
 
3.1 
BACKGROUND AND RATIONALE 
Ovarian Cancer as a Target for Immunotherapy 
Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic 
malignancies in the United States. Over 23,000 new cases are diagnosed annually and an 
estimated 14,000 deaths each year result from EOC (2). Ovarian cancer is the fifth leading cause 
of cancer death in women in the United States. Furthermore, more than 70% of patients have 
stage III or IV disease at the time of diagnosis (3). 
The standard therapy for advanced ovarian cancer is surgical removal of as much 
malignant tissue as possible, followed by cytotoxic chemotherapy that includes a platinum- 
coordination complex and a paclitaxel derivative. Although a temporary response rate to 
chemotherapy of 70% is anticipated, ovarian cancer tends to recur, even in patients who achieve 
a complete response (4). Despite improvements in surgery and chemotherapy,  the  5-year 
survival rate remains at about 20-30% for advanced ovarian cancer (5). 
With current treatment approaches, the natural history of early stage ovarian cancer is 
also one of complete clinical remission, followed by relapse, with a return to remission in many 
patients. Although overall response rates of close to 80% are achieved in patients receiving 
platinum-based combination chemotherapy, only 47% of patients who are clinically free of 
disease will have no evidence of disease at second-look laparotomy (performed after initial 
chemotherapy) (6). Almost half of these patients will eventually recur with a mean interval of 24 
months from second-look surgery to recurrence, with 60% of these recurrences occurring in the 
peritoneal cavity (7). 
Given the high recurrence rate, new therapeutic approaches need to be developed for the 
treatment of advanced ovarian cancer, particularly tolerable approaches which could be 
introduced and sustained for targeted long-term eradication or control of minimal residual 
disease. Over the last decade, advances and clinical successes with the use of tumor-selective 


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monoclonal antibodies, such as rituximab in the treatment of lymphomas (8), CD33 specific 
antibodies in myeloid leukemias (9), the HER/neu-specific monoclonal antibody, Herceptin, in 
breast cancer (10), the GD-2-specific monoclonal 3F8 in neuroblastoma (11), and recently, the 
Ca125 specific monoclonal antibodies, Mab-B43.13 (Ovarex) and ACA 125, in the treatment 
of ovarian cancer (12-14), have demonstrated the potential of immunotherapies directed against 
antigens differentially expressed on tumor cells. 
Monoclonal antibody therapy has been successful in the treatment of ovarian cancer. 
Mab-B43.13, a monoclonal antibody therapy targeting the Ca125 antigen on ovarian cancer, has 
induced an immune response against both B43.13 and against Ca125 antigen that correlates with 
an improved prognosis. Recently, a randomized trial of low repetitive doses of mAb B43.13 
versus placebo in patients with a clinical remission (CR) after primary therapy for ovarian cancer 
revealed a prolonged time to treatment failure in the treated group. Three hundred and forty-five 
patients with stage III and IV ovarian cancer post chemotherapy were enrolled in the study. 
Progression-free-survivals for treated versus untreated patients were 13.4 and 4.7 months (p 
0.0001), respectively (12). In addition, a paper by Noujaim et al also revealed that patients 
treated with B43.13 had an improved survival and, strikingly, that responding patients appeared 
to have a T cell response to Ca125 antigen (see Figure 1) (13). 
 
Figure 1: Survival Curves of Ovarian Cancer Patients With and Without a T cell Response 
to Ca125 (13) 
 
 
 
 
A two-step study was conducted with ACA 125 in ovarian cancer patients and reported 
by Wagner et al (14). Initially, 18 patients were enrolled in the phase I portion, which was 
extended into a phase II study, with 42 patients treated in total. All patients had tumors that 
strongly expressed Ca125. Twenty-eight of 42 patients (67%) developed specific anti-anti 
idiotype antibodies (Ab3) during ACA 125 administration. The IgG subclass was predominantly 
IgG1 and IgG2. Cell mediated cytotoxicity from PBLs against Ca125 expressing and non- 
expressing human ovarian cancer cell lines in 18 patients were evaluated with an increase in cell 
kill in 9 of 18 patients from 19.6%  11.7% to 52.7%  13.6% at an effector:target ratio of 100:1. 
Cell mediated lysis was accompanied by the induction of Ab3 in 8 of 9 patients prompting only 


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humoral response evaluation in the remaining patients. Overall survival of all patients 
vaccinated with ACA 125 was 14.9  12.9 months; for patients with a positive response to ACA 
125 it was 19.9  13.1 months; and for patients with no detectable response 5.3  4.3 months (p 
0.0001). Once again, improved clinical outcome appeared to correlate with T cell responses to 
ACA 125 vaccination. 
Recently, considerable additional evidence has been generated supporting an important 
potential role for tumor reactive T cells in the targeted therapy of ovarian cancer. Early analyses 
of T-lymphocytes derived from ascites or tumor nodules (tumor infiltrating lymphocytes, TILs) 
of patients with ovarian cancer have documented the presence of cytotoxic T cell clones reactive 
against autologous tumor cells in a proportion of patients (15). Furthermore, the presence of T 
cells in the ovarian tumors has been associated with a significantly improved  disease-free 
survival (15). Early clinical trials exploring the clinical potential of autologous TILs expanded in 
vitro and adoptively transferred to patients with advanced disease also demonstrated clinical 
responses. However, such responses were observed in only a small subset of these patients (16). 
Consistent with this finding, characterization of the expanded TIL cells from these patients 
suggested that the cells generated were predominantly CD4+ T cells and that CD8+ T cells 
capable of lysing autologous tumor cells could be generated only from a minority of patients. 
 
Antigens Differentially Expressed by Ovarian Carcinomas Targetable by T cells 
In pursuit of better strategies to stimulate and sustain effective cytotoxic T cell responses 
against ovarian cancer, subsequent investigations of cell-mediated responses to ovarian cancer 
have focused on three areas: 
1) Identification of proteins differentially expressed by ovarian cancers in comparison 
with normal tissues 
2) Definition of immunogenic peptide epitopes derived from these proteins that could be 
used to elicit effective T cell responses 
3) Exploration of alternative sensitization strategies designed to preferentially stimulate 
the generation of tumoricidal T cells in vitro or in vivo 
 
Several genes are now recognized that encode proteins differentially expressed by 
ovarian carcinoma cells, particularly WT1, HER2/neu (27,28), and MUC1 (34), as well as 
CA125, Folate binding protein (FBP), MUC1 and MAGE3 (22-25). Recent studies also indicate 
that degradation of these proteins yields immunogenic epitopes which bind HLA class I alleles 
and can stimulate the generation of peptide specific, tumor cytotoxic CD8+ T cells (29-33). 
The most extensively studied of the immunogenic peptides derived from proteins 
differentially expressed by ovarian carcinoma are those of the HER2/neu protein. HER2/neu is 
over-expressed in 20-30% of patients with ovarian cancer but its expression is correlated with a 
poor prognosis (27,28). Five HLA-A2 binding peptides and an HLA-A24 binding peptide have 
been identified (29-33). Analysis of tumor associated T cells in HLA-A2+ patients with 
HER2/neu+ ovarian cancer also suggest that T cell responses to the E75 HER2/neu peptides are 
immunodominant (30). Based on these studies, Brossart et al performed a pilot vaccination 
study  with  HER2/neu  peptide-pulsed  autologous  dendritic  cells  (DC)  in  six  patients  with 


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advanced HER2/neu+ breast or ovarian cancer (34). Of these patients, two developed peptide- 
specific immune responses, detectable by quantitation of peptide stimulated T cells generating 
intracellular IFN- and T cells lysing peptide-loaded targets. One of these patients, with rapidly 
progressive ovarian carcinoma, experienced stabilization of disease for more than eight months 
post vaccination. 
The epithelial mucin, MUC1, is highly expressed in more than 90% of ovarian cancers, 
and, to a lesser extent, by normal epithelial cells of the gastrointestinal and genitourinary tracts 
(35). The MUC1 protein, conjugated to KLH, has been evaluated by P. Livingston et al at 
Memorial Sloan-Kettering Cancer Center (MSKCC) as part of a polyvalent vaccine in patients 
with ovarian carcinoma. This vaccine has elicited antigen-specific antibody responses in each of 
seven patients immunized. The MUC1 protein also contains at least  six  nonamer  peptide 
epitopes that bind HLA-A2 (n=6) or HLA-A11 (n=1). Brossart et al has used two of the high 
affinity HLA-A2 binding peptides, loaded on autologous DC to vaccinate 4 patients with 
advanced ovarian (n=2) or breast (n=2) (34). The vaccinations have induced peptide-specific 
cytotoxic responses in ¾ patients. Of the responders, one patient experienced transient 
stabilization of ovarian cancer. A second patient with breast cancer experienced regression of 
subcutaneous and visceral metastases and a striking reduction in circulating Ca125 levels, which 
persisted for over eight months.  Biopsies of tumor sites revealed infiltration of the tumor site 
with CD8+ and CD4+ T cells. 
A third protein highly expressed in a large proportion of ovarian cancer cells but not 
expressed in most adult normal tissues is the Wilms’ tumor gene product (WT1). WT1 was 
initially isolated as a mutated gene responsible for the childhood neoplasm, Wilms’ tumor (36- 
38). WT1 encodes a zinc finger transcription factor, which binds early growth factor gene 
promoters, such as platelet-derived growth factor A chain, colony stimulating factor-1, 
transforming growth factor-β1, and insulin-like growth factor II (37). Unlike tumor suppressor 
genes Rb and p53, which are expressed ubiquitously, the expression of the WT1 gene  is 
restricted to a limited number of normal tissues including fetal kidney, ovary, testis, spleen, 
hematopoietic precursors, and the mesothelial cell lining of visceral organs (38). After birth, 
expression of WT1 in these tissues is still detected but at markedly reduced levels (38). In 
contrast, WT1 is aberrantly expressed at higher levels in most adult leukemias including AML, 
ALL, CML and MDS and in several solid tumors including: mesothelioma, desmoplastic small 
round cell tumor, Wilms’ tumor, colon cancer, renal cell carcinoma, and non-small cell lung 
cancer (39). Importantly, human ovarian cancers also over-express WT1. Quenneville et al 
showed that WT1 is a sensitive and specific marker of serous, but not mucinous, ovarian 
carcinomas (24). We have performed a study of human ovarian tumor microarrays and we have 
determined that 64% of serous ovarian cancers express WT1 while other histological subtypes of 
ovarian cancer express the antigen to a lesser degree (40). 
The basis for the increased expression of WT1 in these tumor types is  unknown. 
Initially, WT1 was categorized as a tumor suppressor gene. Several studies by Haber et al 
demonstrated that activation of WT1 induces normal CD34+ CD38- early hematopoietic cells to 
enter a prolonged G0 phase while concomitantly inducing terminal differentiation of precursor 
cells later in their differentiation (90). However, it is now recognized that WT1 is expressed in 
several non-mutated isoforms, and that the isoform dominantly expressed in leukemias and 


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certain solid tumors differs from that expressed in normal precursor cells (91). Recent studies 
have also demonstrated that high expression of WT1 in AML, MDS and certain solid tumors is 
associated with poor prognosis (92-95). Furthermore, when WT1+ leukemic cells are treated 
with WT1 antisense oligonucleotides, they are consistently induced into apoptosis. These 
findings support the hypothesis that the isoform of WT1 dominant in these tumors is acting as an 
oncogene (96). 
Studies from investigators in Japan, England, and MSKCC (Confer Preclinical Studies 
Section), indicate that peptides derived from the WT1 protein are immunogenic in man (42- 
44,47). Ohminami et al and Oka et al were the first to identify peptides of WT1 which, when 
presented by HLA A2402 and HLA A0201, could elicit WT1 peptide specific T cell clones with 
in vitro leukemocidal activity (42,43). The immunogenic HLA A0201 restricted peptide 
nanomers from WT1 are: 1) 126-134RMFPNAPYL and 2) 187-195SLGEQQYSV.  The HLA A2402 
presented nanomers are: CMTWNQMNL235-243 and RWPSCQKKF417-425. Studies at MSKCC 
demonstrated that the two WT1 peptides presented by HLA A0201 elicited IFN+ cytotoxic WT1 
specific HLA A0201 restricted CD8+ T cells in each of 16 normal HLA A0201+ normal donors 
tested. Furthermore, the T cells generated consistently exhibited cytotoxic activity specific for 
WT1+ HLA A0201+ leukemias, including AML, ALL and CML blasts in vitro, and suppressed 
the growth of HLA A0201+ WT1+ leukemic xenografts in NOD/SCID mice in vivo (47). Studies 
of Gao et al have also shown that a T cell clone specific for one of these peptides was capable of 
selectively inhibiting the clonogenic activity of Ph+ CD34+ CML blasts in vitro but did not affect 
normal Ph- CD34 cells (44). Furthermore, when the T cell clone was co-administered with these 
Ph+ CD34+ blasts into SCID mice, leukemia development was prevented. These studies support 
the hypothesis that WT1 is expressed by clonogenic leukemic stem cells and that such cells are 
susceptible to WT1 specific cytotoxic T cell-mediated lysis. 
 
Scheibenbogen et al have demonstrated that patients with WT1+ malignancies may be 
able to generate T cell responses against these peptides (46). They have detected  T  cell 
responses in up to 30% of patients with AML in remission. Our own studies suggesting that 
WT1 specific T cells can be generated in vitro from a majority of women with ovarian carcinoma 
further support these findings (46). 
 
Ohno et al conducted a small phase II trial of 12 patients with WT1 HLA A2402 positive 
gynecological cancer. Patients received intradermal injections of HLA A2402-restricted 
modified 9-mer WT1 peptide every week for 12 weeks. The treatment was well tolerated with 
only local erythema at the injection site. Three of the patients had stable disease for at least a 3- 
month period. (106) 
 
 
 
 
Adoptive T cell Therapy 
 
Until recently, investigations of immunogenic peptides derived from tumor-associated 
proteins have focused on their use as vaccines, either as isolated peptides administered with 


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adjuvant or as peptides loaded on autologous dendritic cells isolated from the blood, for 
immunization of tumor bearing hosts. Unfortunately, responses to these vaccines have been 
observed in only a small fraction of patients. Furthermore, while cytotoxic T cell responses 
against peptide-loaded targets have been detected, these cells have exhibited limited activity 
against autologous tumor cells (51). These limitations have been ascribed to the types of T cells 
generated and the potential of tumor cells in the host to foster T cell tolerance to inhibit the 
tumoricidal activity of the T cell effectors through modulation of the expression of HLA alleles 
or co-stimulatory molecules, or the secretion of inhibitory cytokines such as IL-10, IL-6 or TGF- 
. 
An alternative approach is to adoptively transfer antigen-specific T cells sensitized and 
expanded in vitro, under conditions promoting the generation of a preponderance of cytotoxic 
CD8+ T cells and Th1 CD4+ T cells. This cellular immunotherapy, explored by our  own 
laboratory and by others, has demonstrated striking efficacy in the treatment of CMV and 
adenoviral infections as well as certain hematologic malignancies (particularly CML and virus- 
associated tumors such as marrow or organ allograft-associated EBV lymphomas) (52, 107). 
Unlike the TIL cells expanded after nonspecific activation with IL-2, these T cells have been 
sensitized with antigenic extracts or immunogenic peptides loaded on antigen-presenting cells 
(APCs) expressing required co-stimulatory molecules, such as isolated dendritic cells or EBV 
transformed cells, or APCs transduced to express the relevant antigenic peptides. When T cells 
are sensitized with such professional APCs loaded with peptides or transduced to express a viral 
or tumor protein, the T cells generated are enriched for antigen-reactive CD8+ T cells which 
exhibit strikingly greater cytotoxic activity against autologous cancer cells (34,52-56). 
Clinical experience with the adoptive transfer of such T cell lines or clones is still very 
limited. However, in allogeneic marrow transplant recipients, adoptive transfer of donor-derived 
CMV-specific T cell clones or EBV-specific T cell lines has reduced or abrogated the incidence 
of CMV infection or EBV lymphoma post transplant (57-58). Studies at MSKCC and others 
have also demonstrated that adoptive transfer of as few as 105-106/kg EBV specific T cells can 
also induce durable regressions of multifocal monoclonal EBV+ lymphomas in both marrow and 
organ allograft recipients (59). Similarly, adoptive transfer of 106 T cells expanded from clones 
specific for an HLA-2 binding peptide of the minor alloantigen HA-1 induced remission of AML 
in a HA-1 disparate host who relapsed post transplant (97). Strikingly, in these allogeneic hosts, 
the transferred, donor-derived T cells have increased in frequency in the circulation following 
transfer, with peak frequencies detected at the initiation of tumor regression. Furthermore, such 
cells have persisted for years post transplant. 
In contrast to this experience with allogeneic donor derived adoptively transferred T 
cells, until recently, studies of adoptive transfer of large numbers (e.g. 1010 T cells) of autologous 
tumor associated antigen-specific T cell clones, expanded in vitro from tumor-bearing patients, 
and transferred back into these patients, have rarely recorded clinical effects. Furthermore, the 
highest frequencies achieved in the circulation have been immediately after infusion. Indeed, the 
persistence or survival of the transferred T cells has been measured at no more than 1-2 weeks 
(54). These findings may be due to one or more of the following: 1) T cells generated from 
tumor-bearing hosts against self antigens, such as gp100, differentially expressed on tumors are 
qualitatively different from those generated against even poorly immunogenic alloantigens like 
HA-1 or viral antigens such as CMV pp65 or EBNA3C; 2) T cells generated in vitro against 


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tumor-associated antigens from tumor-bearing hosts differ qualitatively from those generated 
from normal individuals; 3) autologous T cell lines and clones are often generated over extended 
periods in vitro, and may have a very limited proliferation potential after adoptive transfer; 4) 
autologous T cells reactive against such antigens in the tumor-bearing host may be subject to 
regulatory influences inducing apoptosis and clearance that are less effective or inoperative 
against allogeneic HLA matched cells; or 5) the space available for seeding the autologous 
lymphocytes in an otherwise untreated host may be limited when compared to that available to 
lymphocytes expanding in a lymphoablated and myeloablated marrow transplant recipient. 
 
 
Generation and Characterization of WT1 Peptide Reactive T cells From Donors Inheriting 
HLA Alleles Other Than HLA A0201 or HLA A2402 
 
While T cells specific for WT1 peptides presented by HLA A0201 or HLA A2402 can be 
regularly generated and demonstrate activity against WT1+ leukemias both in vitro and in vivo, 
only 40% of our transplant patients inherit these alleles. To broaden the applicability of adoptive 
therapies specific for this oncofetal protein that is differentially expressed in leukemia cells, we 
have recently explored the use of autologous antigen-presenting cells loaded with a pool of 
overlapping pentadecapeptides (15-mers) spanning the sequence of WT1 for sensitization of T 
cells from individuals inheriting HLA alleles other than HLA A0201 or A2402. This approach is 
based on our recently reported success with this approach in generating T cells specific for 
CMV-pp65 (101). The use of a pool of 15-mers, each overlapping the next by 11 amino acids to 
span the WT1 sequence, has several potential advantages. First, the pool includes the full array 
of epitopes of WT1 including epitopes that can be bound and presented by both HLA class I and 
class II alleles permitting generation of both CD4 helper and CD8 cytotoxic T cells. Secondly, 
the 15-mers do not require intracellular processing, but bind directly to the HLA alleles and are 
then edited to appropriate size (9-mers for class I; 11-12-mers for class II) by ectopeptidases 
(102,103). Third, the use of the pool of 15-mers permits the donor T cells to respond to those 
epitopes that they have recognized as most immunogenic in prior exposures. In our studies of T 
cell responses to pentadecapeptides of the CMV-pp65 protein, this in vitro selection has lead to 
the consistent generation of T cells from CMV seropositive donors that are capable of lysing 
autologous targets infected with CMV virus. Accordingly, we have developed a pool of 141 
overlapping 15-mers spanning the sequence of WT1. These pentadecapeptides are both 95% 
pure and microbiologically sterile. We have subsequently used this pool of peptides, loaded on 
autologous dendritic cells, to sensitize T cells from a series of 15 normal donors inheriting other 
HLA alleles. After 35-42 days of expansion, these T cells specifically generate IFNγ in response 
to the pool of peptides and usually to one or several dominant peptides in the pool, which we 
have identified by an epitope mapping strategy based on secondary responses of these total pool- 
sensitized T cells against a matrix of 15-mer subpools. In the example presented in Appendix A, 
the T cells of the donor sensitized with the pool generated in response to subpools 10 and 16, 
suggesting reactivity against pentadecapeptide 60. Subsequent testing (Appendix B-1) 
confirmed this reactivity. Thereafter, pentadecapeptide 60 was loaded onto a panel of EBV 
transformed B cells sharing single HLA alleles with the donor and used as targets of the WT- 
pool reactive T cells.   In the example shown in Appendix B-2, the T cells recognized penta- 


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decapeptide 60 only when presented by cells expressing HLA B3501. The T cells also exhibited 
HLA B3501 restricted cytotoxic activity against B3501+ acute lymphoblastic leukemia co- 
expressing WT1. The specific nonamer epitope within the pentadecapeptide recognized was 
subsequently identified as HSFKHEDPM300-308. Using the same strategy, we have been able to 
generate WT1 specific CD8 and/or CD4+ T cells from 13 of 15 normal donors. These T cells 
only respond to WT1 peptides; they have no reactivity against autologous or HLA-partially 
matched WT1- cells (Appendix C-1). The epitopes recognized have been mapped and their 
restricting class I and class II HLA alleles identified. Most importantly, the T cells generated 
from 12/13 cases after sensitization with the pool of WT1 peptides, have exhibited peptide- 
specific, appropriately HLA-restricted cytotoxic activity against leukemia cells expressing WT1 
and the restricting HLA allele (Appendix C-2). 
 
Lymphodepletion to Foster Expansion and Persistence of Adoptively Transferred T cells 
 
In order to address possibilities 4 and 5 above, Dudley et al recently conducted trials in 
which melanoma patients were treated with a lymphoablative chemotherapeutic regimen 
consisting of cyclophosphamide 60 mg/kg/day x 2 days followed by fludarabine 25 mg/m2/day x 
5 days(1,54). Thereafter, patients were infused with 1010 of either extensively expanded clones 
of CD8+ T cells specific for either tyrosinase or MART1, or tumor infiltrating T cells expanded 
for a much shorter period by stimulation with a mitogenic antibody OKT3 and IL2, each at doses 
of 1010 cells per patient (1,54). Following adoptive transfer of the cloned CD8+ T cells, the T 
cells persisted for only two weeks, and never achieved levels higher than the peak levels detected 
immediately after infusion (54). Not surprisingly, clinical responses were not observed (54). 
However, in striking contrast to this experience with cloned T cells and all prior studies of 
adoptively transferred TIL cells, infusion of TIL cells into these lymphoablated patients resulted 
in a striking expansion of the cells within the patients (99). Persistence of these T cells for 
periods of at least four months and, in certain patients, up to 16 months was observed (89, 99). 
More importantly, adoptive transfer of TILs after lymphoablation has been associated with a 
higher tumor response rate. In the original report of Dudley et al, 6/13 patients achieved partial 
responses (PRs) extending from 2-24 months (1). In their updated series of 35 patients, 15 
patients had achieved PRs with a mean duration of 11.5 +/- 2.2 months (54). 
 
In this study, eligible patients include those with recurrent ovarian, primary peritoneal or 
fallopian tube cancer that expresses the WT1 antigen. In this phase I trial, these patients will be 
treated with autologous T cells sensitized with WT1 peptides. Initially, WT1 cells will be tested 
alone, specifically to test for any toxicities that such infusions might induce as well as to 
determine the levels of WT1 specific T cells achieved in the blood and the persistence of these 
cells post infusion. In association with these studies, these patients will be evaluated for any 
effects these T cells might have on their disease. Thereafter, two escalated dose levels will be 
tested in patients who have previously received lymphodepletion with cyclophosphamide. Prior 
experience with the proposed cyclophosphamide dose is extensive and indicates that it is well- 
tolerated and effective in lymphodepletion of the host.(104) As described above, this approach 
has permitted expression and long-term survival of the transferred T cells in tumor bearing hosts. 
In part, this may be due to elimination of regulatory T cells and NK cells that the body maintains 


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to suppress T cell clones reactive against a “self antigen” such as WT1. However, 
lymphodepletion may also provide space for expansion of these T cells in a largely evacuated 
niche within the lymphoreticular system. Furthermore, as these T cells undergo homeostatic 
proliferation to fill this niche, they become markedly less susceptible to the tolerogenic activity 
of regulatory cells and cytokines (54). Possibly, as a consequence of these alterations, the 
antigen-specific cytotoxic activity of the transferred cells may be sustained for prolonged 
periods, as has been observed in the patients treated with TIL cells by Dudley et al (1). Since 
WT1 expression, may be essential to the survival and growth of clonogenic tumor cells, the anti- 
tumor activity of the transferred cells may also be prolonged. Furthermore, cyclophosphamide is 
an active agent in ovarian cancer. (105) 
 
Potential Toxicities of WT1 Peptide Specific T cells Following Adoptive Transfer 
 
The short-term and long-term risks of adoptive therapy with autologous WT1 peptide- 
specific T cells are unknown but would be expected to involve those normal tissues which 
express WT1 at low levels in the post-natal period, namely, the kidneys, ovaries and testes and, 
possibly, the hematopoietic system, since normal CD34+ hematopoietic cells express low levels 
of WT1. Preclinical and initial trials with WT1 peptides used as vaccines, however, suggest 
toxicities to these organs will be acceptably low. In studies at MSKCC, the 16 T cell lines 
generated from normal donors against the HLA A0201 presented RMF and SLG peptides of 
WT1 have not induced significant lysis of CD34+ cells isolated from G-CSF mobilized 
peripheral blood stem cells or cord blood mononuclear cells to produce CFUc, BFUE or 
CFUGEMM colonies. Gao et al have also shown that while WT1 peptide-specific T cells lyse Ph+ 
leukemic CD34+ cells and inhibit their clonogenicity, they do not lyse Ph- CD34+ cells from 
normal donors and do not inhibit the growth of long-term colony initiating cells (LTCIC) which 
are considered the earliest clonogenic progenitors of the hematopoietic system that can be 
quantitated in vitro (44). In C57 mice, vaccination with the RMF WT1 peptide which is 
presented by H-2Db has induced high levels of WT1 specific T cells capable of protecting mice 
from leukemia following intraperitoneal inoculation of 3 x 107 leukemia following FBL-3 
leukemia cells (44). In these animals, no pathologic changes were observed in the kidney or 
other organs. Furthermore, no suppression of blood counts was observed. Similarly, in initial 
clinical trials, Oka et al vaccinated 26 patients who had WT1+ malignancies and also inherited 
the HLA A2402 allele, with the HLA A2402 presented WT1 peptide, CMTWNQMNL235-243 and 
a higher affinity analog, CYTWNQMNL emulsified in montanide ISA51 adjuvant (45). In this 
group of patients, which included patients with breast (n=2) and lung (n=10) cancer as well as 14 
patients with MDS or leukemia, 13 of 23 evaluable patients generated peptide specific T cell 
responses quantitated by measuring peptide HLA A2402 tetramer binding T cells in the blood. 
Reduction in the size of metastatic tumors was reported in 2/2 patients with breast cancer and 
2/10 patients with lung cancer. Two patients with MDS developed leukopenia after repeated 
vaccinations. However, no alterations in blood counts were observed in the other patients treated 
including 12 patients with AML treated while in complete response. The leukopenia observed in 
these two patients was interpreted to reflect a reaction against the patient’s WT1+ 
myelodysplastic clone. No alterations in kidney or liver function were observed. These early 
clinical results thus suggest that circulating WT1 peptide-specific T cells generated in response 


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to vaccinations do not initiate autoaggressive response against these normal tissues that express 
low levels of WT1. This phase I trial will test whether WT1 peptide-specific T cells, generated 
in vitro, are associated with a low incidence of comorbidity when administered to either 
untreated or lymphoablated tumor-bearing hosts. 
 
Of interest are recently published Phase I studies of adoptive T-cell therapy in patients 
with metastatic melanoma and ovarian cancer which have demonstrated the safety of this 
experimental treatment (99,100). Mackensen et al. conducted a phase I study assessing 
adoptively transferred Melan-A-specific cytotoxic T lymphocytes in patients with metastatic 
melanoma (99). Clinical adverse effects were mild and consisted of low-grade fever and chills 
(99). Three of eleven patients had anti-tumor responses (99). Kershaw et al. studied gene- 
modified autologous T cell transfer in patients with metastatic ovarian cancer (100). These 
authors generated α-folate receptor specific T-cells for the adoptive transfer into patients with 
metastatic ovarian cancer (100). Patients treated without IL-2 had mild side effects and the 
treatment was well tolerated (100). No patient responses were seen; however, this study, as the 
first description of adoptive transfer of gene-modified tumor-reactive T cells in ovarian cancer 
patients, provides insight into the safety and feasibility of adoptive therapy in these patients 
(100). 
 
Pre-Clinical Studies at MSKCC 
 
WT1 Expression by Ovarian Tumors 
 
In order to define the frequency of WT1 expression in ovarian carcinoma, IHC analyses 
of human ovarian tumor microarrays was performed. The ovarian tumor arrays were tested for 
the presence of WT1 by IHC using a WT1 polyclonal antibody (Santa Cruz Biotech, Santa Cruz 
CA). 163 ovarian cancers were treated. Overall, 65% of ovarian carcinomas were positive for 
WT1. Furthermore, the WT1 expression increased with higher stage disease as 78% of stage II 
and IV tumors expressed WT1. Tumors of low malignant potential (LMP) had less WT1 
expression (22%) (see Table 2). 
 
 
 
 
 
 
Table 2:  WT1 Expression in Human Ovarian Cancer as Determined by Immunohistochemical 
Study of Human Ovarian Tumor Microarrays. 
 
Factor 
Levels 
WT1 Negative 
(N = 63) 
WT1 Positive 
(N = 88) 
P-value 
Diagnosis 
Invasive 
Borderline 
41 
22 
81 
6 
<0.001 
Stage 
1A-1C 
IIA-IIC 
40 
10 
13 
17 
<0.001 


 
 
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IIIA-IIIC 
IV 
Missing 
11 
2 
0 
45 
12 
2 
Grade 
1 
2 
3 
Missing 
17 
23 
23 
0 
6 
17 
64 
1 
<0.001 
Cohort 
Early 
Advanced 
50 
13 
27 
61 
<0.001 
N = 151 
*Fischer Exact test 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Amended: 10/09/12 


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In vitro Generation of WT1 Specific T cells from Normal Donors. 
In order to induce antigen-specific T cells against WT1 or survivin, two ex vivo APC systems, 
monocyte derived dendritic cells induced by culture with IL4, GMCSF and TNF, and EBV 
transformed B cell lines were explored. These APCs each express both class I and II HLA alleles 
as well as the necessary co-stimulatory molecules. The initial experiments have focused on the 
generation of WT1 reactive T cells. Therefore, the two types of APC have been loaded with 
WT1 peptides irradiated and used to sensitize autologous T lymphocytes. The HLA A0201 
restricted peptide antigens: 1) 252-260RMFPNAPYL and 2) 313-321SLGEQQYSV or 3) an HLA 
A2401 binding peptide, and the recently identified, 428-436RVPGVAPTL, were used to generate 
WT1 specific T cells. 
 
T cell enriched fractions were isolated 
by depleting adherent monocytes and 
CD56+ NK cells from PBMC of 
normal or tumor bearing HLA A0201+ 
or HLA A2402+ hosts. They  were 
then sensitized with the peptide loaded 
APCs at a 20:1 effector:target ratio. 
Cultures were presensitized at seven- 
day intervals over 42 days. Medium 
was supplemented with IL2 at three- 
day intervals beginning on day 11. In 
these initial studies, seven normal 
donors    and    two    patients    were 
evaluated. Beginning at day seven, T cell cultures were monitored for WT1 peptide-reactive T 
cells, by both quantitation of CD3+, CD8+ T cells binding the relevant peptide HLA A0201 
tetramer and by quantitation of CD4+ and CD8+ CD3+ T cells generating IFN after restimulation 
with peptide loaded autologous PBMC. 
 
As shown in Figure 2, the total number of T cells generated was higher at each stage of the 
culture in the T cells stimulated with peptide loaded on EBV BLCL. The absolute number of 
CD8+ RMF-HLA tetramer+ T cells was also higher in the T cell cultures sensitized with 
autologous peptide loaded EBV BLCL. As shown in Figure 3a and 3b, the proportions of 
RMF/HLA A0201 tetramer+ cells and CD8+T cells generating IFN in response to RMF were 
equivalent. These studies provide evidence that peptide-loaded EBV BLCL may have several 
advantages as APCs including: 1) easy generation from patients while providing a continuously 
renewable source of APCs; 2) induce expansion of large numbers of peptide specific T cells; and 
3) preferential stimulation of CD8+ peptide specific cells. 
Figure 2: Growth Kinetics of Stimulated T cells 


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In analyses of 16 normal donors and six patients with WT1+ leukemias and solid tumors 
expressing A0201, the T cells specific for the HLA A0201-binding RMF and SLG peptides 
quantitated by both tetramer binding and/or IFN production can be consistently generated from 
the blood of normal donors after in vitro sensitization with peptide-loaded autologous EBV 
BLCL. Strikingly, as shown in Figure 3, responses to RMF and SLG WT1 peptides, measured 
by quantitating T cells binding peptide-HLA A0201 tetramers, have been equivalent to the 
responses of these T cells against immunogenic peptides of EBV including EBNA3c, BZLF-1, 
BMLF-1 and LMP-2. 
The capacity of the T cells sensitized with HLA-A0201 or HLA-A2402-binding  WT1 
peptide to lyse WT1+ leukemic cell lines and fresh leukemia was assessed. A representative 
analysis of one donor is illustrated in Figure 3a. The WT1-specific T cells cultured from each 
normal donor lysed HLA A2+ leukemic cell lines that expressed WT1. The cells killed fresh 
WT1+ leukemia cells as well as WT1 peptide loaded targets. The tumor specificity of these T 
cells was confirmed by the fact that these T cells did not kill WT1- HLA A2+ leukemia cells or 
normal hematopoietic cells or PHA blasts derived from the autologous donor. They also failed 
to kill HLA mismatched targets loaded with the relevant WT1 peptide or HLA disparate 
leukemic cells expressing WT1 indicating that these T cells are both peptide-specific and HLA 
restricted. 
Figure 3a: HLA A2 (anti-WT1 RMF) CTL 
Figure 3b: Proportion of HLA A2 WT1 and EBV CTL 


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IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
The cytolytic capacity of the WT1-specific CTLs against human ovarian tumor cells has 
been tested in the laboratory. The levels of HLA A2 expression and WT1 positivity have been 
analyzed by FACS analysis for a number of human ovarian tumor cell lines. The specific 
sensitivity of WT1+ HLA-A2+ ovarian cancer cells is demonstrated in Figure 4. 
 
Figure 4: Anti-Tumor Activity Exhibited by HLA-A2+ CTL Stimulated in vitro by (A) Autologous 
Monocytes or (B) EBV BLCL Loaded With HLA-A2 Binding WT1 Peptide (RMF) Against 
Ovarian Cancer (OC) and B-Cell Acute Lymphoblastic Leukemia (B-ALL) (E:T Ratio 50:1) 
 
 
WT1 specific CTLs stimulated in vitro with autologous monocytes loaded with a WT1 
HLA-A2 binding peptide can lyse ovarian tumor cell lines that express WT1 antigen in a HLA 
restricted manner. The ovarian tumor cell line SKOV-3 is HLA-A2 negative and expresses low 
level (9%) of WT1 antigen (by FACS analysis). There is only 5% cytotoxicity seen in the 51Cr 
Release Assay after a ten-hour incubation. However, SKOV-3 that has been transduced with 
HLA-A2 is lysed to a greater extent (18%). BA25 is an HLA-A2+ human leukemia cell line with 
high WT1 expression (62%) and this was killed more (30%) by the WT1-CTL. When T cells 
restimulated with WT1 loaded EBV BLCL were assayed, their specific cytotoxic activity against 
HLA-A2+ WT1+ ovarian cancer cells was higher (50%). Thus, these experiments indicate that 
HLA-restricted WT1-specific CTL can lyse human ovarian tumor cells in vitro.  The additional 
cytotoxicity experiments with primary ovarian tumor cells that express WT1 are currently being 
performed to assess their sensitivity. 
A raised concern regarding the potential use of WT1 peptide-specific T-cells for adoptive 
immunotherapy has been that such T cells could lyse normal CD34+ hematopoietic progenitor 
cells expressing WT1. Accordingly, the cytolytic activity of WT1 peptide-specific T cells 
against HLA A0201+ normal cord blood progenitors and marrow PBMC fractions enriched for 
CD34+ hematopoietic cells by positive selection for CD34+ cells on Isolex columns further 
depleted of T cells by E-rosette depletion have been comparatively evaluated. Similar 
experiments were performed with a series of primary leukemias, neuroblastomas and sarcomas 
that do or do not express WT1. In these analyses, each target cell population was evaluated for 
its level of WT1 expression. Results are summarized in Figure 5. As illustrated, the level of 
cytotoxicity against the WT1+ leukemias and solid tumors is 


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IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
Figure 5 - Tumor-restricted activity of the WT1-specific T cells 
 
 
high and consistent with the high proportion of WT1+ bright cells in the tumor target cells. In 
contrast, the proportion of cells expressing WT1 and the susceptibility of CD34+ precursor 
enriched marrow fractions and cord blood samples to peptide-specific T cells lysis is low. 
Indeed, the proportion of cells lysed by WT1 peptide-specific T cells in these normal 
hematopoietic precursor fractions does not exceed background levels, and is comparable to the 
cytotoxicity generated against PHA blasts, EBV BLCL and selected leukemias which do not 
express WT1 (Figure 5B). To further ascertain that the small proportion of WT1+ cells in the 
CD34+  marrow and cord blood were not affected by the cytolytic T cells, collaboration with 
M.A.S. Moore, PhD was performed to assess the effects of WT1 peptide-specific T cells on their 
clonigeneity. As shown in Figure 5A, co-incubation with these T cells did not affect the yields 
of CFU-GM, BFU-E, or CFU mix from the CD34+ marrow cell function. 
 
Induction of WT1 Specific CTL from Ovarian Cancer Patients 
 
Autologous EBV BLCL from six ovarian cancer patients (four of these patients are HLA 
A0201) have been established. The precursor frequencies of WT1 RMF-peptide tetramer specific 
CTLs in these patients ranges from 0.03-0.13%. WT1 specific (RMF peptide) CTLs could be 
generated from all patients with pulsed autologous EBV BLCL and the results of the tetramer 
staining and cytotoxicity experiments are illustrated in Figure 6. Interestingly, higher WT1 CTL 
precursor frequencies were seen in patients with WT1 positive tumors (IHC testing performed by 
the Pathology Core). High precursor frequencies resulted in higher percentages of WT1-CTLs 
after stimulation with peptide pulsed EBV BLCL. Of note, ovarian cancer patient WT1-CTLs 
kill WT1+, HLA-A2+ ovarian tumor cells in vitro. 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
 
Amended: 10/09/12 
 
Figure 6:  Tetramer and Cytolytic activity of WT1 Specific (RMF peptide) CTLs generated from 
Four Ovarian Cancer Patients 
 
 
Preclinical Evaluation of WT1 Peptide-Specific T cells In Vivo 
In order to assess the therapeutic potential of the antigen-specific CTLs, a SCID mouse 
model was established which was permissive to the growth of primary human myeloid and 
lymphoid leukemias (90,91). Initial experiments evaluated the effects of T cells sensitized with 
WT1 peptide loaded EBV BLCL isolated from a series of HLA A0201+ marrow allograft donors 
against subcutaneous xenografts of either their sibling’s HLA matched leukemia or against 
allogeneic acute myeloid or lymphoid leukemias sharing the HLA A0201 allele but varying in 
their expression of WT1. 
 
In the example shown in Figure 7, a single dose of 20x106  T cells sensitized with EBV BLCL 
loaded with the RMF peptide 
of WT1 significantly inhibited 
and delayed growth of WT1+ 
HLA A0201+ AML xenografts 
when injected three weeks 
after initial tumor inoculation. 
A second dose of 35x106 T 
cells administered at 9 weeks, 
when the AML xenografts 
again began to expand, led to 
complete 
and 
durable 
regressions of disease. As 
shown in Figure 7, T cells 
sensitized with EBV BLCL 
loaded with either the RMF or 
the SLG HLA A0201 binding 
peptide of WT1 were also able 
Figure 7: 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
to inhibit the growth of a WT1+ HLA A0201 T cell ALL. 
 
The WT1 specificity of the in vivo response observed is demonstrated in the experiment 
presented in Figure 8.  In this study, groups of animals were inoculated subcutaneously (sc) with 
a HLA A0201+ WT1+ B ALL in one thigh and a separate HLA A0201+ WT1- B-ALL in the 
contralateral thigh. Thereafter, at day 6, 12 and 18, they received intravenous infusions of T 
cells sensitized with either autologous EBV BLCL loaded with the RMF peptide of WT1 or 
autologous EBV BLCL alone. As can be seen, the WT1 peptide sensitized T cells specifically 
inhibited the growth of the WT1+ B-ALL, and did not affect the growth of the WT1- B-ALL in 
the same animals. Furthermore, the T cells sensitized with autologous EBV BLCL had no effect 
on either xenograft. A second important feature of the specificity in the adoptively transferred T 
cells is illustrated in Figure 8, namely, the selective infiltration of the T cells into the WT1+ 
BALL xenografts, but not the WT1- BALL. This infiltration results in a striking level of 
apoptosis and eradication of the WT1+ population. 
 
This in vivo model for human ovarian cancer has also been established. SCID mice were 
injected subcutaneously with two human ovarian tumor cell lines that express the WT1 protein: 
SKOV-3 and SKOV-3-A2 (these cells are transduced with human HLA A0201 to express this 
molecule). After two weeks, the mice were treated with either WT1 CTL and IL-2 or IL2 alone. 
In the animals treated with WT1 CTL, the SKOV-3-A2 tumors regressed and their growth was 
inhibited (Figure 8). Pathologic evaluation of the human tumors from the mice revealed that 
human CD8+ T cells were present in the WT1+ A2+ tumor but not in the WT1+ A2- tumor. 
This suggests that WT1 CTL home to tumors if the WT1 antigen in expressed in the context of 
the correct HLA. 
 
Figure 8: The Influence of WT1 peptide specific CTL (RMF) on the Size of Subcutaneous WT1 
Positive, HLA A2.1 Ovarian Xenografted tumors in SCID Mice. 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
 
 
 
Tumor-Directed Migration of HLA-Restricted WT1 Specific T Cells In Vivo 
 
In order to assess the homing of antigen specific CTLs to tumor targets in an in vivo 
system, a new technique was developed permitting sequential in vivo PET imaging of antigen- 
specific CTLs in SCID mice bearing human tumor xenografts (63). 
 
In order to image the antigen-specific T cells, a retroviral vector was developed that can 
efficiently transduce human T lymphocytes and direct the expression of a Herpes-Simplex 
Thymidine Kinase green fluorescence protein fusion gene (HSV-TK/GFP). The transduced cells 
can be isolated based on their expression of GFP. Transduced cells also express HSV-TK. As a 
result, these transduced cells will selectively incorporate 131I or 124I-labeled 2’-fluoro-2’deoxy-1- 
b-D arabinofuransyl-5-iodo-uracil (124I-FIAU) into their DNA. Consequently, transduced T cells 
can be selectively labeled either in vitro, or in vivo at sequential times after their adoptive 
transfer into animals bearing tumor xenografts. 
 
In subsequent experiments, this technique was evaluated as a method for tracking WT1 
specific T cells after adoptive transfer. In the study illustrated in Figure 9, SCID mice were 
inoculated with a HLA A0201+ WT1+ BALL in the left shoulder (T1), an autologous EBV 
BLCL in the right shoulder (T2), and a HLA A0201+ homozygous EBV BLCL (T3) and a WT1- 
HLA A0201+ BALL (T4) in left and right thighs, respectively. After development of the tumors, 
groups of animals were treated with T cells sensitized either with WT1 peptide loaded 
autologous EBV BLCL or autologous EBV BLCL alone. The T cells were transduced with the 
HSV-TK/GFP vector 42 days thereafter.  Post sorting, 3.1% of the transduced T cells sensitized 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
with WT1 peptide loaded EBV BLCL were WT1 specific and the rest reactive against EBV. In 
vitro, both the EBV-specific and the WT1 and EBV-specific transduced and sorted GFP+ T cells 
were restricted by HLA A0201. One and seven days after intravenous infusion of either the 
WT1/EBV dual specific HSV-TK/GFP+ T cells or the EBV-specific, HSV-TK/GFP+ T cells, the 
animals received an infusion of [131I]-FIAU and twenty-four hours after each dose, the animals 
were imaged by scintigraphy (PET could not be used due to the temporary unavailability of 124I). 
As shown in the control animal (Figure 9) infused with non-transduced T cells followed twenty- 
four hours later by [131I]-FIAU, label was detected by scintigraphy only in the thyroid, consistent 
with the absence of HSV-TK+ T cells in the animals. Mice treated with the EBV-specific TK- 
GFP+ T cells accumulated [131I]-FIAU selectively in the EBV+ HLA A0201+ lymphomas (T2 and 
T3) and spleen both at day two and day eight post T cell infusion (Figure 9B,E). In contrast, the 
TK-GFP+ T cells containing both WT1 peptide specific and EBV reactive T cells accumulated in 
the WT1+ BALL xenografts (T1) and the EBV+ lymphoma xenografts in proportion to the 
representation of WT1-specific and EBV-specific T cells in the sorted TK-GFP+ T cells infused, 
but did not infiltrate into the WT1- BALL xenografts (Figure 9A,D). The scintigraphic findings 
were further corroborated by quantitation of the [131I]-label in autopsied tissues and by FACS 
analysis of these tissues for transduced, GFP+ cells obtained from mice sacrificed 24 hours after 
either the day 1one or day seven dose of [131I]-FIAU (Figure 9). 
 
In a separate experiment, purified TK/GFP vector transduced T cells sensitized with 
RMF peptide loaded EBV BLCL were infused into mice bearing four established tumor 
xenografts. At one, eight and 15 days post T cell infusion, the animals were injected with 18F- 
labeled FEAU and then imaged by PET four hours thereafter. As shown in the cross sectional 
images at the shoulders and thighs and the tissue accumulation data presented in Figure 9D, the 
HLA-A2 restricted RMF-specific T cells selectively accumulated in a WT1+ rhabdomyosarcoma 
(T1) and the WT1+ BA-25 ALL (T2) xenograft, each of which also strongly expresses HLA- 
A0201. In contrast, the transduced T cells did not accumulate in the HLA A0201+ WT1- BALL 
tumor (T4). Moreover, they failed to accumulate in a WT1+ rhabdomyosarcoma (T3) which was 
genotypically HLA-A0201 but failed to express HLA A0201 detectable by an allele-specific 
antibody as measured by FACS. These studies suggest that down regulation of HLA expression 
which has been shown to protect tumor cells from T cell mediated lysis in vitro, can also prevent 
the targeted migration to and accumulation of tumor-peptide specific T cells in tumors in vivo. 
Taken together, these in vitro analyses and preclinical evaluations of WT1 peptide-specific T 
cells in vivo in SCID mice bearing human WT1-+ tumor xenografts support the potential of such 
T cells to lyse WT1+ tumors in vitro and inhibit their growth in vivo. Based on these studies and 
other reports summarized in the background, these WT1 peptide-specific T cells will be 
evaluated in  a phase I trial in patients with relapsed WT1+ ovarian, primary peritoneal or 
fallopian tube carcinoma. 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
Th 
T1 
T2
Spl
T3 
T4 
Th 
T1 
T2
Spl
T3 
T4 
Th 
T1 
T2
Spl 
T3 
T4 
Th 
T1 
T2
Spl 
T3 
T4 
Th 
T2
T1 
Spl 
T3 
T4 
Th 
T2
T1 
Spl 
T3 
T4 
Th 
T1 
T2
Spl 
T3 
T4 
Th 
T1 
T2
Spl 
T3 
T4 
h 
T1 
T2 
Spl 
T3 
T4 
h 
T1 
T2 
Spl 
T3 
T4 
Th 
T1 
SplT2 
T3 
T4 
Th 
T1 
SplT2 
T3 
T4 
 
Figure 9: In vivo imaging of WT1 CTL homing to WT1 HLA A0201 Specific tumors using 
FIAU 
 
 
 
A. [131I]FIAU gamma scintigraphy: 
B. 
Day 2 
 
[131I]FIAU tissues accumulation:  
Day 8 
 
 
 
48 hours 
After 
T cell 
EBV-TG-CTL 
Th 
WT1/EBV-TG-CTL 
Th 
 
 
T1 
T2 
Control 
Th 
 
T2 
T1 
T2 
T3 
T4 
Spleen 
Plasma 
T1 
T2 
T3 
T4 
Spleen 
Plasma 
infusion 
T2 
Spl 
Spl 
T1 
Spl 
Muscles 
Stomach 
Thyroid 
Duodenum 
Muscles 
Stomach 
Thyroid 
Duodenum 
T3 
T4 
EBV-TG-CTL 
T3 
T4 
WT1/EBV-TG-CTL 
T3 
T4 
Control 
Colon 
 
0  0.1   0.2  0.3 0.4  0.5 0.6 0.7 0.8 
% dose/gram 
Colon  
 
0  0.1 0.2   0.3  0.4  0.5 0.6 0.7 0.8 
% dose/gram 
8 days 
after 
Th 
C. 
Tumor infiltration with eGFP+ human T-cells 
Day 8 
T cell 
infusion 
 
 
 
 
 
 
D. [18F]FEAU microPET images: 
T2 
 
T1 
Spl 
 
 
T3 
T4 
T1 
 
 
 
T4 
0.1 
1 
10 
% of eGFP+ cells 
T1 
T2 
T3 
T4 
0.1 
1 
10 
% of eGFP+ cells 
Day 1 after CTL infusion 
Day 8 after CTL infusion 
 [18F]FEAU  tissue accumulation 
 
T1 
T1 
T2 
T2 
 
 
 
 
 
 
 
 
thighs 
 
T3 
T4 
Plasma 
 
Muscle 
Colon 
Duodenum 
Liver 
 
T4 
T3 
Plasma 
Muscle 
Colon 
Duodenum 
 
Liver 
 
0 
2 
0 
% Dose/g 
 
 
 
 
 
 
 
 
 
 
 
% Dose/g 
2 
 
 
 
 
Experience with Infusion of WT-1 peptide specific T-cells and the Rationale for Repeated 
Dosings: 
 
The Results to date of Phase I trials at MSKCC 
 
At present, two Phase I trials of WT-1 peptide specific T-cells are in progress: 
IRB 06-155 A2 is evaluating autologous WT-1 specific T-cells in women with advanced 
ovarian carcinomas. This protocol has accrued only two patients at the lowest dose, 5 x 106 T- 
cells/ m2. Both patients ultimately had progression of disease. However, one patient had 
stabilization of disease for 2 months and exhibited a significant reduction in Ca-125 levels 
following infusion of WT-1 specific T-cells. No toxicities were observed. In particular, neither 
patient exhibited count depression or evidence of renal toxicity. Accrual to this trial has been 
limited due to the unwillingness of potential candidates to receive only one dose of the T-cells 
and be observed until disease progression. 
 
Protocol 07-055 A (4) is evaluating WT-1 specific T-cells generated from normal HLA- 
matched  HSCT  donors  in  the  treatment  of  patients  transplanted  for  WT-1+   hematologic 
Th 
T 
1 
T2 
Spl 
T3 
T4 
T1 
T2 
T1 
T2 
T3 
T3 
colon     T4 
colon  T4 
T1 
T2 
colon 
T1 
T2 
T3 
Th 
T1 
T2 
Spl 
T3 
T4 
T1 
Day 2 
Tissues 
Tissue 
Tissues 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
malignancies who have relapsed or have persistent or recurrent minimal residual disease. To 
date, 10 patients have been treated, 4 at dose level 1 (1 x 106 T-cells/ m2), 3 at dose level 2 (5 x 
106 T-cell/m2 ) and 3 at dose level 3 (1 x 107 T-cells/m2). No patient in this trial has experienced 
a grade III toxicity attributable to the infusion of cells. One patient experienced moderate 
diarrhea with streaks of blood in the stool attributable to an ongoing infection. No patient 
experienced renal or hematologic toxicity. No patient developed new acute or chronic GVHD, 
and existing GVHD was not exacerbated by this treatment. 
 
As shown in Figure 10, of a patient with CML was treated with WT-1 specific T-cells 
containing both CD4+ and CD8+ T-cells recognizing epitopes in pentadecapeptides #20 
presented by HLA DRB, 0102 and B4701 respectively. The infusion of WT-1 specific T-cells 
was followed by clearance of cells with WT-1 transcripts, as measured by quantitative RTpcr 
measuring WT-1 mRNA copy numbers in the blood. The infusions were also followed by the 
emergence of detectable WT-1 peptide specific CTL precursors (CTLp), as quantitated by 
limiting dilution analyses in 8 of 8 patients thus far studied. It is important to note that these 
patients have been treated within 1 year post transplant, at which time, they were still relatively 
immunodeficient by virtue of the myeloablative therapy administered for cytoreduction pre- 
transplant, and the T-cell depleted graft they received. Despite this, however, the increases in 
WT-1 specific CTLp were transient, persisting only for 7- 28 days in each of the patients tested. 
Increases in tetramer+ T-cells were also transient except in one patient treated for CNS 
recurrence in whom increments in tetramer+ cells could be detected as late as 100 days post 
infusion. 
 
Based on these preliminary results, we suggest that infusions of autologous or allogeneic 
WT-1 peptide specific T-cells are associated with minimal or no toxicity. However, because 
these T-cells and T-cells specific for other “self” antigens that have been administered without 
lymphodepletion in other clinical trials survive and proliferate for periods of only 1-2 weeks after 
infusion, we hypothesize that repeated infusions of these cells will be required if they are to be 
able to exert a sustained and significant anti-tumor effect in vivo. Accordingly, the protocol has 
been amended to include 4 repeated doses of T-cells, at two week intervals, to better test the anti- 
tumor effects of the T-cells administered. The patients in dose level II will also receive 
cyclophosphamide prior to T-cell infusion to induce a state of lymphodepletion conducive to the 
growth of populations of WT-1 specific T-cells following adoptive transfer. 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
FIGURE 10 
 
 
Case # 000-00-140-08: 
patient with CML treated with WT1 CTL at the dose 5x10^6/m^2 (total dose 10x10^6) 
Identification of the immunogenic epitope by IFN FACS analysis 
 
 
2 
1.5 
1 
0.5 
0 
 
 
CD8+IFN+WT1 CTL 
CD4+IFN+ WT1CTL 
Subpools #8/#13 - Peptide #20: RSGPGCLQQPEQQGV 
PBMC   1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24    WT1 
WT1 subpools 
total 
pool 
HLA restriction of the WT1 specific cytotoxic activity of the WT1 CTL against the EBV BLCL matching one of the HLA alleles of the T 
cell donor loaded with WT1 pool. The same unloaded targets were not lyzed by these T cells. 
30 
25 
20 
15 
10 
5 
0 
A0301/ 
WT1 
 
A0201/ 
WT1 
 
B4701/ 
WT1 
 
B1501/ 
WT1 
 
C06/ 
WT1 
 
C0303/ 
WT1 
targets 
 
DRB10102/ 
WT1 
 
DRB11301/ 
WT1 
 
DQB10501/ 
WT1 
 
DQB10601/ 
WT1 
 
Monitoring of the WT1 PCR and WT1 CTLp in the peripheral blood of the patient after WT1 CTL infusion 
15 
WT1 CTL 
 
10 
 
 
5 
 
 
0 
 
 
WT1 PCR 
 
 
WT1 CTLp 
 
 
5000000 
4000000 
3000000 
 
2000000 
1000000 
 
0 1 
9 
14 
23 
30 
42 
56 
76 
 
days post WT1 CTL infusion 
WT1 DNA 
copy number/ml 
% of IFNg+  cells 
WT1 CTLp 
frequencies 
cells/ml 
% of cytotoxicity 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
Level I 
3-6 
NO 
5 x 106/m2 
Level II 
3-6 
YES 
2 x 107/m2 
Level III 
3-6 
YES 
5 x 107/m2 
 
4.1 
OVERVIEW OF STUDY DESIGN/INTERVENTION 
 
4.2 
Design 
 
This is a non-randomized phase I clinical study. The target population in this study are 
patients with recurrent or persistent, measurable WT1+ ovarian, primary peritoneal, or 
fallopian tube carcinomas. Furthermore, the patients’ ovarian, primary peritoneal or 
fallopian tube carcinoma must express the WT1 protein detectable by IHC analysis of 
the patient’s tumor. 
 
4.3 
Intervention 
 
Once the requisite dose of T cells has been generated, the patients will receive WT1 
reactive T cells by intravenous infusion. Patients will be treated in 4 sequential groups 
of 3-6 patients. Dose escalation will be based on dose limiting toxicity (DLT). In cohort 
1 patients will receive WT1 specific T cells dosed at 5 x 106/m2 (level I) without 
cyclophosphamide lymphodepletion. Patients in dose level II, III, and IV will receive a 
standard lymphodepletive regimen consisting of a single dose of cyclophosphamide 
750mg/m2 (99,104,105), administered intravenously, 48 hours prior to the first T cell 
infusion. No further doses of cyclophosphamide will be administered to these patients 
during the study. After a 48 hour rest period to permit elimination of 
cyclophosphamide, each patient will receive a dose of autologous T cells by an 
intravenous infusion from the WT1 peptide-specific T cell line by intravenous infusion. 
 
Sequential groups of four patients will be accrued to each treatment group. The T cell 
and conditioning chemotherapy to be evaluated in each group are summarized in Table 
3: 
 
Table 3: Dose Levels of T Cells and Conditioning Chemotherapy 
Level 
Patients 
Cyclophosphamide  WT1 Specific T cells 
Level I 
3-6 
NO 
5 x 106/m2 
m2 
Level III 
3-6 
Yes 
5 x 107/m2 
Level IV 
3-6 
Yes 
1 x 108/m2 
 
The first two patients in cohort 1 only received a single administration of WT1 specific 
T cells, whereas all subsequent patients treated in cohort 1 are to receive specific T cell 
infusions every two weeks for four doses.     Patients in cohorts II, III and IV may 
Level II 
3-6 
Yes 
2 x 107/ 


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IRB Protocol 
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Amended: 10/09/12 
 
 
 
receive additional infusions of WT1 peptide-specific T cells once every 2 weeks for a 
total of 4 doses. Each cycle comprises of 28 days or two doses of WT1 specific T cells. 
 
There will be a finite number of WT1 peptide-specific T cells and therefore a finite 
number of infusions possible. If, after 4 infusions, the patient ( in cohort I, II,III, and 
IV) has a clinical response or at least stable disease, additional infusions of WT1 
peptide sensitized T cells may continue to be administered once every 2 weeks until the 
stock has been exhausted, DLT, withdrawal of consent or disease progression. These 
additional infusions of WT1 peptide-specific T cells will be administered at the same 
dose level as the patient’s initial infusion. 
 
5.1 
THERAPEUTIC/DIAGNOSTIC AGENTS 
 
5.2 
WT1 Specific T Lymphocytes 
 
These are the patient’s own (i.e. autologous) T cells (CD4 and CD8 cells) that have 
been sensitized with WT1 peptide loaded on autologous irradiated EBV-transformed B 
cell lines and expanded ex vivo as described in Section 9.1. These T cells are pre- 
tested to ascertain that: 1) they contain WT1 specific cytotoxic T cells; 2) they do not 
react against lymphocytes from the patient; 3) they are microbiologically sterile and 
free of mycoplasma; and 4) they contain less than five EU of endotoxin per dose of 
cells. One infusion of T cells was administered to the first two patients treated at dose 
level I. For all subsequent patients treated at dose level I and for patients treated at level 
II-IV infusions of T cells will be administered to each patient once every 2 weeks, 
according to their assigned dose level, for 4 doses. From week 2 onwards a potential +/- 
72hour treatment window is permissible provided that necessary assessments and 
laboratory tests can still be carried out. If, after 4 T cell infusions, the patient has a 
clinical response or at least stable disease, additional infusions of WT1 peptide 
sensitized T cells may continue to be administered once every 2 weeks until the stock 
has been exhausted, DLT, withdrawal of consent or disease progression. 
 
5.3 
Cyclophosphamide (Cyclophosphamide (Cytoxan®, Neosar®, CTX)) 
 
• Nitrogen mustard derivative-alkylating agent 
 
• Common Indications: Hodgkin's disease, lymphoma, multiple myeloma, chronic 
lymphocytic leukemia, acute lymphocytic leukemia, acute myeloblastic leukemia, 
mycosis fungoides, neuroblastoma, retinoblastoma, ovarian and breast carcinoma 
 
• Supplied as: 200 mg, 500 mg, 2000 mg vials 
 
 
 
DOSAGE, DOSE MODIFICATIONS AND ADMINISTRATION 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
• As previously indicated, the standard dose of cyclophosphamide will be 750mg/m2. 
Baseline renal and hepatic function will be assessed prior to the single 
cyclophosphamide administration. No dose-reductions are permitted. Table  4 
describes the parameters for standard lymphodepletive regimen consisting of a single 
dose of cyclophosphamide 750mg/m2: 
Table 4: Single Dose Parameters of Cyclophosphamide (750mg/m2): 
 
Absolute 
Neutrophil 
Count 
Platelets 
Creatine 
Bilirubin 
AST 
ALT 
≥1.5 K/mcL 
≥100 
K/mcL 
≤1.5 mg/dl 
≤1.5xULN 
≤ 2.5 x 
ULN 
≤ 
2.5 
x 
ULN 
 
 
 
6.1 
CRITERIA FOR SUBJECT ELIGIBILITY 
 
 
6.2 
Subject Inclusion Criteria 
 
• Pathologically confirmed diagnosis of ovarian, primary peritoneal, or fallopian 
tube carcinoma. 
• Recurrent or persistent disease following treatment with platinum-based 
chemotherapy. 
• Evaluable disease as demonstrated by Ca125, radiologic, or pathologic studies 
conducted. 
• Patients’ carcinoma must express the WT1 protein detectable by IHC analysis of 
banked (paraffin embedded) or freshly biopsied tumor nodules. 
▪ IHC evidence of WT1 expression will be performed according to the 
technique described by Dupont and Soslow et al (40). Expression will be 
graded according to an adaption of the German Immunoreactive Score 
(IRS). 
▪ Only WT1 tumors with moderate to strong IRS scores (4-12) will be 
considered positive. 
• Karnofsky Performance Status score of 70% or greater (WHO status 0-1). 
• Life expectancy of at least six months. 
• Adequate bone marrow, renal, and hepatic function: 
▪ Absolute neutrophil count (ANC)  1500/mm³ 
▪ Platelets  100,000/mm³ 
▪ Creatinine  1.5mg/dL or creatinine clearance > 60ml/min 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
▪ ALT and AST all  2.5 x the institutional upper limit of normal (ULN) 
and total bilirubin  1.5 x the institutional ULN 
• Adequate pulmonary and cardiac function. 
▪ No clinical evidence of cardiopulmonary disease, which, in the opinion of 
the investigator, precludes enrollment. 
• Age greater than or equal to 18 years. 
• Written Informed Consent from each patient or the patient’s representative prior 
to study entry. 
• No anti-cancer therapy (chemotherapy, biologic therapy, or immunotherapy) 
within three weeks prior to the initial T cell infusion (Levels I), or 
cyclophosphamide (Levels II, III, and IV). 
• Ability to keep scheduled visits. 
 
6.3 
Subject Exclusion Criteria 
 
• Known hepatitis B, C or HIV infection. 
• History of whole abdominal radiation therapy (WART). 
• Evidence of bowel obstruction. 
• Active brain metastases or history of brain metastases. 
• Clinically significant heart disease (NYHA class III or IV). 
• Active infections requiring antibiotics within two weeks of registration. 
• Serious intercurrent illness, requiring hospitalization. 
• Primary or secondary immunodeficiency or active autoimmune disease. 
• Other cancers (excluding non-melanomatous skin cancer) within 5 years. 
• Women who are pregnant or lactating. 
• Any other issue which is the opinion of the treating physician would make the 
patient ineligible for the study. 
 
 
7.0 
RECRUITMENT PLAN 
 
This is a single institution trial. All patients will be recruited through the outpatient 
gynecologic medical oncology clinic of MSKCC. Patients with recurrent or  persistent 
ovarian, primary peritoneal or fallopian tube cancer following treatment with platinum-based 
chemotherapy will be recruited to this study following evaluation in the outpatient or inpatient 
setting. MSKCC has filed HHS 441 (civil rights), HHS (handicapped individuals), 639-A 
(sex discrimination), and 680 (age discrimination). We also take due notice of the 
NIH/ADAMHA policy concerning inclusion of women and minorities in clinical research 
populations. Women and members of all ethnic groups are eligible for this trial. 
 
The ethnic composition of the subject population reflects patients referred to MSKCC. 
Approximately  70%  of  the  patients  are  Caucasians,  7%  Black,  10%  Hispanic  and  4% 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
Oriental. No patient is excluded from participation in studies on the basis of ethnicity or race. 
Only women develop ovarian carcinoma. 
 
Before protocol specific procedures are carried out, the investigator will explain the full 
details of the protocol and study procedures and the risks involved to each patient prior to the 
patient’s inclusion into the trial. The patient will be informed that she is free to withdraw from 
the study at any time. The patient will sign an IRB approved consent form indicating her 
consent to participate. This consent form will conform to the applicable requirement of 21 
CFR 50.25 elements of informed consent. The original consent will become part of the 
patient’s medical record. Each patient will receive a copy of the informed consent. 
 
8.1 
PRETREATMENT EVALUATION 
 
Informed Consent 1: After signing Informed Consent 1 (Screening Informed Consent), the 
patients’ WT1 tumor expression will be determined as previously described. In order to be 
eligible for this protocol, the patients’ ovarian, primary peritoneal or fallopian tube carcinoma 
must express the WT1 protein detectable by IHC analysis of banked (paraffin embedded) 
tumor. After signing Informed Consent 1, several weeks will elapse while the patients’ 
banked tumor is tested for WT1 expression. Once patients are deemed eligible, a 
leukapheresis will be performed for the collection of PBMC to establish autologous EBV 
BLCL and generation of WT1-specific T cell lines. The screening phase, as described above 
and outlined in Table 5, will take approximately 12-16 weeks. Patients will also have blood 
drawn to assess EBV serology. 
 
Table 5: Screening Phase (Informed Consent One) 
 
Sign Informed Consent One 
X 
WT1 Tumor Testing 
X 
HLA Testing & complete blood count 
X 
 
 
Eligible patients 
 
 
 
Collection of 4 green top tubes for the 
establishment of EBV BLCL if a significant time 
lapse before patient can undergo leukapheresis & 
Collection of 1 red top tube for EBV serology 
X 
Leukapheresis for the Establishment of 
Autologous EBV BLCL if has not been 
established yet and generation of WT1-specific T 
cell lines 
X 
• Screening will take approximately 12-16 weeks. 


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IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
 
Informed Consent 2: Eligible patients will then receive Informed Consent 2 (Treatment 
Informed Consent). Patients will then be treated with the following: T cell infusion alone (dose 
levels I) or T cell infusion with prior cyclophosphamide (dose levels II, III, and IV). Within 30 
days prior to starting the T cell infusion, all patients will undergo the following: 
 
• History and physical examination (including performance status, height, weight) 
 
• Hematopoietic: Complete blood count and PT, aPTT 
 
• Biochemical: Comprehensive metabolic panel, Magnesium, Ca-125 level 
 
• Others: Urinalysis, creatinine clearance 
 
• CT Scan of the chest, abdomen, and pelvis 
 
• EKG 
 
• 
Research Blood samples: Obtained prior to adoptive therapy to quantitate 
baseline levels of WT1 reactive T cells, by quantitation of WT1 specific CTLp 
by LDA, T cells secreting IFN in response to peptide and, in HLA A0201+ 
patients, T cell binding WT1 peptide HLA A2 tetramers. 
9.1 
TREATMENT/INTERVENTION PLAN 
Treatment with WT1 specific T cells: 
9.2 
Generation and Isolation of WT1 Specific T cells for Adoptive Therapy 
 
The patients qualifying and consenting to participate in this study (i.e. after 
signing Informed Consent One) will donate 4 green top tubes of peripheral blood 
to establish autologous EBV BLCL if the leukapheresis cannot be collected 
immediately which will be cryopreserved for potential use as peptide-loaded 
sensitizing cells and peptide loaded targets in assays of T cell reactivity against 
antigenic peptides. If the leukapheresis product is collected the 4 green top tubes 
are not needed. Once the EBV BLCL are established the patient will undergo 
leukapheresis to collect PBMC for the generation of autologous CAMs and 
autologous WT1-specific T cell lines. The patient’s EBV  BLCL or  CAMs, 
loaded with peptide, will be used for autologous T cell sensitization. Please see 
Appendix A for methodology. 
 
The use of EBV BLCL as APCs has advantages in that they can be propagated in 
large numbers once the EBV BLCL have been established. In addition, T cells 
sensitized to peptide loaded EBV BLCL also generate EBV-specific T cells, 
which should replenish EBV-restricted T cells in the immunosuppressed host. 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
Prior to use as sensitizers, the CAMs or EBV BLCL will be irradiated to 3000rads 
or 9,000 rads respectively in a calibrated certified blood irradiator to eradicate the 
capacity of these cells to grow within co-cultures. In addition, EBV BLCL will 
be cultured in media containing acyclovir to eliminate the risk of transferring 
infectious EBV in the T cells transferred. CTLs from the EBV seronegative 
individuals will be generated from the CD3 enriched B cell depleted lymphocytes. 
 
To prepare T cells for sensitization and in vitro expansion, mononuclear cells will 
initially be isolated from heparinized leukapheresed white cell preparations by 
centrifugation on Ficoll-Hypaque density gradient. After washing, T cells will be 
enriched by initially depleting monocytes by adherence to sterile plastic tissue 
culture flasks or by the immunomagnetic depletion using the CD14 microbeads. 
Thereafter, B and NK cells will be depleted by incubation with clinical grade anti- 
CD19 and CD56 microbeads Miltenyi reagent. The T cell enriched cell fractions 
will be washed and suspended in medium containing 5% prescreened heat- 
inactivated Ab serum in preparation for sensitization. 
 
Autologous T cells (CD4 and CD8 cells) will be sensitized in vitro to recognize 
WT1 peptides loaded on autologous irradiated CAMs or EBV BLCL and 
expanded ex vivo. After initial sensitization, the WT1/EBV peptide-sensitized T 
cells are cultured for 35-42 days. They are restimulated with CAMs or EBV 
BLCL loaded with the total pool of pentadecapeptides spanning the whole 
sequence of WT1 protein overlapping by 11aa at 7-day intervals. In addition, 
after 10 days of culture, IL2 (Chiron) is added every 3 days and IL15 (Miltenyi) is 
added every 7 days. After 32-42 days of culture, the T cells are washed and then 
acceptance tested for T cell content and for specific reactivity against targets 
loaded with WT1 total pool of pentadecapeptides, sterility, mycoplasma, HLA 
type and endotoxin. Autologous WT1 specific T cell populations will be 
generated in quantities sufficient for adoptive therapy. 
 
After sensitization, the cells will be expanded in large-scale cultures with IL2 and 
OKT3 according to a modification of the technique of Dudley and Rosenberg, 
using irradiated autologous WT1 peptide loaded EBV BLCL and autologous WT1 
peptide-loaded PBMC as irradiated feeders (1) The T cells will be assessed for 
WT-1 reactive T cells after sensitization and expansion by quantitation of T cells 
generating IFN in response to secondary restimulation with WT1-peptide loaded 
autologous peripheral blood mononuclear cells. In HLA-A0201+ patients, T cells 
binding WT1 peptide-HLA A0201 tetramers will also be quantitated, where 
applicable. The WT1 specific cytolytic activity will be assessed against 
autologous WT1 peptide loaded targets. 
 
Prior to infusion, T cells will be tested and certified as microbiologically sterile 
and free of mycoplasma. Products that do not meet sterility testing requirements 
will not be released for use in patients.  Each dose of cells  will contain < 5 EU 


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IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
__________________________________________________________________ 
 
endotoxin/dose of cells. Certified T cell doses will be cryopreserved until use. 
For administration, the cell dose is suspended in approximately 25-60 ml sterile 
preservative free saline for injection, and infused intravenously over 5-10 
minutes. If the T cells are not used for treatment, they may be used for research 
purposes in the laboratory. 
 
T cell infusions will be administered in the Adult Same Day Hospital, an 
outpatient center, at the main campus of MSKCC. Conditioning chemotherapy 
will be administered at our outpatient chemotherapy center. 
 
 
9.3 
Preparative Immunosuppression and Treatment with WT1 T cells 
Patients will be treated with the cells in sequential groups of three  patients 
varying by T cell dose and the addition of lymphodepletive therapy prior to T cell 
infusion. Level I will receive WT1 specific T cells without prior 
cyclophosphamide. Patients in Levels II, III and IV will undergo pre-infusion 
lymphodepletion with intravenous cyclophosphamide, 750mg/m2. After a 48- 
hour rest period to permit elimination of cyclophosphamide, each patient in levels 
II, III, and IV will receive a dose of autologous T cells from the WT1 specific T 
cell line by intravenous infusion according to the table below. 
 
In this phase I trial, one dose level of T cells will be tested in patients without 
other treatment. An escalated T cell dose will then be evaluated in the next three 
groups (levels II,III, and IV) that will undergo lymphodepletion first with 
cyclophosphamide prior to the initial T cell infusion. Only a single dose of 
cyclophosphamide will be administered to patients on level II, III, and IV during 
the study. The T cell doses evaluated in this dose escalation are listed in Table 6. 
 
Table 6 Dose Levels of T Cells and Conditioning Chemotherapy 
Level 
Patients 
Cyclophosphamide 
WT1 Specific T cells 
Level I 
3-6 
NO 
5 x 106/m2 
Level II 
3-6 
Yes 
2 x 107/m2 
Level III 
3-6 
Yes 
5 x 107/m2 
Level IV 
3-6 
Yes 
1 x 108/m2 
 
The first two patients treated at dose level I received a single infusion of the WT1 
peptide-specific T cells. All subsequent patients may receive additional WT1 peptide- 
specific T cells infusions, once every two weeks for 4 doses. If, after 4 infusions, the 
patient has a clinical response or at least stable disease, additional infusions of WT1 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
peptide sensitized T cells may continue to be administered once every 2 weeks until the 
stock has been exhausted, DLT, withdrawal of consent or disease progression. 
 
9.4 
Dose Escalation and Dose Limiting Toxicity (DLT): 
 
In this phase I trial, dose escalation will be based on the dose limiting toxicity 
(DLT). For this trial, DLT will be defined as a grade III or greater toxicity 
developing following infusion of the T cells. Only toxicities that are possibly, 
probably, or definitely related to T cell infusions will be considered DLTs. For 
dose Levels II, III, and IV (who receive cyclophosphamide), hematopoietic DLT 
will not be included in the first 21 days of the first cycle of treatment, since 
cytopenia is an expected toxicity of the pretreatment course of cyclophosphamide. 
However, severe grade 3 cytopenia extending beyond these 21 days  will  be 
graded as a severe toxicity. The dose escalation scheme for this study is as 
follows: 
 
• If 0 of 3 patients experience DLT at a given dose level, then proceed to the next 
starting dose level. 
 
• If 1 of 3 patients experience DLT at a given dose level, 3 additional patients will 
be treated at that level. If 0 of those 3 develop DLT, the dose will be further 
escalated. 
 
• If 2 or more of the 6 patients experience DLT at a given dose level, the maximum 
tolerated dose (MTD) is defined as the previous dose level. If only 3 patients are 
treated at the MTD, an additional 3 patients will be treated at that level. If among 
these 6 patients, 2 or more experience DLT, the MTD will be lowered and the 
same criterion will be applied to the previous dose level. 
 
If any patient at a dose level is not evaluable through the first twenty-eight days 
post the initial T cell infusion, the patient must be replaced before dose escalation 
to the next level occurs so that at least three patients are evaluated at a dose level 
prior to dose escalation. 
 
 
 
10.1 
EVALUATION DURING TREATMENT/INTERVENTION 
 
• Patients will be evaluated clinically with vital signs pre-and six hours post 
infusion of T cells. 
 
• Patients will receive physical examinations and toxicity assessment (using 
CTCAE version 4.0) on the day after each T cell infusion. All patients will be 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
examined at least once weekly during cycle 1. Patients will also have a safety 
visit four and eight weeks after the final T cell infusion, when applicable. 
 
• Patients will have at least weekly CBCs with differentials and platelets, 
biochemical screens and creatinine clearances to monitor for renal or hepatic 
toxicity for the first four weeks (i.e., cycle 1) and then every two weeks 
thereafter (i.e., cycle 2 or greater) until the final T cell infusion. Patients will 
also have these bloods assessed at 4 and 8 weeks following the final T cell 
infusion, when applicable. 
 
• Serum CA125 levels will be measured at baseline, prior to each T cell 
infusion, and at 4 and 8 weeks after the final T cell infusion. 
 
• In addition, during cycle 1 patients will be tested for circulating levels and 
phenotype of T cells as well as for proportion of T cells or generating 
intracellular IFN in response to WT1 or binding WT1/HLA A2 tetramers 
prior to chemotherapy (if applicable), prior to the first T cell infusion, 24 
hours post the first T cell infusion and at 1 week post the initial infusion. For 
patients receiving multiple T cell infusions frequencies of T cells specific for 
WT1 will also be assessed prior to each infusion of WT1 specific T cells and 
24-hours post the 4th, 5th, 6th and 8th when applicable infusions by LDA. 
Furthermore, frequencies of T cells specific for WT1 will also be assessed 4 
and 8 weeks following the final infusion the T cells. 
 
• Concurrent quantitation of EBV specific T cell precursors by LDA and T cells 
generating IFN in response to autologous EBV BLCL will be performed as a 
second marker of the persistence and expansion of adoptively transferred T 
cells, as well as recovery of autologous immune function. 
 
• Tumor responses will also be monitored radiographically by CT of the chest, 
abdomen and pelvis performed at baseline (prior to the initial T cell infusion 
and if applicable cyclophosphamide) and approximately every 8 weeks (i.e. 
following every 2 cycles) whilst on study. 
 
• Four weeks after the final T cell infusion will be considered the Safety 
Follow-Up visit and an additional follow-up visit will occur at 8 weeks post 
final T cell infusion. 
 
• This study will also monitor patients for alterations in circulating levels of 
WT1 specific T cells at sequential intervals post adoptive transfer. In 
addition, patients will be monitored for alterations in tumor size and levels of 
circulating tumor antigens in the post treatment period. These evaluations will 
be observational and descriptive only, since the study group size is insufficient 
to provide a statistically significant estimate of the proportion of patients 


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IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
exhibiting significant alterations in WT1 specific T cells, biochemical indices 
of tumor burden or radiologic evidence of tumor response. However, this data 
will provide initial qualitative estimates of the T cell infusions effects, which 
are critical in planning subsequent phase II trials. 
 
Timelines with all of the evaluations, tests and procedures occurring after each signed 
informed consent are depicted in Tables 7A-C below: 
 
Table 7A: Screening Phase (Informed Consent One) 
 
 
Sign Informed Consent One 
X 
WT1 Tumor Testing 
X 
HLA Testing & complete blood count 
X 
 
 
Eligible patients 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
Table 7B: Treatment Phase (Informed Consent Two) 
 
Sign Informed Consent Two 
X 
Patients will then follow Table 7C 
X 
 
 
 
Table 7C: Treatment Phase (Informed Consent Two)* 
Collection of 4 green top tubes for the establishment of 
autologous EBV BLCL, if a significant time lapse 
before patient can undergo leukapheresis & 
collection of 1 red top tube for EBV serology 
X 
Leukapheresis for the Establishment of Autologous 
EBV BLCL if has not been established yet and 
generation of WT1-specific T cell lines 
X 
• Screening will take approximately 12-16 weeks 
• 
X 


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IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
a Research Labs = 4 green- top tubes with heparin (WT1 CTLp and EBV CTLp by LDA, TET, Intracellular IFN)  with 
labels, date and time.  These should be drawn prior to infusion on treatment days and 24 hours after the first, 
fourth, fifth, sixth and eighth infusion, if applicable. 
b Standard MSKCC guidelines to be followed for laboratory studies pre-cyclophosphamide. These should include a 
cbc and comprehensive panel within 7 days prior to cyclophosphamide. 
c Phone assessments have a +72hour window to contact patients, if permissible 
d From cycle 1 day 8  onwards a potential +/- 72 hour treatment window is permissible provided that 
necessary assessments and laboratory tests can be still carried out. 
* The first 2 patients on study were treated per protocol versions 06-155A(3) and 06-155A(4).  One patient was 
treated per protocol 06-155A(5). The next 3 patients were treated per protocol version 06-155A(6). 
 
X = All Dose Levels (i.e. 1, 2, 3, 4)   Y = Dose Levels 2, 3 and 4 
 
11.1 
TOXICITIES/SIDE EFFECTS 
 
 
11.2 
Risks of WT1 peptide-specific T cell infusions 
 
• Since WT1 is expressed at low levels on normal kidney cells and marrow 
stem cells, there is a small but real risk of inducing nephritis or extended 
marrow suppression. Patients with AML in remission often have detectable 
levels of WT1 specific CTLs, yet do not have renal impairment or marrow 
 
Prior to Cycle 1 
Cycle 1d 
Cycle 2+ 
Tumor 
Assessment 
Follow-up Visits 
 
Time D=Day 
Within 30 
days of D1 D-1 D0 D1 D2 D3 D8 D15 D16 D17 D22 D1 D2 D3 D15 D16 D17 
After every 
2 Cycles 
4 weeks 
after final 
infusion 
8 weeks 
after final 
infusion 
WT1 peptide- 
specific T cell 
infusion 
X 
X 
X 
X 
Cyclophosphamide 
Y 
History, Physical & 
Toxicity 
Assessment 
X 
Y 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Phone Assessment c 
X 
X 
X 
X 
CBC 
X 
Yb 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
COMP 
X 
Yb 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
MAG 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PT/PTT 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Creatinine 
Clearance 
(estimated) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Ca-125 
X 
X 
X 
X 
X 
X 
X 
Research Labs: 
4 Green-Top tubes 
(RNB) 
Y 
X 
X 
X 
X 
X 
X 
Xa 
X 
Xa 
X 
X 
EKG 
X 
CT Scan 
X 
X 


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suppression (46). Furthermore, initial trials in 26 patients evaluating 
vaccination with two of the WT1 peptides for future use in this study have not 
detected kidney or marrow dysfunction in those patients who generated WT1 
specific responses, except in two patients with MDS who developed 
leukopenia. In these patients, the leukopenia was ascribed to suppression of 
the WT1+ malignant MDS clone (98). Based on these studies, the risks of 
adoptively transferred WT1 peptide specific T cells are expected to be 
acceptably low. However, in this phase I trial, patients will be closely 
monitored by serial creatinine clearances and serum chemistries to detect renal 
toxicities and by serial blood counts to assess for myelosuppression. If 
extended (>20 days) count depressions are observed and are associated with 
high levels of WT1 specific CTL in the circulation, the patients may be 
considered for treatment with steroids and/or ATG (anti-thymocyte globulin) 
to eliminate the T cells. 
• There is a finite risk that the T cells generated in vitro could transmit an 
infection. Each dose of the T cells is tested extensively for microbial 
contaminants prior to release. In addition, the doses are tested for endotoxin. 
These tests are in full compliance with FDA guidelines. Therefore, the risk is 
expected to be small. Furthermore, T cells that do not meet sterility testing 
requirements will not be released for use. 
 
• During infusion of activated T cells, a small proportion of patients may 
develop fever or hypotension. This will be treated with Tylenol and fluid 
boluses as required. There is a small risk of hypersensitivity and/or 
anaphylaxis during infusion of the T cells. If this occurs, the infusion would 
be  stopped  and  appropriate  measures  taken  to  treat  the  symptoms  (fluid 
boluses, steroids, antihistamines, etc.). 
 
11.3 
RISKS OF CYCLOPHOSPHAMIDE 
 
• Nausea, vomiting (patients will receive antiemetics prior to and during 
treatment to prevent this side-effect) and diarrhea 
 
• Acute water retention due to inappropriate antidiuretic hormone release 
(hydration will be closely monitored and acute water retention treated with 
diuretics; hydration will be maintained to insure adequate urine output) 
 
• Cardiomyopathy (pretreatment cardiac function will be assessed to define 
eligibility; patients will be monitored during treatment to insure against 
cardiac 
dysfunction; 
at 
these 
doses, 
cyclophosphamide 
induced 
cardiomyopathy is rare) 
 
• Hemorrhagic cystitis 


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• Fatigue, loss of fertility, nail changes, mouth sores, secondary malignancies 
 
• Infection or bleeding induced during count nadirs. This anticipated risk is low 
because the count depressions that observed with this protocol rarely extend 
10 days post treatment. 
 
• Risk of opportunistic infections due to viruses such as CMV, EBV or 
Pneumocystis. 
The 
level 
of 
immunosuppression 
induced 
by 
cyclophosphamide is transiently severe. Cumulative evidence suggests that 
the risk of opportunistic infections is moderate. Patients will be closely 
monitored by PCR amplified techniques for viral antigenemia and treated if it 
develops. 
 
Table 8: Common Toxicities Related to Cyclophosphamide 
 
Hematologic 
leukopenia, thrombocytopenia, anemia 
Gastrointestinal 
anorexia, nausea and vomiting (common with doses > 600mg/m2), diarrhea 
Hepatic 
hepatitis, elevations in SGOT and SGPT 
Renal 
hemorrhagic cystitis 
Cardiac 
cardiotoxicity with high doses (> 120 mg/kg) 
Respiratory 
high doses may cause interstitial pulmonary fibrosis 
Dermatologic 
alopecia, facial flushing 
Miscellaneous 
nasal stuffiness, syndrome of inappropriate anti-diuretic hormone (SIADH) 
induced hyponatremia, fever after high-dose therapy 
 
 
 
 
 
11.4 
RISKS OF LEUKAPHERESIS 
 
• The risks of leukapheresis can involve the occasional vasovagal responses to 
venipuncture and the minimal hemodynamic alterations associated with single 
unit phlebotomies. To protect against these risks, leukaphereses will be 
conducted in the Blood Bank Donor Room with full medical and nursing 
supervisors and support systems to prevent and/or address adverse events. 
 
 
12.1 
CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT 
 
Response: We will evaluate each patient for evidence of tumor response by RECIST 1.1 
criteria, as a preliminary indication of this treatment’s therapeutic potential.  Parameters 


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IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
Please refer to the following reference for complete details: Eisenhauer EA, Therasse P, 
Bogarerts J, et al. New response evaluation criteria in solid tumours: Revised RECIST 
guideline (version 1.1).  European Journal of Cancer 2009; 45:228-247. 
• 
Measurable disease (“Target”) is defined as at least one lesion that can 
be accurately measured in at least one dimension (longest diameter to be 
recorded).  Each lesion must be  10 mm when measured by CT (CT scan 
slice thickness no greater than 5 mm*); > 10 mm caliper measurement by 
clinical exam (lesions which cannot be accurately measured with calipers 
should be recorded as non-measurable); and > 20 mm by chest x-ray. 
 
*If CT scan with slice thickness > 5 mm is used, the minimum lesion size must have a 
longest dimension twice the actual slice thickness. 
 
• An  assessment  of  response  will  be  made  based  on  the  radiographic 
imaging studies (CT scans). 
 
Response: 
 
Complete Response (CR): Disappearance of all target and non-target lesions. Any 
pathological lymph nodes (whether target or non-target) must have reduction in short axis 
to <10 mm. Normalization of CA125, if elevated at baseline. 
 
Partial Response (PR): At least a 30% decrease in the sum of diameters of target lesions, 
taking into reference the baseline sum diameters. 
 
Progressive Disease (PD): At least a 20% increase in the sum of diameters of target 
lesions, taking as reference the smallest sum on study (this includes the baseline sum if 
that is the smallest on study). In addition to the relative increase of 20%, the sum must 
also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or 
more NEW lesions is also considered progression. Guidance on when a lesion is to be 
considered new is provided in the above cited reference). Unequivocal progression of 
existing non-target lesions is also considered progression (a detailed description and 
examples of unequivocal progression of existing non-target lesions is provided in the 
above cited reference). 
 
For equivocal findings of progression (e.g. very small and uncertain new lesions; cystic 
changes or necrosis in existing lesions), treatment may continue until the next scheduled 
assessment.  If at the next scheduled assessment, progression is confirmed, the date of 
progression should be the earlier date when progression was suspected. 
 
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum diameters while on study. 


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Not evaluable (NE) is when no imaging/measurement is done at all at a particular time 
point.  The patient is not evaluable (NE) at that time point. 
 
Early death is defined as having NO repeat tumor assessments following initiation of 
study therapy resulting from the death of the patient due to disease or treatment. 
 
Patients with global deterioration of health status requiring discontinuation of treatment 
without objective evidence of disease progression at that time will be recorded as 
‘symptomatic deterioration’. Every effort should be made to document objective 
progression even after discontinuation of treatment. 
 
Confirmation of response (for both CR and PR): Complete or partial response may 
only be claimed if the criteria for each are met at a subsequent time point (> 4 weeks 
later) in studies with a primary endpoint that includes response rate. When response rate 
is a secondary endpoint (e.g. randomized phase II or III studies with progression-free 
survival or overall survival as primary endpoint) confirmation is NOT required. 
 
Special note on lymph nodes: Lymph nodes identified as target lesions should always 
have the actual short axis measurement recorded (measured in the same anatomical plane 
as the baseline examination), even if the nodes regress to below 10 mm on study. This 
means that when lymph nodes are included as target lesions, the ‘sum’ of lesions may not 
be zero even if complete response criteria are met, since a normal lymph node is defined 
as having a short axis of < 10 mm. For PR, SD and PD, the actual short axis 
measurement of the nodes is to be included in the sum of target lesions. 
 
Special note on target lesions that become ‘too small to measure’: While on study, all 
lesions (nodal and non-nodal) recorded at baseline should have their actual measurements 
recorded at each subsequent evaluation, even when very small (e.g. 2 mm). However, 
sometimes lesions or lymph nodes which are recorded as target lesions at  baseline 
become so faint on CT scan that the radiologist may not feel comfortable assigning an 
exact measure and may report them as being ‘too small to measure’. When this occurs it 
is important that a value be recorded on the D2M form. If it is the opinion of the 
radiologist that the lesion has likely disappeared, the measurement should be recorded as 
0 mm.  If the lesion is believed to be present and is faintly seen but too small to measure, 
a default value of 5 mm should be assigned (Note: It is less likely that this rule will be 
used for lymph nodes since they usually have a definable size when normal and are 
frequently surrounded by fat such as in the retroperitoneum; however, if a lymph node is 
believed to be present and is faintly seen but too small to measure, a default value of 5 
mm should be assigned in this circumstance as well). 
 
Evaluation of best overall response is according to Tables 9.0-9.1: 
 
Table 9.0: Time point response: patients with target (+/– non-target) disease 


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Target lesions 
Non-target lesions 
New lesions Overall response 
CR 
CR 
No 
CR 
CR 
Non-CR/non-PD 
No 
PR 
CR 
Not evaluated 
No 
PR 
PR 
Non-PD or not all evaluated 
No 
PR 
SD 
Non-PD or not all evaluated 
No 
SD 
Not all evaluated Non-PD 
No 
NE 
PD 
Any 
Yes or No 
PD 
Any 
PD 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
Table 9.1: Time point response: patients with non-target disease only 
 
Non-target lesions 
New lesions 
Overall response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD 
Not all evaluated 
No 
NE 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
 
Duration of response is defined as the time measurement criteria are first met for CR/PR 
until the first date that recurrent or progressive disease is objectively documented (taking 
as reference for progressive disease the smallest measurement recorded on study). 
 
Duration of stable disease is measured from the start of the treatment (in randomized 
trials, from the date of randomization) until the criteria for progression are met, taking as 
reference the smallest sum on study (if the baseline sum is the smallest, this is the 
reference for calculation of PD). 
 
Progression-Free Survival is the period from study entry until recurrent or progressive 
disease is objectively documented (taking as reference for progressive disease the 
smallest measurement recorded on study), death or date of last contact. 


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Survival is the observed length of life from entry into the study to death or the date of last 
contact. 
 
 
13.1 
CRITERIA FOR REMOVAL FROM STUDY 
 
In accordance with the Declaration of Helsinki, ICH Good Clinical Practice Guidelines, 
and the US FDA Regulations, a patient has the right to withdraw from the study at any time 
for any reason without prejudice to his/her future medical care by the physician or at the 
institution. The Investigators also have the right to withdraw patients from the study (see 
below). Should a patient (or a patient’s legally authorized representative) decide to withdraw, 
all efforts will be made to complete and report the observations as thoroughly as possible. 
A complete final evaluation should be made at the time of the patient’s withdrawal with an 
explanation of why the patient is withdrawing, and an attempt should be made to perform a 
follow-up evaluation. 
 
Patients may be removed from study if one or more of the following events occur: 
• 
Significant noncompliance by the patient 
• 
Patient refusal to continue treatments or observations 
• 
Unacceptable toxicity 
• 
Progressive disease that in the Investigator’s opinion requires therapeutic 
intervention that would interfere with the interpretation of results from the study 
• 
Decision by the Investigator that termination is in the patient’s best medical 
interest 
• 
The stock of the patient-specific WT1 specific T cells has been 
exhausted 
• 
Unrelated medical illness or complication 
• 
Lost to follow-up 
 
14.0 
BIOSTATISTICS 
 
This is a phase I dose escalating trial designed to identify tolerable, clinically active doses 
of Wilms’ tumor gene (WT1) peptide sensitized T cells when administered alone or with 
non-myelosuppressive chemotherapy in patients with recurrent or persistent, measurable 
WT1+ ovarian, primary peritoneal, or fallopian tube carcinomas. The dose escalation 
scheme is detailed above.   Using this dose escalation scheme, escalation to the next 
treatment Level (Levels I → II → III→ IV) is probable if the risk of DLT is low, and the 
likelihood of escalations decreases as the risk of DLT increases, as demonstrated in Table 
10: 


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Table 10: Dose Escalation/Toxicity Risk 
 
 
 
 
 
True risk of Toxicity 
.10 
.20 
.30 
.40 
.50 
.60 
Probability of Escalation 
.91 
.71 
.49 
.31 
.17 
.08 
Six patients (dose levels I and II) have already been treated on the protocol. A maximum 
of 18 patients will be treated. It is anticipated that this study will accrue 4-6 patients per 
year and will last approximately 4 years. At the end of this study, a full phase II trial will 
be initiated with the dose of WT1 specific T cells selected on the results of this phase I 
study. 
 
The secondary aims of the study, 3 and 4, will be addressed by descriptive exploratory 
statistical analyses, since the sample size is not known in advance and there are no formal 
hypotheses being tested. These analyses may include descriptions of time patterns for 
continuous variables measured repeatedly, both on the individual level and aggregated by 
dose level. 
 
15.1 
SUBJECT REGISTRATION AND RANDOMIZATION PROCEDURES 
 
15.2 Subject Registration 
 
 
Confirm eligibility as defined in the section entitled Criteria for Patient/Subject 
Eligibility. 
 
Obtain informed consent, by following procedures defined in section entitled Informed 
Consent Procedures. 
 
During the registration process registering individuals will be required to complete a 
protocol specific Eligibility Checklist. 
 
All participants must be registered through the Protocol Participant Registration (PPR) 
Office at Memorial Sloan-Kettering Cancer Center. PPR is available Monday through 
Friday from 8:30am – 5:30pm at 646-735-8000. The PPR fax numbers are (646) 735- 
0008 and (646) 735-0003. Registrations can be phoned in or faxed. The completed 
signature page of the written consent/verbal script and a completed Eligibility Checklist 
must be faxed to PPR. 
 
16.1 
DATA MANAGEMENT ISSUES 
 
A Research Study Assistant (RSA) will be assigned to the study. The responsibilities of 
the RSA include project compliance, data collection, abstraction and entry, data 
reporting, regulatory monitoring, problem resolution and prioritization, and coordinate 


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the activities of the protocol study team. 
 
The data collected for this study will be entered into a secure database. Source 
documentation will be available to support the computerized patient record. 
 
16.2 
QUALITY ASSURANCE 
Weekly registration reports will be generated to monitor patient accruals and 
completeness of registration data. Routine data quality reports will be generated to assess 
missing data and inconsistencies. Accrual rates and extent and accuracy of evaluations 
and follow-up will be monitored periodically throughout the study period and potential 
problems will be brought to the attention of the study team for discussion and action. 
 
Random-sample data quality and protocol compliance audits will be conducted by the 
study team, at a minimum of two times per year, more frequently if indicated. 
 
 
16.3 
DATA AND SAFETY MONITORING 
 
The Data and Safety Monitoring (DSM) Plans at Memorial Sloan-Kettering  Cancer 
Center were approved by the National Cancer Institute in September 2001. The plans 
address the new policies set forth by the NCI in the document entitled “Policy of the 
National Cancer Institute for Data and Safety Monitoring of Clinical Trials” which can be 
found at: http://cancertrials.nci.nih.gov/researchers/dsm/index.html. The DSM Plans at 
MSKCC were established and are monitored by the Office of Clinical Research. The 
MSKCC Data and Safety Monitoring Plans can be found on the MSKCC Intra net at:  
http://mskweb2.mskcc.org/irb/index.htm 
 
There are several different mechanisms by which clinical trials are monitored for data, 
safety and quality. There are institutional processes in place for quality assurance (e.g., 
protocol monitoring, compliance and data verification audits, therapeutic response, and 
staff education on clinical research QA) and departmental procedures for quality control, 
plus there are two institutional committees that are responsible for monitoring the 
activities of our clinical trials programs. The committees: Data and Safety Monitoring 
Committee (DSMC) for Phase I and II clinical trials, and the Data and Safety Monitoring 
Board (DSMB) for Phase III clinical trials, report to the Center’s Research Council and 
Institutional Review Board. 
 
During the protocol development and review process, each protocol will be assessed for 
its level of risk and degree of monitoring required. Every type of protocol (e.g., NIH 
sponsored, in-house sponsored, industrial sponsored, NCI cooperative group, etc.) Will 
be addressed and the monitoring procedures will be established at the time of protocol 
activation. 


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17.1 
PROTECTION OF HUMAN SUBJECTS 
 
17.1 Privacy 
 
 
MSKCC’s Privacy Office may allow the use and disclosure of protected health information 
pursuant to a completed and signed Research Authorization form. The use and disclosure of 
protected health information will be limited to the individuals described in the Research 
Authorization form. A Research Authorization form must be completed by the Principal 
Investigator and approved by the IRB and Privacy Board. 
 
17.2 Serious Adverse Event (SAE) Reporting 
The NCI Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0 will be used 
to identify and grade adverse events. All serious and non-serious adverse events occurring from 
the time of signing the treatment phase informed consent (consent #2) until 30 days following 
the patient’s final T cell infusion will be reported. In addition, any adverse events occurring 
outside of this window that are considered, by the investigator, related to study tests/procedures 
should also be reported. 
 
A serious adverse event (SAE) is any sign, symptom or medical condition that emerges during 
treatment or during a post-treatment follow-up period that (1) was not present at the start of 
treatment and it is not a chronic condition that is part of the patient’s medical history, OR (2) was 
present at the start of treatment or as part of the patient’s medical history but worsened in 
severity and/or frequency during therapy, AND that meets any of the following serious criteria: 
• Results in death 
• Is life-threatening 
• Requires or prolongs inpatient hospitalization 
• Is disabling 
• Is a congenital anomaly/birth defect 
• Is medically significant or requires medical or surgical intervention to prevent 
one of the outcomes listed above. 
 
Any seriousadverse events as defined above should be reported to IRB as soon as possible, but 
no later than five calendar days after notification of event. The IRB requires a CRDB Adverse 
Event (AE) report be submitted electronically to the SAE Office at sae@mskcc.org containing 
the following information: 
Fields populated from the CRDB: 
• Subject’s name 
• Medical record number 
• Disease/histology (if applicable) 
• Protocol number 


Memorial Sloan-Kettering Cancer Center 
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Amended: 10/09/12 
 
 
 
Data to enter: 
• The date the adverse event occurred 
• The adverse event 
• Relationship of the adverse event to the treatment (drug, device, or intervention) 
• If the AE was expected 
• The severity of the AE 
• The intervention 
• Detailed text that includes the following information: 
o A explanation of how the AE was handled 
o A description of the subject's condition 
o Indication if the subject remains on the study 
o If an amendment will need to be made to the protocol and/or consent form 
 
The NCI CTCAE Version 4.0 will be used to grade all adverse events. Furthermore, all 
adverse events that occur while the patients are on study and are considered possibly, 
probably, or definitely related to study treatment and/or procedures, will be collected. 
The severity and relationship to the study drug will be characterized. 
 
The PI’s signature and the date that the report is signed are required on the completed 
form. 
 
For IND/IDE protocols: 
 
The CRDB AE report should be completed as above and the FDA assigned IND/IDE 
number written at the top of the report. If appropriate, the report will be forwarded to the 
FDA by the SAE staff through the IND Office. 


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Amended: 10/09/12 
 
 
 
18.1 
INFORMED CONSENT PROCEDURES 
 
Before protocol-specified procedures are carried out, consenting professionals will 
explain full details of the protocol and study procedures as well as the risks involved to 
participants prior to their inclusion in the study. Participants will also be informed that 
they are free to withdraw from the study at any time. All participants must sign an 
IRB/PB-approved consent form indicating their consent to participate. This consent form 
meets the requirements of the Code of Federal Regulations and the Institutional Review 
Board/Privacy Board of this Center. The consent form will include the following: 
 
1. 
The nature and objectives, potential risks and benefits of the intended study. 
2. 
The length of study and the likely follow-up required. 
3. 
Alternatives to the proposed study. (This will include available standard and 
investigational therapies. In addition, patients will be offered an option of 
supportive care for therapeutic studies.) 
4. 
The name of the investigator(s) responsible for the protocol. 
5. 
The right of the participant to accept or refuse study interventions/interactions 
and to withdraw from participation at any time. 
Before any protocol-specific procedures can be carried out, the consenting professional 
will fully explain the aspects of patient privacy concerning research specific information. 
In addition to signing the IRB Informed Consent, all patients must agree to the Research 
Authorization component of the informed consent form. 
 
Each participant and consenting professional will sign the consent form. The participant 
must receive a copy of the signed informed consent form. 
 
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59. Haque T, et al.  Complete regression of posttransplant lymphoproliferative disease using 
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71. Champagne P, et al.  Skewed maturation of memory HIV-specific CD8 T lymphocytes. 
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72. Sallusto F, et al.  Two subsets of memory T lymphocytes with distinct homing potentials 
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73. Koehne G, et al.  Quantitation, selection and functional characterization of EBV-specific 
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74. Manz R, et al.  Analysis and sorting of live cells according to secreted molecules, 
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75. Otten G and RN Germain.  Split anergy in a CD8+ T cell: receptor dependent cytolysis in 
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they encounter their target antigens.J Virol. 67:44-52.  1993. 


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77. Castilleja A et al.  Accelerated HER2 degradation enhances ovarian tumor recognition by 
CTL.  Implications for tumor immunogenicity. Mol Cell Biochem 217:21-33. 2001. 
78. Baral E, et al.  Antiestrogens sensitize human ovarian and lung carcinomas for lysis by 
autologous killer cells.  Anticancer Res 20:2027-31.  2000. 
79. Knabel M, et al.  Reversible MHC multimer staining for functional isolation of T-cell 
populations and effective adoptive transfer. 
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80. Bourgault I, et al.  Limiting-dilution analysis of the HLA restriction of anti-Epstein-Barr 
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81. Picker IJ, et al.  Direct demonstration of cytokine synthesis heterogeneity among human 
memory/effector T cells by flow cytometry.  Blood. 86:1408-19.  1995. 
82. Morel PA, et al.  Crossregulation between Th1 and Th2 cells.  Crit. Rev. Immunol. 
18:275-303.  1998. 
83. Allen HJ, et al.  Isolation and morphologic characterization of human ovarian carcinoma 
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84. Crickard K, et al.  Borderline malignant serous tumors of the ovary maintained on 
extracellular matrix: evidence for clonal evolution and invasive potential. Cancer Genet. 
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85. Counter, CM, et al.  Telomerase activity in human ovarian carcinoma.  PNAS.  91:2900- 
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86. Van Gool SW, et al.  CD28 ligation by monoclonal antibodies of B7/BB1 provides an 
accessory signal for the cyclosporin A-resistant generation of cytotoxic T cell activity. J 
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87. Dohring C, et al. T-helper- and accessory-cell-independent cytotoxic responses to human 
tumor cells transfected with B7 retroviral vector.  Int J Cancer 57:754-9.  1994. 
88. Ghamande S, et al.  Recombinant CD40 ligand therapy has significant antitumor effects 
on CD40-positive ovarian tumor xenografts grown in SCID mice and demonstrates an 
augmented effect with cisplatin.  Cancer Res  61:7556-62.  2001. 
89. Schumacher U, et al.  Transplantation of a human ovarian cystadenocarcinoma into 
severe combined immunodeficient (SCID) mice--formation of metastases without 
significant alteration of the tumour cell phenotype. 
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90. Haber DA, Buckler AJ, Glaser T, et al. An internal deletion within an 11p 13 zinc finger 
gene contributes to the development of Wilms tumor. Cell 1990;61:1257-69. 
91. Alberta JA, Springett GM, Rayburn H, Natoli TA, Loring J, Kreidberg JA, Housman 
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1996;88:2267-78. 
93. Inoue K, Sugiyama H, Ogawa H, et al. WT1 as a new prognostic factor and a new marker 
for the detection of minimal residual disease in acute leukemia. Blood 1994;84:3071-9. 
94. Schmid D, Heinze G, Linnerth B, et al. Prognostic significance of WT1 gene expression 
at diagnosis in adult de novo myeloid leukemia.  Leukemia 1997;11:639-43. 


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95. Cilloni D, Gottardi E, Messa F, et al; Piedmont Study Group on Myleodysplastic 
Syndromes.  Significant correlation between the degree of WT1 expression and the 
International Prognostic Scoring System Score in patients with myelodysplastic 
syndromes.  J Clin Oncol 2003;21:1988-95. 
96. Mi Y, Wang L, Bian S, Meng Q, Chen G, Wang J.Effect of WT1 gene expression on cell 
growth and proliferation in myeloid leukemia cell lines. 
Chin Med J (Engl). 1999 Aug;112(8):705-8. 
97. Faber LM, van der Hoeven J, Goulmy E, Hooftman-den Otter AL, van Luxemburg-Heijs 
SA, Willemze R, Falkenburg JH.  Recognition of clonogenic leukemic cells, remission 
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histocompatibility antigen-specific cytotoxic T lymphocytes. 
J Clin Invest. 1995 Aug;96(2):877-83. 
98. Oka Y, et al.  Wilms tumor gene peptide-based immunotherapy for patients with overt 
leukemia from myelodysplastic syndrome (MDS) or MDS with myelofibrosis. 
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99. Mackensen A, Meidenbauer N, Vogl S, et al. Phase I study of adoptive T-cell therapy 
using antigen-specific CD8+ T cells for the treatment of patients with metastatic 
melanoma. J Clin Oncol. 2006 Nov;24(31):5060-9. 
100. Kershaw MH, Westwood JA, Parker LL, et al. A phase I study on adoptive 
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101. Trivedi D, Williams RY, O’Reilly RJ, Koehne G. Generation of cytomegalovirus 
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overlapping pentadecapeptides for adoptive immunotherapy.  Blood. 2005April 1 ; 105 
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that bind class I major histocompatibility molecules.  Eur J Immunol 30:1676-1682, 
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103. Kozlowski S, Corr M, Shirai M, et al. Multiple pathways are involved in the extracellular 
processing of MHC class I-restricted peptides.  J Immunol 151:4033-4044. 
104. Weiss MA, Glenn M, Maslak, et al. Consolidation therapy with high-dose 
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105. Thigpen JT, Blessing JA, Vance RB, Lambuth BW. Chemotherapy in ovarian carcinoma: 
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106. Ohno, S., S. Kyo, et al. (2009). "Wilms' tumor 1 (WT1) peptide 
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transplantation." Seminars in immunology 22(3): 162-172. 


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Appendix A: The T cells of the donor sensitized with the pool generated in response to 
subpools 10 and 16, suggesting reactivity against pentadecapeptide 60. 
 
 
 
 
 
Appendix A 


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Appendix B-1 and B-2 
 
 
 
Appendix B-1 
Appendix B-2 
Appendix B-1 


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Appendix C-1 and C-2 
 
 
 
Appendix C 
C-2 
C-1 


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APPENDIX D:  Generation of WT1-Specific Cytotoxic T-Lymphocytes (CTL) 
 
STANDARD OPERATING PROCEDURE 
ADOPTIVE IMMUNE CELL THERAPY FACILITY 
Memorial Sloan-Kettering Cancer Center 
Document No. 
03-II-1.02. 
Effective Date: 
11/06/2009 
Previous Date: 
09/20/2007 
Page 1 of 10 
Title: Generation of WT1-Specific Cytotoxic T-Lymphocytes (CTL) 
Approval: 
Facility Director 
QA Manager 
 
1. Purpose: 
 
1.1 WT1-specific CTL lines are generated from normal donors or recipients of T cells by co- 
culture with irradiated autologous cytokine activated monocytes (CAM) or EBV- 
transformed B cell lines (LCL). Under the culture conditions applied, outgrowing CTL 
should be WT1 or WT1/EBV specific. The CTL lines exhibiting WT1-specific responses 
are infused into patients with WT1-expressing malignancies. 
 
1.2 Responsibility: 
 
1.2.1. Technicians are responsible for the sample receipt, appropriate sample labeling, exact 
accomplishing of the procedures listed in the SOP, reporting the results to the 
Facility Director, keeping the records of all the procedures applied to the 
component during its processing/culturing. 
 
1.2.2. 
Facility Supervisor is responsible for maintaining the equipment to meet the 
requirements of the Quality Management Plan, ordering the necessary equipment, 
supplies and reagents, designate and re-distributes the work load in the facility, 
assures that the planned processes are performed in the facility according to the 
schedule. 
 
1.2.3. 
QA Manager is responsible for the control of all the procedures to be performed 
according to the Standard Operating Procedures, data entries, inventory, revision of 
the batch records, evaluation of the quality of the components produced, validation 
of the procedures, revision of the SOPs,, validation of the reagents, training of the 
personnel, evaluation of the personnel professional education level. QA Manager 
 
insures quality and safety of reagents and supplies used in the manufacturing 
procedures by obtaining the appropriate documentation (certificates of analysis and 
recipes of formulation where applicable) and conducting functional assays (where 
applicable). 


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1.2.4. Facility Director is responsible for the dedication of the procedures to be applied to 
the samples received, individual changes in the protocols, if needed for the special 
cases, reports of the results to the attendings and Medical Director of the AICT 
Facility. 
 
2. Definition and Abbreviations: 
 
2.1. CTL 
- Cytotoxic T-lymphocytes 
 
2.2. PBL 
- Peripheral blood lymphocytes 
 
2.3. PBMC 
- peripheral blood mononuclear cells 
 
2.4. LDS 
- Low density separation 
 
2.3. EBV 
- Epstein Barr Virus 
 
2.4. EBV BLCL 
- EBV transformed B lymphocyte cell line 
 
2.5. WT1 
- Wilm’s tumor antigen 1 
 
2.6. BLCL 
- B Lymphoblastoid Cell Lines 
 
2.7. CAM 
- Cytokine-activated monocytes 
 
2.8. LSM 
- Lymphocyte separation media 
 
2.9. YH5 
- YSSEL’s medium containing 5% Human serum 
 
2.10. YH10 
- YSSEL’s medium containing 10% Human serum 
 
 
3. Materials and Equipment: 
 
3.1. Specimen 
 
3.1.1. NK depleted PBL from normal donor or T cell recipient 
 
3.1.2. PBMC from the same normal donor or T cell recipient 
 
3.1.3. CD3 enriched PBL from the same donor or T cell recipient 


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3.1.4. Lymphoblastoid Cell Lines (LCL) prepared from the PBMC of the same individual 
according to procedure “GENERATION OF B95-8 EBV TRANSFORMED 
LYMPHOBLASTOID CELL LINES (LCL)” and 
cultured for >2 weeks in 100M acyclovir. 
 
3.1.5. CAMs  prepared from the PBMC of the same donor according to SOP 02-I-1.02. 
“Generation of cytokine activated monocytes” 
 
3.2. Materials 
 
3.2.1. YSSEL’s 
Gemini 
 
3.2.2. Characterized human serum 
Gemini 
 
3.2.3. PBS 
Gibco 
 
3.2.4. Accuprep 
Accurate Chemical & Scientific Corporation 
 
3.2.3. 75cm flask 
NUNC 
 
3.2.4. 25cm flask 
Falkon 
 
3.2.5. Interleukin-2 (Proleukin) 
Chiron Corporation 
 
3.2.6. 15ml and 50ml centrifuge tubes 
Falcon 
 
3.2.7. Disposable pipets 
Fisher 
 
3.2.8. Conical cryovials with vented caps 
Fisher 
 
3.2.9. Synthetic overlapping pentadecapeptides 
spanning the sequence of CMV pp65 protein 
Invitrogen 
 
3.2.10. DMSO 
Edwards Lifesciences 
 
3.2.11. Interleukin-15 
Miltenyi Biotech 
 
3.3. Equipment 
 
3.3.1. Biological safety cabinet, certified 
Nuare 
3.3.2. Centrifuge 
Sorvall 
3.3.3. Incubator 
Nuare 


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3.3.4.   Irradiator (maintained by the Blood Bank of MSKCC) 
ILB437C 
 
4. Procedure: 
 
4.1.Preparation  of the total  pool  of the 141 synthetic WT1 derived pentadecapeptides 
spanning the whole sequence of WT1 protein and overlapping by 11aa: 
4.1.1. Each peptide is delivered from the manufacturer in the lyophilized form in several 
vials. The lyophilized peptides are kept at -80C until diluted. Dilute one of the 
vials of each synthetic pentadecapeptide in DMSO at a concentration 10mg/ml. 
Aliquot into conical cryovials at 35µl/vial, label each vial and freeze ay -80C. 
Use one aliquot (25µl) of each peptide for preparation of total pool and subpools 
as described below. Store the leftover volume of each peptide at -80C for future 
single peptide testing. 
 
4.1.2. Take 25µl of each peptide (250µg) and transfer into 50ml tube. 
 
4.1.3. Add 10.575ml of DMSO. Mix well. 
 
4.1.4. Label 282 conical cryovials: total pool of WT1 pentadecapeptides Invitrogen, 
50µg/20µl, 50µl/vial, date of aliquoting. 
 
4.1.5. Aliquot prepared total pool at 50µl/vial and freeze at -80C. 
 
4.2.Preparation of 24 subpools of synthetic WT1 derived pentadecapeptides: 
4.2.1. Each pool (##1-23) is made to contain 12 pentadecapeptides except pool #24 which 
is made to contain 9 pentadecapeptides only. 
 
4.2.1.1. To prepare each of 23 pools (##1-23) take 25l (250g) of each 
pentadecapeptide comprising the pool from the dilutions prepared in step 
4.1.1.and pool into one tube to have total 3000g in 300l. Then add 
900l of DMSO to make final concentration of 50g in 20l, containing 
~4.2µg of each pentadecapeptide. 
 
4.2.1.2. To prepare pool#24 take 30µl (300µg) of each peptide comprising the 
pool from the dilution prepared in step 4.1.1. and pool into one tube to 
have total 2700µg in 270µl. Add 810µl of DMSO to make the final 
concentration of 50g in 20l. This dilution contains ~5.6µg of each 
pentadecapeptide. 
 
Pool the following peptides into the following pools using the map: 
 
Pool 
# 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 


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13 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
14 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
15 
25 
26 
27 
28 
29 
30 
31 
32 
33 
34 
35 
36 
16 
37 
38 
39 
40 
41 
42 
43 
44 
45 
46 
47 
48 
17 
49 
50 
51 
52 
53 
54 
55 
56 
57 
58 
59 
60 
18 
61 
62 
63 
64 
65 
66 
67 
68 
69 
70 
71 
72 
19 
73 
74 
75 
76 
77 
78 
79 
80 
81 
82 
83 
84 
20 
85 
86 
87 
88 
89 
90 
91 
92 
93 
94 
95 
96 
21 
97 
98 
99 
100 
101 
102 
103 
104 
105 
106 
107 
108 
22 
109 
110 
111 
112 
113 
114 
115 
116 
117 
118 
119 
120 
23 
121 
122 
123 
124 
125 
126 
127 
128 
129 
130 
131 
132 
24 
133 
134 
135 
136 
137 
138 
139 
140 
141 
 
 
 
 
 
The upper horizontal row and the left vertical column contain the numbers of the pools 
made and the content of each pool is defined by the pentadecapeptides which numbers are 
presented in the corresponding horizontal or vertical rows aligned together with each pool 
number. 
 
4.3.Preparation of the antigen-presenting cells: 
 
Either CAM or EBV BLCL loaded with the same stimulating peptide(s) can be used for the 
in vitro sensitization of WT1 specific T cells 
 
4.3.1. Preparation of CAM: 
 
4.3.1.1.CAMs are generated according to the SOP 02-I-1.02. If CAMs are frozen 
thaw them in Yssel’s containing 10% of HS by spinning at 1000rpm +4C 
for 7 minutes. Discard supernatants. If fresh CAMs are used they should be 
spun down at the same conditions and supernatant should be discarded. If 
the BLCL are used for sensitization the cells also should be spun down at 
the same conditions and the supernatant should be discarded. 
 
4.3.1.2. Add 5ml of Yssel’s medium and count the cells. 
 
4.3.1.3. Calculate the number of CAMs or BLCL required for initial sensitization 
at 20:1 E:T ratio. CAMs required for subsequent restimulations are used at 
10:-4:1 E:T ratio. BLCL are used at 4:1 ratio.. 
 
4.3.1.4. To load the antigen wash CAMs or BLCL in serum free Yssel’s at 
1000rpm +4C for 7 minutes. 


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4.3.1.5. Discard  supernatant.  Add  serum-free Yssel’s media to have final cell 
concentration 1x106cells/ml. 
4.3.1.6. Add 
50g/ml 
(20µl/ml) of complete pool of synthetic WT1 
pentadecapeptides. 
 
4.3.1.7. Incubate CAMs or BLCL with peptide for 3 or 2 hours accordingly at RT 
shaking every 15-20 minutes. 
 
4.3.1.8. After 2-3 hours of incubation irradiate peptide-loaded CAM with 6000rads 
and BLCL with 9000rads using the irradiator specified for this protocol. 
 
4.3.1.9. Wash the cells with 50ml of Yssel’s for 7 minutes at 1000rpm +4C before 
adding to the responder cells. 
 
4.3.10. Re-suspend in 1 ml of YH5 
 
4.3.2. Preparation of EBV BLCL: 
4.3.2.1.Prepare the EBV BLCL from the same preselected donor of WT1 specific 
T cells as described in the SOP “GENERATION OF B95-8 EBV 
TRANSFORMED LYMPHOBLASTOID CELL LINES (LCL)”. 
 
4.3.2.2. Count the EBV BLCL. 
 
4.3.2.3. BLCLs are used only for secondary restimulations at 4:1 E:T ratio. Take 
EBV BLCL in the amount sufficient to re-stimulate T cells at 4:1 
responder:stimulator ratio and spin down in YH5 at 1200rpm RT. 
 
4.3.2.4. Discard   supernatant.   Add   serum-free   Yssel’s   medium   to   a   cell 
concentration 1x106cell/ml. 
 
4.3.2.5. Add 50g/ml/1x106 cells of the stimulating WT1 peptide pool. 
 
4.3.2.6. Incubate at RT for 2 hours shaking every 20 minutes. 
 
4.3.2.7. Irradiate with 9000rads. 
 
4.3.2.8. Spin down in YH5 at 1200rpm RT. Discard supernatant. 
 
4.4. Preparation of “responder” cells 
 
4.4.1. Wash NK- and monocyte-depleted PBL prepared in the SOP ## IV-1.02 
and IV-2.02 or CD3 separated T cells prepared in the SOP ## III-1.02 
and III-1.01 with YH5 for 7 minutes at +4C 1200rpm. 


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4.4.2. Decant the supernatant and re-suspend the cells in YH5. 
 
 
 
4.4. 3. Remove 20l of cells (if needed, add 20l of 50% red cell lysis buffer) 
and count using a hemocytometer according to standard operating 
procedure. If cell concentration is 1x106 /ml, adjust to 1x106 /ml by 
adding YH5. If cell concentration is 1x106 /ml, pellet and re-suspend at 
1x106 /ml in YH5. 
4.4.4. Put the cells in 25cm2 flask. 
4.5. Mix responders and stimulators prepared in section 4.3. and 4.4. respectively in 
this SOP by adding WT1 peptide loaded irradiated CAMs and BLCL into the 25cm2 flask with 
prepared responders at 20:1 responder:stimulator ratio. 
 
4.6. Culture at +37C 5%CO2 in air for 5 days 
 
4.7. Feed on day 5 by adding the same volume of YH5. 
 
4.8. Day 7: count the CTL. If the viability of the T cells <60% and the total number of 
cells 107 the dead cells should be removed by Low Density separation: cells are 
diluted in PBS at 1:1 ratio and layered over Accuprep in 50 ml tube at 1:2 ratio. 
Cells are centrifuged at 2300rpm RT for 25 minutes. The buffy coat is collected 
into a separate 50ml tube. Tube is filled up with PBS and centrifuged at 1200rpm 
at RT for 7 minutes. Supernatant is discarded. The cell pellet is resuspended in 
YH5. Cells are counted 
 
4.9. Adjust the cell concentration with YH5 up to 1.0x106 cells/ml. Add interleukin-2 
(IL-2) at the final concentration of 10Un/ml. 
 
4.10. 1st re-stimulation is done on day 7: if the CTL line was initiated by stimulation on 
CAMs restimulate on day 7 with autologous peptide-loaded CAM at 10:1-4:1 
responder :stimulator ratio. Prepare the CAMs as described in step 4.3.1. of this 
SOP. Upon Facility Director’s decision EBV BLCL can be used as APC for 
restimulation at 4:1 R:S ratio if the number of CAMs is insufficient or the 
proliferation of T cells is slow. If the WT1 CTL line has been started by 
stimulation on BLCL all subsequent restimulatons should be done by co- 
incubation with the BLCL loaded with the WT1 total pool. In this case prepare 
the stimulators as described in step 4.3.2. of this SOP. Repeat re-stimulations 
every 7-8 days as described in this section of the SOP. 


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4.11. Culture at +37C 5%CO2 in air for 3 days and then feed with YH5 to adjust the 
concentration to 1x106 cells/ml. Add 10 units/ml of recombinant human 
Interleukin-2 (IL-2). Repeat this step every 2-3 days. If the viability of the cells 
decreases and is below 75% - increase the dose of IL-2by 5 Un/ml. Count the 
cells every 2-3 days. If the viability increases keep feeding with the same dose of 
Il-2. If the viability remains below 75% - increase the dose of IL-2 by another 5 
Un/ml. The increase of the IL-2 dose should be approved by the Facility Director 
or, in the absence of the Facilkity Director by the designated experienced senior 
technician. 
 
4.12. Starting from day 14 of the CTL line add interleukin-15 (IL-15) to the feeding 
cocktail at 10ng/ml weekly. 
 
4.13. Culture at +37C 5%CO2 and feed with YH5 and IL-2 every 2-3 days and IL-15 
and restimulation every 7 days as described in steps 4.8.-4.12. 
 
4.14. Test the cells for Interferon-gamma production and cytotoxicity approximately on 
day 34 (between days 32 and 34 ) against uutologous EBV BLCLs unmodified or 
loaded with the WT1 total pool pf pentadecapeptides autologous and recipient- 
derived PHA blasts or CAM and the same targets loaded with the stimulating WT1 
peptides as well as 
 
PHA blasts or EBV BLCL from HLA mismatched donor unmodified and loaded 
with the same WT1 peptides at 50:1 and 25:1 E:T ratio according to SOP 
“Determination of Specific Cytotoxicity of the Cytotoxic T lymphocytes”. 
 
4.15. If the cells are cytotoxic and specific, but the total cell number is not sufficient for 
the therapeutic dose expand them as described in Step 4.10-4.12. for another week 
to achieve the therapeutic dose and repeat testing between days 40 and 42 as 
described in section 4.14. of this protocol. 
 
4.16. When the sufficient number of cells for patient infusion is obtained and control 
tests meet the quality criteria, cells should be characterized and frozen according to 
SOP ”Characterization and Freezing of the Cytotoxic T lymphocytes”. Note! If 
the T cells are cytotoxic but the total dose obtained by day 42 is not sufficient for 
the therapeutic dose, the generated T cell line should be frozen for the infusion and 
super-expansion with OKT3 antibodies should be initiated according to the 
appropriate SOP. If sufficient number of PBMC/PBL is available another WT1 
specific T cell line should be initiated according to this protocol to achieve the 
final total therapeutic dose required. 
 
5. 
Notes: 
5.1.Since all cells prepared are intended for infusion to patients, it is essential to adhere to 
proper procedures to prevent misidentification or contamination of patient samples. 


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5.2. ALL flasks and centrifuge tubes must be labeled with a sticky label including patient 
name, patient AICT number, type of the culture and culture start date. UNLABELED 
MATERIAL WILL BE DISCARDED. 
 
5.3. NEVER work with more than one patient cell line at any one time. 
 
5.4. ALWAYS use medium prepared and labeled specifically for each patient’s cells. 
 
5.5. NEVER use medium to feed more than one patient’s cells 
 
5.6. Perform all steps in a certified biological safety cabinet, using aseptic technique. 
 
5.7. CHANGE gloves between the cultures for different patients 
 
5.8. 
Procedure should be validated annually for the efficiency of the generation of WT1 
specific cytotoxic T cells based on the retrospective analysis of the results of 
cytotoxicity assay, immunophenotyping and cell counts. 
 
6. References: 
6.1. Rooney CM, Smith CA, et al. Use of gene-modified virus-specific T lymphocytes to 
control Epstein-Barr-virus-related lymphoproliferation, the Lancet, 1995; 543:9-13. 
 
6.2. Heslop HE, Ng CYC, Li C, Smith CA, Loftin SK, Krance RA, Brenner MK, Rooney 
CM. Long term restoration of immunity against Epstein-Barr virus 
infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nature 
Medicine 2:551-555, 1996. 
7. Attachments: 
Forms: 
AICT-F-008 
AICT-F-009 
AICT-F-010 
AICT-F-011 
AICT-F-012 
AICT-F-013 


Memorial Sloan-Kettering Cancer Center 
IRB Protocol 
IRB#: 06-155 A(10) 
Amended: 10/09/12 
 
 
 
PROCEDURE AUTHOR, IMPLEMENTATION DATE, 
REVISION FORM 
 
 
 
 
PROCEDURE: Generation of WT1-Specific Cytotoxic T-Lymphocytes (CTL) 
 
 
 
AUTHOR 
  
(Signature) Ekaterina S.Doubrovina, M.D., PhD. 
 
Laboratory Director 
(Position) 
 
 
IMPLEMENTATION DATE:    
 
PREVIOUS VERSION:  _ 
 
REVISIONS: 
I have reviewed this protocol  
 
 
I have reviewed this protocol  
 
 
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I have reviewed this protocol  
 
 
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