A Phase I/IIa Study of TGFß Blockade in TCR-Engineered T-Cell Cancer Immunotherapy in Patients With Advanced Malignancies
Phase: PHASE1
Conditions: Advanced Fallopian Tube Carcinoma, Advanced Malignant Solid Neoplasm, Advanced Melanoma, Advanced Ovarian Carcinoma, Advanced Primary Peritoneal Carcinoma, Advanced Synovial Sarcoma, Clinical Stage III Cutaneous Melanoma AJCC v8, Clinical Stage IV Cutaneous Melanoma AJCC v8, Metastatic Fallopian Tube Carcinoma, Metastatic Melanoma, Metastatic Ovarian Carcinoma, Metastatic Primary Peritoneal Carcinoma, Metastatic Synovial Sarcoma, Pathologic Stage III Cutaneous Melanoma AJCC v8, Pathologic Stage IIIA Cutaneous Melanoma AJCC v8, Pathologic Stage IIIB Cutaneous Melanoma AJCC v8, Pathologic Stage IIIC Cutaneous Melanoma AJCC v8, Pathologic Stage IIID Cutaneous Melanoma AJCC v8, Pathologic Stage IV Cutaneous Melanoma AJCC v8, Platinum-Resistant Fallopian Tube Carcinoma, Platinum-Resistant Ovarian Carcinoma, Platinum-Resistant Primary Peritoneal Carcinoma, Stage III Fallopian Tube Cancer AJCC v8, Stage III Ovarian Cancer AJCC v8, Stage III Primary Peritoneal Cancer AJCC v8, Stage IIIA Fallopian Tube Cancer AJCC v8, Stage IIIA Ovarian Cancer AJCC v8, Stage IIIA Primary Peritoneal Cancer AJCC v8, Stage IIIA1 Fallopian Tube Cancer AJCC v8, Stage IIIA1 Ovarian Cancer AJCC v8, Stage IIIA2 Fallopian Tube Cancer AJCC v8, Stage IIIA2 Ovarian Cancer AJCC v8, Stage IIIB Fallopian Tube Cancer AJCC v8, Stage IIIB Ovarian Cancer AJCC v8, Stage IIIB Primary Peritoneal Cancer AJCC v8, Stage IIIC Fallopian Tube Cancer AJCC v8, Stage IIIC Ovarian Cancer AJCC v8, Stage IIIC Primary Peritoneal Cancer AJCC v8, Stage IV Fallopian Tube Cancer AJCC v8, Stage IV Ovarian Cancer AJCC v8, Stage IV Primary Peritoneal Cancer AJCC v8, Stage IVA Fallopian Tube Cancer AJCC v8, Stage IVA Ovarian Cancer AJCC v8, Stage IVA Primary Peritoneal Cancer AJCC v8, Stage IVB Fallopian Tube Cancer AJCC v8, Stage IVB Ovarian Cancer AJCC v8, Stage IVB Primary Peritoneal Cancer AJCC v8, Unresectable Melanoma, Unresectable Ovarian Carcinoma, Unresectable Synovial Sarcoma
Sponsor: Roswell Park Cancer Institute

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TITLE: A Phase I/IIa Study of TGFβ Blockade in TCR-Engineered T-Cell Cancer 
Immunotherapy in Patients with Advanced Malignancies.
Roswell Park Cancer Institute
Study Number:
I 258514
Initial Date:
Amendment 1:
Amendment 2:
23 July 2014
28 October 2016
21 April 2017
Amendment 3:
Amendment 4:
Amendment 5:
Amendment 6:   
07 August 2017
31 January 2018
 03 April 2018
13 July 2018
Amendment 7:
Amendment 8:
Amendment 9:
Amendment 10:
Amendment 11:
Amendment 12:
Amendment 13:
Amendment 14:
Amendment 15:
21 August 2018
14 December 2018
11 April 2019
13 November 2019
17 December 2019
30 January 2020
18 June 2020
19-August2020
19 February 2021
Principal Investigator (Clinical):
Philip McCarthy, MD
Sponsor:
Funding:
Roswell Park Cancer Institute 
Elm & Carlton Streets
Buffalo, New York 14263
716-845-1444
Philip.McCarthy@roswellpark.org
Roswell Park Cancer Institute
NIH/NCI: R01 CA164333-02
ClinicalTrials.gov# NCT02650986
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Confidentiality Statement
Any and all information presented in this document shall be treated as confidential and shall 
remain the exclusive property of the party (ies) mentioned above. The use of such confidential 
information must be restricted to the recipient for the agreed purpose and must not be disclosed, 
published, or otherwise communicated to any unauthorized persons, for any reason, in any form 
whatsoever without the prior written consent of the party (ies) above.
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List of Abbreviations
ACT, Adoptive cell transfer
AE, Adverse event
CBC, Complete blood count
CFR, Code of federal regulations
CMV, Cytomegalovirus
CR, Complete response
CRF, Case report form
CRP, C reactive protein
CT, Computer tomography
CTL, Cytotoxic T lymphocytes
DC, Dendritic cells
DLT, Dose limiting toxicity
DSMB, Data Safety and Monitoring Board 
EBV, Epstein-Barr virus
ELISPOT, Enzyme linked immunospot assay
FDA, Food and Drug Administration
[18F]FDG, [18F]fluorodeoxy-glucose
GaLV, Gibbon ape leukemia virus
GCP, Good Clinical Practices
GCV, Ganciclovir
GM-CSF, Granulocyte-macrophage colony stimulating factor
GMP, Good Manufacturing Practices
GVHD, Graft-versus-host disease
GVL, Graft-versus-leukemia
HBV, Hepatitis B virus
HCV, Hepatitis C virus
HCT, Hematopoietic cell transplantation
H&E, Hematoxylin eosin staining
HEPA, High-efficiency particulate air
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HIV, Human immunodeficiency virus
HLA, Human leukocyte antigen
ICF, Informed consent form
IBC, Institutional Biosafety Committee
i.d., Intradermal
IHC, Immunohistochemistry
irRECIST, Immune-Related Response Criteria
IU VPF, Indiana University Vector Production Facility
i.v, Intravenous
IQAC, Internal Quality Assurance Committee
IRB, Institutional Review Board
ISPRC, Internal Scientific Peer Review Committee
IL, Interleukin
LTR, Long terminal repeat
MHC, Major histocompatibility complex
MLV, Moloney Leukemia Virus
MRI, Magnetic resonance imaging
N.C.I., National Cancer Institute
NGVL, National Gene Vector Laboratory
N.I.H., National Institutes of Health
PBMC, Peripheral blood mononuclear cells
PCR, Polymerase chain reaction
PD, Progressive disease
PET, Positron emitting tomography
PFT, Pulmonary Function Test
PR, Partial response
PTT, Partial Thromboplastin Time
RAC, Recombinant Advisory Committee
RCR, Replication competent retrovirus
RECIST 1.1, Response Evaluation Criteria in Solid Tumors
RPCI, Roswell Park Cancer Institute
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SAE, Serious adverse event
SD, Stable disease
SUV, Standardized uptake value
TSH, Thyroid stimulating hormone
TCR, T cell receptor
TGFβ, Transforming Growth Factor-beta
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SYNOPSIS
Title / Phase
A Phase I/IIa Study of TGFβ Blockade in TCR-Engineered 
T-Cell Cancer Immunotherapy in Patients with Advanced 
Malignancies.
Roswell Park Cancer Institute Study 
Number
I 258514
Roswell Park Cancer Institute 
Investigator
Grant/Scientific PI: Richard Koya MD, PhD
Clinical PI: Philip McCarthy, MD
Sponsor
National Institutes of Health/ National Cancer Institute
Study Drug(S)
NY-ESO-1 TCR/ dnTGFβRII transgenic T cells
Decitabine (Cohort 4 only)
Objectives
Primary:

To evaluate the safety and feasibility of adoptive 
transfer of autologous NY-ESO-1 TCR/dn TGFβ-RII 
transgenic T-cells in combination with Decitabine
Secondary:

To measure the persistence of these genetically 
modified T cells

To study the T cell populations generated that correlates 
with higher anti-tumor responses

Assess clinical response and progression free survival
Exploratory:

Gut microbiota pre and post treatment to evaluate the 
role of microbiota on the therapeutic efficacy of the 
proposed therapy.
Study Design
This is an open label Phase I/IIa clinical trial. Patients with NY-
ESO-1-positive advanced malignancies who are HLA-A*0201-
positive, and HIV, hepatitis B and C seronegative will be 
enrolled in the study providing they meet the remaining 
eligibility criteria. Upon enrollment, patients will undergo 
leukapheresis on day -6 for T cell collection and their cells will 
be genetically engineered and expanded ex-vivo. 5 days prior to 
ACT, patients will receive a conditioning regimen consisting of 
45 mg/kg IV cyclophosphamide (adjusted body weight if > 120% 
of ideal body weight) for two days as an outpatient or inpatient, 
according to the investigator’s discretion to create space for the 
transgenic cells. The cell product will be infused fresh after lot 
release on day 0 as an inpatient. This is a 3 + (3) design dose 
escalation study with the first cohort receiving 10e7, second 
cohort 10e8 and the third cohort 10e9 NY-ESO-1 TCR/dnTGFb-
RII transgenic cells (0.5e9 or higher, up to 1e9 acceptable) as a 
single dose, calculated based on the transduction efficiency by 
tetramer assay. Cohort 4 will receive the MTD of NY-ESO-1 
TCR/dnTGFb-RII transgenic cells defined in the first 3 cohorts 
in combination with Decitabine and will follow the 3 + (3) rule. 
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There will be at least a 3 weeks staggering of infusions between 
the first two patients of each cohort in order to allow time to 
assess toxicities. Patients will be followed daily while in the 
hospital and discharged when the condition is stable as per 
clinical judgement of the treating physicians with an Absolute 
neutrophil count (ANC) of >500/uL and platelet count of > 
20,000/uL. Thereafter, weekly follow ups for 4 weeks, then at 8 
and 12 weeks, and at months 6, and 9 or until progression, 
whichever comes first.  A biopsy for NY-ESO-1 testing and 
research will be performed at baseline if archival tissue is not 
available for testing.  For cohort 4, with the addition of 
decitabine, NY-ESO-1 positivity will be recorded but is not 
required for eligibility. Optional tumor biopsies at 6 weeks post 
ACT (± 1 week) and upon progression, for research purpose may 
be done. Patients will undergo disease monitoring by CT scan at 
baseline, week 6, week 12, and at 6 months and 9 months, or at 
progression. At 9 months, the interventional portion of the 
protocol ends and long term follow up begins. Subjects who 
responded initially after the T cell infusion may be eligible 
(Section 6.3) for a second infusion at any time after disease 
progression is confirmed.
Target Accrual and Study Duration
A maximum of 27 patients at RPCI will be enrolled. The number 
of patients required is a function of the unknown dose-toxicity 
relationship. Accrual is expected to take up to 4 years.
Study Procedures
Patients who have solid tumors refractory to treatment that are 
either Stage IV or locally advanced will be considered for 
screening in this trial. Patients must be HLA-A*0201 (HLA-
A2.1) and have NY-ESO-1 positive malignancy by IHC and/or 
by RNA expression (NY-ESO-1 positivity not needed for Cohort 
4), age should be greater than or equal to 12 years old with a life 
expectancy of greater than 3 months as assessed by a study 
physician, ECOG performance status (PS) 0 or 1, have a 
minimum of one measurable lesion meeting the criteria for 
measurable disease according to Immune-Related Response 
Criteria (irRECIST), adequate bone marrow, hepatic, cardiac and 
pulmonary function determined within 30-60 days prior to 
enrollment and able to undergo leukapheresis procedure.
Patient or legal representative must understand the 
investigational nature of this study and sign an Independent 
Ethics Committee/Institutional Review Board approved written 
informed consent form prior to receiving any study related 
procedure.
Patients will be excluded from this study if there is previously 
known hypersensitivity to any of the agents used in this study, 
received systemic treatment for cancer, including 
immunotherapy, within one month prior to initiation of dosing 
within this protocol, history of, or significant evidence of risk for 
chronic inflammatory or autoimmune disease, have a known 
active infection (including HIV, HCV, HBV, CMV), have active 
brain metastasis or are pregnant or breast-feeding. 
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Statistical Analysis
This is a phase I/IIa trial, principally designed to assess the safety 
(defined by dose limiting toxicities, DLT) and feasibility 
(defined by sufficient numbers of circulating, lot-release 
confirmed T cells) of treating patients with advanced 
malignancies with escalating doses of genetically engineered T 
cells expressing NY-ESO-1 TCR together with a dnTGFb-RII 
transgene administered after lympho-depleting conditioning 
chemotherapy and in cohort 4, after decitabine and 
lympho-depleting conditioning chemotherapy. The assessment of 
safety and feasibility is based on studying cohorts of three 
subjects, with the addition of 3 subjects in case of toxicities 
meeting the definition of DLT in one of the three subjects in a 
cohort. If two or more subjects have DLT in a cohort, that cohort 
will be considered beyond the MTD and an intermediate dosing 
may be explored to better define the MTD of this combination. A 
minimum of three evaluable subjects will be treated at each of 
the three cohorts. In cohort 4, a total of  9 patients will be 
enrolled to receive the MTD of NY-ESO-1 TCR/dnTGFb-RII 
transgenic cells defined in the first 3 cohorts, in combination 
with decitabine. There is limited power to establish efficacy and 
therefore efficacy analysis will be primarily descriptive.
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INVESTIGATOR STUDY ELIGIBILITY VERIFICATION FORM
Patient Name: _______________________________________________________
Medical Record No.: _________________________________________________
Title: A Phase I/IIa Study of TGFβ Blockade in TCR-Engineered T-Cell Cancer 
Immunotherapy in Patients with Advanced Malignancies
INCLUSION CRITERIA
Yes
No
N/A
All answers must be “Yes” or “N/A” for patient enrollment.
Date
1.
Patients with solid tumors as described below: 
a)
Inoperable or metastatic (advanced) melanoma:
-
Has received, is intolerant, or refused a CTLA-4 inhibitor 
(Ipilimumab) or a PD-1 inhibitor (Nivolumab or 
Pembrolizumab) as monotherapy and/or a combination of 
Ipilumumab and Nivolumab.
-
Has received or is intolerant of a BRAF inhibitor or the 
combination of BRAF and MEK inhibitors for BRAFv600 
mutant melanoma and a PD-1 inhibitor as monotherapy or 
in combination.
b)
Inoperable or metastatic (advanced) ovarian, primary peritoneal 
or fallopian tube carcinoma:
-
Has received platinum containing chemotherapy and has 
platinum refractory or
resistant disease that has progressed on second line therapy
-
If platinum sensitive disease, should have received ≥2 lines 
of chemotherapy.
-
May have received PARP inhibitors, bevacizumab or other 
targeted VEGF inhibitor therapy
c)
Inoperable or metastatic (advanced) synovial sarcoma:
-
Should have received and progressed on ≥ two lines of 
systemic therapy
d)
Subjects with other histologies:
Must have previously received two lines of systemic standard care (or 
effective salvage chemotherapy regimens) for metastatic disease, if 
known to be effective for that disease, and have been deemed 
either non-responders (progressive disease) or have recurred.
2.
For Cohorts 1, 2 and 3 only: Patient's tumor must be positive by 
histological or molecular assay for NY-ES0-1, according to the 
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INCLUSION CRITERIA
Yes
No
N/A
All answers must be “Yes” or “N/A” for patient enrollment.
Date
screening algorithm as described in Section 9.1 under “tumor biopsy”.  
Historical results may be used.
For Cohort 4, NY-ESO-1 results will be noted but NY-ESO-1 positivity 
not required for eligibility.
3.
HLA-A*0201 (HLA-A2.1) positivity by molecular subtyping (blood 
test or buccal swab, historical documentation acceptable)
4.
Age ≥ 18 years old (Cohort 4: Age ≥ 12 years old)
5.
Life expectancy greater than 3 months assessed by a study physician
6.
Have been informed of other treatment options.
7.
A minimum of one measurable lesion defined as:

Meeting the criteria for measurable disease according to Immune-
related Response Criteria (irRECIST)

For patients with skin metastases, lesions selected as non-completely 
biopsied target lesion(s) that can be accurately measured and 
recorded by color photography with a ruler to document the size of 
the target lesion(s)
8.
No restriction based on prior treatments, but at least 4 weeks from prior 
immunotherapy or prior investigational agents. Note: Patients who have 
suffered >grade 2 irAEs during previous checkpoint inhibitor therapy 
should be excluded.
9.
ECOG Performance Status (PS) of 0 or 1 (Appendix A).
10. Must have adequate venous access for apheresis.
11. Women of childbearing potential and men must agree to use effective 
methods of birth control for the duration of the study and 6 months 
after. Methods for effective birth control include: condoms, diaphragm 
or cervical cap with spermicide, intrauterine device, and hormonal 
contraception. It is recommended that a combination of two methods be 
used.
12. Patients must have normal organ and marrow function as defined 
below:

Leukocytes:  ≥ 3,000/mcl

Absolute Neutrophil Count: ≥ 1,000/mcl

Platelets: ≥ 100,000/mcl

Total bilirubin: ≤ 1.5 ULN

AST (SGOT)/ALT (SGPT): ≤ 2.5 x institutional upper limit of 
normal

Creatinine: ≤ 2 X ULN; if creatinine > 2 X ULN, Creatinine 
clearance must be > 60 ml/min 
13. Must be willing and able to accept the leukapheresis procedure.
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INCLUSION CRITERIA
Yes
No
N/A
All answers must be “Yes” or “N/A” for patient enrollment.
Date
14. At screening, must have tissue available for NY-ESO-1 testing (if not 
previously performed) or be willing and able to undergo a fresh tissue 
biopsy.  
15. Patient or legal representative must understand the investigational 
nature of this study and sign an Independent Ethics 
Committee/Institutional Review Board approved written informed 
consent form prior to receiving any study related procedure.
16. Participant must agree to and arrange for a caregiver (age ≥ 18 years 
old) available 24 hours a day/7 days a week and arrange for lodging 
within a 45 minute drive to Roswell Park and transportation for a period 
of time after discharge from the hospital.  The exact amount of time will 
depend on the individual status as determined by the treating physician,
Investigator Signature: ___________________________________
Date: __________
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INVESTIGATOR STUDY ELIGIBILITY VERIFICATION FORM
Patient Name: _______________________________________________________
Medical Record No.: __________________________________________________
Title: A Phase I/IIa Study of TGFβ Blockade in TCR-Engineered T-Cell Cancer 
Immunotherapy in Patients with Advanced Malignancies
EXCLUSION CRITERIA
Yes
No
N/A
All answers must be “No” or “N/A” for patient enrollment.
Date
1.
Previously known hypersensitivity to any of the agents used in this 
study
2.
Currently receiving any other investigational agents. Note: Patients 
who have suffered >grade 2 irAEs during previous checkpoint inhibitor 
therapy should be excluded.
3.
Uncontrolled intercurrent illness including, but not limited to, ongoing 
or active infection, symptomatic congestive heart failure, unstable 
angina pectoris, cardiac arrhythmia, or psychiatric illness/social 
situations that would limit compliance with study requirements.  EKG 
will be done at screening.  Cardiac stress test will be done as clinically 
indicated, the specific test to be chosen at the discretion of the treating 
physician. 
4.
History of severe autoimmune disease requiring steroids or other 
immunosuppressive treatments.
5.
History of inflammatory bowel disease, celiac disease, or other chronic 
gastrointestinal conditions associated with diarrhea or bleeding, which 
in the investigator’s opinion would place the patient at an increased risk 
for adverse effect or current acute colitis of any origin. Treated cases 
with no active disease are eligible.
6.
Potential requirement for systemic corticosteroids or concurrent 
immunosuppressive drugs based on prior history or received systemic 
steroids within the last 4 weeks prior to enrollment (inhaled or topical 
steroids at standard doses or isolated use of steroids as premedication 
for medical procedures to minimize allergic reaction [e.g. CT scan dye] 
are allowed)
7.
Known active infection with HIV, Hepatitis B, Hepatitis C, or CMV. 
8.
Known cases of clinically active brain metastases (brain MRI as 
clinically indicated). Prior evidence of brain metastasis successfully 
treated with surgery or radiation therapy will not be exclusion for 
participation as long as they are deemed under control at the time of 
study enrollment.
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EXCLUSION CRITERIA
Yes
No
N/A
All answers must be “No” or “N/A” for patient enrollment.
Date
9.
Dementia or significantly altered mental status that would prohibit the 
understanding or rendering of compliance with the requirements of this 
protocol even with caregiver support.
10. Pregnancy or breast-feeding. Female patients must be surgically sterile 
or be postmenopausal for two years, or must agree to use effective 
contraception during the period of treatment and 6 months after. All 
female patients with reproductive potential must have a negative 
pregnancy test (serum/urine) within 48 hours from starting the 
conditioning chemotherapy.
11. Lack of availability of a patient for immunological and clinical follow-
up assessment.
Patient meets all entry criteria:
  Yes
  No
If “NO”, do not enroll patient in study.
Investigator Signature: ___________________________________
Date: __________
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TABLE OF CONTENTS
1
Background.........................................................................................................................21
1.2
Rationale for Immune-based Therapies for Advanced Malignancies ................................21
1.3
Lessons Learned from Targeting Tumor Associated Antigens with Immunotherapy .......21
1.4
Why Gene Therapy.............................................................................................................22
1.5
TCR Gene Therapy.............................................................................................................22
1.6
Retroviral Vector-based Human Gene Medicine ...............................................................22
1.7
NY-ESO-1 TCR-engineered Adoptive Cell Transfer (ACT) Clinical Experience ............22
1.7.1 Use of Other TCRs in Humans...........................................................................................22
1.7.2 Use of the NY-ESO-1 TCRs in Humans ............................................................................23
2
Preliminary Data.................................................................................................................24
2.1
Origin and Functionality of the NY-ESO-1 TCR...............................................................24
2.2
Use of the NY-ESO-1 TCR Retroviral Vector (MSGV1-A2aB-1G4A-LY3H10) in Human 
Subjects at the Surgery Branch/NCI..................................................................................24
3
Rationale.............................................................................................................................24
3.1
Rationale for TCR Transgenic Adoptive Cell Transfer Therapy .......................................24
3.2
Rationale for Incorporating dnTGFβRII.............................................................................25
3.3
Rationale for adding decitabine..........................................................................................25
3.4
Rationale for lowering the eligible age to ≥ 12 years old...................................................26
4
Objectives ...........................................................................................................................27
4.1
Primary Objectives .............................................................................................................27
4.1.1 Safety ..................................................................................................................................27
4.1.2 Feasibility ...........................................................................................................................28
4.2
Secondary Objectives .........................................................................................................28
4.2.1 NY-ESO-1 TCR/ dnTGFβRII Transgenic T Cell Persistence............................................28
4.2.2 Study of transgenic T cell differentiation. ..........................................................................28
4.2.3 Functional studies of transgenic T cells..............................................................................29
4.2.4 Clinical Response ...............................................................................................................29
4.3
Exploratory Objectives .......................................................................................................29
4.3.1 Gut Microbiota....................................................................................................................29
5
Methodology.......................................................................................................................30
5.1
Study Design.......................................................................................................................30
5.2
Target Accrual and Study Duration....................................................................................32
6
Patient Selection .................................................................................................................32
6.1
Inclusion Criteria ................................................................................................................32
6.2
Exclusion Criteria ...............................................................................................................34
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6.3
Eligibility Criteria for a Second Transgenic T cell infusion...............................................35
6.4
Inclusion of Women and Minorities...................................................................................35
6.5
Subject Recruitment............................................................................................................36
7
Treatment Plan....................................................................................................................36
7.1
Dosing and Administration.................................................................................................36
7.1.1 Leukapheresis for NY-ESO-1 TCR PBMC Product ..........................................................37
7.1.2 Cyclophosphamide: ............................................................................................................37
7.1.3 Decitabine...........................................................................................................................38
7.1.4 Transgenic NY-ESO-1 TCR/dnTGFβRII PBMC Infusion: Day 0.....................................38
7.1.5 Second Transgenic NY-ESO-1 TCR/dnTGFβRII PBMC Infusion....................................39
7.2
Cohort Management ...........................................................................................................39
7.3
Definition of Dose-Limiting Toxicity ................................................................................40
7.4
Dose Modifications.............................................................................................................40
7.4.1 Treatment Delay .................................................................................................................41
7.5
General Concomitant Medication and Supportive Care.....................................................41
7.5.1 Supportive Therapy on Day 0, Administration of T cells ..................................................41
7.5.2 Potential Toxicities Warranting Ablation of Adoptively Transferred T Cells ...................41
7.5.2.1 Treatment with Corticosteroids .........................................................................................42
7.5.2.2 Treatment with Tocilizumab or Infliximab/Etanercept .....................................................42
7.6
Permissible Systemic Therapies During the Protocol On-Study Period ............................49
7.7
Study Duration....................................................................................................................49
7.8
Study Discontinuation ........................................................................................................49
8
Investigational agents .........................................................................................................50
8.1
PBMC Expressing NY-ESO-1 TCR/ dnTGFβRII..............................................................50
8.1.1 Retroviral Vector NY-ESO-1 TCR/ dnTGFβRII ...............................................................50
8.1.2 PBMC Transgenic for NY-ESO-1 TCR / dnTGFβRII.......................................................52
8.2
Decitabine: Drug Information.............................................................................................56
8.3
Cyclophosphamide: Drug Information...............................................................................56
8.3.1 Mesna: Drug Information ...................................................................................................58
8.4
Laboratory Reagents...........................................................................................................59
8.4.1 OKT3 ..................................................................................................................................59
8.4.2 Interleukin-2 .......................................................................................................................59
8.4.3 RetroNectin®......................................................................................................................60
9
Study Procedures ................................................................................................................60
9.1
Screening Evaluations ........................................................................................................61
9.2
Day (-6): Leukapheresis for NY-ESO-1 TCR T cell Product. ...........................................62
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9.3
Cohort 4 only: Days -6 to -4 Decitabine.............................................................................62
9.4
Cohorts 1, 2 &3: Days –5 & –4; Cohort 4: Days -3 &-2 Chemotherapy Administration..62
9.5
Day 0: NY-ESO-1 TCR/dnTGFβRII T cell Infusion .........................................................63
9.6
Post Infusion Follow-Up (the week of Infusion)................................................................63
9.7
Hospital Discharge..............................................................................................................64
9.8
Post Infusion Follow-Up (Weeks 1 to 4)............................................................................64
9.9
Post Infusion Follow-up (week 6) ......................................................................................65
9.10
Post Infusion Follow-Up (Week 8 and Week 12) ..............................................................65
9.11
Post Infusion Follow-Up (6 months and 9 Months) ...........................................................65
9.12
Re-Staging Evaluation: Week 12........................................................................................66
9.13
Long-Term Follow-up Plan (Years 1 to 15).......................................................................66
9.14
Definitions ..........................................................................................................................71
9.14.1 Medical History and Physical Examination .......................................................................71
9.14.2 Previous Medications..........................................................................................................71
9.14.3 Concomitant Medications...................................................................................................71
9.14.4 ECOG Performance Status .................................................................................................71
9.14.5 Targeted Physical Examination and Vital Signs ................................................................71
9.14.6 Complete Metabolic and Hepatic Panel .............................................................................71
9.14.7 CBC with Differential.........................................................................................................71
9.14.8 HLA A201 Screening .........................................................................................................71
9.15
Research Blood Sample Collection and Processing ...........................................................72
9.15.1 Day 6 in the Morning..........................................................................................................72
9.15.2 Immune Monitoring Analysis (blood for cell analyses).....................................................72
9.15.3 Serum Analyses ..................................................................................................................72
9.16
Pathology ............................................................................................................................73
9.16.1 Screening Tumor Tissue Requirements..............................................................................73
9.16.2 On Study Tumor Tissue Requirements...............................................................................74
9.17
Gut Microbiota....................................................................................................................75
9.17.1 Fecal microbiome collection...............................................................................................75
9.17.2 Questionnaires ....................................................................................................................76
10
Efficacy Evaluations...........................................................................................................76
10.1
Objective Tumor Response.................................................................................................76
10.2
Definition of Target and Non-Target Lesions ....................................................................76
10.2.1 Target Lesions ....................................................................................................................76
10.2.2 Non-Target Lesions ............................................................................................................77
10.3
Lesions................................................................................................................................77
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10.3.1 Follow-Up...........................................................................................................................78
10.4
irRECIST Overall Tumor Assessments..............................................................................79
10.5
Confirmation Measurement................................................................................................80
10.6
Guidelines for Evaluation of Measurable Disease..............................................................80
11
Safety Evaluation................................................................................................................81
11.1
Adverse Events ...................................................................................................................81
11.1.1 Definition............................................................................................................................81
11.1.2 Grading and Relationship to Drug......................................................................................82
11.1.3 Reporting Adverse Events ..................................................................................................83
11.2
Routine AE’s will be reported from the start date of intervention.  Refer to section 9.12 
for duration of follow-up AE reporting.  Serious Adverse Events....................................83
11.2.1 Definition............................................................................................................................83
11.2.2 Reporting Serious Adverse Events .....................................................................................84
11.3
Investigator Reporting: Notifying the Study Sponsor ........................................................84
11.4
Follow-Up for Serious Adverse Events..............................................................................85
11.5
Unanticipated Problems......................................................................................................85
11.5.1 Definition............................................................................................................................85
11.5.2 Reporting Unanticipated Problems.....................................................................................85
11.6
FDA Reporting ...................................................................................................................86
11.7
Study Stopping and Non-stopping Rules............................................................................87
11.8
Gene Therapy Study Reporting of Serious Adverse Events...............................................87
12
Data and Safety Monitoring................................................................................................88
13
Statistical Methodology......................................................................................................88
13.1
Sample Size Determination ................................................................................................89
13.2
Definition of Evaluable Patients.........................................................................................89
13.3
Primary Study Endpoints....................................................................................................89
13.3.1 Safety ..................................................................................................................................89
13.3.2 Feasibility ...........................................................................................................................89
13.4
Secondary Study Endpoints................................................................................................89
13.4.1 Transgenic Cell Persistence................................................................................................89
13.4.2 Evaluation of NY-ESO-1 TCR Transgenic Cell Tumor Trafficking .................................91
13.4.3 Antitumor Activity..............................................................................................................91
13.5
Efficacy Analysis................................................................................................................91
13.6
Safety Analysis ...................................................................................................................91
13.7
Adverse Event.....................................................................................................................92
13.8
Interim Analysis and Criteria for Early Termination of the Study.....................................92
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14
Ethical and Regulatory Standards.......................................................................................92
14.1
Ethical Principles................................................................................................................92
14.2
Informed Consent ...............................................................................................................93
15
Study Responsibilities ........................................................................................................93
15.1
Data Collection ...................................................................................................................93
15.2
Maintenance of Study Documents......................................................................................94
16
Administrative Rules ..........................................................................................................94
16.1
Revisions to the Protocol....................................................................................................94
16.2
Termination of the Study....................................................................................................94
16.3
Confidentiality ....................................................................................................................94
17
Appendices .........................................................................................................................95
18
References.........................................................................................................................112
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IN-TEXT TABLES
Table 1
Dose Escalation for NY-ESO-1 TCR / dnTGFβRII Transgenic Cells........................40
Table 2
Schedule of Procedures and Observations ..................................................................68
Table 3
Guidelines for Routine Adverse Event Reporting for Phase 1 Studies (Regardless of 
Expectedness) ................................................................................................................................83
Table 4
Guidelines for Routine Adverse Event Reporting Phase 2 Studies (Regardless of 
Expectedness) ................................................................................................................................83
Table 5
Criteria to Declare the Study Unfeasible.....................................................................89
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APPENDICES
Appendix A
ECOG Performance Status Scores.......................................................................96
Appendix B
Procedure for Generation of Experimental Cell Therapies..................................97
Appendix C
Clinical Grade Retrovirus Lot Release Criteria ...................................................98
Appendix D
Retrovirus Transduction and Cell Expansion Method.......................................100
Appendix E
In-process Testing and Final Product Lot Release Testing................................102
Appendix F
Final Product Testing and Lot Release Criteria.................................................103
Appendix G
Stool Sample Collection Questionnaire .............................................................105
Appendix H
Microbiome Collection Questionnaire...............................................................107
Appendix I
    Stool Collection Instructions..............................................................................109
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1
BACKGROUND
1.1
NY-ESO-1 Antigen as a Target for Immunotherapy
NY-ESO-1 belongs to a family of genes referred to as Cancer-Testis Antigens (1). CTAs are 
generally only expressed embryonically and are not expressed in adult somatic tissues, but can be 
re-expressed in malignancies(2). This is precisely the case for NY-ESO-1. Studies have indicated 
that the NY-ESO-1 protein is only expressed in male spermatogonia, but evidence for its 
expression in all other adult somatic tissues has not been found (3-6). On the other hand, the NY-
ESO-1 protein has been observed to range in expression from 10-40% across a wide range of 
malignancies, and >80% in synovial sarcoma (5, 7-9). At Roswell Park Cancer Institute, Dr. Kunle 
Odunsi (co-investigator for this grant), detected expression of NY-ESO-1 in epithelial ovarian 
cancer by RT-PCR and/or IHC in 82 of 190 (43%) specimens (8). Together, these observations 
indicate that NY-ESO-1 is an ideal target for cellular based immunotherapies. In addition, a lack 
of MHC-I in the testes greatly reduces the potential for TCR based immunotherapeutics to induce 
orchitis (10).
1.2
Rationale for Immune-based Therapies for Advanced Malignancies
Progress in our understanding of how the immune system recognizes and kills cancer cells has led 
to the notion that high levels of circulating, tumor-antigen-specific T cells, may be required for 
effective antitumor responses. Active immunotherapy with several forms of cancer vaccines has 
shown that antigen-specific T cells can be activated and occasionally lead to antitumor responses 
(11, 12). However, the circulating levels of antigen-specific T cells and tumor response rates are 
much lower than desirable (13).
Adoptive transfer of clonally-expanded, tumor antigen-specific lymphocytes to lymphopenic hosts 
after nonmyeloablative conditioning chemotherapy has resulted in cell proliferation and persistent 
clonal repopulation (14). This approach has resulted in an objective response rate of over 50% in 
patients with metastatic melanoma, that holds up in larger groups of patients (15). The major 
limitation for the broad clinical applicability of this approach is the requirement for large-scale ex 
vivo lymphocyte culture expansion (up to 1011 cells) (14), which restricts this approach to a highly 
select group of patients.
1.3
Lessons Learned from Targeting Tumor Associated Antigens with Immunotherapy
Multiple lines of evidence suggest that most tumor responses to immunotherapy involve the 
activation of tumor antigen-specific CD8+ cytotoxic T lymphocytes (CTL) (16). These immune 
cells have a high affinity receptor, the TCR that specifically recognizes an 8-10 amino acid peptide 
of its ligand in a complex within an MHC molecule. Recently a trial conducted at the NCI utilizing 
ACT directed against the NY-ESO-1 peptide demonstrated a clinical response in 5/11 melanoma 
patients (with 2 patients experiencing a complete response with 20+ and 22+ months of follow-
up) and a clinical response in 4/6 patients with synovial sarcoma (17). This provides an impetus 
for extending this type of therapy to other solid tumors without viable treatment options.
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1.4
Why Gene Therapy
The adoptive transfer of large numbers of antigen-specific T cells leads to the highest percentage 
of tumor regressions reported in patients with melanoma, but this approach is only feasible in a 
minority of patients where TIL can be cloned and expanded from melanoma metastases (14, 15). 
Genetic engineering of T cells with optimal TCRs would make this approach more broadly 
applicable. Currently, there is no other approach that would allow the generation of large numbers 
of uniformly specific T cells directed against tumor associated antigens in a short (less than one 
week) ex vivo cell manipulation. Therefore, gene transfer techniques are the only approach that 
would allow testing this concept in human subjects, as supported by preliminary data from a 
published clinical trial at the Surgery Branch/NCI (18).
1.5
TCR Gene Therapy
The transfer of TCR genes is necessary and sufficient to endow recipient T cells with the specificity 
of donor T cells (19). Genetically modified T cells carrying foreign TCRs respond to target antigen 
recognition through the transgenic TCR both in vitro and in vivo, leading to effective immune 
responses to viral and tumor challenges in murine adoptive transfer models (20). T cells redirected 
by TCR gene transfer are fully functional after transfer into mice, and have been shown to expand 
dramatically (over three logs) after encounter with their cognate antigen in vivo (21). Preliminary 
data in humans provides evidence that the same is true in patients with metastatic melanoma and 
synovial sarcoma (17, 18, 22).
1.6
Retroviral Vector-based Human Gene Medicine
Retroviral-mediated gene transfer is an efficient means for the expression of transgene into actively 
dividing primary cells. Up to 99% of T lymphocytes can be transduced if cells are non-specifically 
activated with anti-CD3 and IL-2 (18, 22).
1.7
NY-ESO-1 TCR-engineered Adoptive Cell Transfer (ACT) Clinical Experience
1.7.1
Use of Other TCRs in Humans
The Surgery Branch/NCI has published the first report of genetically engineered NY-ESO-1 TCR 
therapy for melanoma and synovial sarcoma. In addition, there are 4 currently open trials 
employing this mode of therapy.
o
NCI trial 08-C-0121 treated 17 patients using the MSGV-1G4-α95:LY retroviral vector, a 
similar vector, and the same packaging cell line proposed in the current study. This study 
enrolled patients with synovial cell sarcoma and metastatic melanoma. 5/11 melanoma 
patients experienced a clinical response, with 2 patients experiencing a long term complete 
regression with 20+ and 22+ months of follow up. In addition, 4/6 synovial cell sarcoma 
patients experienced a clinical response; one patient had a partial response that lasted 18 
months (17).
o
Currently, there are 4 accruing clinical trials which employ genetically engineered TCRs 
against NY-ESO-1 (NCI trials: 01343043, 01350401, 00670748, 01352286 and 
01697527). These trials are being conducted in patients with metastatic melanoma and 
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synovial sarcoma and are occurring at the NCI, the University of Pennsylvania, 
Washington University in St. Louis and, the University of California, Los Angeles.
1.7.2
Use of the NY-ESO-1 TCRs in Humans
NCI trial 08-C-0121 (OBA# 0712-886) uses the same NY-ESO-1 TCR and retroviral vector 
construct proposed in the current protocol (22, 23). Below we list the major similarities and 
differences between both clinical trials.
Major similarities:
o
TCR: Both clinical trials use the same MSGV-1G4-α95:LY generated by mutagenesis of 
particular TCR residues.
o
Retrovirus: Both clinical trials use the same retrovirus vector backbone.
o
Target cells: Both experiences include the transduction of activated PBMC. Since a TCR 
can only be expressed and functional on the cell surface in cells that are CD3 positive, this 
allows the TCR engineering of both CD8+ CTL and CD4+ T helper cells. Therefore, CD4+ 
T cells acquire a TCR that redirects them to recognize antigen presented by MHC class I 
molecules.
o
Clinical trial design: Primary endpoints of both are safety and feasibility with a secondary 
end point of antitumor activity. 
Major differences:
o
Incorporation of dnTGFβRII.
o
Rapid Expansion Protocol (REP): The NY ESO TCR clinical trial at the Surgery 
Branch/NCI included a 2-week ex vivo expansion after TCR engineering of lymphocytes 
following the REP protocol described by Riddell and colleagues (24). The clinical trial 
proposed will not include this forced ex vivo T cell expansion. The main reason is that 
several preclinical models suggest that extended ex vivo expansion of lymphocytes before 
adoptive transfer results in terminally differentiated cells with limited in vivo proliferation 
ability (25, 26). Therefore, TCR transduced PBMC will only be kept for up to 96 hours in 
culture after the first transduction and before re-infusing into patients.
o
Conditioning regimen: Both use a non-myelodepleting but lympho-depleting 
chemotherapy conditioning regimen but NCI uses cyclophosphamide and fludarabine and 
we propose to use only cyclophosphamide at a lower dose to allow homeostatic 
proliferation of the adoptively transferred TCR transgenic cells.
o
Cell dose for adoptive transfer: a dose escalation 3 cohort design ranging from 107 to 109 
cells, the highest dose being two logs lower than in the clinical trial conducted at the 
Surgery Branch/NCI (17), where up to 1011 TCR transgenic cells were infused to patients.
o
IL-2 dosing: The initial NCI trial used high dose IL-2 post-adoptive transfer as means to 
provide helper cytokines to support the proliferation of adoptively transferred TCR 
transgenic cells. Our trial will not include IL-2. This modification is based on preliminary 
data from subsequent trials (as presented and discussed at National Institutes of Health 
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(NIH), Office of Biotechnology Activities - T Cell Immunotherapy – Optimizing Trial 
Design, Bethesda, MD, September 10-11, 2013), which indicate that patients can achieve 
clinical responses without the need of IL-2 administration in TCR-engineered T cell ACT. 
Furthermore, administration of IL-2 has been associated with increased toxicity and there 
is evidence that high dose IL-2 may impair the generation of effective memory cells (27).
2
PRELIMINARY DATA
2.1
Origin and Functionality of the NY-ESO-1 TCR
The original retroviral vector, MSGV1-A2aB-1G4A-LY3H10, expressing the NY-ESO-1 TCR 
was developed by Drs. Steven Rosenberg and Paul Robbins from the NCI. The parental 1G4 clone 
recognizing NY-ESO-1157-165 was originally derived from phage display libraries (28). 
Modifications of the CDR regions in the alpha and beta chains of the TCR indicated that higher 
affinity NY-ESO-1157-165 recognizing TCRs could be generated. However, this development came 
with a loss of antigen specificity (29).
However, some substitutions, e.g. the (alpha95:LY) in the CD3alpha region did not engender a 
loss of antigen specificity while increasing sensitivity after being transferred into CD4+ or CD8+ 
cells. Moreover, functionally, these substitutions led to an increased responsiveness in in vitro 
assays (29). As such, the modified 1G4 clone with the alpha chain mutation was selected to be 
used in an ACT trial at the NCI in tumors expressing NY-ESO-1.
2.2
Use of the NY-ESO-1 TCR Retroviral Vector (MSGV1-A2aB-1G4A-LY3H10) in 
Human Subjects at the Surgery Branch/NCI 
Investigators at the Surgery Branch of the NCI recently reported on 17 patients with NY-ESO-1+ 
metastatic melanoma and synovial sarcoma treated with the ACT of lymphocytes expressing the 
NY-ESO-1 TCR. The Surgery Branch protocol differs with the UCLA protocol in that up to 1.3 x 
1011 TCR transgenic cells were administered, and that no DC vaccine was included. Patients in the 
NCI trial were conditioned with cyclophosphamide and fludarabine (25 mg/m2/day x 5 days). The 
protocol was feasible and safe, with no reported grade 3 or 4 toxicities. Objective clinical responses 
were seen in 4/6 patients with synovial sarcoma and 5/11 patients with melanoma (17). In this 
protocol, we will use lower cell dose and 2 days low dose cyclophosphamide regimen with no IL2.
3
RATIONALE 
3.1
Rationale for TCR Transgenic Adoptive Cell Transfer Therapy
The adoptive transfer of large numbers of clonally-expanded antigen-specific T cells into patients 
with melanoma that have received a conditioning regimen to deplete endogenous lymphocytes 
(non-myeloablative but lympho-depleting), together with high doses of interleukin-2 (IL-2), 
results in the highest rate of melanoma responses (up to 70%) reported to date (14, 15 Dudley, 
2008 #4580, 30, 31). This approach provides a proof-of-principle for adoptive transfer 
immunotherapy for metastatic melanoma, but it is difficult to implement outside of pilot studies 
given its requirement for extensive ex vivo manipulations. This roadblock may be overcome and 
may also be extended to other malignancies by the ex vivo transduction of specific TCR genes that 
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recognize tumor associated antigens. In this project we will test the hypothesis that human PBMC 
engineered to express a high affinity TCR specific for the human cancer testes antigen NY-ESO-
1 coupled with dnTGFβRII is safe, feasible and can induce objective tumor responses in human 
subjects with advanced malignancies.
3.2
Rationale for Incorporating dnTGFβRII
Tumor secreted TGFβ is a major contributor to the immunosuppressive properties of the tumor 
microenvironment(32, 33). TGFβ1 inhibits T cell function (proliferation and activation), antigen-
presentation by professional antigen-presenting cells, and promotes maintenance of T regulatory 
(Treg) cells. Most human cancers produce large amounts of active TGFβ, which also support tumor 
growth, invasion and metastasis. Based on a preclinical animal model (pmel-1/B16 melanoma), 
and an analysis of public and our data so far for patients treated with TCR-engineered ACT, we 
hypothesize that incorporating a dominant-negative TGFβ receptor II (dnTGFβRII) into the NY-
ESO-1 TCR retroviral vector used to transduce patient T cells will render these effector cells 
insensitive to TGFβ, resulting in an enhancement of their effector function in the tumor 
microenvironment.
3.3
Rationale for adding decitabine
The ability of the immune system to recognize and reject tumors is dependent on several factors 
that include the expression of immunogenic target antigens on tumor cells, generation of high 
frequencies of tumor antigen (TA) specific T cells with potent effector function, and capacity to 
overcome several mechanisms by which tumors escape immune attack. Although several in vitro 
and in vivo studies have demonstrated that the amount and duration of antigen stimulation can 
influence antigen-specific T cell responses (34, 35), the majority of cancer immunotherapy studies 
have focused on the generation of TA-specific T cells without concomitant manipulation of antigen 
expression. This is a critical consideration because (i) TAs are generally not expressed at 100% 
frequency in cancers; (ii) even when expressed, the antigen density may be low; and (iii) 
expression is often heterogeneous with areas of tumor that are distinctly positive or negative for 
the TA. Moreover, in the latter scenario, the generation of tumor specific immunity may result in 
tumor “immunosculpting” that could influence the ability to evade immune eradication. In this 
regard, a number of human immunotherapy clinical trials, have demonstrated evidence of 
immunoediting with either loss of antigen expression (e.g. MART-1) or MHC class I expression 
(15, 36, 37) 
NY-ESO-1 expression is regulated by DNA methylation in EOC cells and tumor tissues, and 
decitabine treatment induces NY-ESO-1 expression in EOC cell lines (38). Additionally, 
heterogeneous intratumor expression of NY-ESO-1, which frequently occurs in EOC and other 
tumor types, is associated with the DNA methylation status of the NY-ESO-1 promoter and the 
global methylation status of the tumor cells (38). These observations and other work suggest that 
antigen expression is an important limitation for CT antigen immunotherapy, and epigenetic 
therapy is a means to overcome this limitation. 
We therefore hypothesize that the combination of epigenetic modification (Decitabine) and 
adoptive T cell therapy (ACT) will be safe and improve progression free survival (PFS) and/or 
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overall survival (OS) in patients with advanced malignancies. Epigenetic therapy using a 
demethylating agent 5-aza-2’ –deoxycytydine (Decitabine) will increase cancer-testis antigen 
(CTA) expression, minimize emergence of antigen loss variants, and induce an immune signature 
that would further sensitize patients for ACT. Furthermore, this combinatorial strategy would 
allow inclusion of patients without NY-ESO-1 expression.
3.4
Rationale for lowering the eligible age to ≥ 12 years old
The dose escalation portion of this study has been completed with a total of 9 adult subjects infused 
with the study drug, NY-ESO-1 TCR/dn TGFβ-RII transgenic T-cells, without DLT (dose limiting 
toxicity). The last 3 patients in Cohort 3 received the maximum target dose for this study. We also 
treated a pediatric patient on a compassionate basis with the same combination therapy of NY-
ESO-1 TCR/dn TGFβ-RII transgenic T-cells and decitabine as per Cohort 4 of this study. 
Therefore, the eligible age for this next Cohort 4 is being lowered to ≥ 12 years old to include 
adolescent patients with cancers that are relapsed after or refractory to standard therapy with no 
curative options. 
Fixed cell dose justification
The same fixed dose (MTD) of NY-ESO-1 TCR/ dnTGFβRII cells at 1 x 109 cells iv (0.5e9 or 
higher, up to 1e9 acceptable) will be utilized for the pediatric patients as well. In the previously 
reported trial of NY-ESO-1 TCR T-cells for patients with advanced synovial sarcoma 
(NCT01353043)(39), eligible patients were ≥ 4 years old and patients ≥40 kg received the 
minimum cell dose of at least 1x10⁹ transduced NY-ESO-1ᶜ.⁵⁹T cells with a maximum of 6x10⁹ 
transduced cells. The target dose for this protocol was 5x10⁹ transduced NY-ESO-1ᶜ.⁵⁹T cells, 
which is 5 times more than the MTD in our study. Patients <40 kg were dosed per body weight 
with a minimum of 0.025x10⁹ transduced cells/kg, with a target dose of 0.125 x10⁹ transduced 
cells/kg. With this, for a 20 kg child, the dose would be 2.5 x10⁹ cells, which is still 2.5 times 
higher than the MTD in our study.
Relapse of solid tumors in pediatrics 
Solid tumors make up about 30% of all pediatric cancers. Children with therapy-refractory or 
relapsed malignant solid tumors such as sarcomas, wilm’s tumor, and neuroblastoma have minimal 
treatment options (40). The use of multiagent chemotherapy or radiotherapy results in only 
temporary control of disease. Efforts have been made in recent years to find novel approaches for 
treatment. NY-ESO-1 has been found to be variably expressed in a variety of pediatric solid tumors 
including osteosarcoma, synovial sarcoma, rhabdomyosarcoma, melanoma and neuroblastoma 
with expression identified in 25-90% of tumors (40-42). Ongoing and published trials on targeted 
NY-ESO-1 therapies and immunotherapy mainly concern adult patients, but the somatic biology 
of pediatric sarcomas have many similarities to adult disease. A phase I trial in 2015 showed that 
a peptide vaccine targeting cancer tesis antigens including NY-ESO-1 was well tolerated in 
children with relapsed neuroblastoma and sarcomas with 1/10 patients having a complete response 
to therapy (41). Of the other nine patients, one had an initial tumor response with subsequent 
progression and a second who had multiply relapsed neuroblastoma had prior tumor resection, but 
remained disease free for >2 years from receipt of the vaccine. Additionally, in a previously 
reported trial of NY-ESO-1 TCR T-cells for patients with advanced synovial sarcoma 
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(NCT01353043), at least one pediatric aged patient experienced a partial response lasting 32 weeks 
(39)(Ramachandran 2019). This highlights the potential therapeutic efficacy of targeting cancer 
testis antigens including NY-ESO-1 in pediatric solid tumors.
Pediatric experience with NY-ESO-1 TCR/dn TGFβ-RII transgenic T-cells in combination 
with decitabine
On a compassionate basis, we have treated one pediatric patient with NY-ESO-1 TCR/ dnTGFβRII 
transgenic T cells and decitabine as proposed in the Cohort 4 of this current protocol. The patient 
is a 15 years old, with multiply relapsed osteosarcoma who developed progressive disease while 
undergoing prior salvage chemotherapy. The patient was HLA-A*0201 positive, but tumor was 
NY-ESO-1 negative, thus he was treated with inclusion of decitabine to induce NY-ESO-1 
expression. Following apheresis for collection of T-cells, he underwent conditioning 
chemotherapy with decitabine and cyclophosphamide. He was then infused with the successfully 
engineered transgenic T-cells. The patient tolerated the conditioning well with only mild-moderate 
chemotherapy induced nausea controlled with antiemetic therapies. He also tolerated the T-cell 
infusion without complication. He was admitted for observation following the T-cell infusion. 
Markers of inflammation and cytokine release syndrome were monitored throughout his 
admission. The patient remained clinically stable though did develop myelosuppression believed 
to be secondary to the decitabine and cyclophosphamide regimen. He also developed a 
coagulopathy with Grade 1 prolongation of the PTT, elevated D-dimer (though with no clinical 
evidence of DIC) and an elevated CRP, but otherwise his lab evaluations remained normal. The 
only grade 3 or greater toxicity was hematologic with grade 3 febrile neutropenia that developed 
day +16 following T-cell infusion, grade 3 anemia requiring PRBC transfusion and grade 4 
thrombocytopenia requiring a single platelet transfusion. Blood counts had recovered within 1 
month of the therapy. Though he tolerated the therapy well, this patient developed progressive 
disease 1 month following the T-cell therapy. Considering the potential, there is a need to explore 
this type of therapy in adults as well as pediatric patients.
4
OBJECTIVES
4.1
Primary Objectives
To evaluate the safety and feasibility of adoptive transfer of autologous NY-ESO-1 TCR/dn 
TGFβRII transgenic T cells.
4.1.1
Safety
The NY-ESO-1 TCR and the retroviral vector MSGV1-A2aB-1G4A-LY3H10 has already been 
tested in a completed and reported phase 2 clinical trial by investigators at the Surgery Branch/NCI 
(17). In this trial, no grade 3 or 4 side effects were reported in 17 melanoma and synovial cell 
sarcoma patients.  In addition, CD4 cells trained to recognize NY-ESO-1 were used successfully 
in one patient with metastatic melanoma without any acute or long term toxicities (26 months) 
(43). At Roswell Park Cancer Institute, Dr. Kunle Odunsi (co-investigator for this grant) has been 
running a clinical trial with ACT utilizing NY-ESO-1 TCR transgenic cells after a 
lymphodepletion regimen and collection of experimental/clinical data is ongoing. At my previous 
institution, UCLA, we ran a clinical trial targeting another melanoma antigen with ACT utilizing 
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MART1 F5 TCR transgenic cells after a lymphodepletion regimen, followed with DC vaccination 
and high dose IL-2. Three out of 14 patients had a DLT; one patient experienced marrow aplasia 
that has been ascribed to the conditioning regimen of the protocol and extensive bone marrow 
infiltration by melanoma, and two other patients required intubation for bilateral pneumonia 
worsened by high-dose IL-2-induced capillary leak syndrome. Because of these DLTs, future 
patients have received a less intensive conditioning regimen with 4 instead of 5 days of fludarabine 
conditioning, the regimen of high dose IL-2 has been changed to low-dose IL-2 (37, 44), and the 
number of infused transgenic CTLs has decreased to 109. As such, patients in this trial will receive 
a less intensive low dose cyclophosphamide conditioning regimen, IL-2 will not be administered, 
and dose escalation will start with very low numbers of cells (107). Altogether this setting will 
substantially minimize the potential toxicity, improving the safety level for our patients.
After establishing the MTD for the NY-ESO-1 TCR/dnTGFβRII transgenic cells, combination 
with decitabine will be tested in cohort 4 using the 3+3 rule. 
4.1.2
Feasibility
This protocol attempts to administer a cell-based therapy that requires laboratory manipulation 
within the setting of Good Manufacturing Practices (GMP). This will require strict lot release 
criteria of the final product before administration. Feasibility will be determined at every cohort, 
and if 2 out of 6 patients cannot receive the intended cellular therapies, or there is suboptimal TCR 
transgenic cell persistence in vivo, further accrual will not be warranted. The feasibility assessment 
will be based on:
i)
Potential problems in the manufacturing of NY-ESO-1 TCR/ dnTGFβRII engineered 
PBMC.
ii) Potential problems in the delivery of the proposed NY-ESO-1 TCR/ dnTGFβRII cell 
therapy.
4.2
Secondary Objectives
4.2.1
NY-ESO-1 TCR/ dnTGFβRII Transgenic T Cell Persistence
The persistence of NY-ESO-1 TCR/ dnTGFβRII-engineered cells will be determined by analyzing 
serial peripheral blood samples (described in Section 8.4.3.2) for the presence of T cells with the 
NY-ESO-1 TCR by tetramer analysis. In addition, and when technically feasible, samples from 
tumor sites will be biopsied and assessed for the presence of the NY-ESO-1 transgenic cells. V5 
tagged 2A sequence linked to the dnTGFβRII and quantitative PCR will allow the assessment of 
NY-ESO-1 TCR/dnTGFβRII transgenic cells. This consecutive analysis from various time-points 
after infusion will allow us to test if TGFβ signaling blockage in T cells will allow survival, 
expansion and persistent NY-ESO-1 TCR engineered cells.
4.2.2
Study of transgenic T cell differentiation.
In order to study T cell differentiation that correlates with higher anti-tumor responses, we will 
study the different T cell subpopulations by immunophenotypic assays of NY-ESO-1 
TCR/dnTGFβRII transgenic cells, using multicolor flow cytometry and immunohistochemistry. 
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PBMC and tumor infiltrating lymphocytes will be harvested at determined time points. We will be 
able to test the hypothesis that NY-ESO-1 TCR/ dnTGFβRII T cells will generate T memory cells, 
since blockage of immuno-suppressive signals by TGFβ would potentially allow better cell 
survival and hence facilitate further development of T memory phenotypes.
4.2.3
Functional studies of transgenic T cells
In order to test the hypothesis that NY-ESO-1 TCR/dnTGFβRII will constitute in cells more 
efficient in inducing tumor regression, we will perform specific T cell functional assays. PBMC 
and tumor infiltrating lymphocytes will be harvested at determined time points. We will use our 
methodology based on combination of NY-ESO-1 (157-165) tetramer and peptide stimulation to 
analyze TIL and PBMC samples with intracellular effector cytokine staining of TNFα and IFNγ, 
and Granzyme B/CD107a,b for cytotoxic T cell function. With our expertise in polychromatic 
flow cytometry, we will also be able to perform simultaneous multifunctional assessment of the 
transgenic cells. We will include bar-coded carrier cells, if necessary, to minimize cell loss.
4.2.4
Clinical Response
Objective tumor responses will be determined by irRECIST Immune-Related Response Criteria. 
The rate of irCR plus irPR will be used to explore antitumor activity. Response assessment will be 
performed by comparing standard CT imaging scans and photographs of target lesions from 
baseline with repeated imaging tests obtained at day +90 after the TCR transgenic PBMC adoptive 
transfer.
4.3
Exploratory Objectives

Gut microbiota pre and post treatment to evaluate the role of microbiota on the therapeutic 
efficacy of the proposed therapy.
4.3.1
Gut Microbiota
Microbiome sequencing and bacterial 16s RNA expression will be studied to explore the 
therapeutic efficacy on changes in gut microbiota due to the proposed therapy.  After initial 
eligibility has been determined, stool samples will be collected within 1 week of apheresis (day -
6) and 12 weeks post T cell infusion.  Samples will be stored at the DBBR laboratory at Roswell 
Park Cancer Institute and microbiome sequencing will be done at the Genomics Shared Resource 
(GSR), Roswell Park Cancer Institute. 
The human microbiome plays an essential role in digestion and nutrient absorption, epithelial 
homeostasis, angiogenesis, and in the proper function of nervous and immune systems (45, 46). 
Disruption of the microbiome could lead to chronic inflammation, altered immune responses and 
carcinogenesis (47, 48). Most recent research shows that there is a complex reciprocal modulation 
between gut microbiota and the human brain, which involves interaction of the central nervous 
system, sympathetic and parasympathetic branches of the autonomic nervous system, the enteric 
nervous system, and elements of the neuroendocrine and neuroimmune systems. For example, 
some microbiota synthesize neurotransmitters directly (e.g., gamma-amino butyric acid) while 
many modulate the synthesis of neurotransmitters, such as serotonin, dopamine and 
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norepinephrine, and brain-derived neurotropic factor (49). Microbiotic abnormalities have been 
linked to reductions in neurotransmitter levels, cognitive deﬁcits, anxiety, depression, chronic pain 
and fatigue (50, 51).
The composition of the microbiota also regulates the levels of tryptophan in the systemic 
circulation as well levels and nature of tryptophan catabolites (TRYCATs). These catabolites 
influence epithelial barrier integrity and the presence of an inflammatory or tolerogenic 
environment in the intestine and beyond (52). The composition of the microbiota also determines 
the levels and ratios of short chain fatty acids, such as butyrate and propionate, which are key 
energy source for colonocytes (53). Dysbiosis (altered microbiome) can lead to reduced levels of 
butyrate, and therefore may have adverse effects on epithelial barrier integrity, increase bacterial 
translocation into the systemic circulation, effects energy homeostasis, and the T helper 
17/regulatory/T cell balance. (49, 53).
Increasing evidence suggests that different gut, skin, and vaginal microbiota profiles may be 
related to the etiology of certain gynecological cancers, such as cervical cancer, uterine cancer, 
and ovarian cancer (54). Patients’ severity of side effects and response to certain cancer treatments 
can also be impacted by gut microbiome composition (47). In a recent study by Vetizou et al. it 
was shown that anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota and 
the authors showed the key role for Bacteroidales in the immunostimulatory effects of CTLA-
4 blockade both in mice and patients (55). In another recent landmark paper by Sivan et al. (56) 
was shown that Bifidobacterium as associated with the antitumor effects and oral administration 
of Bifidobacterium alone improved tumor control to the same degree as PD-L1-specific antibody 
therapy, and combination treatment nearly abolished tumor outgrowth. Thus, the authors suggested 
that manipulating the microbiota may modulate cancer immunotherapy (56). Since data is more 
mature for gut microbiome and much less is known on pelvic/peritoneal or vaginal microbiome, 
only samples for gut microbiota will be collected in his study.
We will also ask our patients to complete basic surveys (refer to Appendix G and Appendix H) on 
their nutrition and lifestyle in order to gather a more comprehensive data on the interaction of 
microbiome and the immune system.
5
METHODOLOGY
5.1
Study Design
This is a single center, open label, Phase I/IIa study to assess the safety, feasibility, and objective 
tumor response of NY-ESO-1 coupled with dnTGFβRII in human subjects with advanced 
malignancies. Using the MTD of NY-ESO-1 TCR/dnTGFβRII transgenic cells determined in the 
first 3 cohorts, a fourth cohort (Cohort 4) will test the combination with decitabine.
Patients will undergo an initial leukapheresis to collect PBMC. The manufacture of transgenic T 
cells will be started on the day of leukapheresis. Immediately after processing the PBMC, 1x108 
to 3x109 PBMC will be put in activation media containing OKT3 (anti-CD3 antibody) and IL-2 
for two days, and the rest will be cryopreserved. Following activation, cells are transduced twice 
on two consecutive days with NY-ESO-1 TCR/ dnTGFβRII retrovirus in RetroNectin®-coated 
plates. Transduced cells will be expanded ex vivo for 4 days after the first transduction and then 
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put into infusion bags to be infused intravenously, fresh, on the day of harvest as soon as the lot 
release is cleared. 
For Cohorts 1, 2 and 3, the next day after leukapheresis, patient will start receiving the non-
myeloablative conditioning chemotherapy on Day -5 and -4 (45 mg/kg/day IV cyclophosphamide) 
to create space for the transgenic T cells. On day 0, patient will receive the cell infusion.
For Cohort 4, after leukapheresis, on the same day, Day -6, the patient will start receiving 
decitabine (20 mg/m2 /day IV) for a total of 3 days (Day -6 to -4) followed by non-myeloablative 
conditioning chemotherapy on Day -3 and -2 (45 mg/kg/day IV cyclophosphamide). On day 0, 
similar to cohorts 1, 2 and 3, patient will receive the cell infusion. 
In the event the attending physician decides that the patient will not be clinically fit to receive the 
adoptive cell transfer within 24 hours of the scheduled day of cell harvest, the cells will be 
cryopreserved on the harvest day for later use. Patients will undergo repeated peripheral blood 
sampling, CT scanning and biopsies of tumor deposits (see Figure 1).
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Figure 1
Study Schematic (Cohorts 1, 2, and 3)
The schema for cohort 4 is the same as above except Decitabine will be given after leukapheresis 
on Days -6 to -4 (total 3 days) and Cyclophosphamide will be given on Days -3 and -2.
All patients will sign an informed consent prior to study related tests. All patients will meet the 
inclusion and exclusion criteria summarized in Section 6.1and Section 6.2. Conditioning chemo 
could be as an outpatient or inpatient, according to the investigator’s discretion but ACT will be 
done as an inpatient.
Subjects in whom disease control was observed after the T cell infusion may be eligible for a 
second transgenic T cell infusion if the subject still meets the eligibility criteria in Section 6 below.
5.2
Target Accrual and Study Duration
A maximum of 27 patients at RPCI will be enrolled. Accrual is expected to take up to 4 years.
6
PATIENT SELECTION
6.1
Inclusion Criteria
To be included in this study, patients must meet the following criteria:
1. Patients with solid tumors as described below: 
a) Inoperable or metastatic (advanced) melanoma:
 
transduc on
Leukapheresis
Ac va on
(OKT3+IL-2)
RV-NYESO1-TCR/dnTGFβRII
Cyclophosph.
condi oning
Lot release test
1 7 
60 
90 
120 
30 
14 
FDG PET or CT (screening, wk. 6, 12)
Tumor Biops. /TIL harvest (screening, wk.6, at progression)
PBMC collec ons
LTR
LTR
TCR
TCR
dnTGFRII
P2A
T2A
ACT (T cells)
day:
Figure 1: Schema c of clinical trial procedures. Pa ents will undergo leukapheresis to
collect PBMC, which will be ac vated in vitro followed by transduc on with retrovirus
NY-ESO-1 TCR//dnTGFβRII. A er passing
lot release tests, pa ents pre-condi oned
with lymphodeple ng chemotherapy will receive the transgenic T cells. We will collect
peripheral blood, perform PET/CT scanning and biopsies of tumors.
0 
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-
Has received, is intolerant, or refused a CTLA-4 inhibitor (Ipilimumab) or a PD-1 
inhibitor (Nivolumab or Pembrolizumab) as monotherapy and/or a combination of 
Ipilimumab and Nivolumab.
-
Has received or is intolerant of a BRAF inhibitor or the combination of BRAF and 
MEK inhibitors for BRAFv600 mutant melanoma and a PD-1 inhibitor as 
monotherapy or in combination.
b) Inoperable or metastatic (advanced) ovarian, primary peritoneal or fallopian tube 
carcinoma:
-
Has received platinum containing chemotherapy and has platinum refractory or
resistant disease that has progressed on second line therapy
-
If platinum sensitive disease, should have received ≥2 lines of chemotherapy.
-
May have received PARP inhibitors, bevacizumab or other targeted VEGF 
inhibitor therapy
c) Inoperable or metastatic (advanced) synovial sarcoma:
-
Should have received and progressed on ≥ two lines of systemic therapy
d) Subjects with other histologies:
-
Must have previously received two lines of systemic standard care (or effective 
salvage chemotherapy regimens) for metastatic disease, if known to be effective for 
that disease, and have been deemed either non-responders (progressive disease) or 
have recurred.
2. For cohorts 1, 2 and 3 only: Patient's tumor must be positive by histological or molecular 
assay for NY-ESO-1, according to the screening algorithm as described in Section 9.1 
under “tumor biopsy”.  Historical results may be used.
For Cohort 4, NY-ESO-1 results will be noted but NY-ESO-1 positivity is not required for 
eligibility.
3. HLA-A*0201 (HLA-A2.1) positivity by molecular subtyping (blood test or buccal swab, 
historical documentation acceptable).
4. Age ≥ 18 years old, (Cohort 4: Age ≥ 12 years old).
5. Life expectancy greater than 3 months assessed by a study physician.
6. Have been informed of other treatment options.
7. A minimum of one measurable lesion defined as:

Meeting the criteria for measurable disease according irRECIST criteria.

For patients with skin metastases, lesions selected as non-completely biopsied 
target lesion(s) that can be accurately measured and recorded by color photography 
with a ruler to document the size of the target lesion(s).  
8. No restriction based on prior treatments but at least 4 weeks from prior immunotherapy, or 
prior investigational agents. Note: Patients who have suffered >grade 2 irAEs during 
previous checkpoint inhibitor therapy should be excluded.
9. ECOG performance status (PS) 0 or 1 (Appendix A).
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10. Must have adequate venous access for apheresis.
11. Women of childbearing potential and men must agree to use effective methods of birth 
control for the duration of the study and 6 months after. Methods for effective birth control 
include: condoms, diaphragm or cervical cap with spermicide, intrauterine device, and 
hormonal contraception. It is recommended that a combination of two methods be used.
12. Patients must have normal organ and marrow function as defined below:

Leukocytes: ≥ 3,000/mcl

Absolute Neutrophil Count: ≥ 1,000/mcl

Platelets: ≥ 100,000/mcl

Total bilirubin: ≤ 1.5 ULN

AST (SGOT)/ALT (SGPT): ≤ 2.5 x institutional upper limit of normal

Creatinine: ≤ 2X ULN; if creatinine > 2X ULN, creatinine clearance should be > 60 
ml/min.
13. Must be willing and able to accept the leukapheresis procedure.
14. At screening, must have tissue available for NY-ESO-1 testing (if not previously 
performed) or be willing and able to undergo a fresh tissue biopsy. 
15. Patient must understand the investigational nature of this study and sign an Independent 
Ethics Committee/Institutional Review Board approved written informed consent form 
prior to receiving any study related procedure.
16. Participant must agree to and arrange for a caregiver (age ≥ 18 years old) available 24 hours 
a day/ 7 days a week and arrange for lodging within a45 minute drive to Roswell Park and 
transportation for a period of time after discharge from the hospital.  The exact amount of 
time will depend on the individual status as determined by the treating physician.
6.2
Exclusion Criteria
Patients will be excluded from this study for the following:
1. Previously known hypersensitivity to any of the agents used in this study.
2. Currently receiving any other investigational agents. Note: Patients who have suffered 
>grade 2 irAEs during previous checkpoint inhibitor therapy should be excluded.
3. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situations that would limit compliance with study requirements.  
EKG will be done at screening. Cardiac stress test will be done as clinically indicated, the 
specific test to be chosen at the discretion of the treating physician.
4. History of severe autoimmune disease requiring steroids or other immunosuppressive 
treatments.
5. History of inflammatory bowel disease, celiac disease, or other chronic gastrointestinal 
conditions associated with diarrhea or bleeding, which in the investigator’s opinion would 
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place the patient at an increased risk for adverse effect or current acute colitis of any origin. 
Treated cases with no active disease are eligible.
6. Potential requirement for systemic corticosteroids or concurrent immunosuppressive drugs 
based on prior history or received systemic steroids within the last 4 weeks prior to 
enrollment (inhaled or topical steroids at standard doses or isolated use of steroids as 
premedication for medical procedures to minimize allergic reaction [e.g. CT scan dye] are 
allowed).
7. Known active infection with HIV, Hepatitis B, Hepatitis C or CMV.
8. Known cases of clinically active brain metastases (brain MRI as clinically indicated). Prior 
evidence of brain metastasis successfully treated with surgery or radiation therapy will not 
be exclusion for participation as long as they are deemed under control at the time of study 
enrollment.
9. Dementia or significantly altered mental status that would prohibit the understanding or 
rendering of compliance with the requirements of this protocol even with caregiver support.
10. Pregnancy or breast-feeding. Female patients must be surgically sterile or be 
postmenopausal for two years, or must agree to use effective contraception during the 
period of treatment and 6 months after. All female patients with reproductive potential 
must have a negative pregnancy test (serum/urine) within 48 hours from starting the 
conditioning chemotherapy. 
11. Lack of availability of a patient for immunological and clinical follow-up assessment.
6.3
Eligibility Criteria for a Second Transgenic T cell infusion
Subjects in whom disease control was observed after the T cell infusion may be eligible for a 
second transgenic T cell infusion if the subject meets the following criteria:

Patient attained confirmed disease control (either CR, PR or SD) after the first transgenic 
T cell infusion.

Patient still meets the eligibility criteria in Section 6.1 and 6.2.

A second T cell infusion is discussed and the patient agrees to receive it.

Either cryopreserved extra cells from the first T cell product is available or cryopreserved 
autologous PBMC is available for the manufacture of a second transgenic T cell product.
6.4
Inclusion of Women and Minorities
Both men and women and members of all races and ethnic groups are eligible for this study. 
Subjects of any gender and ethnicity with advanced solid tumor expressing NY-ESO-1 will be 
considered in the proposed clinical trials (NY-ESO-1 positivity not required for participants 
enrolling in cohort 4). There has been and will certainly be no discrimination due to gender or 
ethnicity, but the epidemiology of the disease and HLA haplotypes may result in uneven minority 
population distribution.
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6.5
Subject Recruitment
Study subjects will be invited to participate by the study investigators at RPCI as part of the 
discussion of standard and experimental therapy for locally advanced or metastatic melanoma, 
epithelial ovarian cancer, synovial sarcoma or other advanced malignancies expressing NY-ESO-1 
for which no suitable standard of care exists. Patients may access the study investigators through:
a) Subjects may be referred to RPCI from outside clinics. No patient will be approached 
without a referral or agreement from the patient’s physician in order to uphold patient 
confidentiality
b) Subjects may be identified by the study investigators from their own patient pools while 
discussing standard and experimental options for advanced malignancies.
c) Study flyers will be posted in public places, including the oncology clinic waiting room, 
for subject self-referral.
d) Subjects may self-refer to this study when inquiring about clinical trial options at RPCI.
e) This study will be listed at the NIH web site listing active clinical trials, which also assures 
trial registration at a public site.
7
TREATMENT PLAN
7.1
Dosing and Administration
Cell therapy will be administered on an inpatient basis. All patients will receive the same combined 
therapy of: 1) a lympho-depleting preparative regimen (outpatient or inpatient), 2) infusion of 
autologous PBMC retrovirally-transduced with NY-ESO-1 TCR/ dnTGFβRII. Cohort 4 will 
receive decitabine prior to lympho-depleting preparative regimen and NY-ESO-1 TCR/ 
dnTGFβRII transgenic T cells.
All patients will undergo leukapheresis to collect PBMC [study Day (-6)]. The manufacture of 
transgenic T cells will be started on the day of leukapheresis (transduced cells will be expanded 
ex vivo for 4 days after the first transduction and then put into infusion bags to be infused 
intravenously, fresh, on the day of harvest as soon as the lot release is cleared).
Patients in cohorts 1, 2 and 3 will receive the non-myeloablative conditioning chemotherapy with 
cyclophosphamide [intravenous injection of 45 mg/kg/day (use adjusted body weight if > 120% 
of ideal body weight; Male: IBW= 50 + 0.91 (Ht in cm -152), Female: IBW= 45 + 0.91 (Ht in cm- 
152)) study Day (-5) and study Day (-4)]. On study Day 0, the patient will receive a single dose of 
the transgenic NY-ESO-1 TCR/dnTGFβRII PBMC cell infusion as an inpatient.
Patients in cohort 4 will receive Decitabine at a dose of 20 mg/m2 /day IV daily on days -6 after 
leukapheresis to -4 of treatment, for a total of 3 days. On days -3 and -2, patients will receive the 
conditioning regimen consisting of 45 mg/kg/day IV cyclophosphamide for two days to create 
space for the transgenic T cells. On study Day 0, the patient will receive a single dose of the 
transgenic NY-ESO-1 TCR/dnTGFβRII PBMC cell infusion as an inpatient.
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Reported adverse events (AEs) and potential risks are described in Section 8.1.2.1. Appropriate 
dose modifications are described in Section 7.4.
7.1.1
Leukapheresis for NY-ESO-1 TCR PBMC Product
On Day (-6), patients will undergo a 12 liter leukapheresis at the RPCI Blood Donor Facility, 
which is performed as an outpatient procedure. The leukapheresis product will be transferred to 
the GMP Suite for cell processing, at the Therapeutic Cell Production Facility, Center for 
Immunotherapy for the manufacture of the PBMC expressing NY-ESO-1 TCR/ dnTGFβRII cells 
for infusion on Day 0 (Appendix B). 
7.1.2
Cyclophosphamide:
For Cohorts 1, 2 and 3, non-myeloablative chemotherapy with cyclophosphamide 45 mg/kg in 500 
cc NS IV (CTX) is administered in accordance with Institutional standards on Day-5 and Day-4  
(adjusted body weight will be used if > 120% of ideal body weight, Male: IBW= 50 + 0.91 (Ht in 
cm -152), Female: IBW= 45 + 0.91 (Ht in cm- 152)). 
For Cohort 4, non-myeloablative chemotherapy with the same dose of cyclophosphamide 45 
mg/kg in 500 cc NS IV (CTX) is administered over 2 hours or in accordance with Institutional 
standards on Day-3 and Day-2 (adjusted body weight will be used if > 120% of ideal body weight, 
Male: IBW= 50 + 0.91 (Ht in cm -152), Female: IBW= 45 + 0.91 (Ht in cm- 152)). This shift is 
because cohort 4 receives decitabine. Note: For pediatric patients, the following Traub equation 
for IBW will be utilized: for children shorter than 5 ft, the IBW in kilograms is calculated as 
([height in inches]2~ 1.65)/1000; for boys taller than 5 ft, the IBW in kilograms is calculated as 39 
+ (2.27 x [height in inches − 60]); for girls taller than 5 ft, the IBW in kilograms is calculated as 
42 + (2.27 x [height in inches − 60]).
Treatment will be administered on an outpatient or inpatient basis, according to the investigator's 
discretion. Premedication is recommended and may be given in accordance with Institutional 
standards. Aspirin may be substituted for acetaminophen for patients allergic to acetaminophen. 
Hydration with normal saline (250 cc/ hour) is given for 2 hours, or a bolus infusion if suitable or 
in accordance with institutional standards. Mesna (15 mg/kg in 50 cc NS) can be administered IV 
over 30 minutes for 2 doses starting immediately prior to and at 4 hours after the completion of 
cyclophosphamide. Premedication with anti- emetics, chosen as determined by the investigator, is 
provided and as needed after the CTX.
At the investigators' discretion, patients may be hospitalized for the cytoreductive chemotherapy.
7.1.2.1
Toxicities Associated with Cyclophosphamide
Hematologic toxicity occurring with cyclophosphamide usually includes leukopenia and 
thrombocytopenia. Anorexia, nausea and vomiting, rash and alopecia occur, especially after high-
dose cyclophosphamide; diarrhea, hemorrhagic colitis, infertility, and mucosal and oral ulceration 
have been reported. Sterile hemorrhagic cystitis occurs in about 20% of patients; severity can range 
from microscopic hematuria to extensive cystitis with bladder fibrosis. Although the incidence of 
hemorrhagic cystitis associated with cyclophosphamide appears to be lower than that associated 
with ifosfamide, mesna (sodium 2-mercaptoethanesulfonate) has been used prophylactically as an 
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uroprotective agent in patients receiving cyclophosphamide. Prophylactic mesna is not effective 
in preventing hemorrhagic cystitis in all patients. Patients who receive high dose 
cyclophosphamide may develop interstitial pulmonary fibrosis, which can be fatal. Hyperuricemia 
due to rapid cellular destruction may occur, particularly in patients with hematologic malignancy. 
Hyperuricemia may be minimized by adequate hydration, alkalization of the urine, and/or 
administration of allopurinol. If allopurinol is administered, patients should be watched closely for 
cyclophosphamide toxicity (due to allopurinol induction of hepatic microsomal enzymes). At high 
doses, cyclophosphamide can result in a syndrome of inappropriate antidiuretic hormone (SIADH) 
secretion; hyponatremia with progressive weight gain without edema occurs. At high doses, 
cyclophosphamide can result in cardiotoxicity. Deaths have occurred from diffuse hemorrhagic 
myocardial necrosis and from a syndrome of acute myopericarditis; in such cases, congestive heart 
failure may occur within a few days of the first dose. Other consequences of cyclophosphamide 
cardiotoxicity include arrhythmias, potentially irreversible cardiomyopathy, and pericarditis. 
Other reported adverse effects of cyclophosphamide include headache, dizziness, and myxedema; 
faintness, facial flushing, and diaphoresis have occurred following IV administration. Mesna 
(sodium 2-mercaptoethanesulphonate; given by IV injection) is a synthetic sulfhydryl compound 
that can chemically interact with urotoxic metabolites of cyclophosphamide (acrolein and 4-
hydroxycyclophosphamide) to decrease the incidence and severity of hemorrhagic cystitis.
7.1.3
Decitabine
Only Cohort 4 receives Decitabine (20 mg/m2 IV) after leukapheresis, administered daily on 
Days -6 to -4 by IV infusion over 1 hour. Decitabine treatment is planned to be administered on 
an outpatient basis, however, patient may be hospitalized if clinically indicated.
7.1.3.1
Toxicities Associated with Decitabine
Expected toxicities of decitabine are listed in the product label. Most commonly occurring adverse 
reactions are neutropenia, thrombocytopenia, anemia, febrile neutropenia, leukopenia, 
lymphadenopathy, fatigue, pyrexia, nausea, cough, petechiae, constipation, diarrhea and 
hyperglycemia. 
7.1.4
Transgenic NY-ESO-1 TCR/dnTGFβRII PBMC Infusion: Day 0
The TCR genetically engineered PBMC product will be transported to the in-patient unit on ice in 
a rigid-walled container with a tight fitting lid (i.e. Tupperware; Nalgene container etc.) and 
labeled with a biohazard label. The T cell Infusion will be administered through a peripheral IV 
without lymphocyte filter, a Hohn catheter or central line by rapid infusion to gravity over 
approximately 15 minutes. Vital signs are recorded prior to, during, and at 15 minutes, 30 minutes, 
1 hour, 2 hours, 3 hours, and 4 hours post-infusion. If any patient develops a temperature> 38.5ºC 
(101.3ºF), the Principal Investigator should be notified. Patients will be hospitalized for the cell 
infusion and discharged when patient condition is stable and approved by the attending physician.
In the event that any patient develops sepsis or bacteremia following the T cell infusion, 
appropriate cultures and medical management should be initiated. If a contaminated T cell product 
is suspected, the cell product can be re-tested for sterility using archived samples.
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7.1.5
Second Transgenic NY-ESO-1 TCR/dnTGFβRII PBMC Infusion
Eligible patients who showed initial response/disease control may receive a second T cell infusion 
at any time after progression is confirmed. Bridging therapy is allowed before the second T cell 
dosing. For this second T cell infusion, all cohort patients will receive the same highest study dose 
(Cohort 3 dose of 0.5e9 transgenic T cells or higher, up to 1e9). The Chemo-conditioning, 
decitabine (for Cohort 4), and T cell infusion will follow a similar procedure and schedule as 
described in Sections 7.1.2, 7.1.3 and 7.1.4 respectively and Section 9 Study Procedures. The 
second T cell infusion may be administered without chemo-conditioning in the following 
conditions:

Evidence of marrow involvement that may cause count recovery failure after chem-
conditioning

Patient had delayed count recovery after the chemo-conditioning for the first T cell 
infusion (prolonged neutropenia: ANC <500 for >15 days and prolonged 
thrombocytopenia: platelet <50,000/μL for >15 days, with growth factor support) 

Presence of any other risk for delayed count recovery as determined by the principal 
investigator or the treating physician

Presence of transgenic T cells in the peripheral blood
Note: Biopsy tissue may be collected before and after the 2nd T cell infusion when a biopsy is 
clinically indicated. Blood/serum sample collections will follow the same schedule as for the first 
infusion. With the re-start of this collection schedule after the second infusion, the initial schedule 
after the first infusion will be stopped. The starting point of LTFU for up to 15 years will start 
from the second T cell infusion date. Apheresis and Microbiome/stool samples will not be 
collected for this second cycle. 
7.2
Cohort Management
The Phase 1 part of the study will have three cohorts of three patients, with three patients added to 
any cohort with a dose limiting toxicity (DLT). If 2 out of 3 to 6 subjects have a DLT, dose 
escalation will be halted and the cohort will be declared beyond the maximum tolerable dose 
(MTD). Subsequently, an additional cohort # 4 with a total of 9 patients will receive NY-ESO-1 
TCR/dnTGFb-RII transgenic cells at the highest tolerated dose in combination with decitabine. 
Overall, accrual is expected to be between 18-27 patients [18 if no DLT occurs and 27 if DLT 
occurs in all dose levels (Cohort 1 has been completed with 3 patients), it will be 9 subjects plus 3 
extra per dose level, and the possibility of an expanded cohort of 9 patients at the highest tolerated 
dose level determined (MTD)].
Participants who withdraw early after receiving the T-cell infusion will be considered evaluable 
and will not be replaced in their position in the cohort. Participants who have not yet been infused 
will not be considered evaluable and will be replaced with another patient.
Table 1 provides a description of the doses of the proposed combined therapy with a dose 
escalation of NY-ESO-1 TCR / dnTGFβRII transgenic cells in 4 cohorts of patients.
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Table 1
Dose Escalation for NY-ESO-1 TCR / dnTGFβRII Transgenic Cells
Cohort
Number of 
patients
Decitabine
Cyclophosphamide 
Conditioning
NY-ESO-1 TCR/ 
dnTGFβRII cell dose
1
3 (+3)
None
45 mg/kg x 2 days
1 x 107 iv  x 1 infusion
2
3 (+3)
None
45 mg/kg x 2 days
1 x 108 iv  x 1 infusion
3
3 (+3)
None
45 mg/kg x 2 days 
1 x 109 iv x 1 infusion
(0.5e9 or higher, up to 1e9 
acceptable)
4
3 (+3) +6
20 mg/m2 x 3 
days
45 mg/kg x 2 days
MTD: 1 x 109 iv x 1 infusion
(0.5e9 or higher, up to 1e9 
acceptable) *
* After completion of the first 3 cohorts, the Cohort 3 dose of 1 x 109 iv
(0.5e9 or higher, up to 1e9 acceptable) has been declared as the MTD.
7.3
Definition of Dose-Limiting Toxicity
A dose limiting toxicity (DLT) is defined as something which is both:

Any untoward or adverse event which seems at least possibly related to the engineered T 
cell therapy, plus:

Any Grade 3 or higher event that is sustained for more than 1 week, excepting autoimmune 
disorders and chemotherapy related adverse events. For cytokine release syndrome, the 
event must be a grade 4 adverse event and sustain at grade 4 for more than1week to be 
considered a DLT.
Any Grade 3 or higher autoimmune disorder that cannot be resolved to less than or equal to a 
Grade 2 autoimmune toxicity within 100 days should also be deemed a DLT.
Any chemotherapy-related event should not normally be considered a DLT unless the investigator 
is under the reasonable impression that the modified TCR therapy specifically has made the event 
worse in which case it will be considered a DLT.
The DLT time period is 30 days post T cell infusion.
7.4
 Dose Modifications
Each patient receives only a single dose of transgenic T cells and hence dose modification within 
a patient not anticipated.
7.4.1
Treatment Delay
Each patient receives only a single dose of transgenic T cells and hence treatment delay not 
anticipated. In the event the patient is not clinically fit to receive the gene-modified NY-ESO-1 
TCR cell infusion within 24 hours of the scheduled day of cell harvest, the cells will be 
cryopreserved on the harvest day for later use when the physician determines the patient’s 
condition to be fit. If in case a patient already received chemo-conditioning, but not able to receive 
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the cells as scheduled for medical reasons, the cryopreserved cells may be infused within 2 weeks 
after the completion of chemo-conditioning. For more than 2 weeks delay, based on clinical 
judgement and patient’s acceptance, patient may receive a second lymphodepletion (chemo-
conditioning) at a time patient is determined by the investigator to be clinically fit to receive the 
cell infusion.  The first T cell infusion should take place within 6 months of the cell manufacture 
date.
7.5
General Concomitant Medication and Supportive Care
7.5.1
Supportive Therapy on Day 0, Administration of T cells
About 30 minutes to 1 hour prior to Infusion, subjects are given acetaminophen 650 mg p.o. and 
diphenhydramine 50 mg p.o. The T cell Infusion will be administered through a peripheral IV 
without lymphocyte filter, a Hohn catheter or central line by rapid infusion to gravity over 
approximately15 minutes. Vital signs during infusion will be done before, after, and q15 minutes 
if longer than 15 minutes infusion.
Mild transient symptoms have been observed with LAK and TIL cell infusions and, with infusions 
of antigen-specific T cell clones. The management of these symptoms is outlined below or, can be 
done in accordance with institute standards:

Fever, chills and temperature elevations > 101˚F will be managed with acetaminophen 650 
mg p.o. q 4-6 hours. All subjects that develop fever or chills will have a blood culture 
drawn.

Headaches may be managed with acetaminophen following a neurologic examination.

Nausea, vomiting will be treated with a non-steroidal anti-emetic of choice.

Hypotension will initially be managed by intravenous fluid administration and further 
measures as dictated by standard medical practice.

Hypoxemia will initially be managed with supplemental oxygen and further measures as 
dictated by standard medical practice.
7.5.2
Potential Toxicities Warranting Ablation of Adoptively Transferred T Cells
The following will be considered toxicities warranting ablation of transferred T cell clones by 
corticosteroid administration:

Grade 3 or greater toxicity occurring in any other organ system following administration 
of antigen-specific cytotoxic T cells not attributable to underlying metastatic cancer and 
excluding known toxicities of cyclophosphamide.

Any grade 3 or greater toxicities warranting ablation of cells by corticosteroids will be 
submitted to the FDA as an expedited SAE. 
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7.5.2.1
Treatment with Corticosteroids

Patients will receive corticosteroids if treatment-related toxicity warranting abrogation of 
T cells is observed.

The in vivo frequency of infused CD8+ T cells will be assayed immediately prior to and 
48 hours after the start of steroid therapy.

All patients may be hospitalized for the first 48 hours for monitoring. The following dose 
schedule may be used or as per institutional practices:
Day 1
Intravenous methylprednisolone* at 2 mg/kg
Day 2
Intravenous methylprednisolone* at 2 mg/kg
Day 3 - 4
Prednisone at 30 mg po b.i.d.
Day 5 - 6
Prednisone at 15 mg po b.i.d.
Day 7 - 8
Prednisone at 10 mg po b.i.d.
Day 9 - 10
Prednisone at 10 mg po q.d
Day 11 - 12
Prednisone at 5 mg po q.d.
*Consider bismuth, ranitidine and misoprostol ulcer prophylaxis, Dexamethasone may be used 
instead at 0.5 mg/kg.
7.5.2.2
Treatment with Tocilizumab or Infliximab/Etanercept
Engineered T cell therapy is rapidly advancing, and potent clinical responses have been associated 
with acute cytokine release syndrome which often requires intensive care to manage(57, 58). 
Recent studies in adoptive cell therapy and immunotherapy with blinatumomab have revealed that 
the use of the anti-IL 6 humanized antibody, Tocilizumab, and the anti-TNF-α antibody, etanercept 
or infliximab, can resolve CRS within hours and with a single dose (57). There have also been 
well characterized pre-clinical and clinical reports of Tocilizumab making a marked difference to 
patients with intractable/steroid resistant, grade IV (grading is from 1-mild to IV four-severe) graft 
versus host disease after transplantation.
In one case of CRS, the use of steroids did not abrogate CRS, though this was subsequently 
controlled by the anti-cytokine antibodies (57) implying that the antibodies not only provide an 
adequate alternative to steroids but that they are in fact likely a superior alternative. Furthermore, 
with the avoidance of steroids, these patients have been able to go on and have good responses to 
therapy. This is preferable as multiple pre-clinical and clinical reports (59) indicate that high dose 
steroids largely destroy the effects of the T cell therapy on the target. Although these anti-cytokine 
agents are associated with immune suppression when dosed repeatedly in the context of disease 
like arthritis or psoriasis there is little evidence to suggest risk in this on-off dosing context where 
the immediate benefits may well be life-saving (57, 58, 60).
Due to the 1-2 day turn-around time reasonably expected for cytokine results, it is reasonable to 
consider administration of Tocilizumab based on clinical evidence of CRS, and before having the 
cytokine data in hand. Tocilizumab has been used at a dose of 8 mg/kg stat and etanercept at a 
single dose of 50mg.
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To abrogate acute cytokine release treatment with Tocilizumab or Etanercept, the treatment doses 
below are recommended; however, treatment is to be given per investigator's discretion and in 
accordance with institutional practices:

Tocilizumab 8 mg/kg immediately, IV (max 800 mg) 

Etanercept 50 mg one single dose, SC

Infliximab 10 mg/kg, IV
Other additional agents that could be potentially used are Basiliximab (Simulect), an IL-2 
receptor target, Siltuximab (Sylvant), an anti-IL-6 monoclonal antibody, Anakinra (Kineret), an 
IL-1R antagonist.
7.5.2.3
Tocilizumab: Drug Information
Initial U.S. Approval: 2013
U.S. BRAND NAMES: ACTEMRA8 (tocilizumab) ADMINISTRATION: Injection, for 
intravenous infusion.
ADVERSE REACTIONS: Most common adverse reactions (incidence ~ 5%): upper respiratory 
tract infections, nasopharyngitis, headache, hypertension, increased ALT.
WARNINGS AND PRECAUTIONS

Serious Infections
o
Serious and sometimes fatal infections due to bacterial, mycobacterial, invasive fungal, 
viral, protozoa, or other opportunistic pathogens have been reported in patients 
receiving immunosuppressive agents including ACTEMRA for rheumatoid arthritis. 
The most common serious infections included pneumonia, urinary tract Infection, 
cellulitis, herpes zoster, gastroenteritis, diverticulitis, sepsis and bacterial arthritis. 
Among opportunistic infections, tuberculosis, cryptococcus, aspergillosis, candidiasis, 
and pneumocystosis were reported with ACTEMRA. Other serious infections, not 
reported in clinical studies, may also occur (e.g., histoplasmosis, coccidioidomycosis, 
and listeriosis). Patients have presented with disseminated rather than localized disease, 
and were often taking concomitant immuno-suppressants such as methotrexate or 
corticosteroids which in addition to rheumatoid arthritis may predispose them to 
infections.
o
ACTEMRA should not be administered in patients with an active infection, including 
localized infections. The risks and benefits of treatment should be considered prior to 
initiating ACTEMRA in patients:
o
with chronic or recurrent infection;
o
who have been exposed to tuberculosis;
o
with a history of serious or an opportunistic infection who,
o
have resided or travelled in areas of endemic tuberculosis or endemic mycoses; 
or, with underlying conditions that may predispose them to infection.
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o
Patients should be closely monitored for the development of signs and symptoms of 
infection during and after treatment with ACTEMRA, as signs and symptoms of acute 
inflammation may be lessened due to suppression of the acute phase reactants.
o
ACTEMRA should be interrupted if a patient develops a serious infection, an 
opportunistic infection, or sepsis. A patient who develops a new infection during 
treatment with ACTEMRA should undergo a prompt and complete diagnostic workup 
appropriate for an immunocompromised patient, appropriate antimicrobial therapy 
should be initiated, and the patient should be closely monitored.

Tuberculosis
o
Patients should be evaluated for tuberculosis risk factors and tested for latent Infection 
prior to initiating ACTEMRA. Anti-tuberculosis therapy should also be considered 
prior to initiation of ACTEMRA in patients with a past history of latent or active 
tuberculosis in whom an adequate course of treatment cannot be confirmed, and for 
patients with a negative test for latent tuberculosis but having risk factors for 
tuberculosis infection. Consultation with a physician with expertise in the treatment of 
tuberculosis is recommended to aid in the decision whether initiating antituberculosis 
therapy is appropriate for an individual patient.
o
Patients should be closely monitored for the development of signs and symptoms of 
tuberculosis including patients who tested negative for latent tuberculosis infection 
prior to initiating therapy.
o
It is recommended that patients be screened for latent tuberculosis infection prior to 
starting ACTEMRA.
o
The incidence of tuberculosis in worldwide clinical development programs is 0.1%. 
Patients with latent tuberculosis should be treated with standard antimycobacterial 
therapy before initiating ACTEMRA.

Viral Reactivation
o
Viral reactivation has been reported with immunosuppressive biologic therapies and 
cases of herpes zoster exacerbation were observed in clinical studies with ACTEMRA. 
No cases of Hepatitis B reactivation were observed in the trials; however patients who 
screened positive for hepatitis were excluded.

Gastrointestinal Perforations
o
Events of gastrointestinal perforation have been reported in clinical trials, primarily as 
complications of diverticulitis. ACTEMRA should be used with caution in patients who 
may be at increased risk for gastrointestinal perforation. Patients presenting with new 
onset abdominal symptoms should be evaluated promptly for early identification of 
gastrointestinal perforation.

Laboratory Parameters
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o
Neutrophils: Treatment with ACTEMRA was associated with a higher incidence of 
neutropenia. Infections have been uncommonly reported in association with treatment-
related neutropenia in long-term extension studies and postmarketing clinical 
experience. It is not recommended to initiate ACTEMRA treatment in patients with a 
low neutrophil count L [absolute neutrophil count (ANC) <2000/mm3]. In patients who 
develop an absolute neutrophil count <500/mm3 treatment is not recommended. 
Neutrophils should be monitored every 4 to 8 weeks.
o
Platelets: Treatment with ACTEMRA was associated with a reduction in platelet 
counts. Treatment-related reduction in platelets was not associated with serious 
bleeding events in clinical trials.- It is not recommended to initiate ACTEMRA 
treatment in patients with a platelet count below 100,000/mm3. In patients who develop 
a platelet count <50,000/mm3 treatment is not recommended. Platelets should be 
monitored every 4 to 8 weeks.

Liver Function Tests: Treatment with ACTEMRA was associated with a higher incidence 
of transaminase elevations. These elevations did not result in apparent permanent or 
clinically evident hepatic injury in clinical trials. Increased frequency and magnitude of 
these elevations was observed when potentially hepatotoxic drugs (e.g., MTX) were used 
in combination with ACTEMRA. In one case, a patient who had received ACTEMRA 8 
mg/kg monotherapy without elevations In transaminases experienced elevation in AST to 
above 10x ULN and elevation in ALT to above 16x ULN when MTX was initiated in 
combination with ACTEMRA. Transaminases normalized when both treatments were 
held, but elevations recurred when MTX and ACTEMRA were restarted at lower doses. 
Elevations resolved when MTX and ACTEMRA were discontinued. It is not recommended 
to initiate ACTEMRA treatment in patients with elevated transaminases ALT or AST> 
1.5x ULN. In patients who develop elevated ALT or AST> 5x ULN treatment is not 
recommended. ALT and AST levels should be monitored every 4 to 8 weeks. When 
clinically indicated, other liver function tests such as bilirubin should be considered.

Lipids: Treatment with ACTEMRA was associated with increases in lipid parameters such 
as total cholesterol, triglycerides, LDL cholesterol, and/or HDL cholesterol. Assessment of 
lipid parameters should be performed approximately 4 to 8 weeks following initiation of 
ACTEMRA therapy, then at approximately 6 month intervals. Patients should be managed 
according to clinical guidelines [e.g., National Cholesterol Educational Program (NCEP)] 
for the management of hyperlipidemia.

Immunosuppression: The impact of treatment with ACTEMRA on the development of 
malignancies is not known but malignancies were observed in clinical studies. ACTEMRA 
is an immunosuppressant, and treatment with immunosuppressants may result in an 
increased risk of malignancies.

Hypersensitivity Reactions: Serious hypersensitivity reactions, including anaphylaxis, 
have been reported in association with infusion of ACTEMRA. Appropriate medical 
treatment should be available for immediate use in the event of an anaphylactic reaction 
during administration of ACTEMRA.
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
Demyelinating Disorders: The impact of treatment with ACTEMRA on demyelinating 
disorders is not known, but multiple sclerosis and chronic inflammatory demyelinating 
polyneuropathy were reported rarely in clinical studies. Patients should be closely 
monitored for signs and symptoms potentially Indicative of demyelinating disorders. 
Prescribers should exercise caution in considering the use of ACTEMRA in patients with 
preexisting or recent onset demyelinating disorders.

Active Hepatic Disease and Hepatic Impairment: Treatment with ACTEMRA is not 
recommended in patients with active hepatic disease or hepatic impairment.

Vaccinations: Live vaccines should not be given concurrently with ACTEMRA as clinical 
safety has not been established. No data are available on the secondary transmission of 
infection from persons receiving live vaccines to patients receiving ACTEMRA. No data 
are available on the effectiveness of vaccination in patients receiving ACTEMRA. Because 
IL-6 inhibition may interfere with the normal immune response to new antigens, patients 
should be brought up to date on all recommended vaccinations, except for live vaccines, 
prior to initiation of therapy with ACTEMRA.
MECHANISM of ACTION: Tocilizumab binds specifically to both soluble and membrane-
bound IL-6 receptors (sIL-6R and mIL-6R), and has been shown to inhibit IL-6-mediated signaling 
through these receptors. IL-6 is a pleiotropic pro-inflammatory cytokine produced by a variety of 
cell types including T- and B-cells, lymphocytes, monocytes and fibroblasts. IL-6 has been shown 
to be involved in diverse physiological processes such as T-cell activation, induction of 
immunoglobulin secretion, initiation of hepatic acute phase protein synthesis, and stimulation of 
hematopoietic precursor cell proliferation and differentiation. IL-6 is also produced by synovial 
and endothelial cells leading to local production of IL-6 in joints affected by inflammatory 
processes such as rheumatoid arthritis.
7.5.2.4
Infliximab: Drug Information
Initial U.S. Approval: 1998
U.S. BRAND NAMES - Remicade® (Infliximab)
ADMINISTRATION: Lyophilized Concentrate for Injection, for Intravenous Use
ADVERSE REACTIONS 

SIGNIFICANT: Most common adverse reactions (>10%) – infections (e.g. upper 
respiratory, sinusitis, and pharyngitis), infusion-related reactions, headache, and abdominal 
pain.

WARNINGS AND PRECAUTIONS
o
Serious infections – do not give REMICADE during an active infection. If an infection 
develops, monitor carefully and stop REMICADE if infection becomes serious.
o
Invasive fungal infections – for patients who develop a systemic illness on 
REMICADE, consider empiric antifungal therapy for those who reside or travel to 
regions where mycoses are endemic
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o
Malignancies – the incidence of malignancies including lymphoma was greater in 
REMICADE treated patients than in controls. Due to the risk of HSTCL carefully 
assess the risk/benefit especially if the patient has Crohn’s disease or ulcerative colitis, 
is male, and is receiving azathioprine or 6-mercaptopurine treatment. 
o
Hepatotoxicity – rare severe hepatic reactions, some fatal or necessitating liver 
transplantation. Stop REMICADE in cases of jaundice and/or marked liver enzyme 
elevations. 
o
Heart failure –new onset or worsening symptoms may occur.
o
Cytopenias – advise patients to seek immediate medical attention if signs and 
symptoms develop, and consider stopping REMICADE. 
o
Hypersensitivity – serious infusion reactions including anaphylaxis or serum sickness-
like reactions may occur. 
o
Demyelinating disease –exacerbation or new onset may occur.
o
Lupus-like syndrome – stop REMICADE if syndrome develops.
o
Live vaccines or therapeutic infectious agents – should not be given with REMICADE. 
MECHANISM OF ACTION: Infliximab neutralizes the biological activity of TNFby binding 
with high affinity to the soluble and transmembrane forms of TNFand inhibits binding of TNF
 with its receptors. Infliximab does not neutralize TNF (lymphotoxin-), a related cytokine that 
utilizes the same receptors as TNF. Biological activities attributed to TNF include: induction 
of pro-inflammatory cytokines such as interleukins (IL) 1 and 6, enhancement of leukocyte 
migration by increasing endothelial layer permeability and expression of adhesion molecules by 
endothelial cells and leukocytes, activation of neutrophil and eosinophil functional activity, 
induction of acute phase reactants and other liver proteins, as well as tissue degrading enzymes 
produced by synoviocytes and/or chondrocytes. Cells expressing transmembrane TNF
 bound by infliximab can be lysed in vitro or in vivo. Infliximab inhibits the functional activity 
of TNFin a wide variety of in vitro bioassays utilizing human fibroblasts, endothelial cells, 
neutrophils, B and T- lymphocytes and epithelial cells. The relationship of these biological 
response markers to the mechanism(s) by which REMICADE exerts its clinical effects is 
unknown. Anti-TNF antibodies reduce disease activity in the cotton-top tamarin colitis model, 
and decrease synovitis and joint erosions in a murine model of collagen-induced arthritis. 
Infliximab prevents disease in transgenic mice that develop polyarthritis as a result of constitutive 
expression of human TNF, and when administered after disease onset, allows eroded joints to 
heal. 
7.5.2.5
Etanercept: Drug Information
Initial U.S. Approval: 1998
U.S. BRAND NAMES - Enbrel® (etanercept)
ADMINISTRATION: Solution for Subcutaneous Use
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ADVERSE REACTIONS 

SIGNIFICANT: Most common adverse reactions (incidence > 5%): infections and 
injection site reactions.

WARNINGS AND PRECAUTIONS
o
Do not start Enbrel during an active infection. If an infection develops, monitor 
carefully and stop Enbrel if infection becomes serious.
o
Consider empiric anti-fungal therapy for patients at risk for invasive fungal infections 
who develop a severe systemic illness on Enbrel (those who reside or travel to regions 
where mycoses are endemic).
o
Demyelinating disease, exacerbation or new onset, may occur.
o
Cases of lymphoma have been observed in patients receiving TNF blocking agents. 
Congestive heart failure, worsening or new onset, may occur.
o
Advise patients to seek immediate medical attention if symptoms of pancytopenia or 
aplastic anemia develop, and consider stopping Enbrel.
o
Monitor patients previously infected with hepatitis B virus for reactivation during and 
several months after therapy. If reactivation occurs, consider stopping Enbrel and 
beginning anti-viral therapy.
o
Anaphylaxis or serious allergic reactions may occur.
o
Stop Enbrel if lupus-like syndrome or autoimmune hepatitis develops.
MECHANISM OF ACTION: TNF is a naturally occurring cytokine that is involved in normal 
inflammatory and immune responses. It plays an important role in the inflammatory processes of 
rheumatoid arthritis (RA), polyarticular juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA), 
and ankylosing spondylitis (AS) and the resulting joint pathology. In addition, TNF plays a role in 
the inflammatory process of plaque psoriasis (PsO). Elevated levels of TNF are found in involved 
tissues and fluids of patients with RA, JIA, PsA, AS, and PsO. Two distinct receptors for TNF 
(TNFRs), a 55 kilodalton protein (p55) and a 75 kilodalton protein (p75), exist naturally as 
monomeric molecules on cell surfaces and in soluble forms. Biological activity of TNF is 
dependent upon binding to either cell surface TNFR. Etanercept is a dimeric soluble form of the 
p75 TNF receptor that can bind TNF molecules. Etanercept inhibits binding of TNF-α and TNF-α 
(lymphotoxin alpha (LT-α]) to cell surface TNFRs, rendering TNF biologically inactive. In in vitro 
studies, large complexes of etanercept with TNF-α were not detected and cells expressing 
transmembrane TNF (that binds Enbrel) are not lysed in the presence or absence of complement.
7.6
Permissible Systemic Therapies During the Protocol On-Study Period
The on-study period is defined as 9 months from the date of the TCR engineered ACT infusion 
unless there is overt tumor progression or dose limiting toxicities leading to patient 
discontinuation.
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Systemic immune suppressive therapies will be avoided during this time, but they may be 
permissible in the following situations:

Systemic corticosteroids: Used in case there is suspicious or evidence of an autoimmune 
reaction resulting in serious adverse effects.
7.7
Study Duration
Patients may remain on study and continue to receive treatment in the absence of disease 
progression, unacceptable toxicity or withdrawal from study, intercurrent illness that prevents 
further administration of treatment, participant demonstrates an inability/refusal to comply with 
oral medication regime, and participant withdraws from study.
At week 12 post-adoptive cell transfer, patients will be evaluated for antitumor activity after 
undergoing re-staging exams and laboratory tests. Patient with SD, PR or CR will be offered to 
continue on protocol after discussion of the study results. Patients with PD will be offered to 
terminate participation. However, given the phenomenon of delayed responses with tumor 
immunotherapy approaches, the final decision to continue or not on study will be at the discretion 
of the treating physician and the patient, regardless of the tumor response assessment.
7.8
Study Discontinuation
Upon treatment discontinuation all end of treatment evaluations and tests will be conducted. All 
patients who discontinue due to an AE must be followed until the event resolves or stabilizes. 
Appropriate medical care should be provided until signs and symptoms have abated, stabilized, or 
until abnormal laboratory findings have returned to acceptable or pre-study limits. The final status 
of the AE will be reported in the patient’s medical records and the appropriate eCRF.
Reasons for treatment discontinuation should be classified as follows:

Death

Progressive disease

Toxicity; related or unrelated to treatment

Investigator judgment
o
The Investigator may discontinue a participant if, in his/her judgment, it is in the 
best interest of the participant to do so.

Noncompliance

Patient voluntary withdrawal
o
A patient may withdraw from the study at any time, for any reason. If a patient 
discontinues treatment, an attempt should be made to obtain information regarding 
the reason for withdrawal.

Sponsor decision.
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Premature termination of the clinical trial may occur because of a regulatory authority decision, 
change in opinion of the FDA, RAC, IRB, the DSMB, or determination that there are problems in 
the cell product generation or the safety of their administration as descried in the assessment of 
primary study endpoints. Additionally, recruitment may be stopped for reasons of particularly low 
recruitment, protocol violations, or inadequate data recording.
8
INVESTIGATIONAL AGENTS
8.1
PBMC Expressing NY-ESO-1 TCR/ dnTGFβRII
Overview: A leukapheresis product obtained from study patients will be transferred to the GMP 
Suite, at the Therapeutic Cell Production Facility, Center for Immunotherapy. PBMC will be 
isolated by gradient centrifugation, and an aliquot of these cells will be cultured for 48 hours in 
the presence of anti-CD3 (OKT3) and IL-2 in order to stimulate T-cell growth to prepare for viral 
vector transduction. Stimulated PBMC will be transduced with a clinical grade retrovirus vector 
expressing NY-ESO-1 TCR/ dnTGFβRII using RetroNectin®-coated plates on two consecutive 
days and kept in culture for 96 hours from the time of the first retroviral transduction. Transduced 
cells will be harvested and infused fresh as soon as the lot release criteria are cleared. On the 
infusion day, Cohorts 1, 2 and 3 patients will have completed the non-myeloablative but 
lymphocyte depleting preparative regimen consisting of cyclophosphamide and Cohort 4 patients 
will have completed their decitabine and cyclophosphamide regimen.
In the event the patient is not clinically fit to receive the gene-modified NY-ESO-1 TCR cell 
infusion within 24 hours of the scheduled day of cell harvest, the cells will be cryopreserved on 
the harvest day for later use.
8.1.1
Retroviral Vector NY-ESO-1 TCR/ dnTGFβRII
Product Description: This vector contains the 5’ and 3’ LTR of the murine stem cell virus as well 
as a packaging signal containing splicing donor and acceptor sites. In this TCR vector, the alpha 
and beta chains of the NY-ESO-1 TCR are separated by a picornavirus P2A “self-cleaving” 
sequence. The particular vector that is being used in the this trial contains a modified version of 
the non-codon optimized parental 1G4 vector in which the amino acids 95 and 96 of the CDR3 of 
the alpha chain have been modified to leucine (L) and tyrosine(Y) (29). We generated the NY-
ESO-1 TCR/ dnTGFβRII by incorporating a dnTGFβRII into the NY-ESO-1 retrovirus. This new 
clinical grade vector is being manufactured at the Indiana University Viral Production Facility (IU 
VPF, Kenneth Cornetta, Director).
Classification: Immunotherapeutic.
Active Ingredient: This retroviral vector have two active transgenes from a TCR, the alpha and 
beta chains of a high affinity TCR that jointly recognizes the NY-ESO-1157-165 epitope in the context 
of HLA-A*0201, and a dnTGFβRII, which blocks the effect of TGFβ.

NY-ESO-1 TCR: The 1G4 high affinity TCR pair specific for NY-ESO-1157-165 presented 
by HLA-A*0201 was originally cloned from a patient with metastatic melanoma (61). The 
TCR was engineered for higher specificity for NY-ESO-1 by modifying the CDR of the 
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alpha chain of the TCR, leading to the current version of the 1G4 TCR used in this study. 
(29)
Mode of Action: One functional complex will be generated by the active transgenes:

NY-ESO-1 TCR: The NY-ESO-1 TCR is expected to redirect the specificity of the TCR 
transgenic T cells to the NY-ESO-1157-165 peptide presented by HLA-A2*0201 on the surface 
of cancer cells.

Incorporating a dominant-negative TGFβ receptor II (dnTGFβRII) into the NY-ESO-1 
TCR retroviral vector used to transduce patient T cells will render these effector cells 
insensitive to TGFβ, resulting in an enhancement of their effector function in the tumor 
microenvironment.
Manufacturing Information for the Clinical Grade Vector: The clinical grade vector to express 
NY-ESO-1 TCR/ dnTGFβRII is being generated at the Indiana University Vector Production 
Facility (IU VPF). The retroviral vector supernatant will be prepared and preserved following 
cGMP conditions at IU VPF. The physical titer will be determined by RNA dot blot according to 
the manufacturer’s certificate. The supernatant will be stored upon the completion of production 
at least at –70o C at IU VPF. Upon shipment on dry ice, the supernatant will be  stored at –80o C 
at the GMP suite. This facility is equipped with around-the-clock temperature monitoring. 
Supernatant will be thawed and used for in vitro transduction of activated PBMC. There will be 
no re-use of the same unit of supernatant for different patients. The retroviral titer has been shown 
to be stable after immediate thawing and immediate use (tissue culture wells previously coated 
with RetroNectin®). Handling of the vector should follow the guidelines of Biosafety Level-2 
(BSL-2).
How Supplied: The clinical grade vector is supplied by IU VPF following their Standard 
Operating Procedures (SOP) for retroviral vector suspension, labeling and cryopreservation.
Storage: Single use aliquots are stored in a viral bank established in a dedicated –80oC freezer with 
central, computerized monitoring system, with alarm and recording of all GMP storage systems 
located at the restricted access GMP Suite, at RPCI.
Stability: Under the conditions of continuous storage at –80oC without freeze-thawing, it is 
expected that the retroviral vector is stable for at least 5 years. Aliquots of this vector will be 
recertified annually throughout the study period with selected assays for purity, identity and 
potency.
Dose and Schedule: For ex vivo use only.
Route of Administration. All use of this retrovirus vector is ex vivo, and no direct injection will 
be done in human subjects.
Prior Human Experience: This retrovirus vector expressing the NY-ESO-1 TCR similarly used 
to transduce human PBMC has already been administered to humans within a phase 2 clinical trial 
at the Surgery Branch/NCI (22). The prior human experience is as follows:

Retrovirus Vectors: Retroviral vectors have undergone extensive testing in humans. Their 
main safety caveat is the development of insertional mutagenesis, which seems to be a rare 
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event and may be related to the expressed transgene and the immune competency of the 
host. Over 250 patients in over 40 clinical trials have received stem cells genetically 
modified with retroviral vectors (62), and the only cases of malignant transformation are 
the two infants in a clinical trial inserting the gamma C gene (a surface cytokine receptor) 
to HSC of infants with X-linked severe combined immune deficiency (SCID) (63, 64).

NY-ESO-1 TCR: The same NY-ESO-1 TCR alpha and beta gene pair is being tested at 
the Surgery Branch/N.C.I. (Steven A. Rosenberg, P.I.) for its ability to redirect antigen 
specificity of human PBMC adoptively transferred to lymphopenic hosts in a protocol 
design similar to the one proposed herein (22). If we gain knowledge of the development 
of serious adverse events (SAE) related to the NY-ESO-1 TCR transgenic T cell 
administration, our protocol procedures and informed consent form will be modified 
accordingly.
Reported Adverse Events and Potential Risks: The potential adverse events are related to the viral 
vector, transgenes and genetically modified cells, as discussed in the following section.
8.1.2
PBMC Transgenic for NY-ESO-1 TCR / dnTGFβRII
Product Description: PBMC obtained from leukapheresis and activated during 48 hours with 
OKT3 (anti-CD3 antibody) and IL-2, infected by the clinical grade NY-ESO-1 TCR / dnTGFβRII 
retroviral vector supernatant on two consecutive days, maintained in culture for 4 days from the 
start of transduction in IL-2 media. Aliquots of these cells will be used to fulfill the lot release 
criteria.
Classification: Immunotherapeutic.
Active Ingredient: As described for the retrovirus vectors NY-ESO-1 TCR / dnTGFβRII
Mode of Action: The genetically modified PBMC expressing a transgenic surface NY-ESO-1 TCR 
will be expected to be redirected to the NY-ESO-1 antigen expressed by HLA-A2*0201 and NY-
ESO-1 positive cancer cells. Recognition of the cognate antigen will result in the release of effector 
molecules from T cells, like the cytotoxic granules perforin and granzyme B, and activating cytokines 
like interferon-gamma, IL-2 and TNF-alpha.
Manufacturing Information: Genetically modified PBMC expressing NY-ESO-1 TCR / 
dnTGFβRII will be manufactured by transduction of previously activated PBMC (see Appendix E 
and Appendix F) using the clinical grade retroviral vectors.
Storage: PBMC transgenic for NY-ESO-1 TCR / dnTGFβRII will be harvested within 96 hours 
from the first transduction and infused fresh. If cryopreserved, cells will be stored in a centrally-
monitored liquid nitrogen freezer in the GMP suite.
Stability: These genetically modified PBMC will be infused fresh. When stored in liquid nitrogen, 
PBMC are viable for over 2 years with minimal viability loss if not subject to freeze-thawing. Cells 
should be used within 6 months of manufacture. Extra cells cryopreserved from the first T cell 
product may be used within 2 years of manufacture for a second T cell infusion. If preparing a 
second T cell product utilizing cryopreserved PBMC from the first apheresis collection, the PBMC 
may be older than 2 years as long as the viability is >90%.
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Dose and Schedule: Administered once on Day 0 of this protocol. Dose escalation of NY-ESO-1 
TCR / dnTGFβRII transgenic cells will be in 3 cohorts of patients [107,108, and109 cells (0.5e9 or 
higher, up to 1e9 acceptable)].
Administration: Transgenic cells will be transported from the GMP suite to the hospital bed in 
infusion bags with over-wrap, on ice in a rigid-walled container with a tight fitting lid (i.e. 
Tupperware; Nalgene container etc.) labeled with a biohazard label.
Unused Transgenic Cells: Transgenic cells generated but not administered to patients will be 
banked cryopreserved for up to 5 years. Extra cells cryopreserved from the first T cell product may 
be used within 2 years of manufacture for a second T cell infusion. Cells may be used for additional 
in vitro testing beyond this period of time. Disposal of transgenic cells remaining in the bag and i. 
v. tubing after administration to patients will be done in a biohazard container that will be 
autoclaved before disposal.
Prior Human Experience: As described above, PBMC transduced with the same retrovirus vector 
is currently in clinical testing (22).
See Appendix D for the Retrovirus Transduction Method.
8.1.2.1
Reported Adverse Events and Potential Risks
Potential risks are derived from the viral vector, the transgenes it will express and the infusion of 
genetically modified cells:

Retroviral Vectors: There are two potential serious anticipated risks associated with 
retroviral vectors. One is related to its ability to randomly and permanently integrate in the 
genome of target cells, which may lead to altered expression of genes in these cells 
potentially inducing oncogenic changes. The second one is related to the possibility of its 
recombination and generation of replication competent virus that could potentially lead to 
a HIV-like disease.

Risk of Insertional Mutagenesis with Retroviral Vectors. The risks of insertional 
oncogenesis with integrating gene delivery vectors is likely a complex function of several 
factors, related to the type of vector used, the promoter and other transcriptional control 
elements of the vector, the transgene per se, as well as properties of the target cells. 
Moloney Leukemia Virus (MLV)-based gamma-retroviral vectors have been shown to 
have a high predilection for insertion into the 5’ region of actively transcribed genes, which 
may increase the risks for trans-activation of transcription from the cellular gene promoter 
(65). Genes that modulate cell proliferation, such as the gamma C gene used to treat X-
linked SCID may provide one component of a proliferative signal that plays a cooperative 
role in cellular transformation with cellular genes involved in cellular proliferation, such 
as LMO-2, when activated by insertional trans-activation (63). In contrast, effector proteins 
such as TCR and a truncated version of a receptor (TGFβRII) would not, a priori, be 
expected to confer an unregulated proliferative stimulus, although they may play a role in 
clonal expansion upon encounter with the cognate antigen. Furthermore, T cells transduced 
with dnTGFβRII did not lead to spontaneous lymphoproliferation in vivo (66). Recently, a 
decade-long safety/function of retroviral-modified chimeric antigen receptor T cells 
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study(67) showed no evidence of cell immortalization, and neither persistent clonal 
expansion as measured in >500 patient-years of follow-up. Hence given the natural history 
and poor prognosis of advanced cancer patients and large evidence from the retroviral-
based ACT literature, we assess that the risks of leukemogenesis are very small and 
justifiable. Even so, we will monitor insertional mutagenesis events by repeated sampling 
of cells and study if dominant clones of long-term persisting (6 months and over) transgenic 
cells are clonal or polyclonal by analyzing the retroviral insertion sites and ruling out 
common integration sites (CIS)(68). Findings that would suggest proto-malignant behavior 
would lead us to discuss them with the FDA, and consider introducing suicide transgenes 
into the vector, such as sr39Thymidine Kinase (our group recently received a clinical 
GMP-grade virus batch for another future trial) or possible less immunogenic alternatives 
as they become available (69).

Risk of Replication Competent Retrovirus (RCR) with Retroviral Vectors. Another 
risk is that genetic recombination within components of the vector system could lead to the 
generation of RCR. This event would have the potential of generating infective retrovirus 
that may lead to oncogenesis or immune deficiency. Our patients will be screened at 
baseline to be HIV seronegative. Since there will be no wild type HIV virus present, the 
possibility of providing the missing genes for the generation of infective retrovirus in vivo 
would be lower than when retroviral vectors are administered to HIV positive subjects. In 
a recent publication, testing of Master Cell Banks and patient follow-up from 29 clinical 
studies using gene-modified T cells manufactured with gamma-retroviral vectors from five 
different centers showed no RCR in all tests thus far (51), in line with our experience from 
our TCR trial. Nevertheless we will monitor for RCR risk by strictly following FDA 
guidelines.
8.1.2.2
NY-ESO-1 TCR: 
The main potential toxicities from the transgenic alpha and beta NY-ESO-1 TCR genes expressed 
in PBMC are derived from the NY-ESO-1 specificity of the TCR, or the mispairing with 
endogenous TCR genes leading to cells with potential autoimmune specificity.

Autoimmune Toxicity against NY-ESO-1 Positive Cells. Toxic events could be potentially 
derived from cytotoxic activity of the transgenic PBMC against NY-ESO-1 positive cells. 
However, studies to date have not detected expression of NY-ESO-1 in adult somatic 
tissues (3, 4, 6). While spermatogonia express the NY-ESO-1 protein, the testes do not 
express MHC-1, thereby markedly decreasing the potential of a cellular immune response 
against the testes. Furthermore, a recent trial conducted at the NCI in which 17 patients 
underwent ACT with a TCR directed against NY-ESO-1157-165 did not detect any grade 
3 or 4 toxicities.

Autoimmune Toxicity from Mispairing of TCR Chains. The alpha and beta chains of the 
NY-ESO-1 TCR could undergo heterologous pairing with endogenous alpha and beta 
chains of TCR expressed by the transduced T cells, leading to misdirected T cells with 
newly acquired TCR specificities. These would be unpredictable and not subjected to the 
thymic selection process, potentially resulting in T cells with auto-reactivities. If evidence 
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of autoimmunity developed, patients would receive immune suppressive therapy as 
clinically indicated based on the severity of symptoms, using medications like 
corticosteroids, cyclosporin-A, mycophenolate mofetil, anti-TNF-alpha antibodies or anti-
thymocyte globulin .
8.1.2.3
NY-ESO-1/ dnTGFβRII
In addition to the potential toxicities listed above for NY-ESO-1 TCR, autoimmune toxicity from 
dnTGFβRII could occur.
Autoimmune Toxicity from dnTGFβRII: The TGFβ signaling pathway is necessary to maintain T 
cell homeostasis, and abrogation early in T cell development produces T cell hyperproliferation 
and autoimmunity(70). Due to developmental defects, transgenic mice that express dnTGFβRII in 
T cells present with CD8+ lymphoproliferative disorder and autoimmunity. The 
lymphoproliferating T cells were naive and IL-2 receptor negative, and exhibited distinct patterns 
of recirculation and homeostasis. By contrast, our transgenic cells have been activated and 
expanded in vitro and are post-thymic mature T cells that are dependent on antigen and growth 
factors for survival. In communication with Malcolm Brenner at Baylor, there was no evidence in 
vitro and in vivo that long-term expression of the mutant TGF receptor had any deleterious effects 
on the transduced cells, which remained antigen and cytokine dependent. In the event evidence of 
autoimmunity is observed, the patient will receive immune suppressive therapy as clinically 
indicated based on the severity of symptoms (corticosteroids, cyclosporin-A, mycophenolate 
mofetil, anti-TNF-alpha antibodies).
8.1.2.4
NY-ESO-1/ dnTGFβRII Transgenic T Cells
The adoptive transfer of an ex vivo modified autologous cellular product may lead to potential 
toxicities related to the cell manipulation.
Allergic Reaction and Cytokine Release Syndrome: The culture of cells ex vivo in media 
products to maintain lymphocyte viability, the use of reagents to activate cells, the retroviral 
transduction process, and the expression of foreign genes may lead to alterations in the cell product 
resulting in immediate untoward effects. The most serious would be infusion reactions leading to 
severe allergic reactions, or the triggering of a cytokine release syndrome. Therefore, the 
genetically modified cells will be re-infused in an inpatient setting, with continuous monitoring of 
vital signs and by experienced personnel within the bone marrow transplant (BMT) unit.
Contaminated Cell Product: Ex vivo culture and manipulation of cells may result in a cell product 
contaminated by bacteria, fungus, mycoplasma or virus. With the goal of detecting potential 
contaminants, the genetically modified cells will undergo intensive lot release testing prior to 
infusion.
8.2
Decitabine: Drug Information
Decitabine is commercially available and will be supplied by the study.
The date of receipt and the amount of drug received will be documented. Drug shipment records 
will be retained by the investigational pharmacist or designee.
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The package insert will be followed when preparing, handling and disposing Decitabine.
8.3
Cyclophosphamide: Drug Information
Copyright 1978-2009 Lexi-Comp, Inc. All rights reserved.
U.S. BRAND NAMES — Cytoxan®
PHARMACOLOGIC CATEGORY: Antineoplastic Agent, Alkylating Agent
ADMINISTRATION:

I.V. infusions may be administered over 1-24 hours.

Doses >500 mg to approximately 2 g may be administered over 20-30 minutes.

To minimize bladder toxicity, increase normal fluid intake during and for 1-2 days after 
cyclophosphamide dose. Most adult patients will require a fluid intake of at least 2 L/day. 
High-dose regimens should be accompanied by vigorous hydration with or without mesna 
therapy.
ADVERSE REACTIONS 

Significant (Greater than 10%):
o
Dermatologic: Alopecia (40% to 60%) but hair will usually regrow although it may be 
a different color and/or texture. Hair loss usually begins 3-6 weeks after the start of 
therapy.
o
Endocrine & metabolic: Fertility: May cause sterility; interferes with oogenesis and 
spermatogenesis; may be irreversible in some patients; gonadal suppression 
(amenorrhea).
o
Gastrointestinal: Nausea and vomiting occur more frequently with larger doses, usually 
beginning 6-10 hours after administration; anorexia, diarrhea, mucositis, and stomatitis 
are also seen.
o
Genitourinary: Severe, potentially fatal acute hemorrhagic cystitis, believed to be a 
result of chemical irritation of the bladder by acrolein, a cyclophosphamide metabolite, 
occurs in 7% to 12% of patients and has been reported in up to 40% of patients in some 
series. Patients should be encouraged to drink plenty of fluids during therapy (most 
adults will require at least 2 L/day), void frequently, and avoid taking the drug at night. 
With large I.V. doses, I.V. hydration is usually recommended. The use of mesna and/or 
continuous bladder irrigation is rarely needed for doses <2 g/m2.
o
Hematologic: Thrombocytopenia and anemia are less common than leukopenia. Onset: 
7 days, Nadir: 10-14 days, Recovery: 21 days.

From 1% to 10%:
o
Cardiovascular: Facial flushing
o
Central nervous system: Headache
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o
Dermatologic: Skin rash
o
Renal: SIADH may occur, usually with doses >50 mg/kg (or 1 g/m2); renal tubular 
necrosis, which usually resolves with discontinuation of the drug, is also reported.
o
Respiratory: Nasal congestion occurs when I.V. doses are administered too rapidly 
(large doses via 30-60 minute infusion); patients experience runny eyes, rhinorrhea, 
sinus congestion, and sneezing during or immediately after the infusion. If needed, a 
decongestant 
or 
decongestant/antihistamine 
(e.g., 
pseudoephedrine 
or 
pseudoephedrine/triprolidine) can be used to prevent or relieve these symptoms.

Less than 1% (Limited to important or life-threatening):
o
High-dose therapy may cause cardiac dysfunction manifested as CHF; cardiac necrosis 
or hemorrhagic myocarditis has occurred rarely, but may be fatal. Cyclophosphamide 
may also potentiate the cardiac toxicity of anthracyclines.
o
Other adverse reactions include anaphylactic reactions, darkening of skin/fingernails, 
dizziness, hemorrhagic colitis, hemorrhagic ureteritis, hepatotoxicity, hyperuricemia, 
hypokalemia, jaundice, neutrophilic eccrine hidradenitis, radiation recall, renal tubular 
necrosis, secondary malignancy (e.g., bladder carcinoma), Stevens-Johnson syndrome, 
toxic epidermal necrolysis; interstitial pneumonitis and pulmonary fibrosis are 
occasionally seen with high doses.

BMT:
o
Cardiovascular: Heart failure, cardiac necrosis, pericardial tamponade
o
Endocrine & metabolic: Hyponatremia
o
Hematologic: Methemoglobinemia
o
Gastrointestinal: Severe nausea and vomiting
o
Miscellaneous: Hemorrhagic cystitis, secondary malignancy
MECHANISM OF ACTION: Cyclophosphamide is an alkylating agent that prevents cell 
division by cross-linking DNA strands and decreasing DNA synthesis. It is a cell cycle phase 
nonspecific agent. Cyclophosphamide also possesses potent immunosuppressive activity. 
Cyclophosphamide is a prodrug that must be metabolized to active metabolites in the liver.
8.3.1
Mesna: Drug Information
Copyright 1978-2009 Lexi-Comp, Inc. All rights reserved.
U.S. BRAND NAMES — Mesnex®
PHARMACOLOGIC CATEGORY: Antidote; Uroprotectant
ADMINISTRATION: For IV administration, the required dose of mesna should be withdrawn 
from the multidose vial labeled as containing 100 mg/mL and diluted with an appropriate volume 
of a compatible IV solution (i.e., 5% dextrose; 5% dextrose and 0.2, 0.33, or 0.45% sodium 
chloride; 0.9% sodium chloride; lactated Ringer’s) to obtain a solution containing 20 mg/mL The 
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diluted solution may then be given by direct IV injection or infused IV over a period of 15–30 
minutes.
ADVERSE REACTIONS: SIGNIFICANT
Mesna Monotherapy: Headache, injection site reactions, flushing, dizziness, nausea, vomiting, 
somnolence, diarrhea, anorexia, fever, pharyngitis, hyperesthesia, influenza-like symptoms, 
coughing, constipation, flatulence, rhinitis, rigors, back pain, rash, conjunctivitis, arthralgia.
Mesna Alone (frequency not defined):

Cardiovascular: Flushing

Central nervous system: Dizziness, fever, headache, hyperesthesia, somnolence

Dermatologic: Rash

Gastrointestinal: Anorexia, constipation, diarrhea, flatulence, nausea, taste alteration/bad 
taste (with oral administration), vomiting

Local: Injection site reactions

Neuromuscular: Arthralgia, back pain, rigors

Ocular: Conjunctivitis

Respiratory: Cough, pharyngitis, rhinitis

Miscellaneous: Flu-like syndrome
Mesna Alone or in Combination: (Post-marketing and/or case reports) Allergic reaction, 
anaphylactic reaction, hypersensitivity, hyper-/hypotension, injection site erythema, injection site 
pain, limb pain, malaise, myalgia, platelets decreased, ST-segment increased, tachycardia, 
tachypnea, transaminases increased.
MECHANISM OF ACTION: In blood, mesna is oxidized to dimesna which in turn is reduced 
in the kidney back to mesna, supplying a free thiol group which binds to and inactivates acrolein, 
the urotoxic metabolite of ifosfamide and cyclophosphamide.
Cyclophosphamide and mesna will be provided to the study participant, at no cost, by the study 
sponsor.
8.4
Laboratory Reagents
8.4.1
OKT3
OKT3 (eBioscience), is an anti-CD3 antibody used to activate T cells in vitro by engaging the CD3 
complex. In the current clinical trial, OKT3 will be strictly used ex vivo for the activation of PBMC 
for retroviral transduction. A clinical grade OKT3 termed Orthoclone (muromonab-CD3, Ortho-
Biotech) is a therapeutic monoclonal antibody approved by the FDA to treat rejection of 
transplanted organs, including the heart, kidneys and liver. However, this antibody is currently not 
being produced by the clinical grade manufacture. When used in vivo OKT3 antibodies eliminate 
CD3 positive lymphocytes which is beneficial to treat graft rejections. It can induce anaphylactic 
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or anaphylactoid reactions, cytokine release syndrome and a variety of constitutional symptoms 
when administered systemically. The source of OKT3 antibody for the current studies is obtained 
with a certificate of analysis providing its manufacturing and product characterization. In addition, 
the batches used within this study are further characterized by performing the following tests: 
Gram stain, fungal stain, bacterial culture, fungal culture, mycoplasma culture, endotoxin assay.
8.4.2
Interleukin-2
Please refer to package insert for complete product Information. Human recombinant interleukin-
2 is a highly purified protein with a molecular weight of approximately 15,300 daltons. It is a 
lymphokine produced by recombinant DNA technology using a genetically engineered E. coli 
strain containing an analog of the human interleukin-2 gene.
Mode of Action: Immunotherapeutic agent which stimulates T and NK cell responses.
How Supplied: Interleukin-2 (IL-2) is manufactured by Prometheus.
Formulation/Reconstitution: IL-2 is provided as single-use vials containing 22 million IU 
(~1.3 mg) IL-2 as a sterile, white to off-white lyophilized cake. In this protocol, IL-2 will be used 
only for in-vitro stimulation and expansion of PBMC at a concentration of 300 IU/ml. Harvested 
transgenic cells will be washed three times with normal saline before packaging into the infusion 
bag.
Storage: Intact vials are stored in the refrigerator (2ºC – 8ºC) protected from light. Each vial bears 
an expiration date.
Dilution/Stability: Reconstituted IL-2 will be further diluted to prepare a stock solution for in-
vitro use and stored at -80ºC in single use aliquots. One aliquot from this will be subjected to 
sterility tests and endotoxin assay. These aliquots will be used as single use up to the expiry date 
on the original vial.
Dose and Schedule: In-vitro use only at 300 IU/ML in the stimulation media and expansion media 
during the manufacture of the transgenic T cells.
Administration: In-vitro use only. IL-2 will not be administered directly to the patient.
Toxicities: Expected toxicities of IL-2 are listed in the product label. Grade III toxicities common 
to high dose IL-2 include diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, 
mucositis, dysphagia, or constitutional symptoms and laboratory changes; it is not expected that 
these side effects will be manifested by in-vitro use of IL-2 during cell manufacture.
8.4.3
RetroNectin®
RetroNectin® (CH-296, Takara) is a chimeric peptide of recombinant human fibronectin 
fragments produced in E. coli, consisting of three functional domains: a central cell-binding 
domain (type III repeat, 8-10), heparin-binding domain II (type III repeat, 12-14), and a CS1 site 
within the alternatively-spliced IIICS region. It is a 574 amino acid protein with a molecular weight 
of 63 kDa. When coated on the surface of containers such as culture dishes, petri dishes, flasks or 
bags, RetroNectin® significantly enhances retrovirus-mediated gene transduction into mammalian 
cells. This enhancement is hypothetically due to co-localization of retroviral particles and target 
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cells on the molecules of RetroNectin®. Virus particles bind RetroNectin® via interaction with 
heparin-binding domain II, and target cells bind mainly through the interaction of the cell surface 
integrin receptor VLA-4 with the fibronectin CS1 site. Additionally, cells may also bind through 
the interaction of another fibronectin ligand (RGDS in repeat 10) within the central cell-binding 
domain with a corresponding integrin receptor VLA-5 on the cell surface.
8.4.3.1
Clinical Grade Retrovirus Lot Release Testing
Prior to use, the clinical grade retroviral vector will require fulfilling the lot release criteria: (1) 
Certificate of Analysis of the retroviral vector Master Cell Bank (MCB) and, (2) Certificate of 
Analysis of the Clinical grade retrovirus (see Appendix F: C1 & C2).
8.4.3.2
Samples for Transgenic TCR Cell Persistence and Immune Monitoring
TCR and vector presence will be quantitated in PBMC samples, obtained before adoptive transfer 
and at weeks 1, 2, 4, 8, 12, and months 6 and 9. At 9 months, the interventional portion of the 
protocol ends and long term follow up begins. From years 1 to 5, every 6 months and then yearly 
up to 15 years if transgenic T cell is present in the peripheral blood. Analysis will be performed 
both using immune monitoring and molecular techniques. Detection of surface expression of the 
NY-ESO-1 TCR transgenic protein will be analyzed both by MHC tetramer or dextramer analysis 
and staining for the specific region. Molecular analysis of the persistence of cells bearing the NY-
ESO-1 TCR cDNA will be done by real time PCR techniques using primers specific for the 
transgenes and retroviral vector sequences. This testing will provide data to estimate the in vivo 
survival of lymphocytes derived from the infused cells.
9
STUDY PROCEDURES
Informed consent MUST be completed prior to receiving any study related procedures.
To account for the pilot investigational nature of the procedures in this protocol and anticipated 
problems with patient scheduling, timing of events are approximate and the study team will attempt 
to perform them on the dates included in this protocol, but variations on these dates will not be 
considered protocol violations.
9.1
Screening Evaluations
The initial screening period will be 30 days, but patients will be allowed to enroll after the 30-day 
initial screening period, for up to approximately 60 days, using the same screening procedure test 
results to avoid unnecessary assay duplication. A pregnancy test must be completed within 48 
hours prior to dosing, if applicable.
Screening will occur in 2 stages. In the first stage, a patient will provide their written informed 
consent, and will provide a small amount of blood or buccal swab for HLA typing (see section 
9.14.8). In addition, patient will be screened for NY-ESO-1 positivity using historical results, 
archival tissue or fresh biopsy.  
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If the patient is eligible based on HLA type and tumor antigen expression, then the patient will 
undergo the remainder of the screening procedures. This will include the following (see 
Section 9.12 for a more complete definition of assessments):

Informed consent: Must be completed prior to receiving any study-related procedures as 
described above.

Medical history (9.14.1, 9.14.2)

Targeted physical exam (9.14.5)

Determination of performance status (9.14.4)

CBC and differential (9.14.7)

Complete metabolic and hepatic panel (9.14.6)

Baseline CA-125 reading (for ovarian cancer patients)

Urinalysis

Screening for suitability for apheresis

Cardiac function tests (Troponin/EKG)

Cardiac stress test (Cardiac stress test will be done as clinically indicated, the specific test 
to be chosen at the discretion of the treating physician).

Tumor Biopsy or acquisition of archival material (refer to section 9.16.1)
Patients' 
tumor 
will 
be 
screened 
for 
NY-ESO-1 
antigen 
expression 
by 
immunohistochemistry (IHC) and/or molecular analysis by qRT-PCR. At Roswell Park, 
NY-ESO-1 testing will be done in accordance with Protocol I 226412 titled "NY-ESO-1 
Expression in Patients with Solid Tumors" or Protocol I 215512 titled “Analysis of Tumor 
Antigens, Immunity, and Genetic Changes in Gynecological Cancer Patients”.  Data from 
prior IHC and/or molecular tests may be used.

Baseline CT Scan
A baseline CT scan will be done for baseline tumor assessment (An existing scan within 
60 days of enrollment is acceptable).

Brain MRI as clinically indicated

Pregnancy Test
All females of childbearing potential must have a negative serum or urine Pregnancy Test 
within 48 hours from the initial dose of conditioning chemotherapy in order to be eligible 
for participation. Females who have undergone surgical sterilization, or who have been 
postmenopausal for at least 2 years, are not considered to be of childbearing potential.

Concomitant medications
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9.2
Day (-6): Leukapheresis for NY-ESO-1 TCR T cell Product. 

Within 1 week of leukapheresis, after initial eligibility has been determined, the following 
will be performed:
o
Stool Sample Collection Questionnaire (refer to Appendix G)
o
Microbiome Collection Questionnaire (refer to Appendix H)
o
Microbiome Stool Collection (refer to Appendix I)

Collection of blood for research

Apheresis
After enrollment, the leukapheresis date (Day -6) will be scheduled based on the T cell 
manufacture schedule and infusion day (Day 0). All patients will undergo a 12L mononuclear 
cell apheresis procedure to collect steady-state T-cells. Patients will be admitted to the Blood 
Donor facility for leukapheresis that will be performed on an outpatient basis. Antecubital 
veins will be used, or when necessary, a central catheter will be placed. A standard collection 
protocol will be used to obtain the peripheral white blood cells with settings modified 
according to the Cobe Spectra Apheresis System recommendations (or other comparable 
machine) to obtain the best mononuclear cell yield.

Concomitant medications

Adverse events
9.3
Cohort 4 only: Days -6 to -4 Decitabine

Decitabine Administration: as described in Section 7.1.3.

Concomitant medications

Adverse events
9.4
Cohorts 1, 2 &3: Days –5 & –4; Cohort 4: Days -3 &-2 Chemotherapy 
Administration. 

Medical History

Targeted Physical Exam

ECOG Performance Status

CBC, differential

Complete Metabolic and Hepatic Panel

Urinalysis

Chemotherapy Administration: as described in Section 8.1.2.

Concomitant medications
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
Adverse events
9.5
Day 0: NY-ESO-1 TCR/dnTGFβRII T cell Infusion

Subject will be admitted to the BMT ward at RPCI for the infusion. 

T cell infusion: The TCR genetically engineered PBMC product will be transported to the 
in-patient unit on ice in a rigid-walled container with a tight fitting lid (i.e. Tupperware; 
Nalgene container etc.) and labeled with a biohazard label. Infusion and monitoring 
procedures are described in Section 8.1.2.

Research blood will be collected as follows:
o
Prior to infusion (SST: 2 cc)
o
At 1, 2 and 4 hours post infusion (SST: 2 cc)

Medical history

Targeted physical exam

Determination of performance status (ECOG)

CBC and differential

Complete metabolic and hepatic panel

CA-125 level (pre-infusion for ovarian cancer patients only) 

Urinalysis

Concomitant medications

Adverse events

Troponin/EKG if clinically indicated.
9.6
Post Infusion Follow-Up (the week of Infusion)
Patients will be followed daily the week of infusion as inpatient/ outpatient. These visits will 
include the following procedures (urinalysis and EKG/troponin will be done if clinically 
indicated):

Medical history

Targeted physical exam

Determination of performance status (ECOG)

CBC and differential

Complete metabolic and hepatic panel

Ferritin as clinically indicated.

Collection of blood for research (Table 2)
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
Concomitant medications

Adverse events
9.7
Hospital Discharge
Patients will be discharged from the hospital when the following criteria are met and following the 
clinical judgment of the treating physicians:

Absolute neutrophil count (ANC) > 500/μL

Platelet count > 20,000/μL

Hemodynamically stable

Liver function tests stable

Not requiring daily blood product infusion
9.8
Post Infusion Follow-Up (Weeks 1 to 4)
Patients will be followed weekly until week 4. There is a ±2 day window for these visits, which 
will include the following procedures:

Medical history

Targeted physical exam

Determination of performance status (ECOG)

CBC and differential

Complete metabolic and hepatic panel 

Ferritin as clinically indicated

CA-125 reading (Ovarian cancer patients, week 4 only)

Concomitant medications

Adverse events
Collection of blood for research at weeks 1, 2 and 4 (Collection of 100 ml of peripheral blood each 
time (less may be collected depending on patient condition). However, blood draws on these days 
should be performed if considered safe, and may be skipped or delayed because patients are likely 
to have chemotherapy-induced pancytopenia).
9.9
Post Infusion Follow-up (week 6)

Tumor imaging (a restaging CT of the chest, abdomen and pelvis, will be performed to 
track changes in tumors)

Optional biopsy (excisional or core biopsy whenever feasible.)
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9.10 Post Infusion Follow-Up (Week 8 and Week 12)
Patients will be followed at Week 8 (± 2 days) and Week 12 (± 1 week). The reason for the smaller 
window at Week 8 is that this time point is important for staging and monitoring for adverse events. 
These visits will include the following procedures:

Medical history

Targeted physical exam

Determination of performance status (ECOG)

CBC and differential

Complete metabolic and hepatic panel

CA-125 reading (Ovarian cancer patient)

Collection of blood for research

Tumor Imaging (Patients will undergo restaging CT scans of the chest, abdomen and pelvis 
at week 12 ± 1 week)

Concomitant medications

Adverse events

Microbiome Collection Questionnaire week 12 only (refer to Appendix H)

Microbiome Stool Collection week 12 only (refer to Appendix I)
9.11  Post Infusion Follow-Up (6 months and 9 Months)
Patients will be followed at 6 months (± 2 weeks) and 9 months (± 2 weeks). These visits will 
include the following procedures:

Medical history

Targeted physical exam

Determination of performance status (ECOG)

CBC and differential

Complete metabolic and hepatic panel

CA-125 reading (ovarian cancer patients)

Collection of blood for research 

Tumor Imaging

Concomitant medications

Adverse events
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9.12 Re-Staging Evaluation: Week 12
At week 12 post-adoptive cell transfer (± 1 week), patients will be evaluated for antitumor activity 
after undergoing re-staging exams and laboratory tests. Patient with SD, PR or CR will be offered 
to continue on protocol after discussion of the study results. Patients with PD will be offered to 
terminate participation. However, given the phenomenon of delayed responses with tumor 
immunotherapy approaches, the final decision to continue or not on study will be at the discretion 
of the treating physician and the patient, regardless of the tumor response assessment.
9.13 Long-Term Follow-up Plan (Years 1 to 15)
Evaluations will be performed for up to 15 years on all subjects as specified by FDA, in accordance 
with recent guidelines for long-term follow-up (LTFU) set forth by the ASGT and the FDA. Please 
refer to Table 2 for Long Term Follow-up.
The most recently released (November 2006) Guidance on Monitoring for Delayed Adverse 
Events states that for the first 5 years, all subjects should undergo monitoring of vector sequences 
every 6 months, and a full physical examination including a medical history, concomitant 
medications and examination of appropriate organ systems and a hemogram annually. For the final 
10 years, if vector sequences are no longer detected in PBMCs, a one page questionnaire or post 
card may be sufficient for reporting of any adverse events and questions relating to the patient's 
status.
For the LTFU, subjects who have completed the 9 month visit, the first visit will occur at 1 year 
post T cell infusion and then every 6 months until the end of year 5 post T cell infusion. For 
subjects who do not complete the 3 month visit, the first visit will occur at 3 months post T cell 
infusion, the second visit will be 6 month post T cell infusion, the third visit will occur at 9 months 
post T cell infusion and the next visit will occur 1 year post T cell Infusion, and then every 6 
months until the end of year 5 post T cell infusion.
Visits will include a medical history, physical exam, and blood tests for disease monitoring, CBC, 
chemistries, monitoring for persistence of vector modified cells and annually for RCR 
testing/archive. The physical exam and medical history (including concomitant medications and 
adverse events) will be conducted with careful attention to features possibly related to 
oncoretroviral diseases including: (1) New malignancies, (2) New incidence or exacerbation of a 
pre-existing neurologic disorder, (3) New incidence of exacerbation of a prior rheumatologic or 
other autoimmune disorder, and (4) New incidence of a hematologic disorder.
For the next 10 years, subjects will be asked to return for testing only if vector modified cells were 
detected in the previous year, and if so, then blood for persistence of vector modified cells and 
RCR testing {archive) will also be performed/collected. If no vector modified cells were detected 
in the previous year, the follow-up will be conducted by means of a clinical questionnaire 
completed via phone or through the mail by the study coordinator. The questionnaire will include 
the following questions:

When did you last visit your personal physician?

Has there been a change in your condition? Are there any adverse events to report?
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
Did you have a blood test?

Did your physician express any concerns with the test results?
Participants who come off the study prior to 12 months post cell infusion and are started on another 
treatment including standard of care due to disease progression will be asked to come for follow 
up visits at 3, 6 and 9 months when blood for Central Lab Immunemonitoring will be collected (70 
cc: 7 Lavender-Top Tubes, used for PBMC for functional Assays/Immunophenotyping, cell pellets 
for Vector Copy number, RCR & RCL tests). Patients receiving viral vector engineered cells 
are required by the FDA to be tested for RCR/RCL at these time points. For 1 year and later 
time points, the same schedule as in Table 5 will be followed. Biopsy will be done at 3 months 
post infusion if clinically feasible.
The schedule of procedures and observations for this study is summarized in Table 2 below.
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Table 2
Schedule of Procedures and Observations
LTFU
Years 1-5*
Screening
Day -6 
(Cohort 4 only) 
Day -6 to -4
Cohorts 1, 2 &3) 
Day -5 and -4
(Cohort 4 only) 
Day -3 and -2
Day 0
Day 1 to 3 
Day 7 
Week 2 to 4
(weekly ±2 days)
Week 6 
(± 1 week)
Week 8
(±2 days)
Week 12
(±1 week)
6 months
(±2 weeks)
9 months
(±2 weeks)
Years 1,2,3,4,5
(±1 month)
Years 
1.5,2.5,3.5,4.5
(±1 month)
Clinical Assessments14
Informed Consent
X
HLA Typing (Blood-10 ml Lavender 
Top tube or buccal swab)
X
Medical History
X
X
X
X
X
X
X
X
X
X
X
X
X
Targeted Physical Exam
X
X
X
X11
X
X
X
X
X12
X
X
X
X
ECOG Performance Status
X
X
X
X
X
X
X
X
X
X
X
X
X
CBC, differential (2 ml lavender)
X
X
X
X
X
X
X
X
X
X
X
X
Complete Metabolic & Hepatic Panel (2 
ml SST)
X
X
X
X
X
X
X
X
X
X
X
X
Ferritin Level
X13 X13
X13
Urinalysis
X
X
X
X
Troponin/EKG
X2
X2
X2
Leukapheresis screen
X15
CA-125 Monitoring (5 ml SST; ovarian 
cancer patients)
X
X3
X5
X
X
X
X
Pregnancy Test4
X
Concomitant medications (see 9.14.3)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Adverse Event
X
X
X
X
X
X
X
X
X
X
X
X
X
X

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LTFU
Years 1-5*
Screening
Day -6 
(Cohort 4 only) 
Day -6 to -4
Cohorts 1, 2 &3) 
Day -5 and -4
(Cohort 4 only) 
Day -3 and -2
Day 0
Day 1 to 3 
Day 7 
Week 2 to 4
(weekly ±2 days)
Week 6 
(± 1 week)
Week 8
(±2 days)
Week 12
(±1 week)
6 months
(±2 weeks)
9 months
(±2 weeks)
Years 1,2,3,4,5
(±1 month)
Years 
1.5,2.5,3.5,4.5
(±1 month)
Stool Sample Collection Questionnaire16
X16
Microbiome Collection Questionnaire17
X17
X
Procedures14
Cyclophosphamide & Mesna
X
X
Decitabine20
X
Apheresis 12L
X
Inpatient Admission & T cell Infusion
X
CT 
X
X
X
X
X
Brain MRI
X1
Research Assessments14
Biopsy and Tumor Antigen
X6
X7,8
Blood for cell analyses (60 to 100 ml, 
Lavender-Top tube)
X
X9
X
X
X
X
X
X
PBMC (Immunophenotyping, Tetramer 
& functional analyses19
X
X
X9
X
X
X
X
X
X
Serum (2 ml SST)
X10
X
X
X9
X
X
X
X
Microbiome Stool Collection18 
X18
X
*
Follow-up until 15 years post infusion is required, but can be completed by phone call or postcard if gene modified cells are no longer detected 
in the patient.
1
Brain MRI only if clinically indicated.

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2
2EKG will be done at screening.  Cardiac stress test will be done as clinically indicated, the specific test to be chosen at the discretion of the treating 
physician.  Troponin at screening; Troponin and EKG on Day 0, 1, 2, 3 if clinically indicated.
3
Day 0 pre-infusion.
4
Females only: within 48 hours of starting therapy.
5
Week 4 only.
6
Required only if archival is not available for NY-ESO testing (if not previously done) and for research.
7
Optional biopsies at Week 6 (± 1 week) and upon progression.
8
Antigen expression at Week 6 and upon progression if biopsy is performed.
9
Week 2 & 4 only.
10 2 ml pre-infusion and at 1, 2, and 4 hours post infusion.
11 T cells administered IV without lymphocyte filter, by rapid infusion over approximately15 minutes. Vital signs are recorded prior to, during, and at 15 
minutes, 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours post-infusion. Refer to Section 7.5.1for supportive therapy on Day 0.
12 Response assessment for ending participation at Week 12.
13 Ferritin level as clinically indicated.
14 To facilitate scheduling, a (-1 day) window is allowed for assessments and procedures required on first day of cyclophosphamide treatment or Day 0 (day 
of T-cell infusion).
15 Venous access assessment and bone marrow transplant autotransplant serology order set (without CMV testing)
16 Stool Sample Collection questionnaire to be completed at time of stool microbiome collection [within 1 week of apheresis (day -6), after initial eligibility 
has been confirmed]. Refer to section 9.17.2 and Appendix G.
17 Microbiome Collection Questionnaire to be completed at time of stool microbiome collection [within 1 week of apheresis  (day -6), after initial eligibility 
has been confirmed]. Refer to section 9.17.2 and Appendix H.
18 After initial eligibility has been confirmed, stool for microbiome will be collected within 1 week of apheresis (day-6) Refer to section 9.17.1 and Appendix 
I.
19 The PBMC will be processed from the 60 to 100 ml blood collected in lavender top tubes
20 To be started after leukapheresis

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9.14  Definitions
9.14.1
Medical History and Physical Examination
The medical history must include all diagnoses and surgical procedures of major organ systems. 
Any allergies to any medications and their formulations must be documented.
9.14.2
Previous Medications
All prescription and nonprescription medication, vitamins, herbal and nutritional supplements, 
taken by the subject during the 30 days prior to screening will be recorded at the screening visit.
9.14.3
Concomitant Medications
Concomitant medications will be recorded in the medical record and on the appropriate eCRF. In 
addition, any additions, deletions, or changes of these medications will be documented.
9.14.4
ECOG Performance Status
Performance status will be assessed by ECOG. If necessary, for reporting purposes, the scale can 
be converted to Karnofsky using the standard scale shown in Appendix A.
9.14.5
Targeted Physical Examination and Vital Signs
A targeted physical exam will imply assessments in each of the following categories: General 
(weight, height, blood pressure, pulse, respiration rate, and temperature), head, eyes, ear, nose and 
throat examinations, lymph node examination, chest and cardiovascular system examination, 
breasts, respiratory system, abdominal examination, dermatological examination, and neurological 
examination including mental status assessment.
9.14.6
Complete Metabolic and Hepatic Panel
Complete metabolic panel will include measurements of sodium, potassium, chloride, bicarbonate, 
urea nitrogen, creatinine, total protein, albumin, calcium, and lactic dehydrogenase. Hepatic panel 
refers to liver function tests will include measurements of bilirubin, alanine aminotransferase 
(ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP). In the schedule of study 
procedures, chemistries and liver function tests are always performed together.
9.14.7
CBC with Differential
Hematology tests will include hemoglobin, hematocrit, platelet count, red cell count, and white 
cell count with differential count.
9.14.8
HLA A201 Screening
A high-resolution test for the HLA A2 allele is required. At the present time, only HLA A201 
subjects are eligible, as the different subtypes (e.g., HLA A202) do not interact with the engineered 
TCRs. HLA testing will be performed by a CLIA certified laboratory (such as Quest Laboratories) 
as selected by each clinical site.
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9.15 Research Blood Sample Collection and Processing
9.15.1  Day 6 in the Morning
On the morning of the day, a 12 liter leukapheresis will be performed at the Blood Donor Facility 
at RPCI, as an outpatient. Antecubital veins will be used, or when necessary, a central catheter will 
be placed. A standard collection protocol will be used to obtain the peripheral white blood cells 
with settings modified according to the Cobe Spectra Apheresis System recommendations (or 
other comparable machine) to obtain the best mononuclear cell yield. In order to obtain sufficient 
monocytes cells, leukapheresis time may vary from 90 minutes to 240 minutes. Cells will be 
transferred to the cGMP cell production facility at the Center for Immunotherapy (CCC building, 
4th Floor) where the transgenic cells will be manufactured using the procedure described in 
Appendix B and Appendix D.
9.15.2 Immune Monitoring Analysis (blood for cell analyses)
Collection of 60 to 100 ml of peripheral blood (7 to 11 tubes of 10 ml lavender top EDTA tubes) 
is scheduled on study day +7 and study day +14. However, blood draws on these days should be 
performed if considered safe, and may be skipped or delayed because patients are likely to have 
chemotherapy-induced pancytopenia. These blood draws can be done +/- 6 days from the intended 
days.
100 ml of blood samples will also be collected on weeks 4, 8, and 12, and at months 6 and 9 post 
adoptive transfer as listed on Table 2. After 9 months, LTFU starts and during years 1 to 5, blood 
samples will be collected every 6 months until no transgenic cells are detectable in the peripheral 
blood.
Roswell Park Cancer Institute
Immune Analysis Shared Resource (IASR) CCC room 416
Attn: Junko Matsuzaki (I 285416 Samples)
Elm & Carlton Streets
Buffalo, NY 14263
(716) 845-8459
Junko.matsuzaki@RoswellPark.org
Note: All investigator or analyzing research laboratories housing research samples need 
to maintain current Temperature Logs and study-specific Sample Tracking and Shipping 
Logs. The Principal Investigator/Laboratory Manager must ensure that the stated lab(s) 
have a process in place to document the receipt/processing/storage/shipping of study-
related samples/specimens. This is required for both observational and interventional 
clinical studies collecting clinical samples.
9.15.3  Serum Analyses
Serum (2 ml SST) will be collected on infusion day (Day 0) pre-infusion, and at 1, 2 and 4 hours 
post-infusion, Days +1, +2, +3, +7, and weeks +2, +4, +8, +12, and at months +6 and +9.
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The blood samples will be received by the Immune Analyses Facility at the Center for 
Immunotherapy (CCC building, 4th floor).
Roswell Park Cancer Institute
Immune Analysis Shared Resource (IASR) CCC room 416
Attn: Junko Matsuzaki (I 285416 Samples)
Elm & Carlton Streets
Buffalo, NY 14263
(716) 845-8459
Junko.matsuzaki@RoswellPark.org
Note: All investigator or analyzing research laboratories housing research samples need 
to maintain current Temperature Logs and study-specific Sample Tracking and Shipping 
Logs. The Principal Investigator/Laboratory Manager must ensure that the stated lab(s) 
have a process in place to document the receipt/processing/storage/shipping of study-
related samples/specimens. This is required for both observational and interventional 
clinical studies collecting clinical samples.
9.16 Pathology
9.16.1  Screening Tumor Tissue Requirements
NY-ESO-1 Testing 
Historical NY-ESO-1 results may be used.  If not previously done, tumor tissue requirements for 
CT-antigens (e.g., NY-ESO-1) expression in tumor are as follows:

Evaluation by Immunohistochemistry will occur at OmniSeq, LLC if required for 
eligibility (at PNSR if solely needed for research).

A target minimum of 4 x 4 mm block of primary or metastatic tumor.

If unable to release tumor block please submit: 5 unstained sections at 4-5 microns 
thickness on charged glass slides and 8 unstained sections at 10 microns thickness on 
uncharged glass slides, cut with molecular precautions.
A biopsy of a tumor lesion accessible to outpatient sampling either surgically or by image-guided 
biopsy will be collected if adequate archival tissue as described above is not available (collect 2 
cores and process to FFPE, per site usual process, for NY-ESO-1 testing). For cohort 1, 2, and 3: 
This lesion will be required to be NY-ESO-1 positive by IHC or RNA analysis, compared to 
suitable positive and negative controls.  For Cohort 4, NY-ESO-1 results will be noted but NY-
ESO-1 positivity is not required for eligibility.
Tumor Tissue for Research
Confirmation of available archival tissue:

Enough tissue for approximately 5 sections at 5 microns thickness and 15-30 sections at 8 
microns as a block or as unstained charged slides; material to be requested for patients who 
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have a subsequent on-treatment biopsy or biopsy at progression.  If archival material is 
extremely limited, a fresh biopsy will be done at the discretion of the investigator. 
9.16.2  On Study Tumor Tissue Requirements
Optional biopsies, if clinically feasible,  will be performed 6 weeks post ACT (± 1 week, or when 
the treating physicians deem that the patient is stable enough to undergo these procedures), and 
upon progression either as an inpatient or outpatient; optional research tumor tissue at progression 
will only be collected in conjunction with a standard of care procedure needed for patient care..  
For  each optional biopsy, 5 or more cores (or pieces of tumor tissue equivalent to 18 gauge or 
larger needle core) should be collected from a core or excisional biopsy procedure; if fewer than 5 
cores are available, please process in the following order of priority:

Cores 1 and 2: place in 10% NBF 12-24 hours (longer is acceptable), then process and 
embed into 1 block and deliver to IASR

Core 3: place in cryovial and snap freeze in liquid nitrogen, then store at -80°C until 
delivery to IASR on dry ice

Core 4: place in a cryovial of RNAlater, refrigerate overnight, then store at -80°C until 
delivery to IASR on dry ice

Core 5+ : place fresh tissue in media provided by IASR (RPMI +10% FBS) and transport 
on ice pack  as soon as possible to IASR for TIL processing.  
Tumor tissue for research should be labeled as follows:

Study # I 258514

Patient Study ID #

Time Point (Screening, 6W post-ACT, Progression)

Collection Date

Tumor site (e.g. abdominal mass, lymph node, etc.)
For patients having an optional biopsy, the pre-treatment archival “Tumor Tissue for Research” 
described in section 9.16.1 will also be requested at this time and delivered to IASR when ready.
In patients where follow up tumor biopsies are available, the following will be performed to 
attempt to detect the NY-ESO-1 TCR/ dnTGFβRII transduced PBMC. The additional tumor 
biopsies will be analyzed for tumor expression of the tumor antigen target (NY ESO 1, LAGE-1); 
HLA expression, and infiltration by lymphocytes. The qPCR will be performed by the Genomic 
Shared Resource (GSR), and the IHC by the Pathology Network Shared Resource (PNSR) at 
Roswell Park Cancer Institute. TIL analysis will be performed by the Immune Analysis Shared 
Resource (IASR) at the Center for Immunotherapy, Roswell Park Cancer Institute. 
Roswell Park Cancer Institute
Correlative Science Pathology Office, GBSB S-636
Attn: I 285416 Samples
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Elm & Carlton Streets
Buffalo, NY 14263
(716) 845-8917
CRSLabPathTeam@RoswellPark.org
Note: Specimens will be accepted Monday through Thursday only (not on a holiday or day 
before holiday). Please contact the lab for special arrangement if an exception is to be 
requested.
Note: All investigator or analyzing research laboratories housing research samples need to 
maintain current Temperature Logs and study-specific Sample Tracking and Shipping Logs. The 
Principal Investigator/Laboratory Manager must ensure that the stated lab(s) have a process in 
place to document the receipt/processing/storage/shipping of study-related samples/specimens. 
This is required for both observational and interventional clinical studies collecting clinical 
samples.
9.17 Gut Microbiota
9.17.1 Fecal microbiome collection
After initial eligibility has been determined, stool samples will be collected within 1 week of 
apheresis (day -6) and 12 weeks post T cell infusion.  The stool specimen will be self-collected by 
participants.  Patients will be provided with a sample collection kit.  Patients will be provided with 
written instructions (Appendix I), and study staff will explain how to use the kits to collect fecal 
samples in the privacy of their own homes.
We will instruct the participants to return the stool sample within 24 hours of collection.  If 
patient is unable to provide the sample with home collection or enough sample for analysis, a 
digital rectal exam will be performed at the time of their study visit.   All microbiome samples 
will be labeled with the participant’s MR number, participant’s initials, participant’s study 
number, clinical study number, protocol time point, and protocol day.  Collected stool samples 
will be sent to DBBR for de-identification, storage and future DNA extraction.  
Roswell Park Cancer Institute
DBBR Laboratory
GBSB Bldg. 7th Floor, Rm. 726 via Tube station #86
Attn: Study Number-I 258514
Elm and Carlton Streets
Buffalo, NY 14263
Tel: 716-845-1036
Fax: 716-845-1350
warren.davis@roswellpark.org
Note: All investigator or analyzing research laboratories housing research samples need to 
maintain current Temperature Logs and study-specific Sample Tracking and Shipping Logs. The 
Principal Investigator/Laboratory Manager must ensure that the stated lab(s) have a process in 
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place to document the receipt/processing/storage/shipping of study-related samples/specimens. 
This is required for both observational and interventional clinical studies collecting clinical 
samples.
9.17.2 Questionnaires
The Stool Sample Collection Questionnaire (Appendix G) and the Microbiome Collection 
Questionnaire (Appendix H) were designed for patients enrolling in this study in order to broaden 
the information gathered on nutrition and basic lifestyle habits that have been shown to have an 
impact on human microbiome composition.  The Stool Sample Questionnaire will be completed 
only once at screening.  The Microbiome Collection Questionnaire will be completed at the same 
time points as stool collection (within 1 week of apheresis (day -6) and 12 weeks post T cell 
infusion).  Paper questionnaires will be collected by the Clinical Research Coordinator on the day 
of the patient’s visit and will be kept in a secured, locked up study folder in clinic.  Data from 
collected questionnaires throughout the trial will be migrated and stored in a secure REDCap data 
base. 
10 EFFICACY EVALUATIONS
10.1 Objective Tumor Response
All protocol-defined imaging studies must be performed at the investigative site or sponsor-
approved facility using protocol-defined parameters. The same method of assessment and the same 
technique should be used to characterize each identified and reported lesion at baseline and during 
follow-up. Immune-Related Response Criteria (irRECIST) described by Nishino et al, 2013 (71) 
and Bohnsack et al, 2014 (72) will be used to assess objective tumor response.
10.2 Definition of Target and Non-Target Lesions
10.2.1 Target Lesions

All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, 
representative of all involved organs, will be identified as target lesions and recorded and 
measured at baseline

Measurable lesions must be accurately measured in at least one dimension with a minimum 
size of:
o
10 mm in the longest diameter by CT or MRI scan (or no less than double the slice 
thickness) for non- nodal lesions and ≥ 15 mm in short axis for nodal lesions.
o
10 mm caliper measurement by clinical exam (lesions which cannot be accurately 
measured with calipers should be recorded as non-measurable).
o
20 mm by chest X-ray

A sum of the longest diameter (short axis for lymph nodes) of all target lesions will be 
calculated and reported as the baseline sum diameters. This will be used as reference to 
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further characterize the objective tumor response of the measurable dimension of the 
disease.
10.2.2 Non-Target Lesions

Non-target lesions will include:
o
Measurable lesions not selected as target lesions
o
All sites of non-measurable disease, such as neoplastic masses that are too small to 
measure because their longest uninterrupted diameter is < 10 mm (or < two times 
the axial slice thickness), i.e., the longest perpendicular diameter is ≥ 10 and 
< 15 mm.
o
Other types of lesions that are confidently felt to represent neoplastic tissue, but are 
difficult to measure in a reproducible manner. These include bone metastases, 
leptomeningeal metastases, malignant ascites, pleural or pericardial effusions, 
ascites, inflammatory breast disease, lymphangitis cutis/pulmonis, cystic lesions, 
ill-defined abdominal masses, skin lesions, etc.

All lesions or sites of disease not recorded as target lesions (e.g., small lesions and non-
measurable lesions) should be identified as non-target lesions and indicated as present in 
the source documents at baseline. There is no limit to the number of non-target lesions that 
can be recorded at baseline. The general location will also be documented on the images, 
drawing a regularly-shaped Region of Interest. 

Measurements of the non-target lesions will not be performed, but the presence or absence 
of each should be noted throughout follow-up and evaluation.
10.3 Lesions
Target and Non-Target Lymph Node Lesion Definitions

To be considered pathologically enlarged and measurable, a lymph node must be ≥ 15 mm 
in short axis when assessed by CT scan (CT scan slice thickness recommended to be no 
greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and 
followed.
Bone Lesions

Bone scan, PET scan or plain films are not considered adequate imaging techniques to 
measure bone lesions. However, these techniques can be used to confirm the presence or 
disappearance of bone lesions.

Regardless of the imaging modality blastic bone lesions will not be selected as target 
lesions. Lytic or mixed lytic-blastic lesions with a measurable soft tissue component 
≥ 10 mm can be selected as target lesions.
Brain Lesions
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
Brain Lesions detected on brain scans can be considered as both target or non-target 
lesions.
Cystic and Necrotic Lesions as Target Lesions

Lesions that meet the criteria for radiographically defined simple cysts should not be 
considered as malignant lesions (neither measurable nor non-measurable) since they are, 
by definition, simple cysts.

Lesions that are partially cystic or necrotic can be selected as target lesions.

The longest diameter of such a lesion will be added to the Total Measured Tumor Burden 
(TMTB) 1 of all target lesions at baseline. 

If other lesions with a non-liquid/non-necrotic component are present, those should be 
preferred.
Lesions with Prior Local Treatment

During target lesion selection the radiologist will consider information on the anatomical 
sites of previous intervention (e.g. previous irradiation, RF-ablation, TACE, surgery, etc.).

Lesions undergoing prior intervention will not be selected as target lesions unless there has 
been a demonstration of progress in the lesion.
No Disease at Baseline

If a patient has no measurable and no non-measurable disease at baseline the radiologist 
will assign ‘No Disease’ (irND) as the overall tumor assessment for any available follow-
up time-points unless new measurable lesions are identified and contribute to the TMTB.
10.3.1 Follow-Up
Recording of Target and New Measurable Lesion Measurements 

The longest diameters of non-nodal target and new non-nodal measurable lesions, and short 
axes of nodal target and new nodal measurable lesions will be recorded. Together they 
determine the Total Measured Tumor Burden (TMTB) at follow-up.
𝐓𝐌𝐓𝐁 % 𝐜𝐡𝐚𝐧𝐠𝐞=
𝐁𝐚𝐬𝐞𝐥𝐢𝐧𝐞 𝐓𝐮𝐦𝐨𝐫 𝐁𝐮𝐫𝐝𝐞𝐧‒ 𝐂𝐮𝐫𝐫𝐞𝐧𝐭 𝐓𝐮𝐦𝐨𝐫 𝐁𝐮𝐫𝐝𝐞𝐧
𝐁𝐚𝐬𝐞𝐥𝐢𝐧𝐞 𝐓𝐮𝐦𝐨𝐫 𝐁𝐮𝐫𝐝𝐞𝐧
 × 𝟏𝟎𝟎
Definition of New Measurable Lesions 

In order to be selected as new measurable lesions (≤ 2 lesions per organ, ≤ 5 lesions total, 
per time-point), new lesions must meet criteria as defined for baseline target lesion 
selection and meet the same minimum size requirements of 10 mm in long diameter and 
minimum 15 mm in short axis for new measurable lymph nodes. New measurable lesions 
shall be prioritized according to size, and the largest lesions shall be selected as new 
measured lesions.
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Non-Target Lesion Assessment

The RECIST 1.1 definitions for the assessment of non-target lesions apply (i.e., 
measurements of the non-target lesions will not be performed, but the presence or absence 
of each should be noted throughout follow-up and evaluation).

The response of non-target lesions primarily contributes to the overall response 
assessments of irCR and irNon-CR/Non-PD (irNN).

Non-target lesions do not affect irPR and irSD assessments. 

Only a massive and unequivocal worsening of non-target lesions alone, even without 
progress in the TMTB is indicative of irPD.
New Non-Measurable Lesions Definition and Assessment

All new lesions not selected as new measurable lesions are considered new non-measurable 
lesions and are followed qualitatively. 

Only a massive and unequivocal progression of new non-measurable lesions leads to an 
overall assessment of irPD for the time-point.

Persisting new non- measurable lesions prevent irCR.
10.4 irRECIST Overall Tumor Assessments 
The irRECIST overall tumor assessment is based on the TMTB (total measured tumor burden) of 
measured target and new lesions, non-target lesion assessment and new non-measurable lesions.
Time point response assessments will be performed at 4 weeks, 12 weeks, 6 months and 9 months.
Conventional RECIST 1.1 will also be utilized during the trial however; RECIST 1.1 will not be 
used to determine disease progression. The irRECIST will be used for tumor response assessment 
at time of continuing review and will be used for IRB reporting and formal analysis.

irCR: Complete disappearance of all measurable and non-measurable lesions. Lymph 
nodes must decrease to < 10 mm in short axis. 

irPR: Decrease of ≥ 30% in TMTB relative to baseline, non-target lesions are irNN, and 
no unequivocal progression of new non-measurable lesions.

irSD: Failure to meet criteria for irCR or irPR in the absence of irPD.

irNN: No target disease was identified at baseline and at follow-up the patient fails to meet 
criteria for irCR or irPD.

irPD: Minimum 20% increase and minimum 5 mm absolute increase in TMTB compared 
to nadir, or irPD for non-target or new non-measurable lesions. Confirmation of 
progression is recommended minimum 4 weeks after the first irPD assessment.

irNE: Used in exceptional cases where insufficient data exists.

irND: In adjuvant setting when no disease is detected.
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New Lesions: the presence of new lesion(s) does not define progression. The measurements of the 
new lesion(s) are included in the sum of the measurements (the sum of the measurements = the 
sum of the longest diameters of all target lesions and new lesions, if any).
10.5 Confirmation Measurement
A confirmatory assessment is required no less than 6 weeks after a PR or CR is deemed by 
irRECIST
10.6 Guidelines for Evaluation of Measurable Disease
All measurements should be taken and recorded in metric notation. All baseline evaluations should 
be performed as closely as possible to the beginning of treatment and never more than 4 weeks 
before the beginning of the treatment.
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam.

Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are 
clearly defined and surrounded by aerated lung. However, CT is preferable.

Conventional CT and MRI: This guideline has defined measurability of lesions on CT 
scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have 
slice thickness greater than 5 mm, the minimum size for a measurable lesion should be 
twice the slice thickness. MRI is also acceptable in certain situations (e.g., for body scans) 
however, conventional CT scan is the preferred modality to determine response to 
treatment.
Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; 
however, there are many image acquisition variables involved in MRI, which greatly impact image 
quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable 
globally. As with CT, if an MRI is performed, the technical specifications of the scanning 
sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, 
as with CT, the modality used at follow-up should be the same as was used at baseline and the 
lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the 
irRECIST or RECIST guidelines to prescribe specific MRI pulse sequence parameters for all 
scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image 
acquisition protocol should be followed as closely as possible to prior scans. Body scans should 
be performed with breath-hold scanning techniques, if possible.

Ultrasound: Ultrasound is not useful in assessment of lesion size and should not be used 
as a method of measurement. Ultrasound examinations cannot be reproduced in their 
entirety for independent review at a later date and, because they are operator dependent, it 
cannot be guaranteed that the same technique and measurements will be taken from one 
assessment to the next. If new lesions are identified by ultrasound in the course of the study, 
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confirmation by CT or MRI is advised. If there is concern about radiation exposure at CT, 
MRI may be used instead of CT in selected instances.

Tumor Markers: Tumor markers alone cannot be used to assess response. If markers are 
initially above the upper normal limit, they must normalize for a participant to be 
considered in complete clinical response.
11 SAFETY EVALUATION
11.1 Adverse Events
11.1.1 Definition
An adverse event or adverse experience (AE) is any untoward medical occurrence associated with 
the use of a drug in humans, whether or not considered drug related. Therefore, an AE can be ANY 
unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a medicinal (investigational) product, whether or not 
considered related to the medicinal (investigational) product (attribution of ‘unrelated’, ‘unlikely’, 
‘possible’, ‘probable’, or ‘definite’).
An AE is considered “unexpected” if it is not listed in the investigator brochure or is not listed at 
the specificity or severity that has been observed; or if an investigator brochure is not required or 
available, is not consistent with the risk information described in the general investigational plan 
in other study-related documents.
11.1.1.1 Diagnosis Versus Signs and Symptoms
If known, a diagnosis should be recorded on the CRF rather than individual signs and symptoms 
(e.g., record only liver failure or hepatitis rather than jaundice, asterixis, and elevated 
transaminases). However, if a constellation of signs and/or symptoms cannot be clinically 
characterized as a single diagnosis or syndrome at the time of reporting, each individual event 
should be recorded as an AE or SAE on the CRF. If a diagnosis is subsequently established, it 
should be reported as follow-up information.
11.1.1.2 Adverse Events Occurring Secondary to Other Events
In general, AEs occurring secondary to other events (e.g., cascade events or clinical sequelae) 
should be identified by their primary cause. For example, if severe diarrhea is known to have 
resulted in dehydration, it is sufficient to record only diarrhea as an AE or SAE on the CRF.
However, clinically significant AEs occurring secondary to an initiating event that are separated 
in time should be recorded as independent events on the CRF. For example, if a severe 
gastrointestinal hemorrhage leads to renal failure, both events should be recorded separately on 
the CRF.
11.1.1.3 Abnormal Laboratory Values
Only clinically significant laboratory abnormalities that require active management will be 
recorded as AEs or SAEs on the CRF (e.g., abnormalities that require study drug dose 
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modification, discontinuation of study treatment, more frequent follow-up assessments, further 
diagnostic investigation, etc.).
If the clinically significant laboratory abnormality is a sign of a disease or syndrome (e.g., alkaline 
phosphatase and bilirubin 5 x the upper limit of normal associated with cholecystitis), only the 
diagnosis (e.g., cholecystitis) needs to be recorded on the Adverse Event CRF.
If the clinically significant laboratory abnormality is not a sign of a disease or syndrome, the 
abnormality itself should be recorded as an AE or SAE on the CRF. If the laboratory abnormality 
can be characterized by a precise clinical term, the clinical term should be recorded as the AE or 
SAE. For example, an elevated serum potassium level of 7 mEq/L should be recorded as 
“hyperkalemia”.
Observations of the same clinically significant laboratory abnormality from visit to visit should 
not be repeatedly recorded as AEs or SAEs on the CRF, unless their severity, seriousness, or 
etiology changes.
11.1.1.4 Preexisting Medical Conditions (Baseline Signs and Symptoms)
A preexisting medical condition should be recorded as an AE or SAE only if the frequency, 
severity, or character of the condition worsens during the study. When recording such events on 
an Adverse Event CRF, it is important to convey the concept that the preexisting condition has 
changed by including applicable descriptors (e.g., “more frequent headaches”).
11.1.2 Grading and Relationship to Drug
The descriptions and grading scales found in the CTEP Version 4 of the NCI Common 
Terminology Criteria for Adverse Events (CTCAE) will be utilized for AE reporting. CTEP 
Version 4 
of 
the 
CTCAE 
is 
identified 
and 
located 
at: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. AEs not covered by 
specific terminology listed should be reported with common medical terminology, and 
documented according to the grading scales provided in the CTCAE Version 4.
The relationship of event to study drug will be documented by the Investigator as follows:

Unrelated: The event is clearly related to other factors such as the patient’s clinical state, 
other therapeutic interventions or concomitant drugs administered to the patient.

Unlikely: The event is doubtfully related to investigational agent(s). The event was most 
likely related to other factors such as the patient’s clinical state, other therapeutic 
interventions, or concomitant drugs.

Possible: The event follows a reasonable temporal sequence from the time of drug 
administration, but could have been produced by other factors such as the patient’s clinical 
state, other therapeutic interventions or concomitant drugs.

Probable: The event follows a reasonable temporal sequence from the time of drug 
administration, and follows a known response pattern to the study drug. The event cannot 
be reasonably explained by other factors such as the patient’s clinical state, therapeutic 
interventions or concomitant drugs.
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
Definite: The event follows a reasonable temporal sequence from the time of drug 
administration, follows a known response pattern to the study drug, cannot be reasonably 
explained by other factors such as the patient’s condition, therapeutic interventions or 
concomitant drugs; AND occurs immediately following study drug administration, 
improves upon stopping the drug, or reappears on re-exposure.
11.1.3 Reporting Adverse Events
Table 3
Guidelines for Routine Adverse Event Reporting for Phase 1 Studies 
(Regardless of Expectedness)
Attribution
Grade 1
Grade 2
Grade 3
Grade 4
Unrelated
X
X
X
X
Unlikely
X
X
X
X
Possible
X
X
X
X
Probable
X
X
X
X
Definite
X
X
X
X
Table 4
Guidelines for Routine Adverse Event Reporting Phase 2 Studies (Regardless 
of Expectedness)
Attribution
Grade 1
Grade 2
Grade 3
Grade 4
Unrelated
X
X
Unlikely
X
X
Possible
X
X
X
X
Probable
X
X
X
X
Definite
X
X
X
X
11.2 Routine AE’s will be reported from the start date of intervention.  Refer to section 
9.13 for duration of follow-up AE reporting.  Serious Adverse Events
11.2.1 Definition
A serious adverse event (SAE) is any adverse event (experience) that in the opinion of either the 
investigator or sponsor results in ANY of the following:

Death.

A life-threatening adverse event (experience). Any AE that places a patient or patient, in 
the view of the Investigator or sponsor, at immediate risk of death from the reaction as it 
occurred. It does NOT include an AE that, had it occurred in a more severe form, might 
have caused death.

Inpatient hospitalization or prolongation of existing hospitalization (for > 24 hours).
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
A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions.

A congenital anomaly or birth defect.

Important Medical Event (IME) that, based upon medical judgment, may jeopardize the 
patient and may require medical or surgical intervention to prevent one of the outcomes 
listed above.
Progression of the malignancy under trial should not be reported as an adverse event. However, if 
the malignancy has a fatal outcome during the trial or within the safety-reporting period, then 
disease progression must be recorded as a Grade 5 serious adverse event.
Medical and scientific judgment should be exercised in determining whether an event is an 
important medical event. An important medical event may not be immediately life threatening 
and/or result in death or hospitalization. However, if it is determined that the event may jeopardize 
the patient and may require intervention to prevent one of the other outcomes listed in the definition 
above, the important medical event should be reported as serious.
11.2.2 Reporting Serious Adverse Events
All new SAEs occurring from the date the patient signs the study consent until 30 days after the 
last intervention or a new treatment is started, whichever comes first, will be reported. The RPCI 
SAE Source Form is to be completed with all available information, including a brief narrative 
describing the SAE and any other relevant information.
SAEs occurring after the 30 day follow-up period that the investigator determines to be possibly, 
probably or definitely related to the study intervention should be reported.
SAEs identified as an Unanticipated Problem by the Investigator must be reported. Please refer to 
Section 11.5 for details on reporting Unanticipated Problems.
11.3 Investigator Reporting: Notifying the Study Sponsor
Events that meet the SAE definition will be reported within the following timelines based on the 
time of first knowledge of the SAE by a study investigator:

SAE other than resulting in unexpected death:
o
P.I.: Within 24 hours.
o
IRB, ISPRC, DSMB, IBC, NIH/RAC: Within 7 days.
o
FDA: Within 15 days.

Unexpected death:
o
P.I.: Within 24 hours.
o
IRB, ISPRC, DSMB, IBC, NIH/RAC: Within 24 hours.
o
FDA: Within 7 days.
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In general, any SAE will be reported as soon as it is known by the study investigators and enough 
data is gathered for report filing. All SAE reports will be managed through the SAE Compliance 
Officer, working under the JCCC Regulatory Affairs Office. Initial reports may be completed or 
amended with any new relevant additional information.
Copies of all reports will be maintained in the study regulatory file. Copies of any report sent to 
the FDA will also be sent to the study sponsor (Sponsor: NCI).
11.4 Follow-Up for Serious Adverse Events
All related SAEs should be followed to their resolution, until the study patient is lost to follow-up, 
the start of a new treatment, or until the study investigator assesses the event(s) as stable or 
irreversible. New information will be reported when it is received.
11.5 Unanticipated Problems
11.5.1 Definition
An Unanticipated Problem (UP) is any incident, experience, or outcome that meets all of the 
following criteria:
Unexpected (in terms of nature, severity, or frequency) given:
a) The research procedures that are described in the study-related documents, including 
study deviations, as well as issues related to compromise of patient privacy or 
confidentiality of data.
b) The characteristics of the patient population being studied.
Related or possibly related to participation in the research (possibly related means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research).
Suggests that the research places patients or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized and if in 
relation to an AE is also deemed Serious per Section 11.2.
11.5.2 Reporting Unanticipated Problems
The Reportable New Information (RNI) Form will be submitted to the CRS Quality Assurance 
(QA) Office within 1 business day of becoming aware of the Unanticipated Problem. After review, 
CRS QA Office will submit the RNI to the IRB.
When becoming aware of new information about an Unanticipated Problem, submit the updated 
information to CRS QA Office with an updated Reportable New Information Form. The site 
Investigator or designated research personnel will report all unanticipated problems to the IRB in 
accordance with their local institutional guidelines.
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11.6 FDA Reporting
When RPCI is the IND holder the following describes the FDA reporting requirements by timeline 
for AEs and new safety findings that meet the criteria outlined below:
Within 7 Calendar Days
Any adverse event that meets ALL the following criteria:

Related or possibly related to the use of the study drug;

Unexpected; and

Fatal or life-threatening.
Within 15 Calendar Days
Any adverse event that meets ALL the following criteria:

Related or possibly related to the use of the study drug;

Unexpected; and

Serious but not fatal or life-threatening;
Or, meets ANY of the following criteria:

A previous adverse event that is not initially deemed reportable but is later found to fit the 
criteria for reporting (report within 15 days from when event was deemed reportable).

Any findings from other studies, including epidemiological studies, pooled analysis of 
multiple studies, or other clinical studies conducted with the study drug that suggest a 
significant risk in humans exposed to the drug.

Any findings from animal or in vitro testing that suggest a significant risk for human 
participants including reports of mutagenicity, teratogenicity, or carcinogenicity or reports 
of significant organ toxicity at or near the expected human exposure.

Any clinically important increase in the rate of occurrence of a serious, related or possibly 
related adverse event over that listed in the protocol or investigator brochure.
Sponsors are also required to identify in IND safety reports, all previous reports concerning similar 
adverse events and to analyze the significance of the current event in the light of the previous 
reports.
Reporting Process
The principal investigator or designee will complete and submit a FDA Form 3500A MedWatch 
for any event that meets the above criteria. Forms will be submitted to the CRS Compliance Office 
via email to CRSCompliance@RoswellPark.org.
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11.7 Study Stopping and Non-stopping Rules
It is recognized that AEs can occur frequently in this population based on the underlying metastatic 
disease and these can be SAEs. The review of SAEs will form the basis for potential early stopping 
of the study. Only unexpected SAEs that are related to the transduction process/research reagent 
would define a stopping rule. The review of these adverse events, and any decision to prematurely 
stop subject enrollment, will be determined by the DSMB and reviewed by the IRB.
Absolute stopping rules for this clinical trial will be:
1. Any death that is possibly related to the NY-ESO-1 TCR (T cell receptor).
2. Two or more grade 4 events that are possibly related to the NY-ESO-1 TCR (T cell 
receptor).
3. Any event of hematological malignancy must be considered study stopping criteria until 
oncogenesis related to retroviral mutagenesis, EBV lymphoma and Post-Transplant 
Lymphoproliferative Disorder (PTLD) can be excluded.
Premature termination of the clinical trial may occur because of a regulatory authority decision, 
change in opinion of the FDA, RAC, IRB, the DSMB, or determination that there are problems in 
the cell product generation or the safety of their administration as descried in the assessment of 
primary study endpoints. Additionally, recruitment may be stopped for reasons of particularly low 
recruitment, protocol violations, or inadequate data recording.
11.8 Gene Therapy Study Reporting of Serious Adverse Events
Studies that involve Gene Therapy will report SAEs as described above and will be reported to the 
Office of Biotechnology Activities (OBA) / NIH.
Any SAE that is fatal or life-threatening, that is unexpected, and associated with the use of the 
gene transfer product must be reported to the FDA and NIH OBA as soon as possible, but not later 
than 7 calendar days after the Sponsor’s initial receipt of the information.
SAEs that are unexpected and associated with the use of the gene transfer product, but are not fatal 
or life-threatening, must be reported to the FDA and NIH OBA as soon as possible, but not later 
than 15 calendar days after the sponsor’s initial receipt of the information.
If, after further evaluation, an adverse event initially considered not to be associated with the use 
of the gene transfer product is subsequently determined to be associated, then the event must be 
reported to the NIH OBA within 15 days of the determination.
Any follow-up information relevant to a SAE must be reported within 15 calendar days of the 
Sponsor’s receipt of the information. If a SAE occurs after the end of a clinical trial and is 
determined to be associated with the use of the gene transfer product, that event shall be reported 
to the NIH OBA within 15 calendar days of the determination.
The Principal Investigator or delegated research staff will submit a report to the CRS Compliance 
Office via email: CRS Compliance for all gene therapy SAEs. The Compliance office will forward 
the information to ORSP.
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Completed reports may be sent via U.S. mail, courier service, e-mail, or facsimile to:
NIH Office of Biotechnology Activities
6705 Rockledge Drive, Suite 750
Bethesda, Maryland 20892-7985
(For all non-USPS deliveries use Zip Code 20817)
Telephone: 301-496-9838
Fax: 301-496-9839
E-mail address for Reporting Adverse Events: GeMCRIS@od.nih.gov
General E-mail: oba@od.nih.gov
Website: http://oba.od.nih.gov/oba/index.html
Additional information for reporting adverse events, including a report template, can be found at: 
http://oba.od.nih.gov/rdna/adverse_event_oba.html
12 DATA AND SAFETY MONITORING
Phase 1 studies will be reviewed at the scheduled Roswell Park Early Phase Clinical Trials (EPCT) 
meetings and the minutes are forwarded to the IRB for review.
The Research Monitor, Kelvin Lee, MD, PhD, is responsible to oversee the safety of the research 
and report observations/findings to the IRB or a designated institutional official. The Research 
Monitor will review all unanticipated problems involving risks to subjects or others associated 
with the protocol and provide an independent report of the event to the IRB. The Research Monitor 
may discuss the research protocol with the investigators; shall have authority to stop a research 
protocol in progress, remove individual human subjects from a research protocol, and take 
whatever steps are necessary to protect the safety and well-being of human subjects until the IRB 
can assess the monitor's report; and shall have the responsibility to promptly report their 
observations and findings to the IRB or other designated official and the HRPO.
13 STATISTICAL METHODOLOGY
This is a Phase I/IIa clinical trial principally designed to assess the safety (defined by dose limiting 
toxicities, DLT) and feasibility (defined by sufficient numbers of circulating, lot-release confirmed 
T cells) of treating patients with advanced malignancies with escalating doses of infused 
genetically engineered PBMC expressing NY-ESO-1 TCR together with a dnTGFβ-RII transgene 
administered after lympho-depleting conditioning chemotherapy.
The phase I part of the study includes three cohorts of 3 subjects (+ 3 additional subjects in case 
of 1 DLT) will receive, 107, 108 and 109 NY-ESO-1/dnTGFβRII cells, respectively. The 
assessment of safety and feasibility is based on studying cohorts of 3 subjects, with the addition of 
3 subjects in case of toxicities meeting the definition of DLT in one of the three first subjects in a 
cohort. If two or more subjects have DLT in a cohort, that cohort will be considered beyond the 
MTD and an intermediate dosing may be explored to better define the MTD of this combination. 
The Phase II part of the study will include an expanded cohort of 9 patients at the maximum 
tolerated dose. 
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13.1 Sample Size Determination
A maximum of 27 patients at RPCI will be enrolled. A minimum of 3 evaluable subjects will be 
treated at each of the 3 cohorts and 9 patients in cohort 4. Accrual is expected to take up to 4 years.
13.2 Definition of Evaluable Patients
Patients evaluable for the primary analysis are those fully enrolled in the study after meeting the 
inclusion/exclusion criteria. Other patients will be considered un-evaluable and excluded from the 
primary analysis.
13.3 Primary Study Endpoints
13.3.1 Safety
Safety will be assessed by monitoring and recording potential adverse effects of the treatment 
using the Common Toxicity Criteria at each study visit. Subjects will be monitored by  physical 
examinations, and blood studies to detect potential toxicities from the treatment.
In this study, safety evaluations will continue and the same early stopping rules for DLTs will 
apply to this study. Safety remains the priority in all phases and stages of the investigational 
studies.
13.3.2 Feasibility
After entering 3 patients to each cohort, followed up for a minimum of 1 month after the last 
subject has received the infusion of the transgenic cells, an assessment of protocol feasibility will 
be done by the study investigators. The feasibility assessment will be based on potential problems 
in the manufacturing of NY-ESO-1/ dnTGFβRII engineered cells (Table 5).
Table 5
Criteria to Declare the Study Unfeasible
Parameter
Criteria for Unfeasibility
Feasibility of generation transgenic cells that meet the 
lot release criteria.
3 or more preparations not meeting the lot release 
criteria in each cohort
13.4 Secondary Study Endpoints
13.4.1 Transgenic Cell Persistence
TCR and vector presence will be quantitated in PBMC samples, obtained before adoptive transfer 
and at weeks 1, 2, 4, 8, and 12 after transgenic cell adoptive transfer. Thereafter, sampling will be 
every 3 months during the first year, and then every 6 to 12 months as per Schedule of Procedures 
and Observations (refer to Table 2). Analysis will be performed both using immune monitoring 
and molecular techniques. Detection of surface expression of the NY-ESO-1 TCR transgenic 
protein will be analyzed both by MHC tetramer analysis and staining for the specific region. The 
V5 tagged 2A sequence linked to the dnTGFβRII and quantitative PCR will allow the assessment 
of the ratio of NY-ESO-1 TCR versus NY-ESO-1 TCR/dnTGFβRII transgenic cells.
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13.4.1.1 Long Term Monitoring for Replication Competent Retrovirus (RCR)
Analysis for detection of RCR by PCR will be performed at the National Gene Vector 
Biorepository (NGVB) at the Indiana University Viral Production Facility (IU VPF) under the 
supervision of Dr. Kenneth G. Cornetta. Samples taken from cells prior to infusion will be archived 
and blood samples obtained from study patients at 3 and 6 months, and at one year post cell 
administration will be tested. Blood samples will be archived annually thereafter if all previous 
testing has been negative.
If a patient dies or develops a neoplasm (other than cancer recurrence) during this clinical trial, 
efforts will be made to assay a biopsy sample for RCR. If any post-treatment samples are positive, 
then the baseline final product will be tested for RCR. Further analysis of the RCR and more 
extensive patient follow-up will be undertaken, in consultation with the IRB, RAC and FDA.
13.4.1.2 Analysis of Retroviral Insertion Sites in Long Term Persisting NY-ESO-1 
TCR  Clones
It is possible that expansion of specific T-cell clones will be observed as tumor reactive T cells 
proliferate in response to tumor antigens. Therefore, care will be taken to track T cell persistence 
both immunologically and molecularly. Blood samples for persistence of TCR gene transduced 
cells will be obtained at 3, 6, and 12 months, and then annually thereafter. If any patient shows a 
high level of persistence of TCR gene transduced cells at month 6 (by semi quantitative DNA-
PCR using primers specific for vector sequences) the previously archived samples will be 
subjected to techniques that would allow the identification of clonality of persisting TCR gene 
transduced cells. Such techniques may include T cell cloning or LAM-PCR. Clonality analysis is 
available through NGVL (73).
If a predominant or monoclonal T cell clone derived from TCR gene transduced cells is identified 
during the follow-up, the integration site and sequence will be identified and subsequently 
analyzed against human genome database to determine whether the sequences are associated with 
any known human cancers. If a predominant integration site is observed, the T cell cloning or 
LAM-PCR test will be used at an interval of no more than three months after the first observation 
to see if the clone persists or is transient. In all instances where monoclonality is persistent and 
particularly in instances where there is expansion of the clone, regardless of whether or not the 
sequence is known to be associated with a known human cancer, the subject should be monitored 
closely for signs of malignancy.
13.4.1.3 Immunological Monitoring
Immunological monitoring will consist primarily of quantifying T cells bearing surface NY-ESO-1 
TCR by NY-ESO-1(157-165) /MHC tetramer analysis. We will use our methodology based on 
combination of NY-ESO-1(157-165) tetramer and peptide stimulation to analyze PBMC samples 
with intracellular effector cytokine staining of TNF and IFN, and Granzyme B / CD107a,b for 
cytotoxic T cell function. With our expertise in polychromatic flow cytometry (74-77) we will also 
be able to perform simultaneous multifunctional assessment (Almeida et al 2007) of the transgenic 
cells. Functional assays like ELISA and/or multi-cytokine array assays will complement the 
results. Immunological assays will be compared between 1) pre-infusion PBMC, 2) an aliquot of 
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the engineered PBL at the time of infusion, 3) cells recovered from patients’ peripheral blood after 
adoptive transfer.
Additional testing may be performed with other assays, for example intracellular phosphoprotein 
staining for signaling networks, multiplexed microfluidic and nanotechnology-based immune 
monitoring assays (78).
13.4.2 Evaluation of NY-ESO-1 TCR Transgenic Cell Tumor Trafficking
13.4.2.1 Tumor Biopsies
For the numbers and functional phenotype of NY-ESO-1 TCR transgenic T cells accumulating at 
the tumor site, percutaneous or image-guided biopsies of tumor lesions between study days 20-40, 
with allowed variability from this range, will be performed.
The biopsies will be analyzed by H&E, IHC to quantify the numbers of T lymphocytes If the 
sufficient quantity of tissue is available, the study investigators will attempt to monitor the 
phenotype of the TIL obtained from tumor biopsy samples by MHC tetramer, multicolor flow 
cytometry (FACS), and other immune monitoring assays.
13.4.3 Antitumor Activity
13.4.3.1 Malignancy Status
Tumor response is an exploratory endpoint in this clinical trial. All patients who have received an 
infusion of NY-ESO-1 TCR/dnTGFβRII transgenic T cells and have tumor assessments at baseline 
and during the study follow up will be considered evaluable for tumor response.
Potential objective responses to this combinatorial immunotherapy will be recorded following the 
irRECIST (71, 72). Appropriate evaluations, including physical exam, pictures of visible lesions 
and imaging exams, will be evaluated at screening and then between study days 75-90, or at 
discontinuation of study. The duration of patient evaluation may be extended if additional 
outpatient visits are required to assess duration of tumor response or time to progression.
13.5 Efficacy Analysis
The toxicities observed after each TCR transgenic cell infusion will be summarized in terms of 
type (organ affected or laboratory determination such as absolute neutrophil count), severity (by 
Toxicity Table) and nadir or maximum values for the laboratory measures, time of onset (i.e. 
course number), duration, and reversibility or outcome. Tables will be created to summarize these 
toxicities and side effects by dose and by course. Baseline information (e.g. the extent of prior 
therapy) and demographic information will be presented, as well, to describe the patients treated 
in this pilot study. All responses will be reported.
13.6 Safety Analysis
DLTs are the primary endpoint of this study and are used in the estimation of the MTD and the 
accompanying of the dose escalation decisions. However, no formal analyses of DLTs are planned. 
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Patients who do not have a DLT and who do not complete a full cycle of treatment will be 
considered non-evaluable for DLT.
Safety will be assessed by monitoring and recording potential adverse effects of the treatment 
using the Common Toxicity Criteria at each study visit. Subjects will be monitored by medical 
histories, physical examinations, ophthalmologic exams, and blood studies to detect potential 
toxicities from the treatment.
In this study, safety evaluations will continue and the same early stopping rules for DLTs will 
apply to this study. Safety remains the priority in all phases and stages of the investigational 
studies.
13.7 Adverse Event
The frequency of toxicities will be tabulated by grade across all dose levels and cycles. The 
frequency of toxicities will also be tabulated for the dose estimated to be the MTD. All patients 
who receive any study treatment will be considered evaluable for toxicity.
13.8 Interim Analysis and Criteria for Early Termination of the Study
No explicit interim analyses are planned for this study.
However, this is a Phase 1 study and as such will be monitored and discussed by Roswell Park’s 
EPCT Committee, which meets weekly. Drug safety will be monitored and evaluated continuously 
throughout the study including 30 day safety follow-up period by obtaining, reviewing and 
analyzing data on AEs, changes in laboratory values, vital signs, electrocardiograms (ECGs), and 
physical examination findings. Potential early termination decisions are an inherent part of the 
Phase 1 study monitoring.
14 ETHICAL AND REGULATORY STANDARDS
14.1 Ethical Principles
This study will not be initiated until the protocol and informed consent document(s) have been 
reviewed and approved by a properly constituted Institutional Review Board (IRB) or Independent 
Ethics Committee (IEC). Each patient (or legal guardian) shall read, understand, and sign an 
instrument of informed consent prior to performance of any study-specific procedure. It is the 
responsibility of the investigator to ensure that the patient is made aware of the investigational 
nature of the treatment and that informed consent is given.
The Investigator is responsible for the retention of the patient log and patient records; although 
personal information may be reviewed by authorized persons, that information will be treated as 
strictly confidential and will not be made publicly available. The investigator is also responsible 
for obtaining patient authorization to access medical records and other applicable study specific 
information according to Health Insurance Portability and Accountability Act regulations (where 
applicable).
This study will be conducted in compliance with all applicable laws and regulations of the state 
and/or country and institution where the patient is treated, in accordance with the Declaration of 
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Helsinki, Good Clinical Practice, and according to the guidelines in this protocol, including 
attached appendices. 
The IRB approved the consent form and protocol allowing for a legally authorized representative 
to consent on the patient’s behalf if necessary, following IRB and institute policies. This is an early 
phase therapeutic clinical study in which patients have advanced disease that is resistant or 
intolerant to existing therapy, with no standard of care options and therefore has intended potential 
benefit to each individual patient in order to have a favorable benefit-risk relationship. 
14.2 Informed Consent
The Investigator (or IRB approved designee) is responsible for obtaining written consent in 
advance, from each patient or the patient's legally authorized representative in accordance with 
ICH-GCP guidelines using the approved informed consent form, before any study specific 
procedures (including screening procedures) are performed. The informed consent form 
acknowledges all information that must be given to the patient according to ICH-GCP, including 
the purpose and nature of the study, the expected efficacy and possible side effects of the 
treatment(s), and specifying that refusal to participate will not influence further options for therapy. 
Any additional information that is applicable to the study must also be included. Additional 
national or institutionally mandated requirements for informed consent must also be adhered to. 
The patient should also be made aware that by signing the consent form, processing of sensitive 
clinical trial data and transfer to other countries for further processing is allowed.
The Investigator shall provide a copy of the signed consent form to the patient and the signed 
original shall be maintained in the Investigator File. A copy of the signed consent form must be 
filed in the patient file. At any stage, the patient may withdraw from the study and such a decision 
will not affect any further treatment options.
15 STUDY RESPONSIBILITIES
15.1 Data Collection
Data entry into the database is to be completed in a timely fashion (within 30 days) after the 
patient’s clinic visit. If an AE is considered serious it is captured on both the Adverse Event page 
and the Serious Adverse Event Source Form, which is handled in an expedited fashion.
Data management activities will be performed using eClinical. eClinical is a suite of software tools 
that enables the collection, cleaning and viewing of clinical trial data. CRS data management will 
design the study-specific database and facilitate its development by the eClinical Information 
Technology team. Once the database design is approved by the Investigator, Statistician, and 
Clinical Research Coordinator, the database will be put into production and data entry can begin. 
Data can be entered and changed only by those with the rights to do so into the eCRFs (via the 
EXPeRT Module). eClinical is compliant with all relevant technical aspects of relevant GCP 
guidelines.

The system can generate accurate copies of stored data and audit trail information in human 
readable form.
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
System access is limited to authorized individuals through the controlled assignment of 
unique ID and password combinations.

The system is designed to periodically force users to change their passwords and verifies 
that user ID and password combinations remain unique.

The system automatically generates a permanent time-stamped audit trail of all user 
interactions.
When data entry is complete, data management will review the data and will query any missing, 
incomplete, or invalid data points for resolution by the Clinical Research Coordinator and 
Investigator. Once all queries have been resolved, the data can be released to the statistician for 
analysis.
15.2 Maintenance of Study Documents
Essential documents will be retained per RPCI’s policy for 6 years from the study termination 
date. These documents could be retained for a longer period, however, if required by the applicable 
local regulatory requirements or by an agreement with RPCI.
16 ADMINISTRATIVE RULES
16.1 Revisions to the Protocol
RPCI may make such changes to the protocol as it deems necessary for safety reasons or as may 
be required by the U.S. FDA or other regulatory agencies. Revisions will be submitted to the 
IRB/ERC for written approval before implementation.
16.2 Termination of the Study
It is agreed that, for reasonable cause, either the RPCI Investigators or the Sponsor, may terminate 
this study, provided a written notice is submitted within the time period provided for in the Clinical 
Trial Agreement. In addition, RPCI may terminate the study at any time upon immediate notice if 
it believes termination is necessary for the safety of patients enrolled in the study.
16.3 Confidentiality
Any data, specimens, forms, reports, video recordings, and other records that leave the site will be 
identified only by a participant identification number (Participant ID, PID) to maintain 
confidentiality. All records will be kept in a limited access environment. All computer entry and 
networking programs will be done using PIDs only. Information will not be released without 
written authorization of the participant.
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17 APPENDICES
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Appendix A
ECOG Performance Status Scores
Description
Status
Fully active, able to carry on all pre-disease performance without restriction.
0
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light 
or sedentary nature, e.g., light housework, office work.
1
Ambulatory and capable of all self-care but unable to carry out any work activities.
2
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours.
3
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair.
4
Dead
5
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Appendix B
Procedure for Generation of Experimental Cell Therapies
Feasibility is one of the primary endpoints of this protocol. Repetitive deviations to the procedures 
described in this section will be recorded in the SOPs and the protocol modified accordingly and 
highlighted in the next submission to the IRB and FDA.
Generation of NY-ESO-1/ dnTGFβRII Transduced PBMC
Leukapheresis Procedure
Patients will undergo a 12 liters leukapheresis procedure to obtain PBMC for retroviral 
transduction. Any signs and symptoms of citrate toxicity due to apheresis (such as perioral 
paresthesia and muscle cramps) will be treated according to the Hemapheresis center standard 
procedures. The leukapheresis product will be transferred to the cGMP Suite for cell processing.
PBMC Isolation
Following leukapheresis, the product will be separated into mononuclear fraction for PBMC 
isolation using Ficoll and layering the leukapheresis product over this liquid and centrifuging at 
500g for 30 minutes.
PBMC Activation
An aliquot of PBMC obtained from leukapheresis will be cultured in AIMV with 5% heat-
inactivated AB serum at 106 cells/mL and activated for 48 hours in the presence of OKT3 
(50 ng/mL, muromonab anti-human CD3 antibody) and human IL-2 (300 IU/mL, aldesleukin) in 
order to stimulate T-cell growth to prepare for viral transduction.
Clinical Grade Retrovirus Lot Release Testing
Prior to use, the clinical grade retroviral vector will require fulfilling the lot release criteria: (1) 
Certificate of Analysis of the retroviral vector Master Cell Bank (MCB) and, (2) Certificate of 
Analysis of the Clinical Grade Retrovirus (See Appendix C).
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Appendix C
Clinical Grade Retrovirus Lot Release Criteria
C1
Certificate of Analysis of the retroviral vector Master Cell Bank (MCB)
Test
Methods
Limits
Results
Date
Contaminants
Aerobic and anaerobic 
culture for bacteria and 
fungus
No growth within 14 
days
No growth within 14 
days
8/27/2014
Mycoplasma culture 
and vero indicator cells
Negative for the 
presence of 
mycoplasma
Negative
7/15/2014
In-vitro viral assay 
utilizing 3T3, MRC-5 
and Vero cells
No CPE or 
hemadsorption
No CPE or 
hemadsorption
8/27/2014
Bovine viral assay*
Negative for cytopathic 
effect, hemadsorption 
and the presence of 7 
specific bovine viruses 
using IFA 
Negative for cytopathic 
effect, hemadsorption 
and the presence of 7 
specific bovine viruses 
using IFA
8/25/2014
Porcine viral assay*
Negative for cytopathic 
effect, hemadsorption 
and the Presence of 
porcine Viruses, 
BVDV Reovirus and 
rabies
Negative for cytopathic 
effect, hemadsorption 
and the Presence of 
porcine Viruses, BVDV 
Reovirus and rabies
8/25/2014
In-vivo viral assay*
No evidence of viral 
contamination
Negative for the 
presence of 
adventitious viral 
contaminants
8/13/2014
MAP/LCM*
No evidence of viral 
contamination
No evidence of viral 
contamination
8/15/2014
Transmission E.M.*
No Vital particles other 
than retro-virus like 
particles and no other 
identifiable microbial 
agents.
No Vital particles other 
than retro-virus like 
particles and no other 
identifiable microbial 
agents 
8/8/2014
SV40 large T antigen 
(PCR)*
Negative
<10 copies/0.2 ug DNA
8/28/2014
E1a (PCR)*
Negative
<10 copies/0.2 ug DNA
8/28/2014
Human Vital
Contaminants
HTLV (PCR)*
Negative
Negative
7/7/2014
HAV (PCR)*
Negative
Negative
7/7/2014
HCV (PCR)*
Negative
Negative
7/7/2014
HBV (PCR)*
Negative
Negative
7/7/2014
SV40 (PCR)*
Negative
Negative
7/7/2014
HIV-I, HIV-II (PCR)*
Negative
Negative
7/7/2014
HHV 6, HHV 7, HHV 
8 (PCR)*
Negative
Negative
7/7/2014
CMV (PCR)*
Negative
Negative
7/7/2014
EBV (PCR)*
Negative
Negative
7/7/2014
Human parvovirus B19
Negative
Negative
7/7/2014
HSV 1/2
Negative
Negative
7/7/2014
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Test
Methods
Limits
Results
Date
Replication 
Competent 
Retrovirus: 
GAL-V
S+L- (PG-4/293) 
amplification) 5% of 
MCB supernatant
No Evidence of RCR
No Evidence of RCR
8/27/2014
Replication
Competent 
Retrovirus:
GAL-V
S+L- (PG-4/293 co-
culture) 1% of MBC
No Evidence of RCR
No Evidence of RCR
8/27/2014
Identity
ADA Isoenzyme
Cells confirmed to be
Of murine origin
Cells confirmed to
of murine origin
8/28/2014
Vector insert 
stability
Transduction of HeLa 
cells and analysis by 
Southern blotting
Band consistent with 
predicted fragment size
Band consistent with 
predicted fragment size
8/28/2014
C2
Certificate of Analysis of the Clinical grade retrovirus
Test
Methods
Limits
Results
Date
Contaminants
Aerobic and anaerobic 
culture for Bacteria 
and Fungus
No growth 14 days
No growth 14 days
7/17/2015
Mycoplasma culture 
and Vero indicator 
cells
Negative for the 
presence of 
Mycoplasma
Negative for the 
presence of 
Mycoplasma
5/29/2015
In-Vitro viral assay 
utilizing 3T3, MRC-5
And Vero cells
No CPE or 
hemadsorption
No CPE or 
hemadsorption
7/17/2015
Endotoxin
Limulus amebocyte 
lysate 
Less than 0.33 EU/mL
Harvest 1-7: less than 
0.06 EU/mL
7/17/2015
Replication 
competent 
retrovirus: 
GAL-V
S+L-(PG-4)
(293 infection) 5% of
vector supernatant
No Evidence of RCR
No Evidence of RCR
7/10/2015
S+L-(PG-4) (293 co-
culture) 108 cells from 
production run
No Evidence of RCR
No Evidence of RCR
7/17/2015
Transgene 
expression
NY-ESO-1(157-165)  
tetramer staining in 
activated PBMC
> 30% NY-ESO-
1(157-165)  tetramer 
positive cells among 
CD3+ T lymphocytes
Harvest 1-7: 49.6 to 
60% NY-ESO-1(157-
165)  tetramer positive 
cells among CD3+ T 
lymphocytes
2/27/2015
Potency
NY-ESO-1(157-165)  
peptide and antigen-
specific IFN-γ 
production by ELISA
> 30,000 pg/ml of IFN-
γ production upon NY-
ESO-1(157-165)  
peptide stimulation 
using NY-ESO-1(157-
165)  peptide-pulsed 
K562/A2.1 cells
Harvest 1-7: > 100,000 
pg/ml of IFN-γ 
production upon NY-
ESO-1(157-165)  
peptide stimulation 
using NY-ESO-1+ 
HLA2+ cell line
3/6/2015
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Appendix D
Retrovirus Transduction and Cell Expansion Method
The retroviral vectors will be stored in a monitored and locked < = -70ºC freezer in the GMP Suite, 
Transductions will be performed in RetroNectin®-coated 6-well plates.
RetroNectin® Coating. Wells will be pre-coated with RetroNectin® (Takara Bio Inc., Japan), a 
recombinant chimeric fibronectin molecule. 1 mL of 10  mcg/mL RetroNectin® in sterile PBS is 
placed per well in 6-well plates and incubated overnight at 4oC (alternatively at room temperature 
for 2 hours). Wells are blocked with 1.5 ml of HBSS with 2.5% Human serum albumin at room 
temperature for 30 minutes and washed with HBSS containing 2.5% HEPES. 
Retroviral Transduction. Two to four ml of thawed and 1:1 diluted retroviral supernatant in TCR 
medium is applied to each RetroNectin®-coated well of Transduction #1 plates and centrifuged at 
2000g for 2 hours at 32oC. Retroviral supernatant is removed from transduction #1 plates (leaving 
behind one ml), 2 x 106 activated PBMC per well in AIMV plus 5% heat inactivated human AB 
serum supplemented by 300 IU/ml of IL-2 are added and centrifuged at 1000g for 10 minutes at 
32oC. Plates are then incubated at 37oC overnight at 5% CO2. On the following day, after blocking 
and washing, two to four ml of thawed and 1:1 diluted viral supernatant in TCR medium is applied 
to each RetroNectin®-coated well of Transduction #2 plates and centrifuged at 2000g for 2 hours 
at 32oC. Then, 1 ml of viral sup is removed from each well. PBMC are transferred from 
transduction #1 plates to transduction #2 plates and centrifuged at 1000g for 10 minutes at 32oC. 
Plates are incubated at 37oC overnight at 5% CO2. 
Post-transduction Expansion. At the end of the transduction, cells are washed and maintained at 
37oC, 5% CO2 in AIMV plus 5% heat inactivated human AB serum supplemented by 300 IU/ml 
of IL-2 at a density of 0.7 x 106 cells/mL for up to 96 hours from the initial transduction.
Transgenic PBMC Product Washing and Bagging
After completion of the cell transduction procedure and short term ex vivo expansion, the NY-
ESO-1/ dnTGFβRII transduced PBMC products are subjected to two washings, resuspended in 
saline containing 1% HSA, put into infusion bags and kept in a 4 degrees refrigerator until lot 
release clearance and i. v. infusion. Lot release tests are performed on aliquots of this final product. 
In the event patient is not able to receive the infusion within 24 hours of the scheduled harvest, 
cells will be re-suspended in cryopreservation medium and put into an infusion bag. 
Cryopreservation will be at < -130oC in a monitored liquid nitrogen freezer at the GMP suite. 
Product Labeling
The final NY-ESO-1/ dnTGFβRII transgenic PBMC bags will be labeled using a preprinted study 
label with the following information:

Patient ID. Number

Patient’s initials

Subject laboratory clinical trial code

Date of cell preparation, with an expiration date of 24 hours if fresh and of 6 months if 
cryopreserved

Initials of person who prepared the vaccine

Labeled with “For autologous use only”
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
Labeled as “Not Tested for Biohazards” (due to no specific testing for HIV, HepC, HepB 
and other adventitious viruses in the final product)
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Appendix E
In-process Testing and Final Product Lot Release Testing
In Process Testing: We will conduct the following in-process testings:
Appendix E 1 Activated PBMC (Day 2) In-process Testing
Test Item
Acceptability Criteria
Results
Gram Stain
Negative
Negative
Bacterial Culture
No growth
No growth x 14 days
Fungal Culture
No growth
No growth x 14 days
Appendix E 2 Transduced PBMC (Day 5) In-process Testing
Test Item
Acceptable Criteria
MycoAlert test
Negative ;ratio<1 (reading B/reading A)
TCR transgene expression 
>30% of T cells by NY-ESO-1(157-165)  tetramer 
staining 
TCR transgene functionality 
> 5,000 pg/ml/million cells of NY-ESO-1 specific 
IFN- production by ELISA
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Appendix F
Final Product Testing and Lot Release Criteria
The results that will be required before administering the cells to the patients are described (F1), 
along with the results that need to be in process at that time but results may not be available at the 
time of administration to patients are described (F2
Appendix F 1
NY-ESO-1/ dnTGFβRII Transgenic PBMC Lot Release Testing 
with Results Available Before Administration to Patients
Test Item
Acceptable Criteria
Viability 
> 70% 
Gram stain
a
Negative 
Endotoxin Assay b
≤ 5 EU/ kg body weight per dose 
Mycoplasma rapid test
c
Negative ;ratio<1 (reading B/reading A)
TCR transgene expression 
>30% of T cells by NY-ESO-1(157-165) tetramer 
staining 
a.
Performed on the final product. All sterility cultures will be followed up to 14 days. If the cultures become positive 
after the patient has received the cells, the patient will be started on empiric antibiotics.
b.
Performed on the final product using the Limulus amebocyte lysate read using the Quantitative Chromogenic 
Procedure analyzed using a OCL-1000 (Bio Whittaker), or the Endosafe® - PTS™ (Portable Test System: Charles 
River Laboratories International, Inc.) assays. 
c.
If first line MycoAlert test becomes positive, it will be repeated with a second sample from the final product.
Appendix F 2
NY-ESO-1/ dnTGFβRII Transgenic PBMC Lot Release Testing with 
Results Available After Administration to Patients
Test Item
Acceptable Criteria
Bacterial and fungal culture
No growth x 14 days
GalV S+  /L- RCR assay 
Sample archived for later use if required. Not tested 
prospectively in all samples.
TCR transgene functionality 
> 5,000 pg/ml/million cells of NY-ESO-1 specific 
IFN- production by ELISA
Plan in Case of Positive Results of Tests Not Available at the Time of TCR Transgenic Cell 
Administration to Patients. The following will be our plan in case of results different from 
our proposed acceptable criteria:

Bacterial or Fungal culture: An antibiogram will be obtained on the culture to typify the 
contaminant. The patient will be contacted, two peripheral blood draws will be collected 
for culture, and the patient will be started on therapy, first with broad spectrum antibiotics 
or antifungals, and then adapted to the antibiogram of the cultures.

RCR: Since the TCR transgenic cell manufacture process will include ex vivo culture for 
up to 4 days from the first transduction, RCR will not be tested prospectively in the final 
product following the Guidance for Industry document by CBER, FDA from November 
2006. If a sample from a patient tested at 3, 6 or 12 months or later is positive for RCR by 
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PCR test, the stored sample of the final product will be tested. The patient will be contacted, 
two peripheral blood draws will be collected for confirmation, and the patient will be 
started on therapy with combination antiretrovirals used to treat HIV infection.
Notification of Regulatory Agencies for Positive Results of TCR Transgenic Cell 
Administration to Patients.
If a preparation with bacterial, fungal, mycoplasma or RCR contamination has been administered 
to a patient, this event should be reported to the following agencies and committees within 48 
hours of first knowledge:

Human Gene Medicine Program compliance officer.

IRB.

ISPRC.

Study DSMB.

RAC.

FDA.
Additional Optional Testing of TCR Transgenic Cells. The release tests will be used to 
determine whether the final cell product can be released for infusion (see Tables above). In 
addition, the cell product may undergo further characterization apart from the release testing. The 
vector-specific proviral copy number per cell may be determined. The sites of integration may be 
defined as may be the structure of the integrated transgene. Detailed flow cytometry and TCR 
chain usage analysis may be performed. The product may be further characterized for measurable 
transgene products (F3)
Appendix F 3 NY-ESO-1/ dnTGFβRII Transgenic PBMC Optional Additional Testing
Characterization Assays
Comments
Vector proviral copy per 
transduced cell 
Quantification of the copy number of provirus by qRT-PCR 
Final product phenotyping 
CD3, CD4, CD8, CD27, CD28, CD45RA/RO, CD62L, CCR7, PD-1 
Transgene integration site 
analysis 
Determination of integration site(s) 
Structure of integrated 
transgene 
Stability of integrated transgene 
Detailed flow cytometry 
Phenotype of cells 
TCR transgene functionality 
> 30,000 pg/ml/million cells of NY-ESO-1 specific IFN- production by 
ELISA
Other characterizations 
Telomere loss, telomerase expression, Th1 vs Th2 pattern, FoxP3 
expression in pre/post expansion T cells 
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Appendix G
Stool Sample Collection Questionnaire
Patient Name:
MRN:
Date:
Stool Sample Collection Questionnaire 
1. What is your race? (one or more categories may be selected)
o
American Indian or Alaska Native
o
Black or African American 
o
White
o
Asian, please specify:___________
o
Native Hawaiian/Other specific Islander:
o
Other Race, please specify:_______________
2. Are you Hispanic or Spanish origin?
o
No
o
Yes
3. Were you breastfed as an infant?
o
No
o
Yes
o
I don’t know
4. Were you delivered via vaginal delivery or C-section?
o
Vaginal delivery
o
C-section
o
I don’t know
5. Have you had an appendectomy?
o
No
o
Yes
o
I don’t know
6. Have you ever had any bowel surgery/removal a portion of your bowel or 
stomach?
o
No
o
Yes, please specify__________________________
o
I don’t know
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7. Were you ever diagnosed with an autoimmune disease?
o
No __________
o
Yes, please specify_________________________
o
I don’t know _______
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Appendix H
Microbiome Collection Questionnaire
Patient Name:
MRN:
Date:
MICROBIOME COLLECTION QUESTIONNAIRE 
1. Have you had any fevers or infection in the past 2 weeks?
o
No
o
Yes, please specify:______________________________
2. Have you taken antibiotics or antifungal agents in the past 4 weeks?
o
No
o
Yes, please tell us which medication(s):___________________________              
Why were you given antibiotics? __________________________________
For how long did you take it? ______________________________________
3. How many times a week do you eat red meat? 
o
I don’t eat red meat
o
1-3 times a week
o
More than 3 times a week
4. How many times a week you eat cruciferous vegetables such as cabbage, 
broccoli, kale, and cauliflower?
o
I don’t eat cruciferous vegetables
o
1-3 times a week
o
More than 3 times a week
5. Do you eat yogurt? 
o
I don’t eat yogurt
o
1-3 times a week (please specify which kind)________________________________
o
More than 3 times a week (please specify which kind)______________________
6. Do you take probiotics?
o
No
o
Yes, please indicate how often and brand name if known: 
_________________________________________________
7. Are there foods you must avoid?
o
No
o
Yes, please list the food(s):_____________________________________________
o
Foods you avoid for religious, personal, or cultural 
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reasons:__________________________________
o
Foods your Doctor told you to avoid:________________________________________
8. If you are CURRENTLY on a special diet, please indicate below:
o
Not on a special diet
o
Weight loss
o
Weight gain
o
Vegetarian
o
Diet for diabetes
o
Diet for heart disease
o
Diet for kidney disease
o
Other:____________________
9. Are you currently taking prescribed or over-the-counter medications to lose 
weight or maintain your current weight? 
o
No 
o
Yes, I am on these weight loss medications:_________________________________
10.Are you taking laxatives to have a bowel movement on a regular base?
o
No
o
Yes, please describe which kind and how often:_____________________
11.How would you describe your bowel movements in the past 2 weeks:
o
Regular bowel movements every 1-2 days with no change
o
Frequent issues diarrhea, please describe_____________________________
o
Frequent issues with constipation, please describe______________________
12.Do you participate in regular physical activity? 
o
No
o
Yes, please describe what exercise do you like to do?_______________________
How long? __________________________ 
How many times a week? _________________________
13.Do you smoke?
o
No
o
Yes (please indicate how many cigarettes a day)___________________
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Appendix I
Stool Collection Instructions
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