Clinical trial NCT00670748
Conditions: Metastatic Melanoma Metastatic Renal Cell Cancer Metastatic Cancer
Keywords: Metastatic Cancer Gene Therapy Immunotherapy Tumor Regression Metastatic Melanoma Metastatic Renal Cell Cancer
Sponsor: National Cancer Institute (NCI)
Start date: 2008-05-29; Completion date: 2016-06-29
Enrollment: 45 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Background:

-This study uses an experimental cancer treatment that uses the patient s own lymphocytes (type of white blood cell), which are specially selected and genetically modified to target and destroy their tumor.

Objectives:

-To test the safety of the treatment and determine if it can cause the patient s tumor to shrink.

Eligibility:

* Patients greater than 18 years and less than or equal to 66 years of age whose cancer has spread beyond the original site and does not respond to standard treatment.
* Patients have tissue type human leukocyte antigen (HLA)-A\*0201.
* Patients cancer cells have the ESO-1 gene.

Design:

* Workup: Patients have scans, x-rays, laboratory tests, and other tests as needed.
* Patients have leukapheresis to collect cells for laboratory treatment and later reinfusion. For this procedure, whole blood is collected thorough a tube in a vein, the desired cells are extracted from the blood, and the rest of the blood is returned to the patient.
* Chemotherapy: Patients have low-dose chemotherapy for 1 week to prepare the immune system to receive the treated lymphocytes.
* Cell infusion and aldesleukin (IL-2) treatment: Patients receive the lymphocytes by a 30-minute infusion through a vein. Starting within 24 hours of the infusion, they receive high-dose aldesleukin infusions every 8 hours for up to 5 days (maximum15 doses).
* Recovery: Patients rest for 1 to 2 weeks to recover from the effects of chemotherapy and aldesleukin.
* Tumor biopsy: Patients may be asked to undergo a biopsy (surgical removal of a small piece of tumor) after treatment to look at the effects of treatment on the immune cells in the tumor.
* Follow-up: After treatment is completed, patients return to the clinic once a month for several months for physical examinations, a review of side effects, laboratory tests and scans. They may undergo leukapheresis at some visits to look at the effect of treatment on the immune system and check the viability of the infused cells. Patients then return to the National Institute of Health (NIH) clinic once a year for 5 years and then complete a follow-up questionnaire for another 10 years.
* Retreatment: Patients whose tumor shrinks or disappears following treatment and then recurs may receive one additional treatment, using the same regimen of chemotherapy, lymphocyte infusion and IL-2 treatment.

Detailed description:
Background:

* We have constructed a single retroviral vector that contains both alpha and beta chains of a T cell receptor (TCR) that recognizes the NY-ESO-1 (ESO) tumor antigen, which can be used to mediate genetic transfer of this TCR with high efficiency (\> 30%) without the need to perform any selection.
* In co-cultures with human leukocyte antigen serotype within HLA-A A serotype group (HLA-A2) and ESO double positive tumors, anti-ESO TCR transduced T cells secreted significant amount of interferon (IFN)-gamma and additional secretion of cytokines with high specificity.
* Poxviruses encoding tumor antigens, similar to the replication-defective recombinant canarypox virus (ALVAC) ESO-1 vaccine have been shown to successfully immunize patients against these antigens.

Objectives:

Primary objectives:

* Determine if the administration of anti-ESO TCR engineered peripheral blood lymphocytes (PBL) and aldesleukin to patients following a nonmyeloablative but lymphoid depleting preparative regimen will result in clinical tumor regression in patients with metastatic cancer that expresses the ESO antigen.
* Determine if the administration of anti-ESO TCR engineered peripheral blood lymphocytes (PBL), aldesleukin, and ALVAC ESO-1 vaccine to patients following a nonmyeloablative but lymphoid depleting preparative regimen will result in clinical tumor regression in patients with metastatic cancer that expresses the ESO antigen.

Secondary objectives:

* Determine the in vivo survival of TCR gene-engineered cells.
* Determine the toxicity profile of this treatment regimen.

Eligibility:

* Patients who are HLA-A\*0201 positive and 18 years of age or older must have:

  * metastatic cancer whose tumors express the ESO antigen;
  * previously received and have been a non-responder to or recurred to standard care for metastatic disease, except for melanoma patients;
* Patients may not have:

  * contraindications for high dose aldesleukin administration.

Design:

* Peripheral blood mononuclear cells (PBMC) obtained by leukapheresis (approximately 5 X 10(9) cells) will be cultured in the presence of anti-cluster of differentiation 3 (CD3) (OKT3) and aldesleukin in order to stimulate T-cell growth.
* Transduction is initiated by exposure of approximately 10(8) to 5 X 10(8) cells to retroviral vector supernatant containing the anti-ESO TCR genes.
* Patients will receive a nonmyeloablative but lymphocyte depleting preparative regimen consisting of cyclophosphamide and fludarabine followed by intravenous infusion of ex vivo tumor reactive, TCR gene transduced PBMC plus intravenous (IV) aldesleukin (720,000 IU/kg q8h for a maximum of 15 doses) with or without ALVAC ESO-1 vaccine. Subcutaneous injection of ALVAC ESO-1 vaccine will be administered on day 0 approximately 2 hours prior to intravenous infusion of cells and a second dose of ALVAC ESO-1 vaccine is given on day 14 (+/- 2 days).
* Patients will undergo complete evaluation of tumor with physical examination, computed tomography (CT) of the chest, abdomen and pelvis and clinical laboratory evaluation four to six weeks after treatment. If the patient has stable disease (SD) or tumor shrinkage, repeat complete evaluations will be performed every 1-3 months. After the first year, patients continuing to respond will continue to be followed with this evaluation every 3-4 months until off study criteria are met.

Cohorts 1 and 2:

* Patients will be entered into two cohorts based on histology: cohort 1 will include patients with metastatic melanoma or renal cell cancer; cohort 2 will include patients with other types of metastatic cancer.
* For each of the 2 strata evaluated, the study will be conducted using a phase II optimal design where initially 21 evaluable patients will be enrolled. For each of these two arms of the trial, if 0 or 1 of the 21 patients experiences a clinical response, then no further patients will be enrolled but if 2 or more of the first 21 evaluable patients enrolled have a clinical response, then accrual will continue until a total of 41 evaluable patients have been enrolled in that stratum.
* For both strata, the objective will be to determine if the combination of high dose aldesleukin, lymphocyte depleting chemotherapy, and anti-ESO TCR-gene engineered lymphocytes is able to be associated with a clinical response rate that can rule out 5% (p0=0.05) in favor of a modest 20% partial response (PR) + complete response (CR) rate (p1=0.20).

Cohorts 3 and 4:

* For patients receiving ALVAC ESO-1 vaccine, patients will also be entered into two cohorts based on histology: cohort 3 for patients with metastatic melanoma or renal cell cancer and cohort 4 for patients with other histologies and all patients will receive the treatment regimen including the ALVAC ESO-1 vaccine.
* For each of these 2 new strata, the study will be conducted using a phase II optimal design where initially 21 evaluable patients will be enrolled. For each of these two new cohorts of the trial, if 0 or 1 of the 21 patients experiences a clinical response, then no further patients will be enrolled but if 2 or more of the first 21 evaluable patients enrolled have a clinical response, then accrual will continue until a total of 41 evaluable patients have been enrolled in that stratum.
* For both strata, the objective will be to determine if the combination of high dose aldesleukin, lymphocyte depleting chemotherapy, anti-ESO TCR-gene engineered lymphocytes, and ALVAC ESO-1 vaccine is able to be associated with a clinical response rate that can rule out 5% (p0=0.05) in favor of a modest 20% PR + CR rate (p1=0.20).

Arms: arm 1: Patients with melanoma or renal cell cancer (RCC) will receive non-myeloablative lymphodepleting regimen of cyclophosphamide and fludarabine followed by anti-NY ESO-1 T-cell receptor (TCR) peripheral blood lymphocytes (PBL) and high dose aldesleukin. arm 2: Patients with cancers other than melanoma or RCC will receive non-myeloablative lymphodepleting regimen of cyclophosphamide and fludarabine followed by anti-NY ESO-1 TCR PBL and high dose (HD) aldesleukin arm 3: Patients with melanoma or RCC will receive non-myeloablative lymphodepleting regimen of cyclophosphamide and fludarabine followed by replication-defective recombinant canarypox virus (ALVAC) NY-ESO-1 vaccine, anti-NY ESO-1 TCR PBL and high dose aldesleukin arm 4: Patients with cancers other than melanoma or RCC will receive non-myeloablative lymphodepleting regimen of cyclophosphamide and fludarabine followed by ALVAC NY-ESO-1 vaccine, anti-NY ESO-1 TCR PBL and high dose aldesleukin
Interventions: intervention 1: Anti-NY ESO-1 T-cell receptor PBL intervention 2: aldesleukin intervention 3: Cyclophosphamide intervention 4: fludarabine phosphate intervention 5: ALVAC NY ESO-1 vaccine
Intervention descriptions: intervention 1: Patients will receive non-myeloablative lymphodepleting preparative regimen consisting of cyclophosphamide and fludarabine followed by the administration of anti-NY ESO-1 T-cell receptor (TCR) peripheral blood lymphocytes (PBL) and high dose aldesleukin. On day 0,cells (1x10e8 to 1x10e11)will be infused intravenously on the Patient Care Unit over 20-30 minutes. intervention 2: Aldesleukin 720,000 IU/kg intravenous (IV) (based on total body weight)over 15 minutes every eight hours (+/- 1 hour) beginning within 24 hours of cell infusion and continuing for up to 5 days(maximum of 15 doses) intervention 3: Cyclophosphamide 60 mg/kg/day X 2 days IV in 250 ml dextrose 5% in water (D5W) with Mesna 15 mg/kg/day X 2 days over 1 hr. intervention 4: Fludarabine 25 mg/m2/day intravenous piggyback (IVPB) daily over 30 minutes for 5 days. intervention 5: Approximately two hours prior to cell infusion, patients will receive 0.5 mL containing a target dose of 10e7 cell culture infectious dose 50 (CCID50) (with a range of approximately 10e6.4 to 10e7.9 / mL) of the ESO-1 ALVAC virus S.C. in each extremity (total of 4 x 10e7 CCID50/2 mL.

Protocol documents:
Prot_SAP_000.pdf: 
Abbreviated Title: Anti-NY ESO-1 TCR PBL
OBA: 0712-886  IBC: RD-07-XII-17
PRMC Protocol Number: P07308
CC Protocol Number: 08-C-0121N
IRB Submission Date: December 3, 2007
Version Date: July 1, 2015
Phase II Study of Metastatic Cancer that Expresses NY-ESO-1 Using Lymphodepleting Conditioning 
Followed by Infusion of Anti-NY ESO-1 TCR-Gene Engineered Lymphocytes
Principal Investigator:
Steven A. Rosenberg, M.D., Ph.D.
Chief of Surgery, NCI
Building 10, CRC, Room 3-3940
Phone: 301-496-4164
Associate Investigators:  
Paul Robbins, Ph.D., Surgery Branch, NCI
Robert Somerville, Ph.D., Surgery Branch, NCI
Nicholas P. Restifo, M.D., Surgery Branch, NCI**
James C. Yang, M.D., Surgery Branch, NCI
Richard Sherry, M.D., Surgery Branch, NCI
Udai S. Kammula, M.D., Surgery Branch, NCI
Stephanie Goff, M.D., Surgery Branch, NCI
Christopher Klebanoff, M.D., Surgery Branch, NCI
Lori McIntyre, R.N., OCD*
Mary Ann Toomey, R.N., Surgery Branch, NCI**
Marie Statler, R.N., OCD*
Debbie Nathan, R.N., Surgery Branch, NCI**
Monica Epstein, R.N., OCD*
Abigail Johnson, R.N., OCD*
Donald E. White, M.S., Surgery Branch, NCI
Arati Kamath, Ph.D., Leidos Biomedical Research, Inc.
Seth M. Steinberg, Ph.D., BDMS, NCI+
Daniel Zlott, Pharm.D., Pharmacy Department, NIH Clinical Center
Isaac Kriley, M.D., Surgery Branch, NCI
Parisa Malekzadeh, M.D., Surgery Branch, NCI^
Kasia Trebska-McGowan, M.D., Surgery Branch, NCI
Referral Contact:
Linda Williams, R.N., Surgery Branch, NCI
Jessica Yingling, R.N. OCD*
Ellen Bodurian, R.N., OCD*
+Biostatistics Data Management Section, Center for Cancer Research
National Cancer Institute, National Institutes of Health, Bethesda, Maryland
*Office of the Clinical Director
**Not responsible for direct patient care
^Under the Direct Supervision of the PI
Drug 
Name:
PG13-A2aB-1G4A-LY-3H10(anti-
NY ES01 TCR) retroviral vector-
transduced autologous peripheral 
blood lymphocytes (PBL)
NY ES0-1 ALVAC 
Vaccine
IND 
Number:
BB-IND 13620
BB-IND 13450
Sponsor:
Steven A. Rosenberg, M.D., Ph.D.
Sanofi Pasteur Ltd.


0 8- C- 0 1 2 1
2
Précis:
B ac k gr o u n d:
We ha ve 
c hai ns of a T cell 
rece pt or ( T C R) t hat rec o g nizes t he N Y- E S O- 1 ( E S O) t u m or a nti ge n, w hic h ca n be use d t o me di ate 
ge n etic tra nsfer of t his T C R wit h hi g h efficie n c y ( > 3 0 %) wit h o ut t he nee d t o perf or m a n y s electi o n. 
I n c o-c ult ur es wit h H L A- A 2 a n d E S O d o u ble p ositi ve t u m ors, a nti- E S O T C R tra ns d uce d T cells 
secret e d si g nifi ca nt a m o u nt of I F N-
a n d a d diti o nal secreti o n of c yt o ki nes wit h hi g h s pecificit y.
P o x vir uses e nc o di n g t u m or a nti ge ns, si milar t o t he A L V A C E S O- 1 vacci ne ha ve bee n s h o w n t o 
s uccessf ull y i m m u nize patie nts a gai nst t hese a nti ge ns.
O bjecti ves :
Pri mar y o bjecti ves: 
Deter mi ne if t he a d mi nistrati o n of a nti- E S O – T C R e n gi neere d p eri p heral bl o o d l y m p h o c ytes ( P B L)
a n d al desle u ki n t o patie nts f oll o wi n g a n o n m ye l oa blati ve b ut l y m p h oi d de pleti n g pre p arati ve 
re gi me n will res ult i n cli nical t u m or re gressi o n i n patie nts wit h metastatic ca ncer t h at e x presses t he 
E S O a nti ge n.
Deter mi ne if t he a d mi nistrati o n of a nti- E S O – T C R e n gi neere d P B L, al desl e u ki n, a n d A L V A C 
E S O- 1 vacci n e t o patie nts f oll o wi n g a n o n m yel o a blati ve b ut l y m p h oi d de pleti n g pre p arati ve 
re gi me n will res ult i n cli nical t u m or re gressi o n i n patie nts wit h metastatic ca ncer t h at e x presses t he 
E S O a nti ge n.
Sec o n dar y o bjecti ves: 
Deter mi ne t he i n vi v o s ur vi val of T C R ge n e-e n gi n eere d cells.
Deter mi ne t he t o xicit y pr ofile of t his treat me nt re gi me n.
Eli gi bilit y:
Patie nts w h o are H L A- A * 0 2 0 1 p ositi ve a n d 1 8 years of a ge or ol der m ust ha ve 
metastatic ca n cer w h os e t u m ors e x press t he E S O a nti ge n;
Patie nts, ot her t ha n t h ose wit h metastatic mela n o ma, m ust  ha ve pre vi o usl y recei v e d a n d ha v e bee n 
a n o n-res p o n d er t o or r ec urre d t o st a n dar d care f or  metastatic disease;
Patie nts ma y n ot ha ve:
c o ntrai n dicati o ns f or hi g h d os e al desle u ki n a d mi nistrati o n.
Desi g n:
P B M C o btai ne d b y l e u ka p here sis (a p pr o xi matel y 5 X 1 0 9 cells) will be c ult ure d i n t he pres e nce of 
a nti- C D 3 ( O K T 3) a n d al desle u ki n i n or der t o sti m ulate T-cell gr o wt h.
Tra ns d ucti o n is i nitiate d b y e x p os ure of a p pr o xi matel y 1 0 8 t o 5 X 1 08 cells t o retr o viral vect or 
s u per nata nt c o nt ai ni n g t h e a nti- E S O T C R ge n es.
Patie nts will recei ve a n o n m yel oa bl ati ve b ut l y m p h oc yt e de pleti n g pre p arati ve re gi me n c o nsisti n g 
of c ycl o p h os p ha mi de a n d fl u dara bi ne f oll o we d b y i ntra v e n o us i nf usi o n of e x vi v o t u m or reacti ve, 
T C R ge ne-tra ns d uce d P B M C pl us I V al desl e u ki n ( 7 2 0, 0 0 0 I U/ k g q 8 h f or a ma xi m u m of 1 5 d oses)
wit h or wit h o ut A L V A C E S O- 1 vacci n e.  S u bc ut a ne o us i njecti o n of A L V A C E S O- 1 vacci ne will 
be a d mi nistere d o n da y 0 a p pr o xi matel y 2 h o urs pri or t o i ntra ve n o us  i nf usi o n of cells a n d a s ec o n d 
d ose of A L V A C E S O- 1 vacci ne is gi ve n o n d a y 1 4 ( +/- 2 da ys)..
Patie nts will u n der g o c o m plete e val uati o n of t u m or wit h p h ysical e x a mi nati o n, C T of t he c hest, 
a b d o me n a n d pel vis a n d cli nical  la b orat or y e val uati o n f o ur t o si x wee ks after treat me nt. If t he 


08-C-0121
3
patient has SD or tumor shrinkage, repeat complete evaluations will be performed every 1-3
months.  After the first year, patients continuing to respond will continue to be followed with this 
evaluation every 3-4 months until off study criteria are met.
Cohorts 1 and 2:
x
Patients will be entered into two cohorts based on histology: cohort 1 will include patients with 
metastatic melanoma or renal cell cancer; cohort 2 will include patients with other types of 
metastatic cancer.
x
For each of the 2 strata evaluated, the study will be conducted using a phase II optimal design where 
initially 21 evaluable patients will be enrolled. For each of these two arms of the trial, if 0 or 1 of 
the 21 patients experiences a clinical response, then no further patients will be enrolled but if 2 or 
more of the first 21 evaluable patients enrolled have a clinical response, then accrual will continue 
until a total of 41 evaluable patients have been enrolled in that stratum. 
x
For both strata, the objective will be to determine if the combination of high dose aldesleukin, 
lymphocyte depleting chemotherapy, and anti-ESO TCR-gene engineered lymphocytes is able to be 
associated with a clinical response rate that can rule out 5% (p0=0.05) in favor of a modest 20% PR 
+ CR rate (p1=0.20).
Cohorts 3 and 4:
x
For patients receiving ALVAC ESO-1 vaccine, patients will also be entered into two cohorts based 
on histology: cohort 3 for patients with metastatic melanoma or renal cell cancer and cohort 4 for 
patients with other histologies and all patients will receive the treatment regimen including the 
ALVAC ESO-1 vaccine.
x
For each of these 2 new strata, the study will be conducted using a phase II optimal design where 
initially 21 evaluable patients will be enrolled. For each of these two new cohorts of the trial, if 0 or 
1 of the 21 patients experiences a clinical response, then no further patients will be enrolled but if 2
or more of the first 21 evaluable patients enrolled have a clinical response, then accrual will 
continue until a total of 41 evaluable patients have been enrolled in that stratum. 
x
For both strata, the objective will be to determine if the combination of high dose aldesleukin, 
lymphocyte depleting chemotherapy, anti-ESO TCR-gene engineered lymphocytes, and ALVAC 
ESO-1 vaccine is able to be associated with a clinical response rate that can rule out 5% (p0=0.05) 
in favor of a modest 20% PR + CR rate (p1=0.20).


08-C-0121
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Table of Contents
1.0 
Introduction .................................................................................................................................................6 
1.1. 
Study Objectives:...................................................................................................................................6 
1.2. 
Background and Rationale:..................................................................................................................6 
2.0 
Eligibility Assessment and Enrollment.....................................................................................................22 
2.1. 
Eligibility Criteria ...............................................................................................................................22 
2.2. 
Research Eligibility Evaluation..........................................................................................................24 
2.3. 
Patient Registration.............................................................................................................................25 
3.0 
Study Implementation ...............................................................................................................................25 
3.1. 
Study Design:.......................................................................................................................................25 
3.2. 
Drug Administration: .........................................................................................................................27 
3.3. 
On-Study Evaluation...........................................................................................................................31 
3.4. 
Re-Treatment.......................................................................................................................................34 
3.5. 
Post Study Evaluation (Follow-up):...................................................................................................35 
3.6. 
Off Treatment Criteria:......................................................................................................................35 
3.7. 
Off Study Criteria:..............................................................................................................................36 
4.0 
Supportive Care.........................................................................................................................................36 
5.0 
Data Collection and Evaluation.................................................................................................................36 
5.1. 
Data Collection ....................................................................................................................................36 
5.2. 
Response Criteria ................................................................................................................................37 
5.3. 
Toxicity Criteria ..................................................................................................................................39 
5.4. 
Statistical Section.................................................................................................................................40 
5.5. 
Data and Safety Monitoring Plan ......................................................................................................42 
5.6. 
Clinical Trial Monitoring Plan...........................................................................................................42 
5.7. 
Handling of Tissue Specimens for Research Purposes ....................................................................42 
6.0 
Human Subjects Protections......................................................................................................................43 
6.1. 
Rationale for Patient Selection...........................................................................................................43 
6.2. 
Participation of Children....................................................................................................................43 
6.3. 
Evaluation of Benefits and Risks .......................................................................................................43 
6.4. 
Consent Document ..............................................................................................................................44 
7.0 
Data Reporting ..........................................................................................................................................44 
7.1 
Definitions ............................................................................................................................................44 
7.2 
NCI-IRB Reporting.............................................................................................................................46 
7.3 
NIH Office of Biotechnology Activities (OBA)/Institutional Biosafety Committee 
(IBC) Reporting Criteria....................................................................................................................................47 
7.4 
Serious Adverse Event Reporting Requirements to the FDA, OBA and IBC: .............................47 
8.0 
Pharmaceutical Information ......................................................................................................................47 
8.1. 
Aldesleukin (Interleukin-2, Proleukin, Recombinant Human Interleukin 2) ...............................47 
8.2. 
Fludarabine..........................................................................................................................................48 
8.3. 
Cyclophosphamide ..............................................................................................................................49 
8.4. 
Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891)............................50 
8.5. 
Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim,  Neupogen) ...............50 
8.6. 
Trimethoprim and Sulfamethoxazole Double Strength (TMP / SMX DS)....................................50 
8.7. 
Herpes Virus Prophylaxis...................................................................................................................51 
8.8. 
Fluconazole ..........................................................................................................................................51 
8.9. 
Anti-ESO TCR transduced PBL........................................................................................................52 
8.10. 
ALVAC ESO-1 Vaccine......................................................................................................................53 
8.11. 
Support Medications...........................................................................................................................54 


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5
9.0 
References .................................................................................................................................................55 
10.0 
Tables, Figures, and Appendices: .............................................................................................................60 


08-C-0121
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1.0 Introduction
1.1.
Study Objectives:
1.1.1. Primary objective:
x
In Cohorts 1 and 2, to determine if the administration of anti-ESO TCR-engineered 
peripheral blood lymphocytes and aldesleukin to patients following a 
nonmyeloablative but lymphoid depleting preparative regimen will result in clinical 
tumor regression in patients with metastatic cancer expressing ESO.
x
In Cohorts 3 and 4, to determine if the administration of anti-ESO TCR-engineered 
PBL, aldesleukin, and ALVAC ESO-1 vaccine to patients following a 
nonmyeloablative but lymphoid depleting preparative regimen will result in clinical 
tumor regression in patients with metastatic cancer expressing ESO.
1.1.2. Secondary objectives:
x
Determine the in vivo survival of TCR gene-engineered cells.
x
Determine the toxicity profile of this treatment regimen.
1.2.
Background and Rationale:  
Studies in experimental animals have demonstrated that the cellular rather than the 
humoral arm of the immune response plays the major role in the elimination of murine 
tumors1.  Much of this evidence was derived from studies in which the adoptive transfer of T 
lymphocytes from immune animals could transfer resistance to tumor challenge or in some 
experiments, the elimination of established cancer.  Thus, most strategies for the 
immunotherapy of patients with cancer have been directed at stimulating strong T cell 
immune reactions against tumor-associated antigens.
In contrast to antibodies that recognize epitopes on intact proteins, T cells recognize 
short peptide fragments (8-18 amino acids) that are presented on surface class I or II major 
histocompatibility (MHC) molecules and it has been shown that tumor antigens are 
presented and recognized by T cells in this fashion.  The molecule that recognizes these 
peptide fragments is the T-cell receptor (TCR).  The TCR is analogous to the antibody 
immunoglobulin molecule in that, two separate proteins (the TCR alpha and beta chains) are 
brought together to form the functional TCR molecule.  The goal of this protocol is to 
transfer tumor-associated antigen (TAA)-reactive TCR genes into normal peripheral blood 
lymphocytes (PBL) derived from cancer patients and to return these engineered cells to 
patients aimed at mediating regression of their tumors. This trial is similar to previous
Surgery Branch TCR gene transfer adoptive immunotherapy protocols except that we will 
use a TCR that has the potential to treat HLA-A2 melanoma patients as well as patients with 
common epithelial malignancies that express ESO. Table 1.2 briefly summarizes the 
Surgery Branch protocols that demonstrate the progression of applicable adoptive cell 
therapies over time.


08-C-0121
7
Study #/ 
Disease 
Cellular product 
administered
Arms (n)
Chemotherapy, Cytokines and Immunizations
# of Cells
Response
Reference
98-C-0095 in 
patients with 
metastatic 
melanoma
Cloned Peripheral 
Blood Lymphocytes 
(PBL)/ Tumor 
infiltrating lymphocytes 
(TIL)
1. Cloned PBL/TIL 
intravenously (12)
None
1.5 to 34.8 X 
109 cells
NRd
Dudley, ME, 
et al. 2001 
2. IV Cloned PBL/TIL 
intravenously (6a)
SQ IL-2 (125,000 IU/kg/d X 12 d)
3. IV Cloned PBL/TIL 
intravenously (6b)
HD IL-2 (720,000 IU/kg 3X/d to tolerance (max 12)
4. IV Cloned PBL/TIL (3c)
Gp100:209-217(210M)
99-C-0158 in 
patients with 
metastatic 
melanoma
Tumor infiltrating 
lymphocytes (TIL)
1. In vitro expanded cloned 
T cells intravenously (3)
30 mg/kg cyclophosphamide X2 days, 25 mg/kg fludarabine X5 
days
0.9 X 109 to
24.2 X 109 cells 
(ave. 10.4 X 
109)
NR
Dudley, ME, 
et al. 2002 
2. In vitro expanded cloned 
T cells intravenously (3)
60 mg/kg cyclophosphamide X2 days, 25 mg/kg fludarabine X5 
days
NR
3. In vitro expanded cloned 
T cells intravenously (3)
60 mg/kg cyclophosphamide X2 days, 25 mg/kg fludarabine X5 
days plus IV IL-2 (72,000 IU/kg 3X/day X5 days)
0.9 X 109 to
24.2 X 109 cells 
(ave. 10.4 X 
109)
NR
4. In vitro expanded cloned 
T cells intravenously (6)
[60 mg/kg cyclophosphamide X2 days, 25 mg/kg fludarabine X5 
days]g plus IV IL-2 (720,000 IU/kg 3X/day to tolerance (max 12)
NR
5. Autologous tumor reactive 
REP’de bulk TIL cells 
intravenously (35)
Chemotherapyg plus IV HD IL-2 [(720,000 IU/kg 3X/day to 
tolerance (max 15)]h with or without immunization with 
gp100:209-217(210M) or MART-1:26-35(27L) in Montanide 
ISA-51 QD X5, then Qwk X3.
1.1 to 16.0 X
1010 cells (ave. 
6.3 X 1010)
18/35 
(51%)f
3 CRs
15 PRs
Dudley, ME, 
et al. 2003, 
Dudley, ME, 
et al. 2005
6. Autologous tumor reactive 
REP’de bulk TIL cells 
intravenously (5)
Chemotherapyg plus Low Dose IL-2k with or without 
immunization with gp100:209-217(210M) peptide or MART-
1:26-35(27L) peptide in Montanide ISA-51 QD X5, then Qwk 
X3.
19.7  X 109 to
52.9 X 109
cells (ave. 36.3 
X 109)
1 PR
4 TEj
04-C-0181 in 
patients with 
metastatic 
melanoma
Gp100 TCR engineered 
T cells (PBL or TIL)
1. Escalating doses of anti-
gp100 TCR transduced PBL 
(8)
Chemotherapyg plus IV HD IL-2h with 1mg gp100:209-
217(210M) in Montanide ISA-51 QD X5, then Qwk X3
Up to 30 X 109
NR
NA
2. Anti-gp100 TCR CD8+ 
enriched PBL (8)
Chemotherapyg plus IV HD IL-2h with 6X109 pfu rFgp100P209 
IV; rFgp100P209 IV and IL-2 repeated 28 days later.
Up to 30 X 109
NR
3. Escalating doses of anti-
gp100 TCR transduced TIL 
(3)
Chemotherapyg plus IV HD IL-2h with 6X109 pfu rFgp100P209 
IV; rFgp100P209 IV and IL-2 repeated 28 days later.
Up to 30 X 109
1 PRi
04-C-0251 in 
patients with 
metastatic 
melanoma
MART-1 TCR 
engineered T cells (PBL 
or TIL)
1. Escalating doses of anti-
MART-1 TCR transduced 
PBL (18)
Chemotherapyg plus IV HD IL-2h with MART-1:26-35(27L) in 
Montanide ISA-51 QD X5, then Qwk X3.
Up to 30 X 109
2PRsi
Morgan, et al. 
Science, 2006 
Oct 
6;314(5796): 
126-9
2. Escalating doses of anti-
MART-1 CD8+ TCR 
transduced PBL (6)
Chemotherapyg plus IV HD IL-2h with MART-1:27-35 in 
Montanide ISA-51 QD X5, then Qwk X3
Up to 30 X 109
1 PRi
3. Escalating doses of anti-
MART-1 CD8+ TCR 
transduced PBL (8)
Chemotherapyg plus IV HD IL-2h with MART-1:26-35(27L) in 
Montanide ISA-51 QD X5, then Qwk X3
Up to 30 X 109
1 PRi
4. Escalating doses of anti-
MART-1 TCR transduced
TIL (3)
Chemotherapyg plus IV HD IL-2h with MART-1:26-35(27L) in 
Montanide ISA-51 QD X5, then Qwk X3
Up to 30 X 109
NR
Table 1.2


08-C-0121
8
5.  Escalating doses of anti-
MART-1 TCR transduced 
PBL plus 1200 TBI (4)
Chemotherapyg and 1200 TBI plus IV HD IL-2h with MART-
1:26-35(27L) in Montanide ISA-51 QD X5, then Qwk X3
Up to 30 X 109
       NR
07-C-0003 in 
melanoma/renal 
cell or other 
histologies
Anti-p53 TCR 
engineered T cells 
(PBL)
1. Melanoma or renal cell 
cancer: Up to 50 X 109 cells 
(PBL) (2)
Chemotherapyg plus IV HD IL-2h
Up to 50 X 109
NR
NA
2. Other histologies: Up to 
50 X 109 cells (PBL) (10)
Chemotherapyg plus IV HD IL-2h
Up to 50 X 109
1 PRi
07-C-0174 in 
patients with 
metastatic 
melanoma
Anti-gp100(154) TCR 
engineered T cells 
(PBL)
Anti-gp100(154) TCR 
transduced PBL at a dose 
ranging from 0.5 X 109 cells 
up to 300 x109 (10)
Chemotherapyg plus IV HD IL-2h
1.7  X 109 to
19.4 X 109
cells (ave. 6.9 X 
109)
2 NR
10 TEj
NA
07-C-0175 in 
patients with 
metastatic 
melanoma
Anti-MART-1 F5 TCR 
engineered T cells 
(PBL)
Anti-MART-1 F5 TCR 
transduced PBL at a dose 
ranging  from 0.5 X 109
cells up to 300 x109 cells 
(11)
Chemotherapyg plus IV HD IL-2h
1.45  X 109 to
23.3 X 109
cells (ave. 12.4 
X 109)
1 PR
2 NR
8 TEj
NA
07-C-0176 in 
patients with 
metastatic 
melanoma
Young TIL
Young TIL at a dose ranging 
from 1.0 X 109 cells up to 
300 x109 cells (2)
Chemotherapyg plus IV HD IL-2h
54.5  X 109 to
83.2 X 109
cells (ave. 68.9 
X 109)
2 TEj
NA
Data as of 
10/29/07
a Previously treated with cells alone
b Five previously treated with cells alone, one new patient for first cell treatment
c All three patients had previously been treated with cell alone and cells with IL-2
d NR: No Response
e Rapid Expansion Protocol
f Responses defined: CR (complete response) is disappearance of all clinical evidence of disease; PR (partial response) defined as > 50% reduction in the sum of the products of the perpendicular 
diameters for at least one month and no increase in any lesion and no new lesions.
g Chemotherapy at maximum dose: 60 mg/kg cyclophosphamide X2 days, 25 mg/kg fludarabine X5 days
h High dose (HD) IL-2: 720,000 IU/kg 3X/day to tolerance (max 15)
iResponses defined according to RECIST criteria
j TE: Too early to evaluate
k Low dose IL-2: 250,000 IU/kg subcutaneously daily for 5 days.  After a two day rest, IL-2 will be administered at a dose of 125,000 IU/kg subcutaneously daily for 5 days for the next five weeks (2 
days rest per week).


08-C-0121
9
1.2.1. Prior Surgery Branch Trials of Cell Transfer Therapy Using Cloned  
Lymphocytes in Patients with Metastatic Melanoma
Studies in the Surgery Branch, National Cancer Institute, identified genes that encode 
melanoma tumor associated antigens (TAA) recognized by tumor infiltrating lymphocytes 
(TIL) in the context of multiple MHC class I molecules 2-4.  These TAA appeared to be 
clinically relevant antigens responsible for mediating tumor regression in patients with 
advanced melanoma since the TIL used to identify these antigens were often capable of 
mediating in vivo anti-tumor regression.  Two antigens, which were present in virtually all 
fresh and cultured melanomas, were called MART-1 (Melanoma Antigen Recognized by T 
Cells - 1) and gp100 and the genes encoding them have been cloned and sequenced.  The 
MART-1 gene encodes a 118 amino acid protein of 13 kd. The gp100 gene encodes a protein 
identical to that recognized by monoclonal antibody HMB-45. With the exception of 
melanocytes and retina no normal tissues express these antigens and no expression of these 
gene products has been seen on cancers other than melanoma.  These antigens were thus the 
original targets of our initial cell transfer protocols using TIL in patients with metastatic 
melanoma.  
Techniques for the cloning of human lymphocytes enabled the generation, in vitro, 
of greater than 109 cloned lymphocytes with a very high degree of reactivity to T-cell 
recognized antigens as measured by recognition of T2 cells pulsed with very low levels of 
peptide (10-4M) or recognition of tumor cells.  In Surgery Branch protocol 98-0095 we tested 
the feasibility of transferring large numbers of in vitro gp100 peptide stimulated PBL clones 
to melanoma patients.  In this phase I study 5, 12 patients received multiple infusions of anti-
melanoma T cell clones (average 1 X 1010 cells/cycle) with or without IL-2 administration.  
Peripheral blood samples were analyzed for persistence of transferred cells by T-cell 
receptor-specific PCR.  Transferred cells reached a maximum level at 1 hr. post infusion but 
rapidly declined to undetectable levels by 1-2 weeks.  No objective responses were observed 
in the trial arm that included IL-2 administration.  
In the great majority of murine models demonstrating the therapeutic effectiveness 
of the adoptive transfer of lymphocytes mediating tumor regression, immunosuppression of 
the host prior to the adoptive transfer of lymphocytes was required.  Although, the 
mechanism of the improved efficacy of adoptively transferred lymphocytes following 
immunosuppression was not clearly understood, the possible elimination of suppressor 
lymphocytes and elimination of cells that compete with the transferred cells for the 
homeostatic cytokines IL-7 and IL-15 were offered as explanations. The use of 
nonmyeloablative preparative regimens, popular for patients receiving allogeneic bone 
marrow transplants, appeared ideally suited to induce the transient immunosuppression 
required prior to the adoptive transfer of lymphocytes.  This led to a Surgery Branch clinical 
trial (99-C-0158) in which 15 patients received cloned lymphocytes after receiving 
cyclophosphamide-fludarabine nonmyeloablative chemotherapy6. The regimen of 
cyclophosphamide and fludarabine used in this protocol was identical to that used in the bone 
marrow transplant unit of the National Heart, Lung and Blood Institute for patients receiving 
HLA matched allotransplants. Six of these 15 patients had the full-dose chemotherapy and 
high-dose IL-2.
No objective responses were observed in this cohort of 15 patients.  One major 
factor limiting the effectiveness of these cloned lymphocytes appeared to be their shortened 


08-C-0121
10
survival upon adoptive transfer.  Three of 6 patients who received the chemotherapy plus 
high dose IL-2 also received cloned T lymphocytes marked with neomycin 
phosphotransferase resistance gene (Neor).  In none of these three patients, could Neor -
marked cells be detected beyond one week after infusion. The cloned T lymphocytes from 
some of these 15 patients were also labeled with 111Indium for trafficking studies: three 
received chemotherapy without IL-2 and two received chemotherapy with high-dose IL-2.
No enhanced trafficking to the tumor sites of these labeled lymphocytes was observed after 
the chemotherapy.  
No treatment related mortality was observed in this trial, suggesting that this 
approach of nonmyleoablative chemotherapy in combination with antitumor lymphocytes 
plus high-dose IL-2 was safe.  Grade 3-4 toxicities are listed in Table 1.
The combination of cyclophosphamide and fludarabine was myelosuppressive.  
Neutrophils nadired on day 10 after chemotherapy at 6/mm3 and recovered to above 
500/mm3 on day 14.  Lymphocytes nadired at 6/mm3 and recovered to above 500/mm3.
Platelets nadired at 5.5/mm3 on day 8 and recovered to > 20,000/mm3 on day 28 with support 
of filgrastrim (G-CSF). Patients were usually discharged between 2-3 weeks after the 
initiation of the chemotherapy, with neutrophil counts above 500/mm3 and platelet counts 
above 20,000/mm3.  No patients needed a stem cell transfusion to rescue marrow function.  
However, CD4 counts remained persistently low (below 200), which is a known side effect 
of immunosuppression from fludarabine.  Four patients developed herpes zoster.  One patient 
had an RSV pneumonia requiring mechanical ventilatory support.  All patients recovered 
after treatments. Thus we have shown that this approach was safe.  
1.2.2. Prior Surgery Branch Trials of Cell Transfer Therapy Using Heterogeneous TIL 
plus High-Dose IL-2 Following Nonmyeloablative but Lymphodepleting Chemotherapy
In the Surgery Branch, NCI, we next added a cohort to 99-C-0158 to rapidly expand 
heterogeneous TILs for adoptive transfer. This protocol for growing TILs was different from 
the traditional one we developed earlier7. TILs were expanded using the rapid expansion 
protocol (REP) in the presence of OKT3, irradiated allogeneic feeder cells and IL-2. These 
REPed TILs retained highly specific in vitro anti-tumor activity, often contained reactivities 
against several antigenic epitopes and contained both CD8+ and CD4+ lymphocytes.  These 
autologous bulk TIL were re-infused to patients following the same nonmyeloablative 
chemotherapy with cyclophosphamide and fludarabine used in the prior trial.  These patients 
subsequently received high-dose IL-2 and some received peptide immunization when the TIL 
reactivity was against known MART-1 and gp100 peptides.   A total of 35 patients received 
this type of the treatment 8.
This regimen using REPed TIL in protocol 99-C-0158 resulted in objective cancer 
regressions in 51% of patients (18 of 35) with metastatic melanoma (Table 2) patients 
achieved a clonal repopulation of anti-tumor lymphocytes that exceeded 80% of their 
circulating CD8+ lymphocytes that persisted for months after cell infusion.  
Immunohistochemistry studies revealed that specific clonotypes from the infused bulk TIL 
cells infiltrated the regressing tumor nodules.  The toxicities in these 35 patients are shown in 
Table 3.  Non-hematologic and hematologic toxicities were those expected from IL-2 and the 
myelosuppressive chemotherapy.  In addition, autoimmunity was observed in the TIL 
therapy group.  Some patients experienced vitiligo and one patient had an autoimmune 
uveitis easily controlled with steroid eye drops.  One of the patients who had clonal 


08-C-0121
11
repopulation from infused TIL cells and a dramatic response of metastatic melanoma, 
developed Epstein-Barr virus (EBV)-associated B cell lymphoma.  This patient was EBV-
QDȧYHSULRUWRWKHWUHDWPHQWV7KHSRWHQWLDOVRXUFHRI(%9ZDVWKRXJKWWREHPXOWLSOHEORRG
product transfusions.  This patient died several months later of disseminated lymphoma.  One 
patient had an RSV pneumonia requiring transient mechanical ventilatory support and 
recovered completely. One patient developed polyneuropathy manifested by vision 
blindness, motor and sensory defects, approximately two months after chemotherapy.  The 
etiology is unknown for this complication, but possibly related to the fludarabine.  One 
patient who received the chemotherapy regimen plus high-dose IL-2 and bulk peripheral 
blood lymphocytes reactive to the melanoma, developed prolonged respiratory failure 
requiring mechanical ventilation and acute renal failure that required hemodialysis.  Both 
toxicities have been reported with high-dose IL-2 therapy and all the patients recovered from 
the acute toxicities.  The only treatment related death in this series was the patient who 
developed an EBV-associated lymphoma about 6 months after cell infusion.  Responding 
patients survived significantly longer than non-responders (Figure 1) although caution should 
be exercised in interpreting this type of analysis.
1.2.3. Surgery Branch Trials of Cell Transfer Therapy Using Transduction of Anti-
TAA TCR Genes into Non-reactive TIL or PBL 
It is often not possible to isolate sufficient tumor samples from melanoma patients 
and even when tumor is available, only about 60 to 70% generate melanoma reactive TIL 
cultures.  As a potential alternative to the requirement to establish TIL cultures from 
melanoma patients, we sought methods that could be used to easily obtain a polyclonal 
population of T cells with anti-TAA properties.  Transfer of antigen specific TCR genes to 
PBL has recently been described as a potential method of generating large numbers of 
reactive T cells in infectious disease or as anti-cancer cells9.   In a murine model of this 
approach, TCR gene transfer into murine peripheral T-cells was performed using a retroviral 
vector 10.  The engineered T cells were shown to expand in vivo upon viral challenge and 
efficiently homed to effector sites.  In addition, small numbers of TCR-transduced T cells 
promoted the rejection of antigen-expressing tumors in the mice.  Retroviral vector mediated 
gene transfer can be used to engineer human T cells with high efficiency.  In published work, 
the Surgery Branch was among the first to demonstrate that retroviral vector-mediated 
transfer of TCR genes could endow human PBL with anti-tumor reactivity 11.  In this study, 
PBL were engineered with a retroviral vector expressing a TCR gene derived from a MART-
1 reactive CTL.  These engineered cells reacted with MART-1 expressing cells in an HLA-
A201 restricted manner.  
To expand on and potentially improve on these early results, we next isolated highly 
avid TCR genes from both gp100 and MART-1 reactive T cells.  These studies have recently 
been published and are presented briefly here in detail 12;13.  As a source of anti-gp100 TCR 
genes, we used T cell clone R6C12.  T cell line R6C12 is a highly avid CTL clone derived 
from the PBL of a melanoma patient whose PBL were subject to repeated in vitro stimulation 
on gp100 peptide-pulsed autologous PBMC.  The anti-MART-1 TCR genes were derived 
from TIL clone M1F2.  This T cell clone was isolated from the TIL of a patient that 
demonstrated a remarkable in vivo expansion of this clone (as described above).  During the 
period of peak lymphocytosis, 63% of this patients’ CD8+ PBL expressed the TCR 
associated Vȕ12 protein from clone M1F2.  This patient exhibited regression of more than 


08-C-0121
12
95% of his cutaneous and subcutaneous melanoma, ongoing for over 6 months, and 
developed vitiligo on his forearms.
As an initial test of the TCR vectors, we transduced the human T cell line SupT1.  
SupT1 is a human T-cell leukemia cell line with chromosomal translocations involving both 
the alpha and beta TCR genes and therefore, no functional endogenous TCR chains are 
expressed.  SupT1 cells were transduced with the APB vector (expressing the gp100 TCR 
protein) and production of the Vȕ8 chain (from the gp100 TCR) was confirmed by western 
blot analysis.  Functional TCR expression was further tested by assaying for cell surface 
expression of the CD3 protein (without endogenous TCR expression there is no cell-surface 
expression of CD3).  As expected from the lack of endogenous TCR beta gene expression, 
untransduced cells did not stain for CD3.   When vector transduced cells were analyzed in 
parallel, significant CD3 expression was detected in both APB and AIB engineered cells, 
suggesting successful assembly of the full TCR complex.  To determine if the transduced 
cells recognize tumor associated peptides, AIB transduced cells were stained with the 
MART-1 tetramer molecule.  Cells positive for both CD3 protein and the MART-1 tetramer 
were readily detected by FACS analysis of transduced cells.
In patients where it is not possible to obtain viable TIL cultures, we explored whether this 
gene transfer technology could be used to engineer PBL to recognize cancer antigens.  To 
transduce human PBL with the TCR vectors, PBMC from 2 melanoma patients were 
obtained by apheresis and stimulated with anti-CD3 antibody plus IL2 (to induce T cell 
proliferation).  The cells were transduced on days 3 and 4, followed by expansion for 2-8
days post-transduction.  Transduced T cells were stained for CD3 and Vȕ8 (for the gp100 
TCR) or Vȕ12 (for the MART-1 TCR) and subjected to FACS analysis.  The percentage of 
the transduced cell populations ranged from 30-50% Vȕ positive cells without any selection 
for the transduced cells (background staining for Vȕ8 or Vȕ12 was approximately 4%).  
Gene transfer efficiency (as measured by Vȕ8/12 + cells) was equally distributed into both 
CD4 and CD8 positive cells. 
To determine if TCR vector transduced PBL could mediate the release of effector 
cytokines gamma-interferon and GM-CSF, co-culture experiments were setup with peptide 
pulsed T2 cells. T2 cells were pulsed with HLA-A2 specific peptides derived from the 
influenza virus (flu), the MART-1 TAA (Mart), the native gp100 epitope recognized by 
R6C12 (gp100) or an anchor residue modified version of the native 209 peptide (gp100-2M).  
As positive controls for cytokine release, both CTL clones and TIL cultures were used.  The 
engineered PBL secreted very high levels of the cytokines gamma-interferon and GM-CSF
(> 100,000 pg/ml and >10,000 pg/ml respectively).  To determine the relative activity of the 
transduced PBL populations, serial dilutions of the gp100 peptide were incubated with T2 
cells and peptide pulsed cells were co-cultured with APB engineered.  The engineered PBL 
populations were capable of releasing cytokine at low dilutions of peptide (down to 
0.2ng/ml).
To determine melanoma reactivity of the TCR gene-transduced PBL, two HLA-A2 
positive melanoma cell lines and two non HLA-A2 melanoma cell lines were co-cultured 
with TCR and control vector transduced PBL cultures.  HLA-A2 restricted gamma-interferon 
release was demonstrated in both engineered PBL cultures analyzed, with levels comparable 
to control TIL cultures.  As a more stringent assay for effector function in the TCR 
engineered PBL, we determined if these cells were capable of lysing melanoma cell lines.  
The engineered PBL were incubated with 51Cr labeled melanoma cell lines (HLA-A2 


08-C-0121
13
positive and HLA-A2 negative line) at various effector to target cell ratios and the relative 
lysis then determined.  The engineered cells readily lysed the HLA-A2 cell line but not the 
non HLA-A2 line.     
These data demonstrated that using retroviral vector-mediated gene transfer we were
able to transfer avid anti-gp100 and anti-MART-1 TCR genes into a bulk population of both 
TIL and PBL and these cells demonstrated HLA-A2 restricted effector functions including 
cytokine release and cell lysis. 
Based on this technology the Surgery Branch currently has conducted five TCR gene 
transfer trials that have enrolled a total of 71 patients.  In these protocols patients with 
metastatic cancer who are HLA-A2 positive received a nonmyeloablative but lymphocyte 
depleting preparative regimen consisting of cyclophosphamide and fludarabine, and then 
were treated with autologous peripheral blood lymphocytes or TIL that were genetically 
engineered to be reactive with melanoma tumor antigens gp100 or MART-1, or with p53.  
Following adoptive cell transfer, all patients received high-dose IL-2, and some patient 
received peptide vaccination.  In four studies (04-C-0181, 04-C-0251, 07-C-0174, and 07-C-
0175) patients with metastatic melanoma are being evaluated, and in one study (07-C-0003),
patients with metastatic cancer whose tumors overexpress p53 are being evaluated.
To test the in vivo efficacy of the MART-1 TCR engineered T cells in 04-C-0251, 17 
HLA-A*0201 patients with progressive metastatic melanoma (Table 4) were selected for 
treatment 14. All patients were refractory to prior therapy with IL-2.  T cell cultures from all 
17 patients were biologically reactive, with specific secretion of interferon-ȕ following co-
culture with either MART-1 peptide pulsed T2 cells and or melanoma cell lines expressing 
the MART-1 antigen (Figure 2E).  Gene transfer efficiencies measured by staining for Vȕ12
expression in these lymphocytes ranged from 17% to 67% (mean value 42%) (Tables 4 and 
5).  
Patients received adoptive cell transfer (ACT) with MART-1 TCR transduced 
autologous PBL at a time of maximum lymphodepletion.  An initial cohort of three patients 
was treated with cells following an extended culture period of 19 days, at which point they 
had cell doubling times ranging from 8.7 to 11.9 days (Table 4, cohort 1; patients 1, 2a, 3).  
In these patients, less than 10% of the transduced cells persisted across the time points tested 
during the first 30 days post-infusion and 2% or less persisted beyond 50 days (Figure 3A). 
These first three patients showed no delay in the progression of disease. 
In an effort to administer gene-modified lymphocytes that were in their active growth 
phase, the culture conditions were modified to limit the ex vivo culture period to between 6 
and 9 days after stimulation of cells with anti-CD3 antibody (Table 4; cohort 2, doubling 
times two days or less).   In a further cohort, larger numbers of actively dividing cells for 
ACT were generated by  performing a second rapid expansion protocol15 after 8-9 days 
(Table 4, cohort 3; doubling time 0.9 to 3.3 days).  In contrast to the lack of cell persistence 
seen in cohort 1 (Figure 3A), patients in cohorts 2 and 3 (Figure 3B, 3C, 3D), all exhibited 
persistence of the transduced cells at greater than 9% at one and four weeks post-treatment 
(range 9%-56%).  All eight patients providing samples at greater than 50 days exhibited 
persistence at greater than 17%, and this was durable in the seven patients over a monitoring 
period of over 90 days. One patient (patient 14) had > 60% of circulating lymphocytes 
positive for the gene marked cells (Figure 3C).
In 14 patient samples tested at one month post-transfer, quantitative RT-PCR assays 
revealed the presence of vector derived RNA confirming that gene expression continued 


08-C-0121
14
(Table 6).  All but one of 15 patients analyzed had increased levels of CD8+/Vȕ12 cells at 
one week post-treatment and 11 of 15 were higher at one month compared to pretreatment 
levels (Figure 3E) All 13 patients examined had increased MART-1 tetramer-binding cells 
post-treatment (Figure 3F), and 11 of 14 had increased number of elispot positive cells 
(Table 7).  
There was however, a discordance between the mean persistence of transduced cells 
at one month in cohorts 2 and 3 as measured by PCR (mean 26%), compared to the 
measurement of Vȕ12 expressing cells (8.1%) and of MART-1 tetramer-binding cells 
(0.8%).  This discordance is in part due to mispairing of the introduced TCR chains with the 
endogenous chains, as well as, the different sensitivities of the assays.  Tetramer binding 
requires the aggregation of multiple receptor molecules on the lymphocyte surface and is the 
least sensitive of the assays.  The reduced expression of the transgene in the persisting cells 
at one month and later is also a function of the described decrease 16in the transcription of 
retrovially inserted trangenes and the decline in metabolic activity during the conversion of 
activated cells to memory cells. 
Most importantly, four patients demonstrated a sustained objective regression of their 
metastatic melanoma assessed by standard RECIST criteria, two reported in Morgan, et al. 17.
Patient 4, a 52 year old male, had received prior treatment with alpha interferon, lymph node 
dissection, experimental vaccine, and high-dose IL-2. He then developed progressive disease 
in the liver (4.4 X 3.3 cm) and axilla (1.3 X 1.2 cm).  Following treatment in the current 
protocol he experienced complete regression of an axillary mass, and an 89% reduction of a 
liver mass (Figure 4A, 4B)  at which time it was removed. He experienced a sustained partial
response of 23 months in duration. Patient 14, a 30 year old male, received prior treatment 
with lymph node dissection, alpha interferon, and high-dose IL-2.  He developed an 
enlarging 4.0 X 2.5 cm mass in the lung hilum.  Following treatment in the current protocol 
he underwent regression of the hilar mass and is now clinically disease free 30 months later 
(Figure 4C, 4D).  In addition, patient 24, a 56 year old female, who had received prior 
treatment with alpha interferon, lymph node dissection, and high-dose IL-2 and received 36 
X 109 CD8+ purified cells followed by immunization with the native MART-1 (MART-1:27-
35). She has experienced a sustained partial response of retroperitoneal lymph node lesions.  
Also, patient 25, experienced a sustained partial response after treatment with 8.59 X 109
CD8+ purified cells followed by immunization with the modified  MART-1 peptide (MART-
1:27-35 (27L).  This patient is a 49 year old male with history of metastatic melanoma from 
an unknown primary to the right groin/ iliac LN who previously received IL-2.  Thus, four 
patients with rapidly progressive metastatic melanoma were successfully treated with 
genetically engineered autologous PBL.
In responding patients 4 and 14, gene marked cells in the circulation (assumed to be 
1% of total body lymphocytes) expanded 1400 fold and 30 fold respectively compared to the 
infusion cell number.  At one year post-infusion, both responding patients had sustained high 
levels (between 20%-70%) of circulating gene-transduced cells (Figure 4E).  This high level 
of gene marked cells was confirmed in patient 4 by limiting dilution T cell cloning of 
circulating lymphocytes at one year post-treatment which revealed that 33 of 79 (42%) T cell 
clones contained the transgene as assessed by PCR assay. These two patients also displayed 
Vȕ12 cells detectable by antibody staining between 12%-16% when followed out to >300 
days post-treatment (Figure 4F).  The responding patients 4 and 14 were also 2 of 4 patients 
who had greater than 1% circulating tetramer positive cells at greater than 15 days after cell 


08-C-0121
15
infusion (Figure 3F), and these patients demonstrated anti-TAA reactivity in ex vivo co-
culture assays (Table 8).  There were no toxicities in any patient attributed to the gene-
marked cells.
We have thus demonstrated for the first time in humans, that normal autologous T 
lymphocytes, transduced ex vivo with anti-TAA TCR genes and reinfused in cancer patients 
can persist and express the transgene long-term in vivo and mediate the durable regression of 
large established tumors. Although the response rate of 2/15 (13%) seen in cohorts 2 and 3 is 
lower than the 50% response rate we achieved by the infusion of autologous TIL, this method 
can be used in patients for whom TIL are not available and in patients with common 
epithelial cancers (Figure 2A, Table 9).  Two additional patients have recently been 
documented as PR in arm P2A (anti-MART1 TCR PBL, up to 3x10e9 with MART-1:27-35); 
and arm P1H (anti-MART-1 TCR CD8 PBL, up to 1x10e10 with MART-1:27-35 (27L) 
peptide for a total of 4/39 responses.
A similar study was conducted using gp100 TCR gene marked cells, however this 
retroviral vector had a low titer when produced under GMP conditions.  Nineteen patients 
have been treated on this study, and one partial response was observed in a patient treated
with TIL. To date, 12 patients have been treated on the anti-p53 TCR gene marked cells 
study.  Eleven patient’s tumors did not respond to treatment, and one partial response was 
observed in a patient with a parotid gland tumor metastatic to a hilar lymph node and 
multiple metastases throughout the lungs. There have been no grade 5 toxicities observed on 
this study, and all grade 3 and 4 toxicities observed, with one exception, are expected 
toxicities associated with the non-myeloabalative chemotherapy regimen or IL-2.  One 
patient experienced grade 3 buttock pain which was unexpected, however this toxicity was 
due to progression of a gluteal lesion and therefore unrelated to the investigational agent.
Our two newest TCR studies investigate more potent TCRs which target gp100 and 
MART-1.  The anti-gp100(154) TCR transduced PBL study (07-C-0174) has accrued 10 
patients to date.  At this time, 2 patients have been evaluated for response and these patients 
had progressive disease.  Eight patients have been evaluated for toxicities, and most grade 3 
and 4 toxicites are known toxicities of the research, and no toxicities have been attributed to 
the cells. One grade 3 toxicity (headache) was an unexpected event and was possibly related 
to the research.  No grade 5 events have been observed on this study.   The anti-MART-1 F5 
TCR transduced PBL study (07-C-0175) has accrued 11 patients to date.  At this time three 
patients have been evaluated for response with one patient experiencing a partial response.  
Nine patients have been evaluated for toxicities, and most grade 3 and 4 toxicities are known 
toxicities of the research, with no toxicities attributed to the cells.   Two grade 3 toxicities 
(pain, headache) and one grade 4 toxicity (pulmonary embolism) were unexpected events but 
were not related to the research.  No grade 5 events have been observed on this study.   
1.2.4. NY ESO-1 as a Target for Cell Transfer Clinical Studies
The NY-ESO-1 molecule, which was initially identified by screening a cDNA 
expression library with an antiserum from a patient with esophageal squamous cell 
carcinoma, represents a tumor antigen that can be targeted in patients bearing a wide variety 
of malignancies 18. Expression of NY-ESO-1 protein has been observed in approximately one 
third of melanoma, breast, prostate, lung ovarian, thyroid and bladder cancer, but is limited in 
normal tissues to germ cells and trophoblasts 19. A related cancer/testis antigen, LAGE-1, has 


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16
also been identified and shown to possess 84% amino acid similarity to the NY-ESO-1
protein20. Further studies resulted in the identification of an identical peptide corresponding 
to amino acids 157 to 165 of the NY-ESO-1 and LAGE-1 proteins SLLMWITQC as a 
dominant epitope recognized by HLA-A2 restricted, NY-ESO-1 reactive T cells21. An HLA-
A2 restricted epitope representing the first eleven amino acids of an alternative open reading 
frame of the NY-ESO-1 and LAGE-1 transcripts has also been described22 and epitopes 
derived from the normal as well as alternative open reading frames of both gene products in 
the context of HLA-A31 have also been described23. In addition, NY-ESO-1 epitopes are 
recognized in the context of multiple HLA class II restriction elements 24-26. Tumors may 
have heterogeneous expression of NY-ESO-1 as detected by immunohistochemistry though 
this is highly dependent on the affinity of the antibody.  Studies in melanoma have shown 
that only 1-5 peptides on the surface of a cell can stimulate lymphocytes, this is far below the 
sensitivity of most antibodies.
The results of multiple studies indicate that relatively high levels of natural immunity 
directed against NY-ESO-1 may exist in cancer patient. Serum anti-NY-ESO-1 antibodies 
are prevalent in cancer patients, and in one study 10 out of 12 patients bearing NY-ESO-1
positive tumors possessed serum antibodies directed against this antigen 27. This is in contrast 
to other tumor antigens for which antibody responses have been demonstrated in only 5% or 
less of patients 28. Furthermore, the presence of high titers of anti-NY-ESO-1 antibodies in 
the serum of cancer patient has been associated with tumor burden 27 as well as the presence 
of T cell precursors reactive with the HLA class I restricted NY-ESO-1:157-165/HLA-A*02 
epitope in the peripheral blood of cancer patients 29. Clinical trials have been based on 
immunization with the dominant HLA-A2 restricted NY-ESO-1 epitope 30-32, a HLA class II 
restricted peptide 32, as well as recombinant NY-ESO-1 protein 33.  While evidence indicated 
that immunization was capable in some patients of enhancing the frequency of peptide or 
tumor reactive T cells, clinical responses were only observed in a small percentage of 
patients treated in these trials.  
Active immunization strategies directed against a variety of additional tumor antigens 
have resulted in clinical responses in only a small minority of treated patients 34, whereas 
adoptive cell transfer strategies have resulted in response rates of up to 50 percent 8. Another 
strategy now being employed is the adoptive transfer of T cells that have been genetically 
modified to express T cell receptors (TCR) directed against widely expressed tumor antigens.  
As previously discussed, in the first report of a clinical cancer TCR trial, objective clinical 
responses were observed in 17 percent of melanoma patients treated with T cells that were 
genetically modified to express a TCR directed against the immunodominant MART-1 HLA-
A*02 restricted peptide epitope14.
Current attempts to extend cancer therapies based on the genetic modification of T 
cells to patients bearing additional tumor types have focused on the identification of TCRs 
with high functional avidity directed against the dominant NY-ESO-1:157-165 T cell 
epitope. A comparison of two anti-NY-ESO-1 TCRs, designated 1G4 and ET-8F, for their 
ability to mediate peptide reactivity in gene modified CD8+ T cells indicated that the 1G4 
TCR was significantly more active than the ET-8F TCR 35. The 1G4 was also more active 
than the ET-8F, and an additional NY-ESO-1 reactive TCR, when evaluated for their ability 
to mediate specific tumor cell recognition in gene modified CD8+ T cells (unpublished 
results).  


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17
These results indicated that the 1G4 TCR may possess a relatively high functional 
avidity; nevertheless, this TCR, which was determined using a direct binding assay to 
possess an affinity of between 10 and 30 PM, was not able to confer CD4+ T cells with the 
ability to recognize target cells expressing the endogenously processed NY-ESO-1 epitope. 
High affinity variants of the 1G4 TCR generated by bacteriophage display that contained 
multiple amino acid substitutions (AAS) in both the D and E chain TCR complementarity 
determining regions (CDR) 236 were then evaluated in an attempt to identify TCR variants 
that enhanced the function of gene modified CD8+ as well as CD4+ T cells 35. The results 
demonstrated that high affinity variant TCRs possessing affinities of between 5 and 84 nM 
lead to the antigen-specific activation of TCR-gene modified CD4+ T cells, but lead to the
non-specific activation of TCR-gene modified CD8+ T cells by HLA-A*02+ NY-ESO-1-
target cells, whereas TCR variants with affinities nearer to that of the WT 1G4 TCR show 
enhanced reactivity in CD4+T cells without any apparent loss of specificity in CD8+ T cells.
Further studies were carried out to evaluate the role of individual CDR substitutions on the 
function the 1G4 TCR resulted in the identification of variants containing single and dual 
CDR2E chain and CDR3D chain substitutions that enhanced the IFN-J responses of CD4+ as 
well as CD8+ T cells (Robbins, PF et al., submitted for publication). The results indicated 
that substitutions of individual CDR2 E chain residues significantly enhanced the responses 
of gene modified CD4+ T cells to NY-ESO-1+/HLA-A*02+ tumor target cells (Figure 5A)
and modestly enhanced the responses of CD8+ T cells (Figure 5B). A TCR containing dual 
substitutions of alanine and isoleucine residues for the glycine and alanine residues present at 
positions 51 and 52 in the native CDR2 Echaindesignated E51:AI, further enhanced the 
response of transfected CD4+ T cells (Figure 5A). Individual substitutions for CDR3D
residues did not lead to significant enhancement of the response of gene modified CD4+ T
cells; however, the analysis of TCRs containing dual substitutions for the threonine and 
serine residues present at positions 95 and 96 of the 1G4 D chain resulted in the identification 
of two variants, D95:LL and D95:LY, that dramatically enhanced the specific reactivity of 
transduced CD4+ T cells (Figure 6A), and resulted in modest increases in the responses of 
transduced CD8+ T cells (Figure 6B). An increased affinity for the NY-ESO-1/HLA-A*02 
complex appeared to be responsible for enhancing the responses of gene modified T cells, as 
the D95:LY plus WT E 1G4 TCR variant possesses an affinity of 730 nM and the WT D
chain plus E51:AI variants possess an affinity of 280 nM.
These results clearly demonstrated that the responses of CD4+ T cells transduced 
with the 1G4 D95:LY and E51:AI variants to NY-ESO-1+/HLA-A*02+ tumor cells were 
dramatically enhanced relative to those of cells transduced with the WT TCR. An evaluation 
of the results of multiple experiments indicated that CD8+ T cells transduced with the 
D95:LY/WT ETCR released higher levels of IFN-J in response to the NY-ESO-1+/HLA-
A*02+ tumor target cell lines than those expressing the WT D/E TCR (ratio of 2.2+0.34,
mean + SEM), a statistically-significant level of enhancement (p<0.01 using a paired t test, 
36 pairs tested). At the same time, the responses of CD8+ T cells transduced with the WT 
DE51:AI TCR pair did not differ significantly from the WT D/E TCR (ratio of 1.2+0.24,
p>0.4, 34 pairs tested). In addition, the responses of CD8+ T cells transduced with the 
D95:LY/WT E TCR was significantly enhanced relative to those of cells transduced with the 
WT DE51:AI variant (ratio of 3.1+0.67, p<0.01, 34 pairs tested). Additional results 


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18
indicated that the CD8+ T cells produced low but significant levels of IFN-J in response to 
NY-ESO-1-/HLA-A*02+ tumor cells (Robbins, PF et al., submitted for publication).
The relative effectiveness of CD8+ or CD4+ T cells in mediating tumor regression is 
unknown; however, several observations suggest that the presence of CD4+ T cells can 
enhance the function of the CD8+ T cell population. The persistence of CMV-reactive CD4+
T cells is correlated with enhanced control of disease by CMV-reactive CD8+ T cells 37;38.
Co-operative interactions between CD8+ and CD4+ T cells expressing a high affinity CD8-
independent TCR directed against a human p53 epitope have been observed, as assessed by 
their abilities both to secrete IFN-J in response to cognate antigen complex expressing targets 
cell lines and to activate dendritic cells39. In addition, the adoptive transfer of CD4+ T cells 
expressing a CD8-independent TCR has been shown to enhance tumor protection mediated 
by CD8+ tumor reactive T cells 40. Taken together, these results indicate that the D95:LY 
variant of the 1G4 TCR represents a potent TCR that results in high levels of specific anti-
tumor activity in gene modified CD4+ as well as CD8+ T cells and provide evidence that this 
represents an attractive candidate for the further evaluation of TCR-based cancer adoptive 
immunotherapy.
As of October 1, 2009 in the current protocol using transfer of autologous cells 
transduced with the anti-NY-ESO-1 TCR, four patients with synovial cell sarcoma have been 
treated. Three of the four patients have experienced an objective partial response by RECIST 
criteria though the three responders recurred at 10 months, 6 months and 4 months 
respectively. An additional patient treated by compassionate exemption experienced an 
objective response and recurred at 3 months. Eight evaluable patients with metastatic 
melanoma were treated. One experienced a complete response ongoing at 10 months and 3 
others experienced a partial response, one ongoing at 8 months and the other two recurred at 
8 and 3 months respectively. Two patients with metastatic breast cancer were treated (one 
treated as a compassionate exemption) and one patient experienced a PR but recurred at 2 
months.
Thus of the 15 treated patients evaluable as of October 1, 2009, 9 responded (60%) 
but 7 of the 9 have recurred at times varying from 2 to 10 months. This recurrence rate is 
unacceptably high. Our murine models of cell transfer have clearly indicated that the 
administration of a pox virus in conjunction with cell transfer can significantly increase the 
effectiveness of the cell transfer and we are thus amending the protocol to add immunization 
with an ALVAC virus encoding the NY-ESO-1 antigen to the treatment regimen.
In addition, recent studies have emphasized the impact of CD4+, CD25+ regulatory 
T-cells in inhibiting immune reactions.  Our experimental animal data, as well as the studies 
of the PBL of melanoma patients, have revealed a significant incidence of these T regulatory 
cells that can impede anti-tumor responses.  Eliminating T regulatory cells in our pmel 
murine models by eliminating CD4 cells significantly improved the impact of adoptively 
transferred anti-tumor CD8+ cells.  It thus appears likely that the T regulatory cells present in 
peripheral blood lymphocytes may be impeding the effectiveness of the CD8+ anti-tumor 
lymphocytes.  It is possible that removing the cells will improve the response rates in patients 
and may also reduce the toxicities seen by production of cytokines by CD4+ cells.
Therefore, with the approval of amendment E, if after initial treatment with the unselected 
anti- ESO-1 TCR-transduced cells and ALVAC ESO-1 vaccine patients do not respond to 
treatment or progress after a response or have stable disease, they will be allowed retreatment 
with CD8-enriched anti ESO-1 TCR-transduced cells and ALVAC ESO-1 vaccine provided 


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19
they meet the original eligibility criteria. We plan to utilize the GMP quality, CliniMACS 
apparatus from Miltenyi Biotec that is in common use in the NCI and many other centers.  
The exact procedure for depletion utilized in protocol 07-C-0176 to eliminate CD4+ cells 
will be used in this current protocol. In our current study evaluating young TIL (07-C-0176), 
we have observed a 51% clinical response rate (CR and PR) in the 33 patients receiving the 
CD8+ young TIL cells compared to a 20% response rate in the 26 patients receiving young 
TIL containing CD4+ and CD8+ cells.  In addition, patients receiving the CD8+ young TIL 
experienced less adverse events. With approval of amendment J, patients will not be 
retreated with the CD8-enriched cells.  Data from our randomized study comparing CD8-
enriched young TIL and bulk young TIL(containing CD4+ and CD8+ cells), indicates that 
there is no difference in clinical response nor toxicities in patients treated in these two 
randomized cohorts.  Therefore, patients will be retreated with anti-ESO-1 TCR cells similar 
to their initial treatment which contained CD4+ and CD8+ cells. In addition, patients who 
are retreated will only receive the ALVAC vaccine if it was part of their initial treatment and 
it is available. In order to be retreated, patients must have a partial response to treatment that 
then stabilizes and have evaluable disease, or stable disease that subsequently progresses.  
They may be re-treated when progression by RECIST criteria is documented.
1.2.5. Rationale for the current protocol to add immunization with ALVAC pox 
viruses following adoptive transfer of cells. 
In extensive studies utilizing the pmel murine melanoma model it has been clearly 
demonstrated that the adoptive transfer of tumor-reactive T cells into a lymphodepleted 
mouse can mediate the rejection of established melanoma and that the simultaneous 
administration of a pox virus vaccine encoding the antigen recognized by the T cells could 
greatly improve the activity of the T cell transfer 41, 42 Preclinical data has demonstrated that 
peptide vaccination is ineffective whereas vaccination with pox viruses can improve the 
effectiveness of the adoptively transferred cells and could mediate complete tumor 
regression42.Thus, in this animal model, mice bearing large invasive B16 melanomas receive 
the adoptive transfer of transgenic murine T cells reactive with the gp100 epitope presented 
on the B16 melanoma.  Following the adoptive transfer of these cells, mice received the 
administration of IL-2 with or without the simultaneous administration of the recombinant 
fowlpox virus encoding a modified form of the gp100 epitope reactive with the adoptively 
transferred cells.  As shown in Figures 7, 8, and 9 the administration of this fowlpox virus 
substantially improved the effectiveness of the adoptively transferred cells and could mediate 
complete tumor regression of these large tumors in mice.  These anti-tumor effects of the 
vaccine were highly reproducible and were substantially greater in mice that were 
immunosuppressed by whole body irradiation or in highly immunosuppressed RAC knockout 
mice.  These studies of immunization with recombinant pox viruses after cell transfer in the 
immunosuppressed host provided strong evidence that suggests that adoptive transfer of 
lymphocytes in our clinical protocols in patients with melanoma will be substantially 
improved by the simultaneous administration of recombinant pox virus expressing the 
antigen recognized by the transferred T cells.
We have extensive experience in the administration of fowlpox viruses to patients 
with metastatic melanoma in the absence of cell transfer.  Twelve patients with metastatic 
melanoma were treated with Fowlpox virus encoding MART-143.  In three consecutive 
clinical trials a total of 46 patients were immunized with recombinant fowlpox virus 


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20
encoding gp10044.  In these studies viruses incorporating the native gp100 molecule as well
as a “minigene” construct encoding a single modified epitope, gp100:209-217(210M) were 
used for immunization.  We showed that these viruses could be administered safely in doses 
as high as 5-6x109/pfu intravenously or intramuscularly.  Patients could be successfully 
immunized after inoculation with these recombinant viruses although clinical anti-tumor 
responses were only seen when these immunizations were administered in conjunction with 
IL-2.  The current canary pox viruses (ALVAC) being supplied by Sanofi-Pasteur are very 
similar to the fowlpox viruses we used in the prior studies with the exception of the 
introduction of co-stimulatory molecules (TRICOM constructs) into the virus.  We have now 
treated four metastatic melanoma patients with the current ALVAC viruses (including two 
who received the ALVAC MART-1 and two who received the ALVAC gp100 vaccine in 
conjunction with cell transfer) and have seen no toxicity attributable to the virus 
administration.  We have not seen any added toxicities of this combination of cell transfer 
therapy along with the pox viruses and the number of viral particles being used for 
immunization is far lower than that we have previously administered.
1.2.6. NY-ESO-1 ALVAC Vaccine   
Starting with Amendment E, the investigational vaccine to be used in the proposed clinical 
trial, is a viral vector-based vaccine that is comprised of a gene for a tumor-associated 
antigen (TAA).  The vaccine was developed by Sanofi-Pasteur Ltd. who sponsors an IND for 
this agent.  The NY-ESO-1 is one of five TAA chosen for inclusion in the ALVAC virus by 
Sanofi-Pasteur. The NY-ESO-1 gene used in ALVAC(2)-NY-ESO-1(M)/TRICOM 
[vCP2292] was altered by in vitro mutagenesis, changing the encoded amino acid at position 
165 from cysteine to valine. This modification was designed to enhance the immunogenicity 
of the antigen
In addition, the vaccine contains genes that encode three co-stimulatory/adhesion 
molecules: B7.1, ICAM-1, and LFA-3.  B7.1 is an immune response modulating signal, 
while the other two are cellular adhesion molecules.  These molecules, which are collectively 
referred to as TRICOM, are intended to increase the immune response to the TAAs.
The vector is a modified canarypox virus, ALVAC(2), that induces both humoral and 
cellular immune responses to the inserted transgenes.
The genes for the TAA and the genes for the co-stimulatory molecules are included in 
the same construct.  The complete vaccine therefore consists of the following construct:
•
ALVAC(2)-NY-ESO-1(M)/TRICOM (also known as vCP2292 and herein 
called ALVAC ESO-1 vaccine)
The modified canarypox virus ALVAC(2) is used as the antigen-presentation 
platform.  ALVAC is a plaque purified isolate of canarypox virus; ALVAC(2) was derived 
from ALVAC by inserting two coding sequences (E3L and K3L) from vaccinia virus.  These 
additional sequences have been shown to enhance virus-specific gene expression.  Previous 
studies suggest that ALVAC-based recombinants have significant advantages as vector-based 
vaccines, since they are safe for vaccination purposes in animals and humans and induce both 
humoral and cellular immune responses to inserted transgenes.  The safety of ALVAC(2) has 
been established in a Canadian clinical trial conducted from February 2000 to April 2003, 


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21
titled “Recombinant Canarypox Virus Expressing Tumor Antigen gp100 (Modified) and 
Modified gp100 Peptide Combination”.
Poxviruses are strong stimulators of the cellular arm of the immune system, and are 
thus attractive vectors for use in immunotherapeutic strategies of stimulating T-cell responses 
to TAAs. Avian poxviruses, including ALVAC(2), provide additional safety advantages over 
other poxviruses as they do not productively replicate in human cells.  Although they enter 
mammalian cells and express viral gene products from their cytoplasmic replication site, 
infectious progeny virus is not produced.  Virally expressed gene products are available for 
processing and presentation by major histocompatibility complex (MHC) molecules.  
However, poxviruses have a limited life span in mammalian cells such that TAA expression 
is sustained for a limited time.
ALVAC-based vectors engineered to express antigens of choice have been developed 
for active vaccination of patients with infectious diseases or cancer.  Several clinical trials 
using ALVAC-based vaccines have been conducted for melanoma (Canada), colorectal 
cancer (Canada and USA) and Human Immunodeficiency Virus (USA).  In addition, there 
are ALVAC-based vaccines that have been licensed for veterinary applications, including 
canine distemper virus (USA), feline leukemia virus (EU), and rabies (USA).
TRICOM is composed of three co-stimulatory/adhesion molecules:  B7.1, 
Intercellular Adhesion Molecule-1 (ICAM-1), and Lymphocyte Function Associated antigen-
3 (LFA-3).  These proteins are normally expressed at low levels on the surface of 
professional antigen-presenting cells.  They are up-regulated and serve to augment immune 
responses through engagement of their counter-receptors on T-cells, CD28, CD11a/CD18, 
and CD2, respectively, after activation.  There are a number of published reports using 
mouse, monkey, and human systems that demonstrate that the incorporation of TRICOM into 
poxviruses can enhance T-cell responses, and that the three components together are more 
effective than any one or two of the three.  In addition, mice vaccinated with recombinant 
vaccinia and fowlpox viruses containing TRICOM and carcinoembryonic antigen (CEA) 
showed enhanced T-cell responses and tumor protection compared to similar vectors 
containing CEA alone.  It is believed that ALVAC- encoded TRICOM vaccine will infect 
some antigen-presenting cells in the local vicinity after vaccination.  This will result in 
expression of high levels of co-stimulatory molecules in such antigen-presenting cells that 
also express the melanoma tumor-associated antigens.  After injection, these will be highly 
effective at activating both CD8 and CD4 T cells.
With approval of amendment G, cohorts 1 and 2 will be re-opened due to the shortage 
of the ALVAC ESO-1 vaccine at this time. We plan to leave cohorts 3 and 4 open as we 
would like to accrue to these arms if the ALVAC ESO-1 vaccine becomes available in the 
future.  To date, we have seen remarkable clinical responses in patients treated on this study.  
In cohort 1, we have treated 13 patients with melanoma, and 5 of 11 patients with high 
expression of ESO-1 had clinical response including 3 partial responses (PRs) of 3, 8 and 
14+ months, and 2 complete responses (CRs) of 24, and 25 + months.  In cohort 2, we have 
treated 7 patients and 6 of these patients were heavily pretreated synovial cell sarcoma.  Four 
patients with synovial cell sarcoma experienced PRs lasting 10, 14, 4 and 8 months.  
Therefore, the response rate in patients receiving non-myeloablative chemotherapy, anti-
ESO-1 TCR PBL and high dose IL-2 but no vaccine is 45%.  In cohort 3 (with vaccine), we 
have treated 6 patients with melanoma, and of the three evaluable patients, 1 patient has an 
ongoing PR of 6+ months.  In cohort 4 (with vaccine), 5 patients have been treated, and out 


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22
of the 4 evaluable patients, 2 patients with synovial cell sarcoma have ongoing PRs of 7+ 
months.   The preliminary data from cohorts 3 and 4 indicate that clinical response rates 
observed with vaccine in cohorts 3 and 4 are similar to those observed without vaccine in 
cohort 1 and 2.   In addition, the adverse events observed in each arm are comparable with 
most toxicities attributed to the non-myeloablative chemotherapy regimen and high dose IL-
2.
1.2.7. Safety Considerations
Several safety concerns regarding the infusion of large numbers of retrovirally 
modified tumor reactive T-cells have been addressed in our previous clinical studies.  The 
non-myeloablative chemotherapy and the administration of high-dose IL-2 have expected 
toxicities discussed earlier.  The immunomyeloablative chemotherapy used in this protocol 
has been administered to over 80 patients and all have reconstituted their hematopoietic 
systems.  
In other protocols we have administered over 3 X 1011 TIL with widely 
heterogeneous reactivity including CD4, CD8, and NK cells without difficulty.  As discussed 
above, the expansion of tumor reactive cells is a desirable outcome following the infusion of 
antigen reactive T-cells.  Some patients receiving gp100 or MART-1 reactive cells have 
developed vitiligo or uveitis probably due to destruction of normal melanocytes though these 
toxicities have been manageable.  We do not believe the transfer of these gene modified cells 
has a significant risk for malignant transformation in this patient population.  While the risk 
of insertional mutagenesis is a known possibility using retroviral vectors, this has only been 
observed in the setting of infants treated for XSCID using retroviral vector-mediated gene 
transfer into CD34+ bone marrow cells. In the case of retroviral vector-mediated gene 
transfer into mature T-cells, there has been no evidence of long-term toxicities associated 
with these procedures since the first NCI sponsored gene transfer study in 1989.  Although 
continued follow-up of all gene therapy patients will be required, data suggest that the 
introduction of retroviral vectors transduced into mature T-cells is a safe procedure.  While 
we believe the risk of insertional mutagenesis is extremely low, the proposed protocol 
follows all current FDA guidelines regarding testing and follow up of patients receiving gene 
transduced cells.
2.0 Eligibility Assessment and Enrollment
2.1.
Eligibility Criteria
2.1.1.
Inclusion Criteria
a.
Metastatic cancer that expresses ESO as assessed by one of the following methods:
RT-PCR on tumor tissue, or by immunohistochemistry of resected tissue, or serum 
antibody reactive with ESO. Metastatic cancer diagnosis will be confirmed by the 
Laboratory of Pathology at the NCI.
b. Patients with histologies other than metastatic melanoma, must have previously 
received systemic standard care (or effective salvage chemotherapy regimens) for 


08-C-0121
23
metastatic disease, if known to be effective for that disease, and have been either 
non-responders (progressive disease) or have recurred.
c.
Greater than or equal to 18 years of age and less than or equal to 66 years of age.
d. Willing to sign a durable power of attorney
e.
Able to understand and sign the Informed Consent Document
f.Clinical performance status of ECOG 0 or 1.
g. Life expectancy of greater than three months.
h. Patients of both genders must be willing to practice birth control for four months 
after receiving the preparative regimen.
i.Patients must be HLA-A*0201 positive
j.Serology:
1. Seronegative for HIV antibody.  (The experimental treatment being
evaluated in this protocol depends on an intact immune system.  Patients 
who are HIV seropositive can have decreased immune -competence and 
thus be less responsive to the experimental treatment and more susceptible 
to its toxicities.) 
2. Seronegative for hepatitis B antigen and hepatitis C antibody unless antigen 
negative. If hepatitis C antibody test is positive, then patients must be 
tested for the presence of antigen by RT-PCR and be HCV RNA negative.
k. Hematology:
1. Absolute neutrophil count greater than 1000/mm3 without the support of 
filgrastim.
2. WBC (> 3000/mm3).
3. Platelet count greater than 100,000/mm3.
4. Hemoglobin greater than 8.0 g/dl.
l.Chemistry:
1. Serum ALT/AST less or equal to 2.5 times the upper limit of normal.
2. Serum creatinine less than or equal to 1.6 mg/dl.
3. Total bilirubin less than or equal to 1.5 mg/dl, except in patients with 
Gilbert’s Syndrome who must have a total bilirubin less than 3.0 mg/dl.
m. More than four weeks must have elapsed since any prior systemic therapy at the
time the patient receives the preparative regimen, and patients’ toxicities must have 
recovered to a grade 1 or less (except for toxicities such as alopecia or vitiligo). 
n. Six weeks must have elapsed since prior ipilimumabtherapy to allow antibody
levels to decline.
Note: Patients who have previously received ipilimumab or tremelimumab, anti- PD1 
or anti-PD-L1 antibodies, and have documented GI toxicity must have a normal 
colonoscopy with normal colonic biopsies.
2.1.2.
Exclusion Criteria 
a.
Prior vaccination with an ALVAC containing vaccine for patients who will 
receive the ALVAC ESO-1 vaccine (cohorts 3 or 4).
b. Women of child-bearing potential who are pregnant or breastfeeding because of 
the potentially dangerous effects of the preparative chemotherapy on the fetus or 
infant.


08-C-0121
24
c.
Active systemic infections, coagulation disorders or other major medical illnesses 
of the cardiovascular, respiratory or immune system, myocardial infarction, 
cardiac arrhythmias, obstructive or restrictive pulmonary disease.
d. Any form of primary immunodeficiency (such as Severe Combined 
Immunodeficiency Disease).  
e.
Concurrent opportunistic infections (The experimental treatment being evaluated 
in this protocol depends on an intact immune system.  Patients who have 
decreased immune competence may be less responsive to the experimental 
treatment and more susceptible to its toxicities).
f.
Concurrent Systemic steroid therapy
g. Known systemic hypersensitivity to any of the vaccine components, including egg
products or Neomycin for patients who will receive the ALVAC ESO-1 vaccine 
(cohorts 3 or 4).
h. History of severe immediate hypersensitivity reaction to any of the agents used in 
this study.
i.
History of coronary revascularization or ischemic symptoms
j.
Any patient known to have an LVEF less than or equal to 45%.
k. Documented LVEF of less than or equal to 45% tested in patients with:
1. History of ischemic heart disease, chest pain, or clinically significant atrial 
and/or ventricular arrhythmias including but not limited to: atrial 
fibrillation, ventricular tachycardia, second or third degree heart block
2. Age greater than or equal to 60 years old
l.
Documented FEV1 less than or equal to 60% predicted tested in patients with:
1. A prolonged history of cigarette smoking (20 pk/year of smoking within 
the past 2 years).
2. Symptoms of respiratory dysfunction
2.2.
Research Eligibility Evaluation  
Within 4 weeks prior to starting the chemotherapy regimen: 
a.
Complete history and physical examination, including height, weight and vital 
signs, and eye exam noting in detail the exact size and location of any lesions that 
exist. (Note:  patient history may be obtained within 8 weeks)
b. Chest x-ray
c.
EKG
d. Baseline CT of the chest, abdomen and pelvis, and brain MRI to evaluate the 
status of disease. Additional scans and x-rays may be performed if clinically 
indicated based on patients’ signs and symptoms.
e.
Colonoscopy for patients meeting requirements noted in Section 2.1.1.o.
f.
Pulmonary Function Testing for patients with a prolonged history of cigarette 
smoking or symptoms of respiratory dysfunction .  Patients with an FEV1 of less 
than 60% predicted for age will not proceed to treatment. (Testing may be 
performed within 8 weeks of treatment)
g. Cardiac evaluation (stress thallium, echocardiogram, MUGA etc.,) in patients 
who are greater than or equal to age 60, or have a history of  ischemic heart 
disease, chest pain, or clinically significant atrial and/or ventricular arrhythmias 
including but not limited to: atrial fibrillation, ventricular tachycardia, heart 


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25
block..  Patients with a LVEF of less than or equal to 45% will not proceed to 
treatment. (Testing may be performed within 8 weeks of treatment). 
h. HIV antibody titer and HbsAG determination, anti HCV (may be performed 
within 3 months of chemotherapy start date)
i.
Anti CMV antibody titer, HSV serology, and EBV panel. (Note: may be 
performed within 3 months of chemotherapy start date; patients who are known to 
be positive for any of the above do not need to be retested.)
j.
Documentation of results HLA typing done at the NIH (testing is permitted to be
conducted at any time prior to this point).
k. One of the following methods will be used to assess ESO expression: RT-PCR 
analysis of tumor biopsy, or immunohistochemistry, or analysis of serum to 
evaluate for expression of ESO1 antibody reactive with ESO.
Immunohistochemistry verification of ESO expression will be carried out in a 
CLIA-approved pathology laboratory, while RT-PCR and ELISA assay for ESO1 
antibody will be conducted in Dr. Rosenberg’s laboratory using CLIA-approved 
methods. (Testing is permitted to be conducted at any time prior to this point).
Within 14 days prior to starting the chemotherapy regimen:
l.
Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), 
Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium 
total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, 
Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric Acid)
m. Thyroid panel 
n. CBC with differential and platelet count
o. PT/PTT
p. Urinalysis and culture, if indicated 
Within 7 days prior to starting the chemotherapy regimen: 
q. ȕ-HCG pregnancy test (serum or urine) on all women of child-bearing potential
r.
ECOG performance status of 0 or 1 
2.3.
Patient Registration
Patients will be registered on protocol 03-C-0277 (Cell Harvest and 
Preparation for Surgery Branch Adoptive Cell Therapy Protocols) prior to 
transduction of PBL cells (either fresh or cryopreserved samples), by the clinical 
fellow or research nurse within 24 hours of the patient signing the consent by faxing a
completed Eligibility Checklist to the Central Registration Office (CRO) at 301-480-
0757. Once cells exceed the potency requirement and are projected to exceed the 
minimum number specified in the Certificate of Analysis (COA), patients will sign 
the consent document for this protocol and the Eligibility Checklist will be completed 
and faxed to the Central Registration Office at 301-480-0757 within 24 hours of the 
patient signing the consent.  
3.0 Study Implementation
3.1.
Study Design:
3.1.1.
Treatment Phase:


08-C-0121
26
PBMC will be obtained by leukapheresis (approximately 1 X 1010 cells).  Whole 
PBMC will be cultured in the presence of anti-CD3 (OKT3) and aldesleukin in order to 
stimulate T-cell growth.  Transduction is initiated by exposure of approximately 1 X
107 to 5 X 108 cells to supernatant containing the anti-ESO TCR retroviral vector.  
These transduced cells will be expanded and tested for their anti-tumor activity.  
Successful TCR gene transfer will be determined by FACS analysis for the TCR 
protein and anti-tumor reactivity will be tested by cytokine release as measured on 
peptide pulsed T2 cells.  Successful TCR gene transfer for each transduced PBL 
population will be defined as >10% TCR positive cells and for biological activity, 
gamma-interferon secretion must be at least 200 pg/ml.  Patients will receive up to 1.0
X 1011 anti-ESO TCR engineered PBL.  A minimum of approximately 1.0 X 108 cells 
will be given.  The cells administered vary depending on their growth characteristic.  In 
prior protocols over 3 X 1011 T cells have been safely infused to cancer patients.
Prior to receiving the engineered PBL cells, patients will receive a nonmyeloablative 
but lymphocyte depleting preparative regimen consisting of cyclophosphamide and 
fludarabine followed in one to four days by intravenous infusion of in vitro tumor 
reactive, TCR gene-transduced PBL plus IV aldesleukin (720,000 IU/kg q8h for a 
maximum of 15 doses). Patients will receive one course of treatment.  The start date of 
the course will be the start date of the chemotherapy; the end date will be the day of the 
first post-treatment evaluation.  Patients may undergo a second treatment as described 
in Section 3.4.
Patients will be entered into two cohorts based on histology: cohort 1 will include 
patients with metastatic melanoma or renal cell cancer; cohort 2 will include patients 
with other types of metastatic cancer.  All cancers must be shown to express ESO.
Patients accrued to cohorts 3 and 4 will receive a nonmyeloablative but lymphocyte 
depleting preparative regimen consisting of cyclophosphamide and fludarabine 
followed by subcutaneous injection of ALVAC ESO-1 vaccine approximately 2 hours 
prior to intravenous infusion of ex vivo tumor reactive, TCR gene-transduced PBMC 
plus IV aldesleukin (720,000 IU/kg q8h for a maximum of 15 doses).  A second 
vaccination with ALVAC ESO-1 vaccine will be given on day 14 (+/- 2 days).  Cohort
3 will include patients with metastatic melanoma or renal cell cancer and cohort 4 will 
include patients with other types of metastatic cancer. 
3.1.2.
Protocol Stopping Rules:
The study will be halted pending discussions with the FDA and NCI IRB if the 
following conditions are met:
x
Two or more patients develop a grade 3 or greater toxicity at any point in the 
VWXG\JUDGHWR[LFLW\QRWDWWULEXWDEOHWRWKHFKHPRWKHUDS\SUHSDUDWLYH
regimen or aldesleukin therapy (or circumstances unrelated to this study).
x
If 1 of the first 3 patients (OR 2 of the first 6 patients, OR 3 of the first 9 patients, 
OR 4 of the first 12 patients) develop grade 3 autoimmunity, that cannot be 
resolved to less than or equal to a grade 2 autoimmune toxicity within 10 days or 
any grade 4 or greater autoimmune toxicity.


08-C-0121
27
x
Development of EBV lymphoma or polyclonal lymphoproliferative disease 
(PLPD) in an EBV negative subject on this or any other SB adoptive cell therapy 
study.  Accrual of EBV negative subjects will be halted only.
3.2.
Drug Administration:
(see table 3.2)
3.2.1.
Preparative Regimen with Cyclophosphamide and Fludarabine (all cohorts)
(Times are offered as examples and may be changed as long as a similar time 
relationship between administration of the drugs is maintained. Study medication 
start times for drugs given once daily should be given within 2 hours of the scheduled 
time. All other medications should be given +/- one hour of the scheduled time; the 
length of administration is all +/- 15 minutes. Administration of diuretics, electrolyte 
replacement, and hydration and monitoring of electrolytes should all be performed as 
clinically indicated – the doses and times noted below are offered only as examples.)
Day -7 and –6 1 am 
Hydrate: Begin hydration with 0.9% Sodium Chloride Injection containing 10 
meq/L of potassium chloride at 2.6 ml/kg/hr (starting 11 hours pre-
cyclophosphamide and continue hydration until 24 hours after last 
cyclophosphamide infusion). At any time during the preparative regimen, if
urine output <1.5 ml/kg/hr or if body weight >2 kg over pre-
cyclophosphamide value, furosemide 10-20 mg IV maybe administered. 
Serum potassium should be monitored and treated as indicated following 
administration of furosemide.
11 am 
Ondansetron (0.15 mg/kg/dose [rounded to the nearest even mg dose between 
8 mg and 16 mg based on patient weight] IV every 8 hours X 3 days) will be 
given for nausea. 
12 pm (NOON)
Cyclophosphamide 60 mg/kg/day X 2 days IV in 250 ml D5W with mesna 15 
mg/kg/day X 2 days over 1 hr. If patient is obese (BMI > 35) drug dosage will 
be calculated using practical weight as described in Table 10.
1 pm 
Begin mesna infusion at 3 mg/kg/hour intravenously diluted in a suitable 
diluent (see pharmaceutical section) over 23 hours after each 
cyclophosphamide dose. If patient is obese (BMI > 35) drug dosage will be 
calculated using practical weight as described in Table 10.
Day -5 to Day -1:
Fludarabine 25 mg/m2/day IVPB daily over 30 minutes for 5 days. If patient 
is obese (BMI > 35) drug dosage will be calculated using practical weight as 
described in Table 10.
3.2.2.
Cell Infusion and Aldesleukin Administration (cohorts 1 and 2)


08-C-0121
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Cells are delivered to the patient care unit by a staff member from the Tumor 
Immunology Cell Processing Laboratory.  Prior to infusion, the cell product identity 
label is double-checked by two authorized staff (MD or RN), and identification of the 
product and documentation of administration are entered in the patient’s chart, as is 
done for blood banking protocols.  The cells are to be infused intravenously over 20-
30 minutes via non-filtered tubing, gently agitating the bag during infusion to prevent 
cell clumping.
Day 0 (one to four days after the last dose of fludarabine):
x
Cells will be infused intravenously (i.v.) on the Patient Care Unit over 20 to 30 
minutes (between one and four days after the last dose of fludarabine). Note: for 
patients with miliary lung disease up to 5 x 1010 cells will be administered, split
into two equal doses.  The first dose will be administered on day 0, and if there 
are no infusion related toxicities, the second dose will be given on day +1.
Aldesleukin will start after the 2nd dose of cells.
x
Aldesleukin (based on total body weight) 720,000 IU/kg IV over 15 minute 
approximately every eight hours beginning within 24 hours of cell infusion and 
continuing for up to 5 days (maximum of 15 doses).
Day 1-4 (Day 0 is the day of cell infusion): 
x
Beginning on day 1 or 2, filgrastim will be administered subcutaneously at a dose 
of 5 mcg/kg/day (not to exceed 300 mcg/day).  Filgrastim administration will 
continue daily until neutrophil count > 1.0 x109/L X 3 days or > 5.0 x109/L.
x
Aldesleukin (based on total body weight) 720,000 IU/kg IV over 15 minute 
approximately every eight hours for up to 5 days
3.2.3.
ALVAC Vaccine, Cell Infusion and Aldesleukin Administration (cohorts 3 and 
4).
Cells are delivered to the patient care unit by a staff member from the Tumor 
Immunology Cell Processing Laboratory.  Prior to infusion, the cell product identity 
label is double-checked by two authorized staff (MD or RN), an identification of the 
product and documentation of administration are entered in the patient’s chart, as is 
done for blood banking protocols.  The cells are to be infused intravenously over 20-
30 minutes via non-filtered tubing, gently agitating the bag during infusion to prevent 
cell clumping.
Day 0 (one to four days after the last dose of fludarabine):
x
ALVAC ESO -1 Vaccine:  Approximately two hours prior to cell infusion, 
patients will receive 0.5 mL containing a target dose of 107 CCID50 (with a range 
of approximately 106.4 to 107.9 / mL) of the ESO-1 ALVAC virus S.C. in each 
extremity (total of 4 x 107 CCID50/2 mL. If a patient has a compromised extremity 
due to lymphedema, prior radiation or other causes, vaccination may be given on 
the abdominal wall or chest wall proximal to the extremity.
x
Cells will be infused intravenously (i.v.) on the Patient Care Unit over 20 to 30 
minutes (between one and four days after the last dose of fludarabine). Note: for 
patients with miliary lung disease up to 5 x 1010 cells will be administered, split


08-C-0121
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into two equal doses.  The first dose will be administered on day 0, and if there 
are no infusion related toxicities, the second dose will be given on day +1.  
Aldesleukin will start after the 2nd dose of cells.
x
Aldesleukin (based on total body weight) 720,000 IU/kg IV over 15 minute 
approximately every eight hours beginning within 24 hours of cell infusion and 
continuing for up to 5 days (maximum of 15 doses). 
Day 1-4 (Day 0 is the day of cell infusion): 
x
Beginning on day 1 or 2, filgrastim will be administered subcutaneously at a dose 
of 5 mcg/kg/day (not to exceed 300 mcg/day).  Filgrastim administration will 
continue daily until neutrophil count > 1.0 x109/L X 3 days or > 5.0 x109/L.
x
Aldesleukin (based on total body weight) 720,000 IU/kg IV over 15 minute 
approximately every eight hours for up to 5 days
Day 14 (+/- 2 days):
x
ALVAC ESO-1 Vaccine (see day 0 above)
Table 3.2
Day
-7
-6
-5
-4
-3
-2
-1
01
1
2
3
4
Therapy
Cyclophosphamide (60 mg/kg)
X
X
Fludarabine (25 mg/m2)
X
X
X
X
X
Anti-ESO TCR PBL
X1
ALVAC ESO-1 vaccine (cohort 3 
and 4 only)
X7
Aldesleukin
X2
X
X
X
X
Filgrastim3 (5 mcg/kg/day)
X
X
X
X
TMP/SMX4
160mg/800mg (example)
X
X
X
X
X
X
X
X
X
X
X
X
Fluconazole5 (400 mg po)
X
X
X
X
X
Valacyclovir po or Acyclovir IV6
X
X
X
X
X
1One to four days after the last dose of fludarabine
2Initiate within 24 hours after cell infusion
3Continue until neutrophils count > 1X109/L for 3 consecutive days or > 5x109/L.
4The TMP/SMX schedule should be adjusted to QD three times per week (Monday, Wednesday, Friday) and 
continue for at least six months and until CD4 > 200 X 2
5Continue until ANC > 1000/mm3
6In patients positive for HSV continue until CD4 >200 X 2
7Administered to patients treated in cohorts 3 and 4 only.  Vaccine is also administered on day 14 (not shown)
3.2.4.
Infection Prophylaxis
Note:  Other anti-infective agents may be substituted at the discretion of the treating 
investigator.
3.2.4.1 Pneumocystis Jirovecii Pneumonia
All patients will receive the fixed combination of trimethoprim and sulfamethoxazole 
[SMX] as double strength (DS) tab (DS tabs = TMP 160 mg/tab, and SMX 800 mg/tab) 


08-C-0121
30
P.O. daily three times a week on non-consecutive days, beginning between days -5 and -
8.
Pentamidine will be substituted for TMP/SMX-DS in patients with sulfa 
allergies. It will be administered aerosolized at 300 mg per nebulizer within one week 
of chemotherapy start date and monthly thereafter.
3.2.4.2 Herpes Virus Prophylaxis
Patients with positive HSV serology will be given valacyclovir orally at a 
dose of 500 mg daily the day after chemotherapy ends, or acyclovir, 250 mg/m2 IV q 
12 hrs if the patient is not able to take medication by mouth.  Reversible renal
insufficiency has been reported with IV but not oral acyclovir.  Neurologic toxicity 
including delirium, tremors, coma, acute psychiatric disturbances, and abnormal 
EEGs have been reported with higher doses of acyclovir.  Should this occur, a dosage 
adjustment will be made or the drug will be discontinued.  Acyclovir will not be used 
concomitantly with other nucleoside analogs which interfere with DNA synthesis, 
e.g. ganciclovir.  In renal disease, the dose is adjusted as per product labeling.
Prophylaxis for Pneumocystis and Herpes will continue for 6 months post 
chemotherapy.  If the CD4 count is less than 200 at 6 months post chemotherapy, 
prophylaxis will continue until the CD4 count is greater than 200 for 2 consecutive 
measures. 
3.2.4.3 Fungal Prophylaxis 
Patients will start Fluconazole 400 mg p.o. the day after chemotherapy concludes 
and continue until the absolute neutrophil count is greater than 1000/mm3.  The drug 
may be given IV at a dose of 400 mg in 0.9% sodium chloride USP daily in patients 
unable to take it orally.
3.2.4.4 Empiric Antibiotics 
Patients will start on broad-spectrum antibiotics, either a 3rd or 4th generation 
cephalosporin or a quinolone for fever of 38.3°C once or two temperatures of 38.0°C
or above at least one hour apart, AND an ANC <500/mm3.  Aminoglycosides should 
be avoided unless clear evidence of sepsis. Infectious disease consultation will be 
obtained for all patients with unexplained fever or any infectious complications.
3.2.5.
Blood Product Support 
Using daily CBC’s as a guide, the patient will receive platelets and packed red blood 
cells (PRBC’s) as needed.  Attempts will be made to keep Hb >8.0 gm/dl, and plts 
>20,000/mm3.  All blood products with the exception of the stem cell product will be 
irradiated.  Leukocyte filters will be utilized for all blood and platelet transfusions to 
decrease sensitization to transfused WBC’s and decrease the risk of CMV infection.
3.2.6.
Interleukin-2 (aldesleukin): Intravenous Administration
Aldesleukin (NSC #373364) (based on total body weight) will be 
administered at a dose of 720,000 IU/kg as an intravenous bolus over a 15 minute 
period approximately every eight hours (+/- 1hr) beginning within 24 hours of cell 
infusion and continuing for up to 5 days (maximum 15 doses). Doses may be skipped 
depending on patient tolerance.  Doses will be skipped if patients reach Grade 3 or 4


08-C-0121
31
toxicity due to aldesleukin except for the reversible Grade 3 toxicities common to 
aldesleukin such as diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, 
mucositis, dysphagia, or constitutional symptoms and laboratory changes as detailed 
in Appendix1.  Toxicities will be managed as outlined in Appendix 2.  If these 
toxicities can be easily reversed within 24 hours by supportive measures then 
additional doses may be given.  If greater than 2 doses of aldesleukin are skipped, 
aldesleukin administration will be stopped. In addition, dosing may be held or 
stopped at the discretion of the treating investigator (Appendix 3 lists the toxicities 
seen in patients treated with aldesleukin at the NIH Clinical Center). Because 
confusion is a possible side effect of aldesleukin administration, a Durable Power of 
Attorney will be signed by the patient to identify a surrogate to make decisions if a 
patient becomes unable to make decisions.
3.3.
On-Study Evaluation
3.3.1.
Prior to starting the preparative regimen
x
Apheresis as indicated
x
Within 14 days prior to starting the preparative regimen, patients will have a 
complete blood count, serum chemistries performed including electrolytes, 
BUN, creatinine, and liver function tests.  If any results are beyond the criteria 
established for eligibility, the patient will not proceed until the abnormalities 
can be resolved. 
3.3.2.
During the preparative regimen: DAILY
x
Complete Blood Count
x
Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium 
total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, 
ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, 
Total CK, Uric Acid)
x
Urinalysis
3.3.3.
Prior to cell infusion:
x
Blood samples for analysis for detection of RCR by PCR
3.3.4.
After Cell Infusion:
x
Vital signs will be monitored hourly (+/- 15 minutes) for four hours and then 
routinely (every 4-6 hours) unless otherwise clinically indicated.
x
CMV PCR assay will be assessed if clinically indicated (e.g. unexplained 
fevers, pulmonary changes). Every 1-2 days (until values return to normal 
limits/baseline)
o
A review of systems and physical exam as clinically indicated.
o
CBC
o
Chem 20 (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, 
Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline 
Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, 
Total Protein, Total CK, Uric Acid )
o
Other tests will be performed as clinically indicated.
3.3.5.
Additional research evaluations:


08-C-0121
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3.3.5.1 Post cell infusion evaluations:
x
The amount of blood that may be drawn from adult patients for research purposes 
shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any eight 
week period.
x
Once total lymphocyte count is greater than 200/mm3, the following samples will 
be drawn and sent to the TIL lab on Monday, Wednesday and Friday X 5, then 
weekly (while the patient is hospitalized):
x
5 CPT tubes (8 ml each)
x
1 SST tube (8 ml)
x
1 SST tube (4 ml) daily starting on the day of the chemotherapy
x
At other time points, patient peripheral blood lymphocytes (PBL) will be obtained 
from whole blood by purification using centrifugation on a Ficoll cushion. 
Aliquots of these PBMC will be 1) cryopreserved for immunological monitoring 
of cell function and 2) subjected to DNA and RNA extraction for PCR analysis of 
TCR and vector copy number estimation.
3.3.5.2 Biopsies
x
Biopsies of tumor tissue or lymph nodes may be performed but are not required 
during the course of therapy.  These biopsies will only be performed if minimal 
morbidity is expected based on the procedure performed and the granulocyte and 
platelet count.  Biopsy tissue will be processed in the Surgery Branch Cell 
Production Facility in the presence of a Pathology Laboratory pathologist and part 
of all biopsy tissue will go to the Laboratory of Pathology. Studies will be 
performed to evaluate the antigen expression by the tumor and to evaluate the 
reactivity of lymphocytes grown from these biopsies. In addition the presence of 
transduced cells will be quantitated using RT-PCR for vector sequences.
3.3.5.3 Immunological Testing:
x
Apheresis may be performed, prior to and 4-6 weeks after the treatment.  At other 
time points, patient peripheral blood lymphocytes (PBL) will be obtained from 
whole blood by purification using centrifugation on a Ficoll cushion. Aliquots of 
these PBMC will be:
1.
Cryopreserved for immunological monitoring of cell function,
2.
Subjected to DNA and RNA extraction for PCR analysis of TCR and 
vector copy number estimation
x
Lymphocytes will be tested directly and following in vitro culture. Direct 
immunological monitoring will consist of quantifying T cells reactive with ESO
by FACS analysis using tetramer staining.  Ex vivo immunological assays will 
consist of cytokine release by bulk PBL (+/- peptide stimulation) and by other 
experimental studies such as cytolysis if sufficient cells are available. If cell 
numbers are limiting, preference will be given to the direct analysis of 
immunological activity.  Immunological assays will be standardized by the 
inclusion of 1) pre-infusion PBMC and 2) an aliquot of the engineered PBL 
cryopreserved at the time of infusion. In general, differences of 2 to 3 fold in 
these assays are indicative of true biologic differences.  Foxp3 levels will be 
analyzed by semiquantitative RT-PCR to evaluate for mRNA on PBL samples 
obtained prior to cell infusion and at the follow up time point.


08-C-0121
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3.3.5.4 Monitoring Gene Therapy Trials:  Persistence and RCR:
x
Engineered cell survival.  TCR and vector presence will be quantitated in PBMC 
samples using established PCR techniques.   Immunological monitoring using 
both tetramer analysis and staining for the TCR will be used to augment PCR-
based analysis.  This will provide data to estimate the in vivo survival of 
lymphocytes derived from the infused cells. In addition, measurement of CD4 and 
CD8 T-cells will be conducted and studies of these T-cell subsets in the 
circulation will be determined by using specific PCR assays capable of detecting 
the unique DNA sequence for each retroviral vector engineered T-cell.
x
Patients blood samples will be obtained and undergo analysis for detection of 
RCR by PCR prior to cell infusion and RCR PCR will be performed at 3 and 6 
months, and at one year post cell administration.  Blood samples will be archived 
annually thereafter if all previous testing has been negative with a brief clinical 
history. If a patient dies or develops neoplasms during this trial, efforts will be 
made to assay a biopsy sample for RCR. If any post-treatment samples are 
positive, further analysis of the RCR and more extensive patient follow-up will be 
undertaken, in consultation with the FDA. RCR PCR assays detect the GaLV 
envelop gene and are performed under contract by the Indiana University Vector 
Production Facility. The results of these tests are maintained by the contractor 
performing the RCR tests and by the Surgery Branch research team.
x
Due to nature of these studies, it is possible that expansion of specific T-cell 
clones will be observed as tumor reactive T-cell proliferate in response to tumor 
antigens.  Therefore, care will be taken to track T-cell persistence both 
immunologically and molecularly.  Blood samples (5-10 mL) for persistence of 
TCR transduced cells will be obtained 1 month after cell infusion, then at 3, 6, 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.  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, so that treatment, if available, may be initiated early.
x
Blood and tissue specimens collected in the course of this research project may be 
banked and used in the future to investigate new scientific questions related to this 
study. However, this research may only be done if the risks of the new questions 


08-C-0121
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were covered in the consent document. If new risks are associated with the 
research (e.g., analysis of germ line genetic mutations) the principal investigator 
must amend the protocol and obtain informed consent from all research subjects.  
Any new use of samples will require prospective IRB review and approval.
3.4.
Re-Treatment
Patients will be evaluated 4 to 6 weeks after the initial treatment regimen 
(defined as the end of the last aldesleukin dose). If patients have a partial response to 
treatment and then stabilizes and have evaluable disease, or stable disease that 
subsequently progresses, they may be re-treated when progression by RECIST 
criteria is documented after evaluation by principal investigator with the same 
schedule that they had been given safely. Retreatment will consist of the non-
myeloablative chemotherapy regimen, ALVAC ESO-1 vaccine (if available for 
cohorts 3 and 4), cell infusion, and aldesleukin (See table 3.4 below). Patients who 
develop grade 3 toxicity due to cell infusion which is reversible within 24 hours with 
supportive measures may be retreated, but patients who develop grade 4 toxicity due 
to cell infusion will not be retreated. Patients must continue to meet the original 
eligibility criteria to be considered for retreatment. Toxicity related to 
cyclophosphamide or fludarabine should be stable and resolved to less than grade 1 
prior to retreatment.  Re-treatment benefits and risks will be carefully explained to the 
patient. Patients must be reconsented prior to retreatment. Retreatment would not 
begin prior to 6 to 8 weeks after the last aldesleukin dose for each of the cohorts.  A 
maximum of 2 retreatment courses may occur.
Table 3.4:  Retreatment with anti-ES0-1 PBL
Day
-7
-6
-5
-4
-3
-2
-1
01
1
2
3
4
Therapy
Cyclophosphamide (60 mg/kg)
X
X
Fludarabine (25 mg/m2)
X
X
X
X
X
anti-ESO TCR PBL
X1
ALVAC ESO-1 vaccine (cohort 3 
and 4 only, if available)
X7
Aldesleukin
X2
X
X
X
X
Filgrastim3 (5 mcg/kg/day)
X
X
X
X
TMP/SMX4
160mg/800mg (example)
X
X
X
X
X
X
X
X
X
X
X
X
Fluconazole5 (400 mg po)
X
X
X
X
X
Valacyclovir po or Acyclovir IV6
X
X
X
X
X
1One to four days after the last dose of fludarabine
2Initiate within 24 hours after cell infusion
3Continue until neutrophils count > 1X109/L for 3 consecutive days or > 5x109/L.
4The TMP/SMX schedule should be adjusted to QD three times per week (Monday, Wednesday, Friday) and 
continue for at least six months and until CD4 > 200 X 2
5Continue until ANC > 1000/mm3
6In patients positive for HSV continue until CD4 > 200 X 2
7Administered to patients treated in cohorts 3 and 4 only, if available.  Vaccine is also administered on day 14 (not 
shown)


08-C-0121
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3.5.
Post Study Evaluation (Follow-up):
3.5.1
All patients return to the NIH Clinical Center for evaluation 6 weeks (+/- 2 weeks) 
following the administration of the cell product. 
3.5.2
Patients who experience stable disease, a partial response, or a complete response or 
have unresolved toxicities will be evaluated as noted below:
x
Week 12 (+/- 2 weeks)
x
Every 3 months (+/- 1 month) x 3
x
Every 6 months (+/- 1 month) x 2
x
As per PI discretion for subsequent years
Note:  Patients may be seen more frequently as clinically indicated. 
3.5.3
At each scheduled evaluation, patients will undergo:
x
Physical examination 
x
Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), 
Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium 
total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, 
Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric Acid)
x
Complete blood count
x
Thyroid panel as clinically indicated.
x
Toxicity assessment, including a review of systems.
x
CT of the chest, abdomen and pelvis. This end of course evaluation will be used 
to determine tumor response. If clinically indicated, other scans or x-rays may be 
performed, e.g. brain MRI, bone scan.
x
Visual symptoms will be evaluated and if changes have occurred from baseline, 
i.e. changes in visual acuity, an ophthalmologic consult will be performed.
x
A 5 liter apheresis may be performed.  If the patient is unable to undergo pheresis, 
approximately 96 ml of blood may be obtained.  Subsequently, approximately 60
ml of blood will be obtained at follow up visits (approximately monthly) for at 
least 3 months. Peripheral blood mononuclear cells will be cryopreserved so that 
immunologic testing may be performed. 
x
.
3.5.4
Detection  of RCR and persistence of TCR gene transduced cells: (see section 3.3.7.4)
3.5.5
Long-term follow up of patients receiving gene transfer:  Physical examinations will be 
performed and documented annually for 5 years following cell infusion to evaluate 
long-term safety.  After 5 years, health status data will be obtained from surviving 
patients via telephone contact or mailed questionnaires.  The long term follow up period 
for retroviral vectors is 15 years.
3.5.6
Patients who are unable or unwilling to return for follow up evaluations will be 
followed via phone or e-mail contact.  Patients may be asked to send laboratory , 
imaging, and physician exam reports performed by their treating physician. 
3.6.
Off Treatment Criteria:
Patients will be taken off treatment (and followed until progression of disease) for the 
following
x
Grade 3 autoimmunity that involves vital organs (heart, kidneys, brain, eye, 
liver, colon, adrenal gland, lungs).


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x
Grade 3 or greater toxicity attributable to the ALVAC ESO-1 vaccine, except 
for known toxicities such as local injection site reactions, skin rash, pruritis, 
transient fatigue, transient fever, transient laboratory changes or local 
adenopathy.  
x
If a patient experiences a grade 3 or 4 toxicity due to cell infusion (reaction to 
cellular product or infusion reaction) the patient will receive no further cells 
but may continue to receive aldesleukin if the reaction resolves to grade 2 or 
less within 24 hours.
x
Completion of treatment period.
3.7.
Off Study Criteria:
Patients will be taken off study for the following:
x
The patient voluntarily withdraws
x
There is significant patient noncompliance
x
Radiographic or clinical disease progression, unless eligible for second 
treatment
x
General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment on this study in the judgment of the 
investigator.
x
Death
Note:  patients who are taken off study for progressive disease or study closure maybe 
followed on Protocol 09-C-0161 Follow up Protocol for subjects Previously Enrolled in 
Surgery Branch Studies.
Note:  Patients must be followed until all adverse events have resolved to grade 2 or less with 
the exception of lymphopenia and alopecia.  If an adverse event is not expected to resolve to 
grade 2 or less this will be noted in the patient medical record and the patient may be taken 
off study. 
4.0 Supportive Care
Concomitant medications to control side effects of therapy will be given.  Meperidine 
(25-50 mg) will be given intravenously if severe chilling develops.  Other supportive therapy 
will be given as required and may include acetaminophen (650 mg q4h), indomethacin (50-
75 mg g6h) and ranitidine (150 mg g12h). If patients require steroid therapy they will be 
taken off treatment. Patients who require transfusions will receive irradiated blood products.  
Ondansetron 0.15 mg/kg/dose IV every 8 hours will be administered for nausea and 
vomiting.  Additional antiemetics will be administered as needed for nausea and vomiting 
uncontrolled by ondansetron.  Antibiotic coverage for central venous catheters may be 
provided at the discretion of the investigator.
5.0 Data Collection and Evaluation
5.1.
Data Collection
The investigators will be responsible for the collection, maintenance, quality control 
of the study data. Clinical data will be entered in to the NCI CCR C3D database. 
5.1.1.
Routine Adverse Event Reporting


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Following registration through 30 days after the last treatment, adverse events will be 
recorded in the source documents, reviewed by the designated data manager or 
research nurse or principle investigator and captured in C3D. Events occurring during 
this period must be followed until resolution to grade 2 or baseline. Events that occur 
after this period will only be captured in C3D if they are related to investigational 
agent.
5.1.2.
Exclusions to Routine Adverse Event Reporting
Patients will be receiving multiple agents, which include commercially available 
agents (fludarabine, cyclophosphamide and supportive medications) in combination 
with the investigational agents. Therefore, Grade 2 adverse events ‘unrelated’ or 
‘unlikely related’ to the investigational agent, and ‘possibly’, ‘probably’ or 
‘definitely’ related to the commercially available agents as specified in the package 
inserts do not require reporting/recording. In addition, all grade 1 events and all 
expected grade 2 events not related to the cell product will not be reported/recorded.
5.1.3.
Reporting of Laboratory Events
Laboratory results will be uploaded in C3D however only those grade 3 and 4 events 
that support the diagnosis of a reportable adverse event or that reflect major organ 
function will be considered adverse events. For example grade 3 and 4, creatinine, 
liver function tests, hemoglobin, ANC, ALC, platelets, and lipase and amylase as 
indicated will be captured as adverse events; electrolytes, BUN, albumin, total 
protein, uric acid etc, and the remainder of the CBC differential will not be captured 
as adverse events. For hematological toxicities, the event will not be considered 
resolved until it reaches grade 2 without the support of transfusions or growth factors.
5.1.4.
Reporting of non-Laboratory Events
For reportable expected events: the adverse event start date will be the date the event 
reaches a grade 3; the event will be considered resolved once it reaches grade 2.  The 
highest grade the event reaches in that period will be considered the grade of the 
event.  For unexpected adverse events, the adverse event start date will be the date the 
event reaches grade 2; the event will be considered resolved once it returns to grade 1 
or baseline.  The highest grade the event reaches in that period will be considered the 
grade of the event. 
5.2.
Response Criteria
5.2.1
Evaluation of target lesions1
x
Complete Response (CR):  Disappearance of all target lesions
x
Partial Response (PR):  At least a 30% decrease in the sum of the longest 
diameter (LD) of target lesions taking as reference the baseline sum LD.
x
Progression (PD):  At least a 20% increase in the sum of LD of target lesions 
taking as reference the smallest sum LD recorded since the treatment started or 
the appearance of one or more new lesions.
x
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD taking as references the smallest sum LD.
5.2.2
Evaluation of non-target lesions2
x
Complete Response (CR): Disappearance of all non-target lesions and 
normalization of tumor marker level.


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x
Non-Complete Response:  Persistence of one or more non-target lesions
x
Progression (PD):  Appearance of one or more new lesions.  Unequivocal 
progression of existing non-target lesions
-----------------------------------------------------------------------------------
1
All measurable lesions up to a maximum of 10 lesions representative of all involved organs should be 
identified as target lesions and recorded and measured at baseline.  Target lesions should be selected on 
the basis of their size (lesions with the longest diameter) and their suitability for accurate repetitive 
measurements (either by imaging techniques or clinically).  A sum of the longest diameter (LD) for all 
target lesions will be calculated and reported as the baseline sum LD.  The baseline sum LD will be used 
as reference to further characterize the objective tumor response of the measurable dimension of the 
disease.
2
All other lesions (or sites of disease) should be identified as non-target lesions and should also be 
recorded at baseline.  Measurements are not required, and these lesions should be followed as “present” 
or “absent.”
5.2.3
Evaluation of best overall response
The best overall response is the best response recorded from the start of the 
treatment until disease progression/recurrence (taking as reference for 
progressive disease the smallest measurements recorded since the treatment 
started).  The patient's best response assignment will depend on the 
achievement of both measurement and confirmation criteria.
Target Lesions
Non-Target Lesions
New Lesions
Overall Response
CR
CR
No
CR
CR
Non-CR/Non-PD
No
PR
PR
Non-PD
No
PR
SD
Non-PD
No
SD
PD
Any
Yes or No
PD
Any
PD
Yes or No
PD
Any
Any
Yes
PD
5.2.4
Confirmatory Measurement/Duration of Response
Confirmation
To be assigned a status of PR or CR, changes in tumor measurements must be 
confirmed by repeat studies that should be performed at least 4 weeks after the 
criteria for response are first met.  In the case of SD, follow-up measurements must 
have met the SD criteria at least once after study entry at a minimum interval of  6-8
weeks.
Duration of Overall Response
The duration of overall response is measured from the time measurement 
criteria are met for CR/PR (whichever is first recorded) until the first date that 
recurrent or progressive disease is objectively documented (taking as reference for 
progressive disease the smallest measurements recorded since the treatment started).
The duration of overall complete response is measured from the time 
measurement criteria are first met for CR until the first date that recurrent disease is 
objectively documented.
Duration of Stable Disease


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Stable disease is measured from the start of the treatment until the criteria for 
progression are met, taking as reference the smallest measurements recorded since the 
treatment started. 
5.3.
Toxicity Criteria
This study will utilize the CTC version 3.0 for toxicity and adverse event 
reporting.  A copy of the CTC v3.0 can be downloaded from the CTEP home page 
(http://ctep.cancer.gov).
Over 100 patients have been treated in the Surgery Branch, NCI with tumor 
infiltrating lymphocytes.  Early toxicities related specifically to the infusion of the 
cells (those which are seen immediately following cell infusion and prior to 
aldesleukin administration) are generally mild and include fevers, chills, headache, 
and malaise.  Toxicities which occur following administration of aldesleukin but are 
thought to be related to the cells include immune mediated events such as vitiligo, 
transient uveitis, hearing loss and vestibular dysfunction.  The use of the non-
myeloablative regimen prior to cell administration increases the toxicity of this
treatment as profound myelosuppression occurs in all patients.  In 93 patients treated 
with TIL using the non-myeloablative chemotherapy regimen with or without total 
body irradiation, there was one treatment related death (NMA + 200 cGY TBI) due to 
an unexpected but preexisting diverticular abscess.  
The standard approach to the administration of high-dose aldesleukin in all 
studies is to continue dosing until grade 3 or 4 events occur. The most commonly 
seen grade 4 events are pulmonary, and renal impairment, and mental status changes.  
These toxicities may sometimes require intubation for protection of the patient’s 
airway.  It is important to note that although these patients require significant 
supportive measures during this period, all toxicities are reversible and the 
overwhelming majority of patients have suffered no long term sequelae following this 
treatment regimen.  However, fatal complications are possible and it is therefore only 
appropriate to carry out this experimental treatment in the context of life threatening 
metastatic cancer.
To ensure safety using this treatment, the NCI SB will review safety data on 
all protocols semi-annually at the time of continuing review.  Data will be presented 
for both the recent 6 month period and for the entire length of time the protocol has 
been open.  The toxicity data for review will include all toxicities captured on the 
protocol and will be presented in individual tables as follows: 
x
all toxicities attributed to the cells, 
x
all incidences of intubation including the duration of and reason for intubation,
x
all grade 2 unexpected adverse events, and all grade 3 or greater events 
regardless of attribution.   
Toxicities seen on protocols using this non-myeloablative regimen and 
aldesleukin, that occur during the follow up period are rare but have included EBV 
lymphoma following prolonged lymphopenia, herpes zoster infection, and sensory 
neuropathy likely related to fludarabine.  


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The major discomforts of the research are those of nausea, mucositis, 
anorexia, diarrhea, fever and malaise.  Side effects of common drugs used in this 
nonmyeloablative regimen include:
Cyclophosphamide: Marrow suppression, nausea, mucositis, rash, 
hemorrhagic cystitis, myocardial damage, alopecia, infertility, nausea and vomiting, 
SIADH.  
Fludarabine: Myelosuppression, fever and chills, nausea and vomiting, 
malaise, fatigue, anorexia, weakness, neurologic toxicity including sensory 
neuropathies and blindness, and interstitial pneumonitis.  Serious opportunistic 
infections have occurred in CLL patients treated with fludarabine.
Antimicrobials in general: Allergic reactions, renal impairment, nausea, 
vomiting, hepatic damage, marrow suppression. 
A variety of side effects have been associated with high-dose aldesleukin 
administration in our experience and a listing of these side effects in 652 patients who 
received 1,039 treatment courses are listed in Appendix 1 and Appendix 2.
5.4.
Statistical Section 
For cohorts 1 and 2, the primary objective of this trial is to determine whether
the combination of high dose aldesleukin, lymphocyte-depleting chemotherapy, and 
an infusion of anti-ESO TCR-gene engineered lymphocytes is able to be associated 
with a modest fraction of patients that can experience a clinical response (PR +CR) to 
therapy.  For cohorts 3 and 4, the primary objective is to determine whether the 
combination of high dose aldesleukin, lymphocyte-depleting chemotherapy, and an 
infusion of anti-ESO TCR-gene engineered lymphocytes and ALVAC ESO-1 vaccine 
is able to be associated with a modest fraction of patients that can experience a 
clinical response (PR +CR) to therapy. A secondary objective is to have sufficient 
patients in order to do exploratory evaluations of survival of cells.   
Patients who express ESO will be enrolled into individual strata depending on 
their specific histology.  This stratification is being used to separate patients who have 
been shown historically to be IL-2 sensitive (melanoma and renal cell cancer) from 
those who have not.  In each case, the ESO expression is expected to be so dominant 
in regulating potential for response that differences in clinical response according to 
histology will not be expected but will be evaluated in an exploratory manner to help 
assess this, in a pilot sense.
As of March 2011, the ALVAC ESO-1 is not currently available, therefore 
with approval of amendment G cohorts 1 and 2 will be reopened.  We plan to leave 
cohorts 3 and 4 open as we would like to accrue to these arms if the ALVAC ESO-1
vaccine becomes available in the future.  To date, we have seen remarkable clinical 
responses in patients treated on this study.  In cohort 1, we have treated 13 patients 
with melanoma, and 5 of 11 patients with high expression of ESO-1 had clinical 
response including 3 partial responses (PRs) of 3, 8 and 14+ months, and 2 complete 
responses (CRs) of 24, and 25 + months.  In cohort 2, we have treated 7 patients and 
6 of these patients were heavily pretreated synovial cell sarcoma.  Four patients with 
synovial cell sarcoma experienced PRs lasting 10, 14, 4 and 8 months.  Therefore, the 
response rate in patients receiving non-myeloablative chemotherapy, anti-ESO-1
TCR PBL and high dose IL-2 but no vaccine is 45%.  In cohort 3 (with vaccine), we 


08-C-0121
41
have treated 6 patients with melanoma, and of the three evaluable patients, 1 patient 
has an ongoing PR of 6+ months.  In cohort 4 (with vaccine), 5 patients have been 
treated, and out of the 4 evaluable patients, 2 patients with synovial cell sarcoma have 
ongoing PRs of 7+ months.   The preliminary data from cohorts 3 and 4 indicate that 
clinical response rates observed with vaccine in cohorts 3 and 4 are similar to those 
observed without vaccine in cohort 1 and 2.   In addition, the adverse events observed 
in each arm are comparable with most toxicities attributed to the non-myeloablative 
chemotherapy regimen and high dose IL-2..
For each of the 2 strata, the study will be conducted using a phase II optimal 
design (Simon R, Controlled Clinical Trials 10:1-10, 1989). For both strata, the 
objective will be to determine if the combination of high dose aldesleukin, 
lymphocyte depleting chemotherapy, ALVAC ESO-1 vaccine and anti-ESO TCR-
gene engineered lymphocytes is able to be associated with a clinical response rate that 
can rule out 5% (p0=0.05) in favor of a modest 20% PR + CR rate (p1=0.20).  
In patients in each of the two strata (melanoma or renal carcinoma vs. other 
histologies), the following design will be used.  For each strata, with alpha=0.05 (5% 
probability of accepting a poor therapy) and beta=0.10 (10% probability of rejecting a 
good therapy,), initially 21 evaluable patients will be enrolled.  If 0 or 1 of the 21 
patients experiences a clinical response, then no further patients will be enrolled.  If 2 
or more of the first 21 evaluable patients enrolled have a clinical response, then 
accrual will continue until a total of 41 evaluable patients have been enrolled.  As it 
may take several weeks to determine if a patient has experienced a clinical response, 
a temporary pause of up to 6 months in the accrual to the trial may be necessary to 
ensure that enrollment to the second stage is warranted.  If 2 to 4 of the 41 have a 
clinical response, then this will be considered inadequate for further investigation. If 5 
or more of 41 patients have a clinical response, then this will indicate that this 
strategy provides a new approach that may be worthy of further consideration.  Under 
the null hypothesis (5% response rate), the probability of early termination is 72%.
Further, to help ensure that maldistribution of patients who are either 
particularly responsive or unresponsive in the first stage does not materially interfere 
with the intended use of the two-stage design, we will aim to enroll 4-5 patients of 
each allowed histology among the first 21 enrolled in the ‘other histology’ arm.  
Although this has its own inherent issues due to limited sample size, since we believe 
that these ‘other histologies’ will behave the same clinically, it will permit us to 
evaluate the different response rates in a limited number of subjects and determine if 
they differ markedly or not.  Since this would merely be an exploratory analysis, we 
will also look at minor response as well to help evaluate for hints of efficacy.  If the 
response rates do seem to potentially differ markedly by histology, despite our 
hypothesis that this will not happen, we may consider amending the protocol when 
appropriate to try to restrict enrollment to those histologies with stronger evidence of 
responsiveness.  For patients with breast cancer or other chemotherapy-sensitive 
tumors (i.e. sarcoma), only responses seen at day 28 and maintained at 4 months will 
be considered a positive response for accrual to the second phase of this study.
To complete cohorts 3 and 4, a total of up to 164 patients may be required (2 
with a maximum of 41 apiece).  It is not expected that more than 20 patients will be 
accrued in cohorts 1 and 2 prior to approval of amendment E, therefore, the maximum 


08-C-0121
42
accrual for this study will be 102 patients. Provided that about 4-5 patients per month 
will be able to be enrolled onto this trial, approximately 2.5 years may be needed to 
accrual the maximum number of required patients.  However, as adequate responses 
to proceed to the second stage of accrual may not occur, the trial may end up accruing 
as few as 62 patients if no arm demonstrates sufficient patients to accrue a second 
stage.
5.5.
Data and Safety Monitoring Plan 
Careful evaluation to ascertain the toxicity, immunologic effects and anti-
tumor efficacy of cell infusions will be performed. Due to the nature of these studies, 
it is possible that expansion of specific T-cell clones will be observed as tumor 
reactive T-cell proliferate in response to tumor antigens.  Therefore, care will be 
taken to track T-cell persistence both immunologically and molecularly according to 
plan specified in Section 3.3.7.
The principal investigator will review all serious adverse events and will 
monitor the data and toxicities to identify trends monthly.  The principal investigator 
will be responsible for revising the protocol as needed to maintain safety.  The NCI 
IRB will review submitted adverse events monthly to also evaluate trends and will 
require a follow up plan from the principal investigator whenever a trend is identified.  
A CCR Safety Monitoring Committee will monitor toxicity trends on this study on at 
least an annual basis and report any trends to the NCI IRB and Principal Investigator.
5.6.
Clinical Trial Monitoring Plan
A detailed description of the clinical trial monitoring plan has been included 
in the initial IND submission as required.  This trial will be monitored by personnel 
employed by Harris Technical Services on contract to the NCI, NIH. Monitors are 
qualified by training and experience to monitor the progress of clinical trials. 
Personnel monitoring this study will not be affiliated in any way with the trial 
conduct.
At least 25% of enrolled patients’ will be randomly selected and monitored at least quarterly, 
base on accrual rate. The patients selected will have 100% source document verification done. 
Additional monitoring activities will include: adherence to protocol specified study eligibility, 
treatment plans, data collection for safety and efficacy, reporting and time frames of adverse 
events to the NCI IRB and FDA, and informed consent requirements. Written reports will be 
generated in response to the monitoring activities and submitted to the Principal investigator 
and Clinical Director or Deputy Clinical Director, CCR, NCI.
5.7.
Handling of Tissue Specimens for Research Purposes
Blood and tissue collected during the course of this study will follow the Cell 
Tracking and Labeling System established by the Tumor Immunology Cell 
Processing Laboratory.  The Cell Tracking and Labeling System is designed to 
unambiguously ensure that patient/data verification is consistent.  The patients' cell 
samples (blood or tissue) are tracked by distinct identification labels that include a 
unique patient identifier and date of specimen collection.  Cryopreserved blood and 
tissue samples also bear the date the sample was frozen.  All cryopreserved samples 


08-C-0121
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are tracked for freezer location and storage criteria.  All samples are stored in 
monitored freezers/refrigerators in 3NW Surgery Branch Laboratories at specified 
temperatures with alarm systems in place.  All samples (blood or tissue) are entered 
into a central computer database with identification and storage location, and this 
database is backed up every night.
At the conclusion of this protocol, if additional studies are to be performed on 
any samples obtained during the conduct of this trial, a Request to Conduct Research 
for Stored Human Samples Specimens, or Data Collected in a Terminated NCI-IRB 
Protocol will be submitted.  Otherwise, specimens will be disposed of in accordance 
with the environmental protection laws, regulations and guidelines of the Federal 
Government and the State of Maryland.
Any loss or unintentional destruction of the samples will be reported to the 
IRB, the NCI Clinical Director, and the office of the CCR, NCI.
6.0 Human Subjects Protections
6.1.
Rationale for Patient Selection
The patients to be entered in this protocol have metastatic cancer which is 
refractory to standard therapy, and limited life expectancies.
Subjects from both genders and all racial/ethnic groups are eligible for this 
study if they meet the eligibility criteria.  To date, there is no information that 
suggests that differences in drug metabolism or disease response would be expected 
in one group compared to another.  Efforts will be made to extend accrual to a 
representative population, but in this preliminary study, a balance must be struck 
between patient safety considerations and limitations on the number of individuals 
exposed to potentially toxic and/or ineffective treatments on the one hand and the 
need to explore gender and ethnic aspects of clinical research on the other hand.  If 
differences in outcome that correlate to gender or to ethnic identity are noted, accrual 
may be expanded or a follow-up study may be written to investigate those differences 
more fully.
6.2.
Participation of Children
The use of the nonmyeloablative regimen in this protocol is a major procedure 
which entails serious discomforts and hazards for the patient, such that fatal 
complications are possible.  It is therefore only appropriate to carry out this 
experimental procedure in the context of life threatening metastatic cancer.  Since the 
efficacy of this experimental procedure is unknown, it does not seem reasonable to 
expose children to this risk without further evidence of benefit.  Should results of this 
study indicate efficacy in treating metastatic cancer, which is not responsive to other 
standard forms of therapy, future research can be conducted in the pediatric 
population to evaluate potential benefit in that patient population.  
6.3.
Evaluation of Benefits and Risks
The experimental treatment has a chance to provide clinical benefit though 
this is unknown.  The risks in this treatment are detailed in section 5.2.  The goal of 
this study is to improve upon the number of patients who may benefit from adoptive 
cell therapy by using patients’ own transduced T-cells without the need to identify 


08-C-0121
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anti-tumor T-cells uniquely from each patient as was required in several prior 
protocols.  The success of this effort cannot be predicted at this time.  Because all 
patients in this protocol have melanoma cancer and limited life expectancies the 
potential benefit is thought to outweigh the potential risks.
6.4.
Consent Document
Patients initially sign a consent when they agree to have PBMC obtained for 
study and growth on 03-C-0277, Cell Harvest and Preparation for Surgery Branch 
Adoptive Cell Therapy Protocols.  If the lymphocytes can be generated for infusion 
and the patient meets the thorough screening for eligibility, the patient, with family 
members or friends at the request of the patient, will be presented with a detailed 
description of the protocol treatment.  The specific requirements, objectives, and 
potential advantages and disadvantages will be presented.  The Informed Consent 
document is given to the patient, who is requested to review it and to ask questions 
prior to agreeing to participate in the treatment portion of this protocol.  The patient is 
reassured that participation on trial is entirely voluntary and that he/she can withdraw
or decide against treatment at any time without adverse consequences. The research 
nurse, principal investigator, associate investigator, or clinical associate is responsible 
for obtaining written consent from the patient.
7.0 Data Reporting
This study will utilize the CTCAE 3.0 for toxicity and Adverse Event reporting.  A copy of the 
CTCAE 3.0 can be downloaded from the CTEP home page (http://ctep.cancer.gov).  All 
appropriate treatment areas should have access to a copy of the CTCAE 3.0.  
This study will be monitored by the Internal Monitoring System conducted by Harris Technical 
Services Corporation, with reporting to the QA specialist of the CCR, NCI.  
7.1 Definitions
7.1.1
Adverse Event
An adverse event is defined as any reaction, side effect, or untoward event that occurs during 
the course of the clinical trial associated with the use of a drug in humans, whether or not the 
event is considered related to the treatment or clinically significant. For this study, AEs will 
include events reported by the patient, as well as clinically significant abnormal findings on 
physical examination or laboratory evaluation. A new illness, symptom, sign or clinically 
significant laboratory abnormality or worsening of a pre-existing condition or abnormality is 
considered an AE.  
All AEs, including clinically significant abnormal findings on laboratory evaluations, 
regardless of severity, will be followed until satisfactory resolution. AEs should be reported 
up to 30 days following the last dose of study drug.  AEs that are considered treatment 
related, expected, continuing, but not resolvable by 30 days after treatment completion (e.g., 
alopecia) will not be followed after the 30-day period.
7.1.2
Suspected Adverse Reaction
Suspected adverse reaction means any adverse event for which there is a reasonable 
possibility that the drug caused the adverse event. For the purposes of IND safety reporting, 


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‘reasonable possibility’ means there is evidence to suggest a causal relationship between the 
drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty 
about causality than adverse reaction, which means any adverse event caused by a drug.
7.1.3
Unexpected Adverse Reaction
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed 
in the investigator brochure or is not listed at the specificity or severity that has been 
observed; or, if an investigator brochure is not required or available, is not consistent with the 
risk information described in the general investigational plan or elsewhere in the current 
application. "Unexpected”, also refers to adverse events or suspected adverse reactions that 
are mentioned in the investigator brochure as occurring with a class of drugs or as anticipated 
from the pharmacological properties of the drug, but are not specifically mentioned as 
occurring with the particular drug under investigation.
7.1.4
Serious
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following:
x
Death,
x
A life-threatening adverse drug experience
x
Inpatient hospitalization or prolongation of existing hospitalization
x
Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions
x
A congenital anomaly/birth defect.
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition.
7.1.5
Disability
A substantial disruption of a person’s ability to conduct normal life functions.
7.1.6
Life threatening Adverse Drug Reaction
Any adverse event or suspected adverse reaction that places the patient or subject, in the view 
of the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., 
it does not include a reaction that had it occurred in a more severe form, might have caused 
death.
7.1.7
Protocol Deviation (NIH Definition)
Any change, divergence, or departure from the IRB-approved study procedures in a research 
protocol that does not have a major impact on the subjects’ rights, safety or well-being, or 
the completeness, accuracy and reliability of the data. 
7.1.8
Protocol Violation (NIH Definition)
Any change, divergence, or departure from the study procedures from the IRB-approved 
study procedures in a research protocol that does have a major impact on the subject’s rights, 
safety, or well-being and/or the completeness, accuracy or reliability of the study data.


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7.1.9
Unanticipated Problems
Any incident, experience, or outcome that:
x
Is unexpected in terms of nature, severity, or frequency in relation to 
(a) the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents, 
and
(b) the characteristics of the subject population being studied; AND
x
Is related or possibly related to participation in the research; AND
x
Places subjects or others at a greater risk of harm (including physical,      
psychological, economic, or social harm) than was previously known or recognized
7.2 NCI-IRB Reporting
7.2.1
NCI-IRB Expedited Reporting of Adverse Events, Unanticipated Problems, and 
Deaths
The Protocol PI will report to the NCI-IRB:
x
All unexpected serious adverse events that are possibly, probably, or definitely related to the 
research 
x
All deaths, except deaths due to progressive disease
x
All Protocol Violations or Deviations
x
All Unanticipated Problems
Reports must be received by the NCI-IRB within 7 working days of PI awareness via iRIS.
7.2.2
NCI-IRB Requirements for PI Reporting of Adverse Events at Continuing Review
The PI will provide a summary report of adverse events that have occurred on the protocol since the 
previous continuing review and in aggregate, and interpreted in relation to the risk:benefit of study 
participants in the narrative.
The protocol PI will report to the NCI-IRB: 
1.
All Grade 2 unexpected events that are possibly, probably or definitely related to the 
research; 
2.
All Grade 3 and 4 events that are possibly, probably or definitely related to the 
research; 
3.
All Grade 5 events regardless of attribution; 
4.
All Serious Events regardless of attribution. 
NOTE: Grade 1 events are not required to be reported. 
7.2.3
NCI-IRB Reporting of IND Safety Reports
Only IND Safety Reports that require a sponsor recommended change to the protocol or the consent 
form or in the opinion of the PI increases risks to study participants will need to be reported to the 
NCI IRB.


0 8- C- 0 1 2 1
4 7
7. 3  NI H Office of Bi otec h n ol o g y Acti vities ( O B A)/I nstit uti o n a l Bi os afet y C o m mittee (I B C) 
Re p orti n g Criteri a
7. 3. 1 
Seri o us A d verse E ve n t Re p orts t o O B A/I B C
T he Pri nci pal I n vesti gat or  will n otif y O B A/I B C vi a e mail of a n y u n e x pecte d fatal or life-t hreat e ni n g
e x perie nce ass o ciate d wit h t he use of a nti- N Y- E S O 1 T C R-tra ns d uce d P B L as s o o n as p ossi ble b ut i n 
n o e ve nt later t ha n 7 cale n dar da ys of i nitial recei pt  of t he i nf or mati o n.  Seri o us a d verse  e ve nts t hat
are u ne x pecte d a n d ass ociate d wit h t he use of t he a nti- N Y- E S O 1 T C R-tra ns d uce d P B L, b ut are n o 
fatal or life-t hreat e ni n g, m uc h be re p orte d t o NIH O B A/I B C as s o o n as p ossi ble, b ut n ot later t ha n 
1 5 cale n dar da ys after t h e s p o ns or’s i nitial recei pt of t he i nf or mati o n.  
7. 3. 2  
A n n u al Re p orts t o O B A/I B C
T he st u d y Pri nci pal I n vesti gat or  will s u b mit a brief re p ort a n n uall y of t he pr o gr ess of t he tri al wit hi n 
6 0 da ys of t he a n ni versar y dat e t hat t he I N D we nt i nt o effect u nless t he I N D s p o ns or has bee n 
a ut h orize d t o s u b mit t his re p ort, as per NI H G ui deli nes, A p pe n di x M.
7. 4 Seri o us A d verse E ve nt Re p orti n g Re q uire me nts t o t he F D A, O B A a n d I B C:
D uri n g t h e d urati o n of t his trial, t he pri nci pal i n vesti gat or will ass u me res p o nsi bilit y f or 
re p orti n g a n y seri o us a n d u ne x pecte d a d vers e e v e nts t o t he U. S. F o o d a n d Dr u g A d mi nistrati o n 
( F D A) wit h a p pr o pri ate dea dli nes defi n e d b y t he re g ulati o ns:
T he F D A, O B A a n d I B C will be i nf or me d b y p h o ne or fa x of a n y u n e x pecte d fatal or life 
t hreate ni n g s us pect e d a d verse r eacti o n as s o o n as p ossi ble b ut i n n o e ve nt, later t ha n 7 cale n dar 
da ys after t h e s p o ns or’s i nitial recei pt of t he i nf or mati o n;
T he F D A, O B A a n d I B C will be i nf or me d of a n y ot her u ne x pecte d a n d s eri o us s us pecte d 
a d verse reacti o n b y writte n n otificati o n wit hi n 1 5 cale n dar da ys after t he s p o ns or’s i nitial recei pt 
of t he i nf or mati o n.
All a d verse e ve nts, i ncl u di n g seri o us a d vers e e ve nts, will be re p orte d i n t he a n n ual re p ort 
t o t he F D A,  O B A, a n d I B C as i n dicate d i n 2 1 C F R 3 1 2. 3 3.
8. 0 P h ar m a ce utic al I nf or m ati o n
8. 1.  
Al desle u ki n (I nterle u ki n- 2, Pr ol e u ki n, Rec o m bi n a nt H u m a n I nterle u ki n 2)
H o w S u p plie d: Al desle u ki n is o btai ne d fr o m c o m mercial s o urces b y t he NI H 
Cli nical P har mac y De p art me nt.
F or m ulati o n/ Rec o nstit uti o n:  Al desl e u ki n, N S C # 3 7 3 3 6 4, is pr o vi de d as 
si n gle- us e vials c o ntai ni n g 2 2 milli o n I U (
off- w hite  l y o p hilize d ca ke pl us 5 0 m g ma n nit ol a n d 0. 1 8 m g s o di u m d o dec yl s ulfate, 
b uffer e d wit h a p pr o xi matel y 0. 1 7 m g m o n o basic a n d 0. 8 9 m g di basic s o di u m 
p h os p hate t o a p H of 7. 5 (ra n ge 7. 2 t o 7. 8).  T he vial is rec o nstit ute d wit h 1. 2 m L of 
Sterile Water f or I njecti o n, U S P, a n d t he res ulta nt c o nce ntrati o n is 1 8 milli o n I U/ ml 
or 1. 1 m g/ m L.  Dil ue nt s h o ul d be directe d a g ai nst t he si de of t he vial t o a v oi d e x cess 
f oa mi n g.  S wirl c o nt e nts ge ntl y u ntil c o m pletel y diss ol ve d.  D o n ot s ha ke.  Si nce 
vials c o ntai n n o preser vati ve, rec o nstit ute d s ol uti o n s h o ul d be use d wit h 2 4 h o urs.
St ora ge: I ntact vials are st ore d i n t he refri gerat or ( 2 – 8 ° C) pr otecte d fr o m 
li g ht.  Eac h vial b ears a n e x pirati o n date.
Dil uti o n/ Sta bilit y: Rec o nstit ute d al desle u ki n s h o ul d be f urt her dil ute d wit h 5 0 
m L of 5 % H u ma n Ser u m Al b u mi n ( H S A).  T he H S A s h o ul d be a d de d t o t he dil ue nt 


08-C-0121
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prior to the addition of aldesleukin.  Dilutions of the reconstituted solution over a 
1000-fold range (i.e., 1 mg/mL to 1 mcg/mL) are acceptable in either glass bottles or 
polyvinyl chloride bags.  Aldesleukin is chemically stable for 48 hours at refrigerated 
and room temperatures, 20 – 30°C.
Administration: The final dilution of aldesleukin will be infused over 15 
minutes.  Aldesleukin will be administered as an inpatient.  
Toxicities: Expected toxicities of aldesleukin are listed in the product label 
and in Appendix 1 and 2.  Grade 3 toxicities common to aldesleukin include diarrhea, 
nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or 
constitutional symptoms and laboratory changes as detailed in Appendix 1.
Additional grade 3 and 4 toxicities seen with aldesleukin are detailed in Appendix 2.
8.2.
Fludarabine
Description:  (Please refer to package insert for complete product Information)
Fludarabine phosphate is a synthetic purine nucleoside that differs from physiologic 
nucleosides in that the sugar moiety is arabinose instead of ribose or deoxyribose.  
Fludarabine is a purine antagonist antimetabolite. 
How Supplied: It will be purchased by the NIH Clinical Pharmacy 
Department from commercial sources. Fludarabine is supplied in a 50 mg vial as a 
fludarabine phosphate powder in the form of a white, lyophilized solid cake.  
Stability: Following reconstitution with 2 mL of sterile water for injection to 
a concentration of 25 mg/ml, the solution has a pH of 7.7.  The fludarabine powder is 
stable for at least 18 months at 2-8°C; when reconstituted, fludarabine is stable for at 
least 16 days at room temperature.  Because no preservative is present, reconstituted 
fludarabine will typically be administered within 8 hours.  Specialized references 
should be consulted for specific compatibility information.  Fludarabine is 
dephosphorylated in serum, transported intracellularly and converted to the nucleotide 
fludarabine triphosphate; this 2-fluoro-ara-ATP molecule is thought to be required for 
the drug’s cytotoxic effects.  Fludarabine inhibits DNA polymerase, ribnucleotide 
reductase, DNA primase, and may interfere with chain elongation, and RNA and 
protein synthesis.
Storage: Intact vials should be stored refrigerated (2-8°C).
Administration:  Fludarabine is administered as an IV infusion in 100 ml 0.9% 
sodium chloride, USP over 15 to 30 minutes.  The doses will be based on body 
surface area (BSA).  If patient is obese (BMI > 35) drug dosage will be calculated 
using practical weight as described in Table 10.
Toxicities: At doses of 25 mg/m2/day for 5 days, the primary side effect is 
myelosuppression; however, thrombocytopenia is responsible for most cases of 
severe and life-threatening hematologic toxicity.  Serious opportunistic infections 
have occurred in CLL patients treated with fludarabine.  Hemolytic anemia has been 
reported after one or more courses of fludarabine with or without a prior history of a 
positive Coomb’s test; fatal hemolytic anemia has been reported.  In addition, bone 
marrow fibrosis has been observed after fludarabine therapy.  Other common adverse 
effects include malaise, fever, chills, fatigue, anorexia, nausea and vomiting, and 
weakness.  Irreversible and potentially fatal central nervous system toxicity in the 
form of progressive encephalopathy, blindness, and coma is only rarely observed at 
the currently administered doses of fludarabine.  More common neurologic side 


08-C-0121
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effects at the current doses of fludarabine include weakness, pain, malaise, fatigue, 
paresthesia, visual or hearing disturbances, and sleep disorders.  Adverse respiratory 
effects of fludarabine include cough, dyspnea, allergic or idiopathic interstitial 
pneumonitis.  Tumor lysis syndrome has been rarely observed in fludarabine 
treatment of CLL.  Treatment on previous adoptive cell therapy protocols in the 
Surgery Branch have caused persistently low (below 200) CD4 counts, and one 
patient developed polyneuropathy manifested by vision blindness, and motor and 
sensory defects.
8.3.
Cyclophosphamide 
(Refer to FDA-approved package insert for complete product information)
Description: Cyclophosphamide is a nitrogen mustard-derivative alkylating 
agent.  Following conversion to active metabolites in the liver, cyclophosphamide 
functions as an alkyating agent; the drug also possesses potent immunosuppressive 
activity.  The serum half-life after IV administration ranges from 3-12 hours; the drug 
and/or its metabolites can be detected in the serum for up to 72 hours after 
administration.  
How Supplied: Cyclophosphamide will be obtained from commercially 
available sources by the Clinical Center Pharmacy Department.    
Stability: Following reconstitution as directed with sterile water for injection, 
cyclophosphamide is stable for 24 hours at room temperature or 6 days when kept at 
2-8°C.  
Administration: It will be diluted in 250 ml D5W and infused over one hour.  
The dose will be based on the patient’s body weight.  If patient is obese (BMI > 35) 
drug dosage will be calculated using practical weight as described in Table 10.
Toxicities: Hematologic toxicity occurring with cyclophosphamide usually 
includes leukopenia and thrombocytopenia.  Anorexia, nausea and vomiting, rash and 
alopecia occur, especially after high-dose cyclophosphamide; diarrhea, hemorrhagic 
colitis, infertility, and mucosal and oral ulceration have been reported.  Sterile 
hemorrhagic cystitis occurs in about 20% of patients; severity can range from 
microscopic hematuria to extensive cystitis with bladder fibrosis.  Although the 
incidence of hemorrhagic cystitis associated with cyclophosphamide appears to be 
lower than that associated with ifosfamide, mesna (sodium 2-
mercaptoethanesulfonate) has been used prophylactically as a uroprotective agent in 
patients receiving cyclophosphamide.  Prophylactic mesna is not effective in 
preventing hemorrhagic cystitis in all patients.  Patients who receive high dose 
cyclophosphamide may develop interstitial pulmonary fibrosis, which can be fatal.  
Hyperuricemia due to rapid cellular destruction may occur, particularly in patients 
with hematologic malignancy.  Hyperuricemia may be minimized by adequate 
hydration, alkalinization of the urine, and/or administration of allopurinol.  If 
allopurinol is administered, patients should be watched closely for cyclophosphamide 
toxicity (due to allopurinol induction of hepatic microsomal enzymes).  At high 
doses, cyclophosphamide can result in a syndrome of inappropriate antidiuretic 
hormone secretion; hyponatremia with progressive weight gain without edema 
occurs.  At high doses, cyclophosphamide can result in cardiotoxicity.  Deaths have 
occurred from diffuse hemorrhagic myocardial necrosis and from a syndrome of 


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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. 
8.4.
Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891) 
(Please refer to the FDA-approved package insert for complete product 
information)
Description: Mesna will be obtained commercially by the Clinical Center 
Pharmacy Department and is supplied as a 100 mg/ml solution.  
Storage:  Intact ampoules are stored at room temperature.  
Stability: Diluted solutions (1 to 20 mg/mL) are physically and chemically 
stable for at least 24 hours under refrigeration.  Mesna is chemically stable at room 
temperature for 48-72 hours in D5W, 48-72 hour in D5W/0.45% NaCl, or 24 hours in 
0.9% NaCl.
Administration: Dilute to concentrations less than or equal to 20 mg 
mesna/ml fluid in D5W or 0.9% NaCl and to be administered intravenously as a 
continuous infusion.  If patient is obese (BMI > 35) drug dosage will be calculated 
using practical weight as described in Table 10.  Toxicities include nausea, vomiting 
and diarrhea. 
8.5.
Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim, 
Neupogen)
Filgrastim will be obtained commercially by the Clinical Center Pharmacy 
Department and is supplied in 300 ug/ml and 480 ug/1.6 ml vials.  G-CSF should be 
refrigerated and not allowed to freeze.  The product bears the expiration date.  The 
product should not be shaken.  It is generally stable for at least 10 months when 
refrigerated.  The appropriate dose is drawn up into a syringe.  G-CSF will be given 
as a daily subcutaneous injection.  The side effects of G-CSF are skin rash, myalgia 
and bone pain, an increase of preexisting inflammatory conditions, enlarged spleen 
with occasional associated low platelet counts, alopecia (with prolonged use) elevated 
blood chemistry levels.
8.6.
Trimethoprim and Sulfamethoxazole Double Strength (TMP / SMX DS)
TMP/SMX DS will be obtained by the Clinical Center Pharmacy Department 
from commercial sources.  It will be used for the prevention of PCP pneumonia.  The 
oral dose is 1 tablet PO daily three times a week (MUST be on non-consecutive days) 
beginning on day -7 and continuing for at least 6 months and until the CD4 count is 
greater than 200 on 2 consecutive lab studies.  Like other sulfa drugs, TMP/SMX DS 
can cause allergies, fever, photosensitivity, nausea, and vomiting. Allergies typically 
develop as a widespread itchy red rash with fever eight to fourteen days after 


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beginning the standard dose. Neutropenia, a reduction in the number of neutrophils, 
can also occur.
8.6.1.
Aerosolized Pentamidine in Place of TMP/SMX DS:  
Patients with sulfa allergies will receive aerosolized Pentamidine 300 mg per 
nebulizer with  one week prior to admission and continued monthly until the CD4 
count is above 200 on two consecutive follow up lab studies and for at least 6 months 
post chemotherapy.  Pentamidine Isethionate will be obtained by the Clinical Center 
Pharmacy Department from commercial sources.  It will be used to prevent the 
occurrence of PCP infections.  It is supplied in 300 mg vials of lyophilized powder 
and will be administered via nebulizer.  Toxicities reported with the use of 
Pentamidine include metallic taste, coughing, bronchospasm in heavy smokers and 
asthmatics; increased incidence of spontaneous pneumothorax in patients with 
previous PCP infection or pneumatoceles, or hypoglycemia. 
8.7.
Herpes Virus Prophylaxis
8.7.1.
Valacyclovir (Valtrex)
Valacyclovir will be obtained by the Clinical Center Pharmacy Department 
from commercial sources.  It will be used orally to prevent the occurrence of herpes 
virus infections in patients with positive HSV serology.  It is supplied in 500 mg 
tablets.  Valcyclovir will be started the day after the last dose of fludarabine at a dose 
of 500 mg orally daily if the patient is able to tolerate oral intake.  See package insert 
for dosing adjustments in patients with renal impairment.  Common side effects 
include headache, upset stomach, nausea, vomiting, diarrhea or constipation.  Rare 
serious side effects include hemolytic uremic syndrome and thrombotic 
thrombocytopenic purpura.      
8.7.2.
Acyclovir
Acyclovir will be obtained by the Clinical Center Pharmacy Department from 
commercial sources.  It will be used to prevent the occurrence of herpes virus 
infections in patients who cannot take oral medications.  It is supplied as powder for 
injection in 500 mg/vials.  Reconstitute in 10 mL of sterile water for injection to a 
concentration of 50 mg/mL.  Reconstituted solutions should be used within 12 hours.  
IV solutions should be diluted to a concentration of 7mg/mL or less and infused over 
1 hour to avoid renal damage.  Reversible renal insufficiency has been reported with 
IV but not oral acyclovir.  Neurologic toxicity including delirium, tremors, coma, 
acute psychiatric disturbances, and abnormal EEGs have been reported with higher 
doses of acyclovir.  Should this occur, a dosage adjustment will be made or the drug 
will be discontinued. Stomach upset, headache or nausea, rash or hives; peripheral 
edema; pain, elevated liver function tests; and leukopenia, diarrhea, 
lymphadenopathy, myalgias, visual abnormalities and elevated creatinine have been 
reported. Hair loss from prolonged use has been reported. Acyclovir will not be used 
concomitantly with other nucleoside analogs which interfere with DNA synthesis, 
e.g. ganciclovir. In renal disease, the dose is adjusted as per product labeling. 
8.8.
Fluconazole


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Fluconazole will be obtained by the Clinical Center Pharmacy Department 
from commercial sources.  It will be used to prophylax against fungal infections.  It is 
available in 200 mg tablets.  It can cause headache, nausea, vomiting, diarrhea or 
abdominal pain, and liver damage which may be irreversible.  It can cause rashes and 
itching, which in rare cases has caused Stevens Johnson Syndrome.  It has several 
significant drug interactions.  The package insert should be consulted prior to 
prescribing.  For IV administration in patients who cannot tolerate the oral 
preparation, Fluconazole comes in 2 MG/ML solution for injection, and prepared 
according to Clinical Center Pharmacy standard procedures.  It should be 
administered at a maximum IV rate of 200 mg/hr.
8.9.
Anti-ESO TCR transduced PBL 
The procedure for expanding the human PBL and the Certificate of Analysis 
are similar to those approved by the Food and Drug Administration, and used at the 
NCI in ongoing protocols evaluating cell therapy in the Surgery Branch (most 
recently 07-C-0003, 07-C-0174, 07-C-0175). The CoA is included in Appendix 4.
The PBL will be transduced with retroviral supernatant containing the alpha chain 
and beta chain genes of the anti-ESO TCR.
8.9.1.
Retroviral Vector Containing the anti-ESO TCR Gene 
The retroviral vector supernatant (PG13-A2aB-IG4A-LY3H10) encoding a T 
cell receptor directed against NY-ESO-1, was prepared and preserved following 
cGMP conditions in the Indiana University Vector Production Facility (IUVPF). The 
retroviral vector, MSGV1- A2aB-IG4A-LY3H10, consists of 7,417 bps including the 
5' LTR from the murine stem cell virus (promoter), packaging signal including the 
splicing donor (SD) and splicing acceptor sites, alpha chain and beta chain genes of 
the anti-NY-ESO-1 TCR cloned from the peripheral blood lymphocytes of a patient 
with melanoma, and murine stem cell virus 3'LTR. The alpha and beta chains are 
linked by a T2A peptide. The physical titer will be determined by RNA dot blot 
according to sponsor certificate.  The supernate has been stored in IUVPF upon the 
completion of production at –80 °C at IUVPF. Upon shipment on dry ice, the 
supernatant will be stored at –80 °C in Cryonix, Rockville, MD. This storage facility 
is equipped with around-the-clock temperature monitoring. Upon request, supernatant 
will be delivered on dry ice to be used in in vitro transduction. There will be no re-use 
of the same unit of supernate for different patients. Retroviral titer has been shown to 
be stable after immediate thawing and immediate administration (coating the tissue 
culture wells previously coated with Retronectin). Handling of the vector should 
follow the guidelines of Biosafety Level-2 (BSL-2). The specific guidelines for 
Biosafety Level-2 (BSL-2) can be viewed at http://bmbl.od.nih.gov/sect3bsl2.htm.
8.9.2
OKT3
OKT3 will be obtained by the Surgery Branch Laboratory from commercial 
sources.
Formulation: Muromonab-CD3 (Ortho), NSC #618843, is provided as a 
sterile, clear, colorless solution at a concentration of 1 mg/ml in 5 ml ampoules.  The 
solution may contain a few fine, translucent protein particles.  The antibody is 
dissolved in a buffered solution at pH of 6.5 to 7.5.  The solution contains 2.25 mg of 


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monobasic sodium phosphate, 9 mg of dibasic sodium phosphate, 43 mg of sodium 
chloride and 1 mg of polysorbate 80 per 5 ml of water for injection.
Storage/Stability: Ampules should be stored in a refrigerator at 2-8 °C.  
Solution should not be frozen or shaken.  Each ampule bears an expiration date.
8.10.
ALVAC ESO-1 Vaccine  
The vaccine consists of the following construct: 
•
ALVAC(2)-NY-ESO-1(M)/TRICOM (vCP2292)
The construct is provided in liquid formulation, and is administered by the 
subcutaneous route.
Identity of the Investigational Product:  NY-ESO-1 antigen therapeutic 
vaccine.
Composition:  The investigational vaccine uses ALVAC(2), a modified 
canarypox virus, as a vector for carrying the gene for the NY-ESO-1 tumor-
associated antigen.  The vaccine also contains the genes for three co-stimulatory 
molecules:  B7.1, ICAM-1, and LFA-3, collectively referred to as TRICOM.
The genes for NY-ESO-1 and the co-stimulatory molecules are contained in 
the same construct.  Each vial contains a target dose of 107 CCID50 (with a range of 
approximately 106.4 to 107.9 /mL) of the ALVAC(2) virus.
Other ingredients:  The only excipient used in the final formulation of the 
drug product is 10 mM Tris pH 9.0 with 0.9% NaCl, which may be added if 
necessary to reduce titer.  This type of titer adjustment has not been required for any 
of the clinical lots to date.
Production Site: Sanofi Pasteur Ltd., BB-IND 13450
Batch/lot numbers:
TBD
Formulation: Liquid; 1 vial contains 0.72 mL of vaccine construct, with a 
target dose of 107 CCID50 (with a range of approximately 106.4 to 107.9 /mL) of the 
ALVAC(2) virus in a withdrawable volume of 0.5 mL.
Appearance:  Clear to opalescent solution with white to pink tint.  Some 
settling may occur, which can be resuspended with shaking
Storage:  2–8°C
Injection route:  Subcutaneous
Preparation and Administration:  Patients will receive 0.5 mL of the construct 
in each of the four extremities.  If a patient has a compromised extremity due to 
lymphedema, prior radiation or other causes, vaccination may be given on the 
abdominal wall or chest wall proximal to the extremity. As the construct is provided 
in a 0.5 mL vial, one vial is therefore required for each injection.  Each construct 
must be administered at the same injection site throughout the treatment. All 
injections will be subcutaneous, using a needle not more than 5/8 of an inch in length.
In the event of overdose:  Due to the nature of the product, there is no 
suggested treatment currently available.
Precautions for Use:  Prior to administration, all study products must be 
inspected visually for cracks, broken seals, correct label content and extraneous 
particulate matter and/or discoloration, whenever solution and container permit. If 


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any of these conditions exists, the vaccine should not be administered and a 
replacement dose may be used.
As after any vaccination, subjects must be kept under observation for 30 
minutes after each injection to ensure their safety. Appropriate equipment must be 
available on site in the event of immediate allergic reactions. 
Dose Selection and Timing:  The vaccine construct contains a target dose of 
107 CCID50 (with a range of approximately 106.4 to 107.9 /mL) of the ALVAC(2) virus. 
Earlier studies with ALVAC vaccines have found lower doses to not yield 
satisfactory immune responses, while higher doses present a manufacturing 
challenge, so cannot be used at this stage of development.  Patients will receive two 
sets of vaccinations, two weeks apart (+/- 2 days).
8.11.
Support Medications
Ondansetron hydrochloride
Ondansetron hydrochloride will be obtained by the Clinical Center Pharmacy 
Department from commercial sources.  It will be used to control nausea and vomiting 
during the chemotherapy preparative regimen.  It can cause headache, dizziness, 
myalgias, drowsiness, malaise, and weakness.  Less common side effects include 
chest pain, hypotension, pruritis, constipation and urinary retention.  Consult the 
package insert for specific dosing instructions.
Furosemide
Furosemide will be obtained by the Clinical Center Pharmacy Department 
from commercial sources.  It will be used to enhance urine output during the 
chemotherapy preparative regimen with cyclophosphamide.  Adverse effects include 
dizziness, vertigo, paresthesias, weakness, orthostatic hypotension, photosensitivity, 
rash and pruritis.  Consult the package insert for a complete list of all side effects.


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9.0 References
1. Wunderlich, J. R. and R. J. Hodes. 1991. Principles of tumor immunity:Biology of cellur 
immune responses. Biologic Therapy of Cancer, DeVita, Hellman and Rosenberg, eds.3-21.
2. Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, L. Rivoltini, S. L. Topalian, T. Miki, 
and S. A. Rosenberg. 1994. Cloning of the gene coding for a shared human melanoma antigen 
recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci USA 91:3515-
3519.
3. Kawakami, Y., S. Eliyahu, K. Sakaguchi, P. F. Robbins, L. Rivoltini, J. R. Yannelli, E. 
Appella, and S. A. Rosenberg. 1994. Identification of the immunodominant peptides of the 
MART-1 human melanoma antigen recognized by the majority of HLA-A2 restricted tumor 
infiltrating lymphocytes. J.Exp.Med 180:347-352.
4. Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, K. Sakaguchi, E. Appella, J. R. 
Yannelli, Adema, GJ, T. Miki, and Rosenberg SA. 1994. Identification  of a human melanoma 
antigen recognized by tumor infiltrating lymphocytes from a patient responding to 
immunotherapy. Proc.Natl.Acad.Sci. 54:3124-3126.
5. Dudley, M. E., J. Wunderlich, M. I. Nishimura, D. Yu, J. C. Yang, S. L. Topalian, D. J. 
Schwarztentruber, P. Hwu, F. M. Marincola, R. Sherry, S. F. Leitman, and S. A. Rosenberg. 
2001. Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treament of 
patients with metastatic melanoma. J.Immunotherapy 24:363-373.
6. Dudley, M., J. Wunderlich, J. C. Yang, P. Hwu, D. J. Schwartzentruber, S. L. Topalian, R. 
Sherry, F. M. Marincola, S. F. Leitman, C. A. Seipp, L. Rogers-Freezer, K. E. Morton, S. A. 
Mavroukakis, D. E. White, and Rosenberg SA. 2002. A Phase I study of non-myeloablative 
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32. Khong, H. T., J. C. Yang, S. L. Topalian, R. M. Sherry, S. A. Mavroukakis, D. E. White, and 
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42. Gattinoni, L., D. J. Powell, S. A. Rosenberg, and N. P. Restifo. 2006. Adoptive immunotherapy 
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10.0
Tables, Figures, and Appendices:


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Table 1.  Grade 3-4 non-hematologic toxicities, transfusion requirements, and 
episodes of febrile neutropenia during the first cycle of treatment.
Number of occurrences1
Study group2
Arm 1
Arm 2
Arm 3
Arm 4
Total
Toxicity
Hypotension
0
0
0
1
1
Malaise
0
0
0
0
0
Nausea
2
0
0
2
4
Neurologic
0
0
0
0
0
Febrile neutropenia
0
2
1
3
6
RBC transfusion
1
2
3
6
12
Platelet transfusion
1
3
1
6
11
1Counted once for each patient at the highest grade
2 Arms defined: 
Arm 1: 3 patients received up to 3 X 1011 cloned and/or uncloned lymphocytes plus cyclophosphamide 30 
mg/kg d-7 & -6 and fludarabine 25 mg/m2 d-5 to -1, without IL-2.
Arm 2: 3 patients received up to 3 X 1011 cloned and/or uncloned lymphocytes plus cyclophosphamide 60 
mg/kg d-7 & -6 and fludarabine 25 mg/m2 d-5 to -1, without IL-2.
Arm 3: 3 patients received up to 3 X 1011 cloned and/or uncloned lymphocytes plus cyclophosphamide 60 
mg/kg d-7 & -6 and fludarabine 25 mg/m2 d-5 to -1 + IV IL-2 (72,000 IU/kg q8h for a maximum of 15 doses) 
Arm 4: 6 patients received up to 3 X 1011 cloned and/or uncloned lymphocytes plus cyclophosphamide 60
mg/kg d-7 & -6 and fludarabine 25 mg/m2 d-5 to -1+ IV IL-2 (720,000 IU/kg q8h for a maximum of 12 
doses) 


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Table 2: Clinical Responses to treatment
11/27/06
Patient1
Sites of Disease
Response
Duration (months)2
1
Axillary, Mesenteric, and Pelvic Lymph Nodes
PR
29
2
Subcutaneous and Skin
PR
8
4
Iliac and Inguinal Lymph Nodes, Skin
PR
2
6
Intraperitoneal Lymph Nodes, Lungs, 
Subcutaneous
PR
28
9
Subcutaneous and Skin
PR
12
10
Inguinal Lymph Nodes, Subcutaneous and Skin
PR
14
16
Subcutaneous
CR
52+
17
Bone, Liver, Lung, Subcutaneous
PR
38+
19
Intramuscular
PR
13
21
Lung, Subcutaneous
CR
45+
25
Lung, Subcutaneous
PR
2
26
Inguinal Lymph Nodes, Liver, Subcutaneous
PR
8
28
Axillary Lymph Nodes, Brain
PR
4
30
Axillary and Inguinal Lymph Nodes, 
Intramuscular, Subcutaneous
PR
7
31
Liver, Lung
CR
35+
32
Skin
CR
34+
33
Lung, Subcutaneous
PR
4
34
Intramuscular, Pelvis
PR
5
1Patients 2, 6, and 17 had minor (Patient 6) or mixed (Patient 2 and Patient 17) responses after a first course 
of treatment, and were treated with a second course of treatment consisting of ablation, cell transfer and high 
dose IL-2 therapy prior to achieving an objective clinic response.   
2Measured from treatment date to time of first recurrence.  Patients 16, 17, 21, 31 and 32 had ongoing 
responses at the time of this writing.  


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Table 3
Time in Hospital and Non-hematological Grade 3 and 4 Toxicities Related to 
Lymphodepleting Chemotherapy and Cell Transfer
Attribute measured
Duration, Number or 
Type
Number of Patients 
(%)
Days in Hospital1
6-10
6 (17%)
11-15
18 (51%)
16-20
4 (11%)
21-25
7 (20%)
pRBC Transfusions
0
2 (6%)
1-5
18 (51%)
6-10
13 (37%)
11-15
2(6%)
Platelet
Transfusions
0
6 (17%)
1-5
21 (60%)
6-10
5 (14%)
11-15
2 (6%)
16-20
1 (3%)
Autoimmunity
Uveitis
5 (14%)
Vitiligo
13 (37%)
Opportunistic
Infections
Herpes zoster
3 (9%)
Pneumocystis pneumonia
2 (6%)
EBV-B cell lymphoma
1 (3%)
RSV pneumonia
1 (3%)
Other
Febrile neutropenia
13 (37%)
Intubated for dyspnea 
3 (9%)
Cortical blindness
1 (3%)
1Measured from the day of cell administration to discharge


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Table 4


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Table 5:
Gene transfer efficiency at time of infusion. 
Patient Number 
1
2a
3
4
5
6
7
8
9
10
11
12
13
2b
14
15
16
17 Ave
VE12
(%CD4)
58
63
21
n/d
18
36
45
38
43
52
51
66
43
66
58
47
18
41 45
Tetramer
(%CD4)
8
15
1
n/d
2
10
15
8
15
21
22
33
15
15
7
4
10
7
12
VE12
(%CD8)
49
65
63
24
17
32
23
29
43
42
43
59
44
72
51
51
36
24 42
Tetramer
(%CD8)
16
28
3
23
3
10
6
5
13
15
19
30
16
33
17
19
11
15 17
Samples were taken from transduced T lymphocytes cultures at or near time of infusion.  FACS 
analysis for CD4, CD8, VE12, and MART-1 Tetramer (M27L) was as described in methods.  
n/d, not determined.; Ave, average value.
Table 6:
Q-RT PCR Analysis
Patient Number
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Infusion
5.8
2.2
14.5
10.7
21.5
20.8
4.0
10.3
9.6
11.2
4.0
5.1
24
11.1
Post
Infusion
1.2
0.8
3.5
1.9
0.6
2.8
0.9
1.4
0.6
2.9
2.8
1.1
1.9
2.2
Amount of TCR vector-derived RNA normalized to cellular actin mRNA.  Using RNA derived 
from a stable transduced human T cell line (Sup T1) as reference; total RNA from infused T cells 
or patient PBMC post-infusion were subjected to Q-RT PCR and then normalized to the amount 
of actin mRNA (values are vector/1 x 106 actin).  Samples were 2-5 weeks post-infusion, with 
exception of patients 8, 9, and 11, which were 2 months, 3 months, and 2 weeks respectively. 


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Table 7: Elispot Analysis
Patient Number
1
2a
3
4
5
6
8
9
13
2b
14
15
16
17
Infusion
1523
1783
215
4527
1367
1203
1352
460
3380
4877
2023
2938
1393
190
Pre 
treatment
22
21
1
14
0
1
0
0
0
0
20
1
6
0
Post
infusion
13
45
0
41
7
1
215
20
700
13
530
35
92
108
Number of interferon-J positive Eispots/100,000 cells.  Post-infusion values derived from PBL 
taken at one to four weeks post-infusion and rested overnight in medium (without cytokine) prior 
to assay.
Table 8:
Antigen reactivity of ex vivo cultured PBL.
Patient 4:
Melanoma Cell Line
A2-
A2+
Peptide pulsed T2 cells 
888
938
526
624
None
gp100
MART-1
None
A1,24
A1,24
A2,3
A2,3
1.0 
PM
10 PM
1.0 PM
0.1 
PM
Controls
None
0
0
0
0
0
0
0
0
0
0
AK 1700-3
181
66
9
37
10
46
36
32
35
32
JKF6
0
22
28
10250
10680
1
0
>16910
>15430
7770
JR6C12
0
41
0
4190
4640
0
8000
0
8520
143
Patient 4
Pre-
treatment
130
204
117
98
133
521
421
422
462
520
Day 7
40
65
44
131
165
104
94
>1255
>1480
851
Day 8
87
110
75
92
78
122
190
957
893
537
Day 18
42
27
23
43
55
184
63
108
160
113
Anti-melanoma properties of genetically engineered lymphocytes determined for patient 4 PBL 
following overnight culture in IL-2 (300 IU/ml).  The production of interferon-J (pg/ml) 
following co-culture with peptide pulsed T2 cells (peptide reactivity) and anti-melanoma activity 
(tumor reactivity) for HLA-A2 + lines (526, 624) and HLA-A2- lines (888, 938).  Controls 
included; non-peptide reactive TIL AK 1700-3, MART-1 reactive TIL JKF6, and gp100 reactive 
TIL JR6C12.   JR6C12 recognition of 1.0PM MART-1 peptide was not reproducible in repeated 
assays. Values demonstrating specific release of cytokine are in bold.


08-C-0121
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Table 8 (con’t)
Patient 14:
Melanoma Cell Line
A2-
A2+
Peptide pulsed T2 cells
888
938
526
624
None
gp100
MART-1
None
A1,24
A1,24
A2,3
A2,3
1.0 
PM
10 PM
1.0 PM
0.1 
PM
Controls
None
0
0
43
0
0
0
23
70
0
18
AK 1700-3
35
53
33
21
26
52
43
94
73
105
JKF6
15
11
66
6750
9160
37
337
>2168
>2168
>182
8
JR6C12
0
0
0
4410
3760
0
>1510
23
15
61
Patient 14
Pre-
treatment
368
410
352
284
344
639
645
874
614
471
Day 5
66
53
67
110
170
134
96
918
716
634
Day 7
69
52
45
41
55
33
57
192
221
109
Day 10
79
91
0
80
46
33
52
245
214
108
Day 27
52
76
35
33
55
67
103
282
232
158
Day 88
26
50
31
0
52
75
41
52
43
31
Day 257
105
52
93
53
46
111
83
597
492
399
Anti-melanoma properties of genetically engineered lymphocytes determined for patient 14 PBL 
following overnight culture in IL-2 (300 IU/ml).  The production of interferon-J (pg/ml) 
following co-culture with peptide pulsed T2 cells (peptide reactivity) and anti-melanoma activity 
(tumor reactivity) for HLA-A2 + lines (526, 624) and HLA-A2- lines (888, 938).  Controls 
included; non-peptide reactive TIL AK 1700-3, MART-1 reactive TIL JKF6, and gp100 reactive 
TIL JR6C12. Values demonstrating specific release of cytokine are in bold


08-C-0121
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Table 9


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TABLE 10:
Modification of Dose Calculations* in Patients whose BMI is greater 
than 35
Unless otherwise specified in this protocol, actual body weight is used for dose 
calculations of treatment agents.   In patients who are determined to be obese (BMI > 
35), the practical weight (see 3 below) will be used.
1. BMI Determination:
BMI = weight (kg) / [height (m)]2
2. Calculation of ideal body weight
Male = 50 kg + 2.3 (number of inches over 60 inches)
Example:  ideal body weight of 5’10’’ male
50 + 2.3 (10) = 73 kg
Female = 45.5 kg + 2.3 (number of inches over 60 inches)
Example:  ideal body weight of 5’3’’ female
45.5 + 2.3 (3) = 57 kg
3. Calculation of “practical weight”
Calculate the average of the actual and the 
ideal body weights.  This is the practical 
weight to be used in calculating the doses of 
chemotherapy and associated agents 
designated in the protocol.
*Practical weight will NOT be used in the calculation of dose for aldesleukin.


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Figure 1


0 8- C- 0 1 2 1
7 1
Fi g ur e 2.  Tr a n s d u cti o n a n d a n al y si s of T C R e n gi n e er e d c ell s.  A .  C D 8 + h u m a n l y m p h o c yt e s w er e el e ctr o p or at e d wit h R N A 
e n c o di n g c o ntr ol ( G F P) or cl o n e d T C R s r e a cti v e wit h H L A- A 2 r e stri ct e d e pit o p e s fr o m h u m a n t u m or a nti g e n s M A R T- 1, g p 1 0 0, N Y-
E S O- 1, a n d p 5 3.  Eff e ct or T- c ell s w er e c o- c ult ur e d wit h T 2 c ell s p ul s e d wit h 1
M of t h e i n di c at e d p e pti d e ( v al u e s ar e i nt erf er o n-
,
p g/ ml).  B .  Di a gr a m of r e c o m bi n a nt r etr o vir al v e ct or M S G V 1 AI B u s e d t o e n gi n e er h u m a n l y m p h o c yt e s. C . Tr a n s d u c e d ( T d) 
l y m p h o c yt e s w er e a n al y z e d fi v e d a y s p o st-tr a n s d u cti o n f or t h e e x pr e s si o n of V 1 2 a n d M A R T- 1 t etr a m er i n C D 8 + c ell s i n 
c o m p ari s o n t o u ntr a n s d u c e d ( U n T d) c ell s.  N u m b er s ar e t h e p er c e nt p o siti v e c ell s i n t h at r e gi o n.  D .  T C R v e ct or- e n gi n e er e d c ell s 
fr o m p ati e nt 6 ( T C R) w er e c o- c ult ur e d wit h M A R T- 1 p e pti d e p ul s e d T 2 c ell s, H L A- A 2-  m el a n o m a li n e ( M el 8 8 8), or H L A- A 2 + 
m el a n o m a li n e ( M el 5 2 6) a n d t h e a m o u nt of i nt erf er o n-
a n d M A R T- 1 r e a cti v e TI L J K F 6 ( J K F 6).  E .  A nti- m el a n o m a pr o p erti e s of g e n eti c all y e n gi n e er e d l y m p h o c yt e s w er e d et er mi n e d f or 
all p ati e nt s pri or t o i nf u si o n.  T h e pr o d u cti o n of i nt erf er o n-
( p g/ ml) f oll o wi n g c o- c ult ur e wit h p e pti d e p ul s e d T 2 c ell s ( P e pti d e 
R e a cti vit y) a n d a nti- m el a n o m a a cti vit y ( T u m or R e a cti vit y) f or H L A- A 2 + li n e s ( 5 2 6, 6 2 4) a n d H L A- A 2- li n e s ( 8 8 8, 9 3 8).  V al u e s 
d e m o n str ati n g s p e cifi c r el e a s e of c yt o ki n e ar e i n b ol d.     
Fi g ur e 2


08-C-0121
72
Figure 3:. 
Persistence of gene marked cells.  DNA extracted from PBMC was subjected to real-time quantitative PCR to 
determine the percent of vector-transduced cells in patient circulation at various times post-infusion.  Each line 
represents data from a separate patient.  A, cohort 1; B, cohort 2; C, cohort 3.  D.  Mean value of the % of gene 
marked cells for all patients in each cohort at the given time interval post-treatment.  E. The percentage of CD8+/ 
VE12+ cells in the intermediate gate for patients in cohorts 2 and 3 are shown.  F. The percentage of CD8+/MART-1
tetramer+ cells was determined for patients in cohorts 2 and 3 at the times shown.  Pre-treatment values for each 
patient are plotted as day 0 post-infusion.
Figure 3


0 8- C- 0 1 2 1
7 3
Fi g ur e 4.: C a n c er r e gr e s si o n i n t w o p ati e nt s.  A.  C T i m a g e s of p ati e nt 4 li v er m et a st a si s; pr e-tr e at m e nt, o n e m o nt h 
a n d 1 0 m o nt h s p o st-tr e at m e nt wit h T C R e n gi n e er e d T- c ell s.  B. Si z e of li v er a n d a xill ar y t u m or s a n d t e m p o of 
r e gr e s si o n of t u m or sit e s i n p ati e nt 4 (tr e at m e nt ti m e = d a y 0).  C. C T i m a g e s of p ati e nt 1 4 hil ar l y m p h n o d e 
m et a st a si s; pr e-tr e at m e nt, b e gi n ni n g of tr e at m e nt ( D a y 0), a n d t w o m o nt h s a n d 1 2 m o nt h s p o st-tr e at m e nt.  D.  Si z e 
of t u m or a n d t e m p o of r e gr e s si o n i n p ati e nt 1 4.  E.  Q u a ntit ati o n of g e n e m ar k e d c ell s i n p ati e nt s 4 a n d 1 4 P B M C w a s 
d et er mi n e d b y r e al-ti m e q u a ntit ati v e P C R. D a y of i nf u si o n (I nf.) i n di c at e d b y arr o w.  F.  T h e p er c e nt a g e of 
C D 8 +/ V
1 2 
Fi g ur e 4


08-C-0121
74
Figure 5:
Figure 5. Rapid screening of NY-ESO-1/HLA-A*02-reactive TCR 1G4 CDR2E variants in 
CD4+ and CD8+ T cells. T cells were activated with anti-CD3 antibody and isolated CD4+ (A)
or CD8+ (B) T cells were co-transfected with RNA 1G4 TCR E chain constructs encoding AASs 
within the CDR2 along with a WT 1G4 TCR D chain RNA construct. Transfected T cells were 
incubated overnight with HLA-A*02+ melanoma target cell lines that did or did not also express 
NY-ESO-1 transcript and the release of IFN-J measured the following day.
A2 ESO
+   +
+   +
+   +
+
-
+
-
1G4 CDR2E
51
WT    GAGI
E51A  A---
E52I  -I--
E53A  --A-
E54T  ---T
E51AI AI--
GFP
0
200
400
600
800
1000
1200
1400
1600
IFN-J(pg/ml)
624.38
1363
A375
SK23
526
A                                                                          B                          
                     CD4+ T cells                                                         CD8+ T cells
1G4 CDR2E
51
WT    GAGI
E51A  A---
E52I  -I--
E53A  --A-
E54T  ---T
E51AI AI--
GFP
A2 ESO
+   +
+   +
+   +
+
-
+
-
0
1000
2000
3000
4000
5000
6000
7000
8000
IFN-J(pg/ml)
624.38
1363
A375
SK23
526


0 8- C- 0 1 2 1
7 5
Fi g ure 6:
Fi g ure 6. R a pi d scree ni n g of N Y- E S O- 1/ H L A- A * 0 2-re a cti ve T C R 1 G 4 C D R 3
v ari a nts i n
C D 8 + a n d C D 4 + T cells. T cells wer e acti vat e d wit h a nti- C D 3 a nti b o d y a n d is olate d C D 4 + (A )
or C D 8 + (C ) T cells were c o-tra nsf ecte d wit h R N A 1 G 4 T C R 
A A Ss wit hi n t he C D R 3 re gi o n al o n g wit h a W T 1 G 4 T C R 
c hai n R N A c o nstr uct. Tra nsfecte d T 
cells wer e i nc u bate d o v er ni g ht wit h H L A- A * 0 2 + mela n o ma ( 3 9 7- A 2, 6 2 4. 3 8, 1 3 0 0, 5 2 6 a n d 
8 8 8- A 2) or br east ca ncer ( M D A 4 3 5 S- A 2) tar get ce ll li nes t hat di d or di d n ot als o e x press N Y-
E S O- 1 tra nscri pt a n d t he releas e of I F N-
was meas ure d t he f oll o wi n g da y.
A 2  N Y- E S O-
1
+       +
+       +
+       +
+       +
+
-
+
-
1 G 4  C D R 3
9 5
W T     T S
9 5 : L   L -
9 6 : L 
- L
9 5 : L L  L L
9 5 : L Y  L Y
G F P
0 
2 0 0 0 
4 0 0 0 
6 0 0 0 
8 0 0 0
I F N- ( p g/ ml)
3 9 7 A 2
6 2 4. 3 8
1 3 0 0
M D A 4 3 5 S- A 2
5 2 6
8 8 8 A 2
A 2  N Y- E S O-
1
+       +
+       +
+       +
+       +
+
-
+
-
1 G 4  C D R 3
9 5
W T     T S
9 5 : L   L -
9 6 : L 
- L
9 5 : L L  L L
9 5 : L Y  L Y
G F P
0 
5 0 0 0 
1 0 0 0 0 
1 5 0 0 0 
2 0 0 0 0 
2 5 0 0 0
I F N- ( p g/ ml)
3 9 7 A 2
6 2 4. 3 8
1 3 0 0
M D A 4 3 5 S- A 2
5 2 6
8 8 8 A 2
A                                                                          B                          
                     C D 4+ T c ell s                                                         C D 8 + T c ell s


08-C-0121
76
Figure 7.
Adoptive transfer of tumor-specific T cells combined with vaccination and IL-2 causes 
regression and cure of large, established tumors.  B16 tumor was implanted 
subcutaneously into C57BL/6 mice were treated by adoptive transfer of fresh pmel-1
splenocytes +/- vaccination with recombinant fowlpox virus (rFPV)hgp100 either seven 
(A,B) or fourteen days (C,D) after inoculation with B16 melanoma.  IL-2 was 
administered twice daily for 6 doses.  Fresh or cultured splenocytes were effective I the 
treatment of large, established tumor.  Splenocytes derived from an identically 
constructed TCR transgenic mouse with specificity for beta–galactosidase were used as a 
control in some experiments, (a representative of which is shown in panels C,D) were not 
therapeutic.  Statistically significant tumor regression was seen in mice treated with 
pmel-1 cells given in combination with rFPVhgp100 and IL-2 in over 20 independently 
performed experiments.  There were at least 5 mice/group in all experiments.  Mouse 
survival consistently correlated with tumor growth reduction.


08-C-0121
77
Figure 8.
Long –term (>1 yr) survival of mice bearing large, established B16 tumors after treatment 
with adoptive transfer of tumor-specific T cells combined with vaccination and IL-2 is 
associated with the development of vitiligo.  C57BL/6 mice were treated with adoptive 
transfer of fresh or cultured pmel-1 transgenic splenocytes fourteen days after inoculation 
with B16 melanoma, then vaccinated with rFPVhgp100. IL-2 was administered twice 
daily for 6 doses.  Mice treated with fresh naïve or cultured transgenic T cells were cured 
of B16 and vitiligo was seen at the former tumor site.  At more than 1-year post therapy, 
these mice remain tumor-free.  


08-C-0121
78
Figure 9.
Endogenous host B or T lymphocytes are not required for the treatment of established 
B16 tumors. C57BL/6 Rag-1-/- knockout mice bearing subcutaneous B16 tumors 
established for 14 days were treated with 1 x 105 cultured pmel-1 cells given cells given 
in combination with rFPVhgp100 and IL-2 as described previously.  Similar results were 
obtained in five independently completed experiments.


08-C-0121
79
Appendix 1


08-C-0121
80
Appendix 2
Expected IL-2 Toxicities and their Management
Expected 
toxicity
Expected 
grade
Supportive 
Measures
Stop Cycle*
Stop Treatment **
Chills
3
IV Meperidine 25-50
mg, IV q1h, prn,
No
No
Fever
3
Acetaminophen 650 
mg, po, q4h; 
Indomethicin 50-75
mg, po, q8h
No
No
Pruritis
3
Hydroxyzine HCL 
10-20 mg po q6h, 
prn; 
Diphenhydramine 
HCL25-50 mg, po, 
q4h, prn
No
No
Nausea/ Vomiting/ 
Anorexia
3
Ondansetron 10 mg, 
IV, q8h, prn; 
Granisetron 0.01 
mg/kg IV daily prn; 
Droperidol 1 mg, IV 
q4-6h, prn; 
Prochlorperazine 25 
mg pr, prn or 10 mg 
IV q6h prn
No
No
Diarrhea
3
Loperamide 2mg, po,
q3h, prn; 
Diphenoxylate HCl 
2.5 mg and atropine 
sulfate 25 mcg, po, 
q3h, prn; codeine 
sulfate 30-60 mg, po, 
q4h, prn
If uncontrolled after 
24 hours despite all 
supportive measures
No
Malaise
3 or 4
Bedrest
If other toxicities 
occur 
simultaneously
No
Hyperbilirubinemia
3 or 4
Observation
If other toxicities 
occur 
simultaneously
No
Anemia 
3 or 4
Transfusion with 
PRBCs
If uncontrolled 
despite all 
supportive measures
No
Thrombocytopenia
3 or 4
Transfusion with 
platelets
If uncontrolled 
despite all 
supportive measures
No
Edema/Weight gain
3
Diuretics prn
No
No
Hypotension
3
Fluid resuscitation
Vasopressor support
If uncontrolled 
despite all 
supportive measures
No


08-C-0121
81
Dyspnea
3 or 4
Oxygen or ventilatory 
support
If requires 
ventilatory support
No
Oliguria
3 or 4
Fluid boluses or 
dopamine at renal 
doses
If uncontrolled 
despite all 
supportive measures
No
Increased creatinine
3 or 4
Observation
Yes (grade 4)
No
Renal failure
3 or 4
Dialysis
Yes
Yes
Pleural effusion 
3
Thoracentesis
If uncontrolled 
despite all
supportive measures
No
Bowel perforation
3
Surgical intervention
Yes
Yes
Confusion
3
Observation
Yes
No
Somnolence
3 or 4
Intubation for airway 
protection
Yes
Yes
Arrhythmia
3
Correction of fluid 
and electrolyte 
imbalances; chemical 
conversion or 
electrical conversion 
therapy
If uncontrolled 
despite all 
supportive measures
No
Elevated Troponin 
levels
3 or 4
Observation
Yes
If changes in LV function 
have not improved to 
baseline by next dose
Myocardial 
Infarction
4
Supportive care
Yes
Yes
Elevated 
transaminases
3 or 4
Observation
For grade 4 without 
liver metastases
If changes have not 
improved to baseline by 
next dose
Hyperbilirubinemia
3 or 4
Observation
For grade 4 without 
liver metastases
If changes have not 
improved to baseline by 
next dose
Electrolyte 
imbalances
3 or 4
Electrolyte 
replacement
If uncontrolled 
despite all 
supportive measures
No
Neutropenia
4
Observation
No
No
*Unless the toxicity is not reversed within 12 hours
** Unless the toxicity is not reversed to grade 2 or less by next treatment.


08-C-0121
82
Appendix 3: Interleukin-2 toxicities observed in patients treated at the NIH Clinical Center


08-C-0121
83
Appendix 4
Certificate of Analysis:
Anti-ESO-1 TCR transduced T cells, protocol 08-C-0121
Date of preparation of final product:
Patient:
Allogeneic PBMC
Donor Name:
Pheresis date: 
Tests performed on final product:
Test
Method
Limits
Result
Initials/
Date
Cell viability1
trypan blue exclusion
>70%
Total viable cell 
number1
visual microscopic count
>1 x108
Tumor reactivity2
J-IFN release vs. peptide 
pulsed T2 cells
>200 pg/ml
TCR expression2
FACS analysis of the 
transduced cells
PBL, >10%
Microbiological 
studies 
gram stain1,3, 
no micro-
organisms seen
aerobic culture3,4
no growth
fungal culture3,4
no growth
anaerobic culture3,4
no growth
mycoplasma test5
negative
Endotoxin 
limulus assay1
<5 E.U./kg
RCR
S+L- Assay4
RCR-PCR6
negative
1 Performed on sample of the final product immediately prior to infusion.  Results are available at the time of infusion.
2 Performed 2-10 post transduction. Results are available at the time of infusion.
3 Performed 2-4 days prior to infusion.  Results are available at the time of infusion but may not be definitive.
4 Sample collected from the final product prior to infusion.  Results will not be available before cells are infused into the patient.
5 Performed 2-10 days prior to infusion.  Results are available at the time of infusion.
6 Performed on sample approximately 1-4 days prior to infusion.  Results are available at the time of infusion. 
Prepared by:
_______________________________________
Date: ____________________
QC sign-off:
_______________________________________
Date:  ____________________
James C. Yang, M.D. or designee

ICF_001.pdf: 
MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
INSTITUTE: 
National Cancer Institute 
STUDY NUMBER: 
08-C-0121 
PRINCIPAL INVESTIGATOR: 
Steven A. Rosenberg, MD, PhD 
STUDY TITLE:  
 
Phase II Study of Metastatic Cancer that Expresses NY-ESO-1 Using Lymphodepleting 
Conditioning Followed by Infusion of Anti-NY ESO-1 TCR-Gene Engineered 
Lymphocytes 
 
Continuing Review Approved by the IRB on 06/23/16 
Amendment Approved by the IRB on  07/22/15 (N)  
                    Date posted to web:   06/30/16 
Standard 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent (1) 
 
 
INTRODUCTION 
We invite you to take part in a research study at the National Institutes of Health (NIH). 
First, we want you to know that: 
 
Taking part in NIH research is entirely voluntary. 
 
You may choose not to take part, or you may withdraw from the study at any time.  In 
either case, you will not lose any benefits to which you are otherwise entitled.  However, to 
receive care at the NIH, you must be taking part in a study or be under evaluation for study 
participation. 
 
You may receive no benefit from taking part.  The research may give us knowledge that 
may help people in the future. 
Second, some people have personal, religious or ethical beliefs that may limit the kinds of medical 
or research treatments they would want to receive (such as blood transfusions).  If you have such 
beliefs, please discuss them with your NIH doctors or research team before you agree to the study. 
Now we will describe this research study.  Before you decide to take part, please take as much 
time as you need to ask any questions and discuss this study with anyone at NIH, or with family, 
friends or your personal physician or other health professional. 
 
Description of Research Study 
 
You have been diagnosed with metastatic cancer and the standard treatments available have not 
been effective. We have developed an experimental procedure for treating patients with 
melanoma that uses blood cells found in their tumors or peripheral blood cells.  We genetically 
modify these cells and grow them in the laboratory.  We hope that these cells when infused will 
decrease the size of your tumors.  However, it is possible that these cells will not have this effect.  
We will be using the anti-ESO-1 gene and a type of virus (retrovirus) in making these special 
cells (anti-ESO-1 cells). The anti-ESO--1 cells will be given to you as an intravenous (IV) 
infusion.  ESO-1 is a gene present in cancer cells.  This type of experimental treatment is called 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 2 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
“gene therapy” and is very closely monitored by the FDA and other regulatory agencies.  The 
risks of gene therapy will be described later in this document. 
 
Our studies in animals of cell transfer have indicated that the administration of a pox virus (a 
type of virus that cannot reproduce in mammals) in conjunction with cell transfer may increase 
the effectiveness of the cell transfer.  Therefore, we would like to study the combination of anti-
ESO-1 cells with an experimental vaccine that includes the NY-ESO-1 gene inserted inside a 
virus called ALVAC(2) (ALVAC ESO-1 vaccine).  This virus, which is used as a vaccine 
against disease in canaries, cannot reproduce in mammals, so it cannot cause disease in humans.  
However, when it is injected into a patient, it stimulates specific cells in the immune system.   
 
To date, we have treated 20 patients - 13 with melanoma, 6 with synovial cell sarcoma and 1 
patient with breast cancer with the antii-ESO-1 cells (no ALVAC ESO-1 vaccine) and 11 
patients have experienced clinical response for a response rate of 45%.  We have treated 10 
patients with the anti-ESO-1 cells and ALVAC vaccine and of the 8 patients evaluable for 
response, 4 have experienced clinical responses (50% response rate).  Although the response rate 
seems similar between patients treated with and without the vaccine, we will need to treat many 
more patients to determine if the vaccine actually makes a difference in the treatment.  
 
There are many steps in the production of the  ALVAC ESO-1 vaccine and thus it is not always 
available to us for clinical trials.  Therefore, you will receive the ALVAC ESO-1 vaccine along 
with the anti-ESO-1 cells when the vaccine is available.  If the vaccine is not available at the 
time that we determine you are eligible for this study, you will receive only the anti-ESO-1 cells. 
 
This highly experimental regimen is explained below. 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 3 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Research Study Overview  
This study has 5 stages outlined below: 
 
Stage 
Timeframe 
Location 
Comments & Instructions 
Work up 
1-2 weeks 
Inpatient and 
out patient   
Scans, x-rays, labs leukapheresis other 
tests as needed 
Chemotherapy 
(day -7 to -1) 
1 week 
Inpatient 
Receive IV chemotherapy  to prepare 
your immune system for the cells 
ALVAC 
ESO-1 
vaccine (Days 0 and 
14 
(when 
the 
vaccine is available) 
1 day, then 
again on day 
14 
Inpatient 
Receive vaccine under the skin as 4 
injections (one in each extremity) 
Cells, and IL-2 
(Day 0) 
1-5 days 
Inpatient and 
possibly ICU 
Receive, anti-ESO-1 cells, and then 
IL-2 every 8 hours for up to 15 doses 
Recovery 
1-2 weeks 
Inpatient unit Recover 
from 
the 
effects 
of 
chemotherapy and IL-2. 
Follow -up 
Ongoing 
until disease 
progression 
Outpatient 
Return to clinic for physical exam, 
review of side effects, labs, scans 
every 1-6 months 
 
The major side effects of this experimental treatment (described in detail on pages 5-8) that 
are most severe include:  
• Infection and low blood counts caused by the chemotherapy  
• Confusion and changes in mental status caused by the IL-2 
• Fluid retention, low blood pressure, and high heart rate caused by the IL-2 
 
We will discuss the side effects of this experimental treatment with you. You will be given 
medicines, transfusions, and treatments to prevent or treat the side effects including drugs to 
prevent and/or treat different types of infections.  We will try to make you as comfortable as 
possible.   
 
Women who are breast feeding or pregnant may not participate in this study because of the 
unknown effect on a fetus or nursing baby.  We also ask that you practice an effective form of 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 4 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
birth control while on this trial from prior to receiving chemotherapy, and for four months after 
being treated on this trial.  Women who think they might have become pregnant while on this 
trial should notify their doctor immediately. 
 
Work up 
Prior to receiving the experimental treatment you will under go many tests. These include 
imaging procedures, heart and lung function tests, eye exams, and laboratory tests.  If you have 
received ipilimumab (also called MDX-010) or ticilimumab, you will have a colonoscopy and 
biopsies to make sure your colon is normal since these drugs may cause damage to your colon”.  
If you are a woman, you will undergo a pregnancy test.  You will also have a large catheter 
inserted into a vein and leukapheresis will be performed.  You may be admitted to the hospital 
for these tests. However, you will be allowed to leave on pass on the days that you are not having 
tests performed. 
 
Catheter insertion 
Prior to beginning the experimental treatment, you will have an intravenous (IV) catheter placed 
in your upper chest. The area will be numbed with an anesthetic before the catheter is put in.  
Although rare, putting these catheters in can sometimes cause collapse of a lung or cause 
bleeding.  Lung collapse is treated by putting a tube into your chest for a few days to allow your 
lung to expand.  Pressure is placed on any area that might bleed.  Other IVs may be needed in 
one or both of your arms if we to give you extra fluids, medicines, or nutrition. 
 
Leukapheresis 
Leukapheresis is a procedure that allows us to remove certain types of blood cells from you and 
return the rest of your blood.  It is a very common procedure that is done routinely here at the 
NIH with very few risks.  During leukapheresis,  blood is removed from you through a needle in 
your arm, circulated through a machine that divides whole blood into red cells, plasma (the 
serum part), and lymphocytes (or white cells), and then the plasma and red cells are returned to 
you through a second needle in your other arm.  The white blood cells may be used to help grow 
the cells and after the treatment, you will have leukapheresis so that we can test your cells. 
 
Chemotherapy Regimen (Day -7 through Day -1) 
After we have grown the anti-ESO-1 cells to large numbers in the laboratory, you will be 
admitted to the hospital to begin your experimental treatment. You will be given two 
chemotherapy medicines, cyclophosphamide and fludarabine, to suppress your immune system 
so the anti-ESO-1 cells can work without any interference from the cells in your immune system. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 5 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
(These medicines will not treat your cancer.)  Animal experiments have indicated that this can 
make the cells more effective in fighting cancer tumors, but it is not known whether this is true 
in humans.  The cyclophosphamide will be given into your catheter over 1 hour for two days 
(Day -7 and Day -6) and the fludarabine will be given into your catheter for 30 minutes every 
day for the next five days (Day -5 through Day -1).  The side effects of these medicines are 
described on the following pages. 
After you have completed the chemotherapy regimen you will receive the cell infusion with 
or without the experimental vaccine.  
 
Cell Infusion and IL-2 Regimen (Day 0 through Day 4) (No Vaccine) 
One to four days after the last dose of chemotherapy, you will be given the anti-ESO-1 cells.  
The anti ESO-1 cells will be given in your catheter over 30 minutes.  Within 24 hours after the 
anti-ESO-1 cell infusion, you will be given high dose IL-2 through your catheter. IL-2 is 
approved by the FDA for treatment of metastatic melanoma and metastatic renal cell cancer.  
The purpose of giving the IL-2 with this therapy is to keep the cells we give you active for as 
long as possible so they will fight your tumor.  The IL-2 will be given as a 15-minute infusion 
every 8 hours for up to five days after the cell infusion (maximum number of doses is 15).  Doses 
may be skipped or delayed depending on how well you tolerate the doses.  The risks of the cells 
and IL-2 are described on the following pages. 
 
The day after you receive the anti-ESO-1 cells, we will give you more G-CSF (filgrastim) as a 
shot or injection under the skin. This will continue until your white blood cell counts begin to 
return to normal.   
 
Experimental ALVAC ESO-1 vaccine, Cell Infusion, and IL-2 Regimen (Day 0 through 
Day 4) 
One to four days after the last dose of chemotherapy, you will be given the ALVAC ESO-1 
vaccine and the anti-ESO-1 cells.  The ALVAC ESO-1 vaccine will be given as 4 injections, one 
in each extremity, approximately 2 hours before you receive the cells.  If one or two of your 
extremities is not suitable for vaccination, due to prior radiation, lymphedema or other cause, 
you may be given the vaccine in the stomach or chest. The anti ESO-1 cells will be given in your 
catheter over 30 minutes.  Within 24 hours after the anti-ESO-1 cell infusion, you will be given 
high dose IL-2 through your catheter. IL-2 is approved by the FDA for treatment of metastatic 
melanoma and metastatic renal cell cancer.  The purpose of giving the IL-2 with this therapy is to 
keep the cells we give you active for as long as possible so they will fight your tumor.  The IL-2 
will be given as a 15-minute infusion every 8 hours for up to five days after the cell infusion 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 6 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
(maximum number of doses is 15).  Doses may be skipped or delayed depending on how well 
you tolerate the doses.  The risks of the cells and IL-2 are described on the following pages. 
 
The day after you receive the anti-ESO-1 cells, we will give you more G-CSF (filgrastim) as a 
shot or injection under the skin. This will continue until your white blood cell counts begin to 
return to normal.   
 
Two weeks after the anti-ESO-1 cell infusion you will receive one more dose of the ALVAC 
ESO-1 vaccine, one injection in each of 4 extremities again.  If one or two of your extremities is 
not suitable for vaccination, due to prior radiation, lymphedema or other cause, you may be 
given the vaccine in the stomach or chest.  If you have already been discharged, you will receive 
this second vaccination during a clinic visit. 
 
Recovery 
After your last dose of IL-2, you will recover in the hospital until you are well enough to go 
home.  This usually takes 5 to 10 days. We will continue to give you support medications, do 
laboratory tests, and watch you closely for any side effects until we feel your condition is stable.  
 
In addition to the laboratory tests to monitor your condition, we will remove approximately 9 
teaspoons of blood three times per week to study the effects of this regimen on your immune 
system.  The maximum amount of blood for research is approximately 2.3 cups in 8 weeks. 
 
We may ask you to allow us to perform a biopsy (remove a small piece) of your tumor or lymph 
node after receiving the treatment to look at the effects of the treatment on the immune cells in 
your tumor.  However, this biopsy is not required for you to participate in this experimental 
study.  To obtain cells by a biopsy, a small area of skin is numbed with an anesthetic and a small 
piece of your tumor is removed, either with a needle or by a small cut in the tumor.  The area is 
covered with a bandage for a day or two, during which time we will ask you to keep it dry.  
 
Follow up and Evaluation of Experimental Regimen  
You will need to continue to take Bactrim, an antibiotic, for at least 6 months following your 
treatment. We will ask you to return to NIH 4 – 6 weeks after completing your regimen for 
evaluation.  This visit will probably take 2 days.   If your tumor shows evidence of shrinking, we 
will ask you to return for evaluation every month for several more months.  If your tumor 
appears to be growing, we will look for other investigational therapies you may be eligible for, or 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 7 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
refer you back to the care of your local physician.  At some of your follow up visits, you may 
undergo leukapheresis so that we can see the effect this therapy has had on your immune system 
and if the cells we gave you are still alive.  
 
Because we do not know the long term side effects of gene therapy, we will collect your blood 
over the next several years, frequently at first and then less frequently.  If you return to your 
referring physician after treatment here we will ask you to have your physician send your blood 
specimens here for this testing.   This testing will determine if the cells have grown or changed in 
your body.  We will test your blood immediately after you receive the cells, and then at 3, 6 and 
12 months (2 teaspoons each time).  If all of the tests are normal and show no change, we will 
collect blood from you every year after that to store in case you develop symptoms later. 
According to FDA requirements, we need you to return annually to the NIH for a physical 
examination for five years after you receive the cells.  After that time we will be sending you a 
questionnaire to get information regarding your health for the next ten years, for a total follow up 
time period of 15 years.  For this reason, we ask that you continue to provide us with a current 
address and telephone number, even after you complete this research study.   At the time of your 
death, no matter the cause, we may request permission for an autopsy in order to obtain vital 
information concerning the safety of this experimental treatment approach.  Please advise your 
family of this request. 
 
Retreatment 
If your tumor shrinks or disappears following the initial treatment and then stops shrinking or 
recurs, you may receive additional treatments if you tolerated the treatment well and if all the 
side effects have resolved.  If you are retreated, you will need to sign a new consent form.  The 
second treatment will not begin prior to  6- 8 weeks after your last dose of IL-2.  You may 
receive up to two retreatments.   
 
Risks or Discomforts of Participation 
The risks and discomforts of this research study can be significant.   This experimental treatment 
can lead to long-term decrease in your immune function.  It is also possible that you may loose 
your fertility following this experimental treatment. It is possible, although unlikely, that this 
experimental treatment may cause your death.  
 
During the leukapheresis procedure, you may have some tingling in your face and lips due to the 
medicine used to keep your blood from clotting during the procedure.  The nurses may give you 
a calcium containing antacid, like TUMS to chew that takes away this tingling.  Rarely, people 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 8 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
may experience lightheadedness or dizziness.  We ask that you eat prior to the procedure to 
prevent this.  Rare complications of this procedure are lowered blood pressure or bleeding. 
 
Discomfort due to a biopsy may include pain at the site of the biopsy, swelling, bruising, and 
infection. 
When IL-2 is given through a catheter, it can make you feel like you have the flu.  It can also 
cause confusion and mental status changes making you unable to make sound decisions.  Prior to 
beginning treatment, we will ask you to complete a Durable Power of Attorney so that a person 
of your choosing can make health care decisions for you in case you develop these side effects.  
In our experience giving IL-2 to over 2,000 patients we have found that these side effects go 
away within a few days of stopping the IL-2. 
 
ALVAC ESO-1 vaccine (gene therapy) (only for patients receiving the vaccine) 
This experimental vaccine is made with virus, which is used as a vaccine against disease in 
canaries.  It cannot reproduce in mammals, so it cannot cause disease in humans.  This specific 
vaccine has been given only to a few individuals before so we do not have much information 
about the side effects, however similar experimental vaccines have been given in other clinical 
trials and the following side effects have been observed: 
 
ALVAC ESO-1 Vaccine side effects 
Common 
Less common  
Rare 
 
Swelling of lymph 
nodes near the 
injection site 
 
Bruising, pain and 
redness 
(inflammation) at the 
injection sites 
 
 
Headache 
 
Tiredness 
 
Bumps caused by 
inflammation (granulomas) 
 
Skin ulcerations (which may 
require surgery to be 
removed) 
 
Fever and chills 
 
Muscle aches 
 
Rash, itchy skin 
 
Joint pain 
 
Allergic reaction 
Anti-ESO-1 Cell Infusion (gene therapy)  
The cells we will be giving you have a type of virus (retrovirus) put into them along with the 
anti-ESO-1 protein.  Although this retrovirus is not active, there is the rare possibility that it may 
cause infection.  The cells could also cause you to develop another type of cancer, such as 
leukemia or lymphoma.  These specific gene-modified cells have been given only to a few 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 9 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
individuals before so we do not have much information about the side effects.  Potential risks 
include:  
• Fever, chills and shortness of breath, which may last for a few hours (common) 
• Lung congestion  
• Autoimmune reaction such as loss of skin pigment (known as vitiligo) or 
inflammation of the eye (uveitis) which may require the use of steroid eye drops.  
• There is no data available at this time to guide us in how humans might respond to 
this type of cell infusion.  As this is a new experimental therapy, side effects that we 
do not anticipate that may cause your condition to deteriorate may be encountered.  
Any new information that becomes available during the course of this study will be 
shared with you. 
 
Risk of Cancer:  When retroviral vectors enter a normal cell in the body, the DNA of the vector 
inserts itself into the normal DNA in that cell; this process is called integration.  Most integration 
is expected to cause no harm to the cell or to the patient. However, there is a chance that there 
may be some regions of the normal human DNA where integration of the viral vector=s DNA 
may result in activation of neighboring genes.  For example, if one of these genes were a growth 
factor, this may cause uncontrolled division of the cell, resulting in a cancer.  This type of event 
has occurred in one animal study in mice where the vector integration site correlates with the 
occurrence of cancer in these mice.  Four instances of a similar event have been reported in four 
children who received a retroviral vector in an experimental gene therapy study for X-linked 
Severe Combined Immunodeficiency (SCID) conducted in France, not under the jurisdiction of 
the U.S. Food and Drug Administration (FDA).  While most of the children who participated in 
this clinical trial appear to have been cured of their disease, four children developed leukemia (a 
form of cancer of the blood) approximately after receiving the gene therapy treatment.  The first 
patient had extensive testing done to determine the cause of the leukemia.  A group of experts in 
this field have looked at all the test results, and concluded that the gene therapy caused the 
leukemia in the first child.  One of the children died as a result of their leukemia.    The risk of 
another cancer developing in you, including leukemia, is unknown, but you need to be aware of 
this possible risk.  To monitor you for this risk we will be testing your blood 3 months after cell 
infusion, then at 6 and 12 months, and then annually thereafter.  If we find that the cells we have 
given you grow out of control, chemotherapy will be given to you to kill the cells, given their 
risk of causing leukemia or a second cancer. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 10 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Medications 
The side effects of cyclophosphamide, fludarabine, IL-2 and some of the other medications you 
will receive are listed in the tables below.  
 
Cyclophosphamide and Fludarabine side effects 
Common 
Less Common 
Rare 
 
Changes in blood 
counts including: 
low red cell count 
(causing fatigue and 
shortness of breath), 
low platelet count 
(increasing the risk 
of bleeding and 
bruising), decrease 
in white blood cells 
(increasing the risk 
of infection and the 
need for treatment 
with antibiotics or 
other treatment)  
 
Loss of appetite, 
nausea, vomiting,  
 
Diarrhea, stomach 
pain 
 
Mouth sores 
 
Hair loss 
 
Fatigue 
 
Muscle or joint 
aches  
 
Bleeding 
 
Infection 
 
Bladder irritation with 
bloody urine 
 
Severe allergic 
reaction (difficulty 
breathing/swelling) 
 
Headache or dizziness 
 
Sweating 
 
Swelling of arms or 
legs 
 
Skin changes, rash, 
blisters  
 
Weakness 
 
Hearing loss 
 
Heart damage 
 
Lung damage 
 
Kidney damage 
 
Inflammation of the eye resulting 
in blindness 
 
Inflammation of nervous system 
resulting in death 
 
Epstein Barr Lymphoma (in 
patients who have not been 
exposed to the Epstein-Barr 
virus).  This can be fatal (One 
patient on another study died as a 
result of this.) 
 
Loss of fertility 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 11 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
 
Support Medications – side effects 
 
Common 
Less common 
Rare 
Filgrastim (To increase production of white blood cells) 
 
 
Bone Pain 
 
Severe headache 
 
Severe breathing problems 
 
Rupture of your spleen 
Bactrim (To prevent a specific type of pneumonia) 
 
 
 
Fever 
 
Nausea, vomiting,  
 
Skin rash with itching 
 
reduced number of 
white blood cells  
 
Allergic reaction 
 
Fluconazole:  (To prevent fungal infections)  
 
 
Headache 
 
Nausea, vomiting, 
diarrhea, abdominal pain 
 
Itching  
 
 
 
A skin disorder called Stevens 
Johnson Syndrome, which can 
be fatal 
 
Liver damage which may be 
permanent 
Acyclovir and Valacyclovir 
 
 
 
Temporary decrease 
in kidney function 
which may not cause 
any symptoms 
 
Nausea, vomiting, 
diarrhea, constipation  
 
Pain and irritation at 
place of injection 
 
Skin rash, hives, itching  
 
Tremors, dizziness, 
Confusion, seizures 
 
Fatigue  
 
Blood in the urine 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 12 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
 
IL-2 (aldesleukin) side effects 
Common 
Less common  
Rare 
 
Fever, chills, and fatigue   
 
Lowered platelet and red blood 
cell levels that may require 
transfusions  
 
Significant fluid retention 
causing weight gain (as much 
as 20 pounds).  
 
Low blood pressure 
 
Increased heart rate 
 
Low urine output 
 
Swelling in your extremities,  
 
Fluid in your lungs that can 
require  oxygen  
 
Dry mouth, nausea, vomiting 
and diarrhea; 
 
Rash, itching; and changes in 
skin or hair pigmentation, 
called vitiligo;   
 
Changes in mental status, 
including confusion, difficulty 
sleeping or vivid dreams; this 
can be severe and require 
sedation and monitoring in the 
ICU 
 
 
Decrease in thyroid 
function that may 
require daily thyroid 
hormone 
replacement; 
 
Abnormal kidney 
and liver function 
that can be severe;   
 
Abnormal heartbeats 
or low blood 
pressure that may 
require treatment in 
the ICU.  
 
Breathing problems 
which may need 
monitoring in ICU 
and insertion of a 
breathing tube 
 
 
Bowel perforation (a 
hole) requiring longer 
hospitalization or 
surgery. This is more 
common in patients who 
have previously received 
MDX-010. You will have 
a colonoscopy and biopsy 
before treatment if you 
have previously received 
MDX-010. 
 
Autoimmune disease, 
where your immune 
system attacks cells in 
organs of your body.  
Should this occur, you 
will be treated with 
steroids to stop the 
immune response. 
 
Damage to the heart 
muscle or heart attack 
 
Loss of blood flow to the 
extremities due to 
medicines used to treat 
very low blood pressure 
and shock. In one 
instance a patient had to 
have her lower arm 
amputated after treatment 
with these medicines. 
 
IL-2 is mixed with 
human albumin which 
could cause an allergic 
reaction or potentially 
transmit viral infections, 
although we have not had 
this occur. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 13 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Prior to and throughout this study you will undergo many tests to determine the size and extent 
of your tumor, as well as the impact of the treatment.  Multiple blood tests will be performed and 
some of your serum and lymphocytes will be stored for future testing.  Blood and tissue samples 
collected from you may be stored and used in the future to study scientific questions related to 
this protocol.  If there are any risks to you or your family associated with these future scientific 
studies which are not covered in this consent form, your consent will be obtained before such 
studies are performed. 
 
If your disease progresses or recurs after this experimental treatment, then you will no longer 
receive treatment in this protocol, though you may be eligible to be considered for other 
protocols at the National Cancer Institute, NIH or referred elsewhere for treatment.  
 
Alternative Approaches or Treatment 
If there are effective salvage regimens (regimens used when standard regimens have failed), you 
will be directed to undergo these regimens before participating in this protocol. 
Other options for treatment of your cancer include: 
• standard therapies (such as dacarbazine, or temozolomide for melanoma) 
• experimental vaccines;  
• experimental chemotherapies or biotherapies (such as ipilimumab or ticilimumab);  
• other combination therapies;  or 
• getting no treatment; getting comfort care, also called palliative care. This type of care 
helps reduce pain, tiredness, appetite problems, and other problems caused by cancer. It 
does not treat the cancer directly, but instead tries to improve how you feel. Comfort care 
tries to keep you as active and comfortable as possible. 
 
If you have kidney cancer, you will also have the option of being treated with sorafenib, 
sunitinib, and temsirolimus. These drugs are FDA-approved for kidney cancer, and have been 
shown to keep some people with kidney cancer alive, without the disease getting worse.  These 
drugs have different side effects than the drugs used in this study.  You can take these drugs by 
mouth at home.   
 
Potential Benefits of Participation 
It is possible that your tumors may shrink as a result of this experimental regimen, but it is not 
possible to predict whether this will occur. 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 14 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Research Subject's Rights 
You should understand that this study involves research and that your participation is voluntary. 
Unexpected or unforeseeable side effects may also occur. Your participation in this protocol may 
be terminated without your consent if your physician feels that it would not be safe for you to 
continue.  Any significant new findings that relate to this protocol will be discussed with you. 
 
The sponsor of this study is Dr. Steven A. Rosenberg.  Sanofi-Pasteur is providing the ALVAC 
vaccine.  Your records may be reviewed by NIH organizations and by organizations outside the 
National Institutes of Health, such as the company that supplies the drugs for the study and 
representatives of the US Food and Drug Administration. Every effort will be made to protect 
your privacy in any recording or reporting of this information.  
 
Conflict of Interest: 
The National Institutes of Health reviews NIH staff researchers at least yearly for conflicts of 
interest. 
The 
following 
link 
contains 
details 
on 
this 
process: 
http://ethics.od.nih.gov/forms/Protocol-Review-Guide.pdf. You may ask your research team for 
additional information or a copy of the Protocol Review Guide.  It is possible that the 
information obtained from your participation on this study may become valuable for commercial 
research and development purposes (including patentable inventions), which may be of 
significant benefit to society, the sponsor of this study, individual researchers or other third 
parties.  You will not receive direct financial benefit from such research and development.  The 
NIH and two of the investigators on the research team have developed the cell process being 
used in this research and have a patent pending.  This means that it is possible that the results of 
this study could lead to payments to NIH scientists and to the NIH.  By law, government 
scientists are required to receive such payments for their inventions.  If you refuse to participate 
or withdraw from the protocol or at the completion of the protocol, we will attempt to offer you 
participation in other NIH protocols if these are available, or will refer you to your home 
physician for further management. 
 
Optional Studies 
 
We would like to keep some of the specimens and data that is collected for future research.  
These specimens and data will be identified by a number and not your name.  The use of your 
specimens and data will be for research purposes only and will not benefit you.  It is also 
possible that the stored specimens and data may never be used.  Results of research done on your 
specimens and data will not be available to you or your doctor.  It might help people who have 
cancer and other diseases in the future. 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 15 of 17 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
If you decide now that your specimens and data can be kept for research, you can change your 
mind at any time. Just contact us and let us know that you do not want us to use your specimens 
and/or data.  Then any specimens that remains will be destroyed and your data will not be used 
for future research. 
 
Please read each sentence below and think about your choice. After reading each sentence, circle 
and initial the answer that is right for you.  No matter what you decide to do, it will not affect 
your care. 
1. My specimens and data may be kept for use in research to learn about, prevent, or treat cancer.  
 
Yes              No               Initials_________ 
2. My specimens and data may be kept for u 
se in research to learn about, prevent or treat other health problems (for example: diabetes, 
Alzheimer's disease, or heart disease).  
 
Yes              No               Initials_________ 
 
3. Someone may contact me in the future to ask permission to use my specimen(s) and/or data in 
new research not included in this consent.  
 
Yes              No               Initials_________ 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 16 of 17 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH 
STUDY (Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
 
OTHER PERTINENT INFORMATION 
1. 
Confidentiality.  When results of an NIH research study are reported in medical journals 
or at scientific meetings, the people who take part are not named and identified.  In most cases, 
the NIH will not release any information about your research involvement without your written 
permission.  However, if you sign a release of information form, for example, for an insurance 
company, the NIH will give the insurance company information from your medical record.  This 
information might affect (either favorably or unfavorably) the willingness of the insurance 
company to sell you insurance. 
The Federal Privacy Act protects the confidentiality of your NIH medical records.  However, you 
should know that the Act allows release of some information from your medical record without 
your permission, for example, if it is required by the Food and Drug Administration (FDA), 
members of Congress, law enforcement officials, or authorized hospital accreditation 
organizations. 
2. 
Policy Regarding Research-Related Injuries.  The Clinical Center will provide short-
term medical care for any injury resulting from your participation in research here.  In general, 
no long-term medical care or financial compensation for research-related injuries will be 
provided by the National Institutes of Health, the Clinical Center, or the Federal Government.  
However, you have the right to pursue legal remedy if you believe that your injury justifies such 
action. 
3. 
Payments.  The amount of payment to research volunteers is guided by the National 
Institutes of Health policies.  In general, patients are not paid for taking part in research studies at 
the National Institutes of Health.  Reimbursement of travel and subsistence will be offered 
consistent with NIH guidelines. 
4. Problems or Questions.  If you have any problems or questions about this study, or about 
your rights as a research participant, or about any research-related injury, contact the Principal 
Investigator Steven A. Rosenberg, M.D., Ph.D.; Building CRC, Room 3-3940, Telephone:  301-
496-4164.  If you have any questions about the use of your tissue for future research studies, you 
may also contact the Office of the Clinical Director at 301-496-4251. 
You may also call the Clinical Center Patient Representative at (301) 496-2626. 
5. 
Consent Document.  Please keep a copy of this document in case you want to read it 
again. 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 08-C-0121 
CONTINUATION: page 17 of 17 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH 
STUDY (Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
 
 
 
COMPLETE APPROPRIATE ITEM(S) BELOW: 
A. Adult Patient’s Consent 
B. Parent’s Permission for Minor Patient. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby consent to 
take part in this study. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby give 
permission for my child to take part in this 
study.   
(Attach NIH 2514-2, Minor’s Assent, if 
applicable.) 
 
  
  
 
 
 
Signature of Adult Patient/ 
Legal Representative 
Date 
Signature of Parent(s)/ Guardian Date 
 
 
Print Name 
 
 
Print Name 
C. Child’s Verbal Assent (If Applicable) 
The information in the above consent was described to my child and my child agrees to 
participate in the study. 
 
  
  
 
Signature of Parent(s)/Guardian 
Date 
Print Name 
THIS CONSENT DOCUMENT HAS BEEN APPROVED FOR USE  
FROM JUNE 23, 2016 THROUGH JUNE 22, 2017. 
 
 
 
 
 
 
 
 
Signature of Investigator 
Date 
Signature of Witness 
Date 
 
 
Print Name 
 
 
Print Name