Clinical trial NCT01369875
Conditions: Metastatic Melanoma Skin Cancer
Keywords: Immunotherapy Cell Therapy Metastatic Cancer Melanoma Metastatic Melanoma Skin Cancer
Sponsor: National Cancer Institute (NCI)
Start date: 2011-06-17; Completion date: 2012-02-14
Enrollment: 2 participants; Allocation: NON_RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
Background:

\- Tumor infiltrating lymphocytes (TIL) are white blood cells that have been taken from tumor tissue. The cells are modified to help them kill tumor cells, then given back to the person with cancer. By giving these cells to patients, researchers hope to improve the current treatments available for patients with melanoma that has not responded to standard therapies. The TIL will be given after treatments that will suppress the immune system. This makes it easier for the TIL to attack the cancer cells. The TIL will also be given with aldesleukin (IL-2), which is designed to help keep the TIL cells alive in the body.

Objectives:

\- To study the safety and effectiveness of specially modified tumor infiltrating lymphocytes to treat melanoma that has not responded to other treatments.

Eligibility:

\- Individuals at least 18 years of age who have metastatic melanoma that has not responded to other treatments.

Design:

* Participants will be screened with a physical exam and medical history. They will also have blood tests and imaging studies.
* A piece of tumor will be collected and white blood cells will be separated to make the TIL for the treatment.
* Participants will take drugs to suppress the immune system for 7 days before the start of treatment.
* Participants will receive the TIL in a single dose. Then they will receive IL-2 every 8 hours for up to 15 doses. Participants will remain in the hospital for up to 2 weeks after treatment. They will be monitored with frequent blood tests and other studies.
* After leaving the hospital, participants will have regular followup visits every 1 to 4 months for the first year. Then they will return for followup every 3 to 4 months, as directed by the study researchers.

Detailed description:
Background:

* Tumor Infiltrating Lymphocyte (TIL) administration and high dose interleukin (IL)-2 following lymphodepletion can mediate durable complete responses in patients with refractory melanoma. Obstacles to administration of this therapy include failure to establish TIL in vitro for about 20% of patients, long delays between tumor resection and TIL establishment resulting in poor TIL attributes for therapy and patient ineligibility due to progression, and requirements for large numbers of feeder cells for TIL expansion to therapeutic numbers.
* The K562 cell line was engineered to express the 4-1BBL costimulatory molecule and CD64 (the high affinity Fc receptor for loading with antibodies such as OKT3). The K562.CD64.4-1BBL Engineered Cells with Costimulation Enhancement (ECCE) replaced up to 75% of feeder cells in large scale TIL expansions. ECCE added to tumor cell suspensions provided costimulation in trans resulting in rapid and reliable lymphocyte growth even from tumors with no TIL growth under standard conditions.
* A cloned K562.CD64.4-1BBL-7F11 ECCE line was produced under Good Manufacturing Practice (GMP) conditions and a master cell bank has been generated. An optimized protocol was established to rapidly expand (REP) young TIL using minimum feeders and 7F11. These ECCE REPed TIL retained tumor recognition and some other attributes of standard TIL, but differed from standard TIL by containing fewer CD4+ cells and more natural killer cells.

When 7F11ECCE were added directly to single cell tumor suspensions, young TIL cultures were reliably generated even from patients who otherwise would not have a standard young TIL culture for treatment.

Objectives:

Primary objectives:

* In cohort 1, to evaluate whether young TIL that are rapidly expanded using 7F11 ECCE to replace some feeder cells and administered with IL-2 in patients following a non -myeloablative conditioning regimen will result in clinical tumor regression in patients with refractory metastatic melanoma.
* In Cohort 2, to evaluate whether young TIL generated using in trans costimulation with 7F11 ECCE in patients for whom standard young TIL did not grow can mediate tumor regression after a nonmyeloablative conditioning with high dose IL-2 in patients with refractory metastatic melanoma.
* Determine the toxicity of ECCE young TIL in these treatment regimens

Eligibility:

Patients who are 18 years of age or older must have:

* metastatic melanoma;
* Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1
* One or more lesions 2 cm or greater suitable for resection for TIL culture

Patients may not have:

* Concurrent major medical illnesses;
* Any form of immunodeficiency;
* Severe hypersensitivity to any of the agents used in this study;
* Contraindications for high dose IL-2 administration.

Design:

* Patients will undergo resection to obtain tumor for generation of autologous young TIL cultures.
* Parallel TIL cultures will be established using a) the standard technique with IL-2 only and b) the Engineered Cells with Costimulation Enhancement (ECCE) protocol using irradiated K562.CD64.4-1BBL-7F11 (7F11) cells.
* After 10 to 20 days cultures will undergo evaluation for TIL establishment. Standard TIL will be used preferentially and patients who have TIL established by standard methods will be assigned to Cohort 1
* Cohort 1:

  * TIL will undergo ECCE REP by exposure to OKT-3, IL-2, feeder cells and irradiated 7F11.
  * Patients will receive a non-myeloablative lymphocyte depleting preparative regimen of cyclophosphamide (60 mg/kg/day intravenous (IV)) on days -7 and -6 and fludarabine (25 mg/m\^2/day IV) on days -5 through -1.
  * On day 0 patients will receive the infusion of autologous young TIL and then begin highdose aldesleukin (720,000 IU/kg IV every 8 hours for up to 15 doses).
  * Clinical and immunologic response will be evaluated about 4-6 weeks after treatment.
  * Using an optimal two-stage Phase II design, initially 18 patients will be enrolled, and if three or more of the first 18 patients have a clinical response (partial response (PR) or complete response (CR)), accrual will continue to 35 patients, targeting a 30% goal for objective response.
* If standard young TIL fail to grow then ECCE young TIL will be evaluated and patients who have ECCE TIL available will be assigned to Cohort 2 .
* Cohort 2:

  * Cultures from patients in Cohort 2 will be evaluated for ECCE TIL establishment. If adequate ECCE TIL are available, TIL will undergo ECCE REP by exposure to OKT-3, IL-2, feeder cells and irradiated 7F11.
  * Patients will receive a non-myeloablative lymphocyte depleting preparative regimen of cyclophosphamide (60 mg/kg/day IV) on days -7 and -6 and fludarabine (25 mg/m\^2/day IV) on days -5 through -1.
  * On day 0 patients will receive the infusion of autologous young TIL and then begin highdose aldesleukin (720,000 IU/kg IV every 8 hours for up to 15 doses).
  * Clinical and immunologic response will be evaluated about 4-6 weeks after TIL infusion.
  * Using a small optimal two-stage Phase II design, initially 9 patients will be enrolled, and if one or more of the first 9 patients has a clinical response (PR or CR), accrual will continue to 24 patients, targeting a 25% goal for objective response.
* If TIL cultures were not established by either standard methods or ECCE young TIL protocols, patients will be eligible for re-resection and evaluation of TIL from a different site.

Arms: arm 1: Tumor Infiltrating Lymphocytes : intravenous (IV) over 30 minutes on day 0

Aldesleukin : 720,000 IU/kg IV over 15 min every 8 hours (+/- 1hr) beginning within 24 hours of cell infusion and continuing for up to 5 days (max. 15 doses.)

Fludarabine : 25 mg/m2/day intravenous piggy back (IVPB) daily over 30 minutes for 5 days (days -5 to -1)

Cyclophosphamide : 60 mg/kg/day X 2 days IV over 1 hour on days -7 and -6 arm 2: Tumor Infiltrating Lymphocytes : IV over 30 minutes on day 0

Aldesleukin : 720,000 IU/kg IV over 15 min every 8 hours (+/- 1hr) beginning within 24 hours of cell infusion and continuing for up to 5 days (max. 15 doses.)

Fludarabine : 25 mg/m2/day IVPB daily over 30 minutes for 5 days (days -5 to -1)

Cyclophosphamide : 60 mg/kg/day X 2 days IV over 1 hour on days -7 and -6
Interventions: intervention 1: Cyclophosphamide intervention 2: Fludarabine intervention 3: Aldesleukin intervention 4: Tumor Infiltrating Lymphocytes
Intervention descriptions: intervention 1: 60 mg/kg/day X 2 days intravenous (IV) over 1 hour on days -7 and -6 intervention 2: 25 mg/m\^2/day intravenous piggyback (IVPB) daily over 30 minutes for 5 days (days -5 to -1) intervention 3: 720,000 IU/kg intravenous (IV) over 15 min every 8 hours (+/- 1hr) beginning within 24 hours of cell infusion and continuing for up to 5 days (max. 15 doses.) intervention 4: Intravenous (IV) over 30 minutes on day 0

Protocol documents:
Prot_SAP_000.pdf: 
11-C-0163 
1
ECCE TIL 
CC Protocol Number: 11-C-0163C 
NCI Protocol Number: P10790 
PROTOCOL TITLE 
Phase II Study of Tumor Infiltrating Lymphocytes Generated with Engineered Cells for 
Costimulation Enhancement in Patients with Metastatic Melanoma Following Lymphodepletion. 
 
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:   
Mark E. Dudley, Ph.D., Surgery Branch, NCI 
Steven A. Feldman, Ph.D., Surgery Branch, NCI  
Marybeth Hughes, 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 
Giao Phan, M.D., Surgery Branch, NCI  
Kathleen E. Morton, R.N., Surgery Branch, NCI 
Marie Thompson, R.N., Surgery Branch, NCI 
Debbie Ann Nathan, R.N., Surgery Branch, NCI 
Carolyn M. Laurencot, Ph.D., Surgery Branch, NCI 
Donald E. White, M.S., Surgery Branch, NCI 
Seth M. Steinberg, Ph.D., BDMS, NCI+ 
Mei Li Marie Kwong, M.D., Surgery Branch, NCI 
Jennifer Tseng, M.D., Surgery Branch, NCI 
Caroline Butler, M.D., Surgery Branch, NCI 
 
+Biostatistics and Data Management Section, Center for Cancer Research 
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 
 
Drug 
Name: 
ECCE Young TIL 
IND 
Number: 
IND 14265 
Sponsor: 
Steven A. Rosenberg, M.D., Ph.D. 
IRB Submission Date:  December 27, 2010 
Version Date:  July 2, 2012 


11-C-0163 
2
 
Précis: 
 
Background:
x
Tumor Infiltrating Lymphocyte (TIL) administration and high dose interleukin (IL)-2 following 
lymphodepletion can mediate durable complete responses in patients with refractory melanoma.  
Obstacles to administration of this therapy include failure to establish TIL in vitro for about 20% 
of patients, long delays between tumor resection and TIL establishment resulting in poor TIL 
attributes for therapy and patient ineligibility due to progression, and requirements for large 
numbers of “feeder” cells for TIL expansion to therapeutic numbers.   
x
The K562 cell line was engineered to express the 4-1BBL costimulatory molecule and CD64 (the 
high affinity Fc receptor for “loading” with antibodies such as OKT3).  The K562.CD64.4-1BBL  
Engineered Cells with Costimulation Enhancement (ECCE) replaced up to 75% of feeder cells in 
large scale TIL expansions. ECCE added to tumor cell suspensions provided costimulation “in 
trans” resulting in rapid and reliable lymphocyte growth even from tumors with no TIL growth 
under standard conditions.   
x
A cloned K562.CD64.4-1BBL-7F11 ECCE line was produced under GMP conditions and a 
master cell bank has been generated.  An optimized protocol was established to rapidly expand 
(REP) young TIL using minimum feeders and 7F11.  These ECCE REPed TIL retained tumor 
recognition and some other attributes of standard TIL, but differed from standard TIL by 
containing fewer CD4+ cells and more natural killer cells.    
When 7F11ECCE were added directly to single cell tumor suspensions, young TIL cultures were 
reliably generated even from patients who otherwise would not have a standard young TIL 
culture for treatment.  
Objectives: 
Primary objectives:  
x
In cohort 1, to evaluate whether young TIL that are rapidly expanded using 7F11 ECCE to 
replace some feeder cells and administered with IL-2 in patients following a non -myeloablative 
conditioning regimen will result in clinical tumor regression in patients with refractory metastatic 
melanoma. 
x
In Cohort 2, to evaluate whether young TIL generated using “in trans” costimulation with 7F11 
ECCE in patients for whom standard young TIL did not grow can mediate tumor regression after 
a nonmyeloablative conditioning with high dose IL-2 in patients with refractory metastatic 
melanoma.   
x
Determine the toxicity of ECCE young TIL in these treatment regimens 
 
Eligibility: 
Patients who are 18 years of age or older must have  
x
metastatic melanoma; 
x
ECOG performance status 0 or 1 
x
One or more lesions 2 cm or greater suitable for resection for TIL culture 
Patients may not have: 
x
Concurrent major medical illnesses; 
x
Any form of immunodeficiency; 
x
Severe hypersensitivity to any of the agents used in this study; 
x
Contraindications for high dose IL-2 administration. 


11-C-0163 
3
Design: 
x
Patients will undergo resection to obtain tumor for generation of autologous young TIL cultures. 
x
Parallel TIL cultures will be established using a) the standard technique with IL-2 only and b) the 
Engineered Cells with Costimulation Enhancement (ECCE) protocol using irradiated 
K562.CD64.4-1BBL-7F11 (7F11) cells. 
x
After 10 to 20 days cultures will undergo evaluation for TIL establishment. Standard TIL will be 
used preferentially and patients who have TIL established by standard methods will be assigned 
to “Cohort 1”   
x
Cohort 1:  
o
TIL will undergo ECCE REP by exposure to OKT-3, IL-2, feeder cells and irradiated 
7F11.   
o
Patients will receive a non-myeloablative lymphocyte depleting preparative regimen of 
cyclophosphamide (60 mg/kg/day IV) on days -7 and -6 and fludarabine (25 mg/m2/day 
IV) on days -5 through -1.   
o
On day 0 patients will receive the infusion of autologous young TIL and then begin high-
dose aldesleukin (720,000 IU/kg IV every 8 hours for up to 15 doses).     
o
Clinical and immunologic response will be evaluated about 4-6 weeks after treatment. 
o
Using an optimal two-stage Phase II design, initially 18 patients will be enrolled, and if 
three or more of the first 18 patients have a clinical response (PR or CR), accrual will 
continue to 35 patients, targeting a 30% goal for objective response.   
x
If standard young TIL fail to grow then ECCE young TIL will be evaluated and patients who 
have ECCE TIL available will be assigned to “Cohort 2”.   
x
Cohort 2: 
o
Cultures from patients in Cohort 2 will be evaluated for ECCE TIL establishment.  If 
adequate ECCE TIL are available, TIL will undergo ECCE REP by exposure to OKT-3, 
IL-2, feeder cells and irradiated 7F11.   
o
Patients will receive a non-myeloablative lymphocyte depleting preparative regimen of 
cyclophosphamide (60 mg/kg/day IV) on days -7 and -6 and fludarabine (25 mg/m2/day 
IV) on days -5 through -1.   
o
On day 0 patients will receive the infusion of autologous young TIL and then begin high-
dose aldesleukin (720,000 IU/kg IV every 8 hours for up to 15 doses).     
o
Clinical and immunologic response will be evaluated about 4-6 weeks after TIL infusion. 
o
Using a small optimal two-stage Phase II design, initially 9 patients will be enrolled, and 
if one or more of the first 9 patients has a clinical response (PR or CR), accrual will 
continue to 24 patients, targeting a 25% goal for objective response.  
x
If TIL cultures were not established by either standard methods or ECCE young TIL protocols, 
patients will be eligible for re-resection and evaluation of TIL from a different site.  


11-C-0163 
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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 ................................................................................... 13
2.1. Eligibility Criteria ............................................................................................................. 13
2.2. Research Eligibility Evaluation ....................................................................................... 15
2.3. Patient Registration .......................................................................................................... 15
3.0
Study Implementation ............................................................................................................. 16
3.1. Study Design ...................................................................................................................... 16
3.2. Drug Administration ........................................................................................................ 17
3.3. On-Study Evaluation ........................................................................................................ 20
3.4. Post Study Evaluation (Follow-up) ................................................................................. 22
3.5. Off Treatment Criteria ..................................................................................................... 22
3.6. Off Study Criteria ............................................................................................................. 23
4.0
Supportive Care ...................................................................................................................... 23
5.0
Data Collection and Evaluation .............................................................................................. 23
5.1. Response Criteria .............................................................................................................. 23
5.2. Toxicity Criteria ................................................................................................................ 25
5.3. Statistical Section .............................................................................................................. 27
5.4. Clinical Trial Monitoring Plan ........................................................................................ 28
5.5. Data and Safety Monitoring Plan .................................................................................... 28
5.6. Handling of Tissue Specimens for Research Purposes .................................................. 29
6.0
Human Subjects Protections ................................................................................................ 29
6.1.
Rationale for Patient Selection ............................................................................................ 29
6.2.
Participation of Children ..................................................................................................... 30
6.3.
Evaluation of Benefits and Risks ......................................................................................... 30
6.4.
Consent Document ................................................................................................................ 30
7.0
Data Reporting ...................................................................................................................... 30
7.1
Definitions .......................................................................................................................... 31
7.2
Routine Data Reporting: .................................................................................................. 32
7.3
Expected Adverse Events and Protocol-Specific Expedited Adverse Event Reporting 
Exclusions: ......................................................................................................................................... 32
7.4
NCI IRB Reporting Requirements .................................................................................. 33
7.5
Serious Adverse Event Reporting to the FDA: .............................................................. 34
8.0 
Pharmaceutical Information ................................................................................................ 34
8.1
Interleukin-2 (Aldesleukin, Proleukin, Recombinant Human Interleukin 2) ............. 34
8.2  Fludarabine ....................................................................................................................... 35
8.3
Cyclophosphamide ............................................................................................................ 36
8.4
Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891) .......... 37
8.5  Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim, Neupogen)
 
37
8.6  Trimethoprim and Sulfamethoxazole Double Strength (TMP / SMX DS) ................. 37
8.7
Herpes Virus Prophylaxis ................................................................................................ 37
8.8  Fluconazole ........................................................................................................................ 38
8.9
Cell Preparation ................................................................................................................ 38


11-C-0163 
5
8.10 OKT3 .................................................................................................................................. 39
8.11  Support Medications ......................................................................................................... 39
9.0
References ............................................................................................................................... 40
10.0
Tables, Figures, and Appendices ............................................................................................ 45
 


11-C-0163 
6
 
1.0 Introduction 
 
 
1.1. 
Study Objectives
1.1.1. Primary objectives:  
x
In cohort 1, to determine whether the administration of young TIL generated 
with the ECCE REP process in patients receiving a non myeloablative 
conditioning regimen plus IL-2 will result in clinical tumor regression in patients 
with metastatic melanoma. 
x
In Cohort 2, to evaluate whether ECCE young TIL from patients for whom 
standard young TIL did not grow can mediate tumor regression after a 
nonmyeloablative conditioning with high dose IL-2 in patients with metastatic 
melanoma.   
1.1.2. Secondary objective: 
x
Determine the toxicity of young TIL expanded with the use of ECCE in these 
treatment regimens 
 
 
1.2. 
Background and Rationale
1.2.1. Surgery Branch, NCI Studies of Tumor Infiltrating Lymphocytes for the
Treatment of Patients with Metastatic Melanoma
Melanoma is the sixth leading cancer in both men and women1. Metastatic 
melanoma has a poor prognosis with five-year survival of less than 5%.  FDA-
approved treatments for metastatic melanoma include aldesleukin and dacarbazine 
chemotherapy.  Aldesleukin has an objective clinical response rate of about 16% and 
a complete response rate of 6%2.  It is the only approved treatment known to cure 
some patients with metastatic melanoma.  Dacarbazine-based chemotherapy has a 
clinical response rate of up to 20% but few complete responders or long-term 
survivors3.  With the incidence of melanoma increasing in the United States, more 
effective treatment for metastatic melanoma is needed.  Previous studies in animal 
models demonstrated that the cellular arm of the immune system plays an important 
role in tumor surveillance and may be recruited to destroy tumor4.  Therefore, most 
therapeutic strategies focusing on immunotherapy against metastatic melanoma have 
focused on the ability of effector T-cells to mediate tumor destruction. 
The Surgery Branch of the National Cancer Institute has pioneered Adoptive 
Cell Therapy (ACT) for the treatment of patients with metastatic melanoma.  We 
have reported the results of ACT therapy in 93 patients with metastatic melanoma 
who received autologous TIL following a lymphodepleting regimen plus aldesleukin 
administration5.  Forty-three patients received a non-myeloablative chemotherapy 
consisting of 60 mg/kg cyclophosphamide qd x 2 and 25mg/m2 fludarabine qd x 5 
prior to cell transfer and aldesleukin administration.  Twenty-five patients each also 
received the same chemotherapy agents in conjunction with either 200 or 1200 cGy 
whole body irradiation prior to cell infusion and aldesleukin administration.  The 
overall objective response rate using RECIST criteria in these 93 patients was 56%.  


11-C-0163 
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The clinical results in these three trials are shown in Table 1, and the toxicities shown 
in Table 2 for the total body irradiation (TBI) studies (04-C-0288, 06-C-0136) and 
Table 3 for the initial study without TBI (99-C-0158).  There was one treatment 
related death in these 93 patients which occurred in a patient who had an undetected 
diverticular abscess prior to beginning therapy.  There appeared to be an increase in 
survival with increasing lymphodepletion although the consecutive nature of these 
non-randomized trials and possible selection bias of patients entering the trials cast 
doubt concerning the validity of this conclusion (Figure 1).  Of the 52 responding 
patients in this trial, 42 were previously shown to be refractory to aldesleukin therapy 
and 21 were refractory to prior aldesleukin plus chemotherapy. 
In recent studies we have simplified the techniques for the generation of 
human TIL from melanomas.  In the initial report of the treatment of melanoma 
patients with adoptive transfer of TIL in the absence of lymphodepletion, we noted 
that patients treated with TIL from younger cultures had a higher frequency of 
clinical response to treatment than patients treated with TIL from older cultures (p = 
0.0001)6.  More recent studies in patients receiving rapidly expanded TIL after 
lymphodepletion support these studies 7.  Additional experiments examining the role 
of telomere length of the TIL cultures in the 93 patients who received selected TIL 
support this hypothesis.  Differentiation and expansion of T cells is associated with 
telomere shortening and with altered and loss of cell functions, including 
proliferation.  Zhou et. al. reported that TIL which were associated with objective 
clinical responses in treated patients had longer telomere lengths than TIL that were 
not associated with clinical responses (p <  0.01)8.  TIL which persisted in vivo had 
longer telomere lengths than TIL which did not persist (p < 0.001). 
The hypothesis that younger TIL have improved in vivo survival and anti-
tumor activity was corroborated by studies demonstrating that younger T cells have 
phenotypes with more favorable characteristics for in vivo persistence and antitumor 
activity.  CD27 and CD28 are associated with proliferation and survival of T cells and 
are often used as markers of less-differentiated T cells.  Huang, et al reported that T-
cell clonotypes with long-term in vivo persistence expressed higher levels of CD27 
and CD28 than clonotypes with short-term persistence9.  In a murine model of 
adoptive immunotherapy, Gattinoni et al demonstrated that the differentiation of 
effector T cells in culture had a negative effect on their capacity to function in vivo, 
and T cells expressing higher levels of CD27 had better in vivo antitumor activity 
than CD27-low cells10. 
We thus developed an approach to rapidly generate TIL for treatment that 
involved growth of the entire complement of infiltrating lymphocytes for about two 
weeks and then expanding them to numbers sufficient for cell treatment.  We called 
these cells “young” TIL11.  Matched pairs of young TIL (mean age = 14 days) and 
standard TIL (mean age = 31 days) were generated from 14 tumor specimens.  
Expression of CD27 and CD28 as well as CD3, CD4, and CD8 were measured in 
both sets of TIL by fluorescence-activated cell sorting (FACS).  Young TIL had 
significantly higher expression of CD27 (p < 0.0001) and CD28 (p < 0.003) than 
standard TIL.  In addition, FACS analysis of gated CD8+ cells from these TIL 
populations demonstrated a strong relationship between culture age and the 
expression of CD27 and CD28.  These observations suggested that younger effector T 


11-C-0163 
8
cells have phenotypic expression of CD27 and CD28 which may indicate better in 
vivo proliferation, survival, and antitumor activity compared to older effector T cells. 
An additional potential advantage of the use of this simpler method of producing TIL 
is that they were more heterogeneous in their reactivity than highly selected TIL 
which have grown for longer periods of time.   
 
A clinical trial is being performed in the Surgery Branch, NCI utilizing these 
young TIL to treat patients with metastatic melanoma (07-C-0176).  Twenty-four 
patients were treated in the initial  arm with rapidly expanded young TIL following 
non-myeloablative lymphodepleting chemotherapy (cohort 1).   Five patients in this 
cohort have had confirmed clinical responses, four partial responses of 35+, 13, 4, 
and 3 months , and one complete response of 26+ months duration.  The toxicities  
observed in cohort 1 were mostly expected and related to chemotherapy and 
aldesleukin, but the incidence of serious (grade 3-4) adverse events was higher than 
expected with a total of 7 patients who were intubated and two patients who required 
CVVH support.  The protocol was amended to investigate the hypothesis that the 
removal of CD4+ cells from the infusion would reduce the toxicity and improve the 
effectiveness of the therapy.  CD8+ cell enrichment was performed and 39 patients 
have now been treated with CD8+ enriched young TIL cells  following non-
myeloablative (NMA) lymphodepletion (cohort 2), and an additional 23 patients have 
been treated with CD8+ enriched young TIL plus 600 cGy of total body irradiation as 
a preparative regimen (cohort 3).   In cohort 2, three patients were intubated, and in 
cohort 3, two patients were intubated.  One patient in each of these cohorts died, both 
from sepsis during the neutropenic period.  In addition, one patient in cohort 2 
experienced a subarachnoid bleed from a known brain metastasis and recovered 
without sequelae and is an ongoing responder, and one patient in cohort 3 
experienced renal microangiopathy that resolved. In cohort 2, 18 patients have 
confirmed PRs, and 3 patients have confirmed CRs for an objective response rate in 
the 36 evaluable patients of 58% in this cohort.  Two CRs (26+ and 22+) and 2 of the 
PRs (24+ and 22+) are currently ongoing. In Cohort 3, six patients have confirmed 
partial responses (one on-going at 17+ months), and four patients have confirmed 
complete responses (2 ongoing at 17+ and 16+ months ) for an objective response 
rate in the 22 evaluable patients of 45 % in this cohort. It thus appears that the 
administration of CD8 enriched “young TIL” plus IL-2 following lymphodepleting 
chemotherapy represents a simplified safe and effective treatment for patients with 
metastatic melanoma 
In order to definitively assess the role of CD8 enrichment in potentially 
reducing toxicities and increasing response rates without patient selection bias, in 
November 2009, an amendment was approved in which cohorts 1-3 were closed to 
accrual and cohorts 4 (identical to cohort 1- Young TIL) and 5 (identical to cohort 2- 
CD8 enhanced Young TIL) were opened with accrual to both cohorts occurring in a 
randomized fashion.  To date, 25 patients have been enrolled in cohort 4 and 25 
patients have been enrolled in cohort 5.  It is still too early to evaluate clinical 
response in most patients treated in cohorts 4 and 5.  At this time, in cohort 4, one 
patient has a confirmed complete response, five patients have confirmed partial 
responses and eleven patients were taken off study for disease progression.  In cohort 
5, two patient have ongoing confirmed complete responses, four patients have 


11-C-0163 
9
confirmed partial responses and twelve patients were taken off study for disease 
progression.    No patients in these two cohorts received CVVH and one patient in 
cohort 5 was intubated.  The adverse events observed in patients in these two cohorts 
are comparable. 
 
1.2.2. Rationale for the Use of Engineered Cells with Costimulatory 
Enhancement (ECCE) in TIL Generation.
A major problem for standardization and dissemination of TIL protocols and 
for individual patients is attrition from TIL protocols after surgery.  Between 2002 
and 2007, only 27% of the 386 patients who underwent resection of a metastatic 
melanoma in the Surgery Branch with intent to generate TIL were ultimately treated 
with their antigen-reactive TIL12. Careful retrospective review of all patient charts 
revealed that for 147 patients (38%) an appropriate TIL culture could not be obtained.  
Process improvements implemented with the “young TIL” protocol included more 
patients in the pool that were eligible for treatment.  From 2007 to 2009, 122 
consecutive patients were resected with intent to generate minimally cultured TIL, 
and 21 patients (17%) failed to generate adequate TIL for therapy13.  Retrospective 
analysis of these samples demonstrated that patients whose TIL did not grow in vitro 
had a low level of lymphocytic infiltration in their tumors, with  a median of only 8 % 
lymphocytes in the initial single cell suspension (Figure 2).  Further improvement to 
protocol accrual based on generation of a TIL product for therapy will require new 
approaches that yield therapeutic lymphocyte cultures from tumors with low 
lymphocyte infiltrates. 
Another source of attrition involves patients for whom TIL were obtained but 
therapy was not administered due to disease progression or protocol specific 
ineligibilities subsequent to tumor resection. This category included 62 of 386 
patients (16%) set up for selected TIL through 2007, and 20 of the 122 patients (16%) 
patients resected for generation of minimally cultured TIL between 2007 and 2009.  
Reducing protocol attrition due to patient progression will require further protocol 
simplification and elimination of hurdles to rapid patient treatment.  A significant 
delay in cell production arises due to securing adequate peripheral blood mononuclear 
cells (PBMC) for “feeders” in the rapid expansion protocol (REP).  Large patient 
volumes made recruitment of adequate normal donors to support REPs difficult. 
Recent Surgery Branch efforts have focused on quantifying, standardizing, and 
comparing autologous and allogeneic feeder cells for clinical lymphocyte expansion.  
No contribution of the genotype of the feeder cells (autologous vs. allogeneic cells) 
toward either total expansion of the TIL or to the ultimate clinical response in the 
patient (Figure 3) was found.  Further simplifications to autologous feeder collection 
could shorten the time between resection and treatment for individual patients, and 
help disseminate ACT clinical protocols using TIL.   
To minimize the use of feeder cells in the rapid expansion protocol (REP), and 
maximize the establishment of TIL cultures from tumors with limited lymphocytic 
infiltration, we examined the requirements for optimal human CD8+ lymphocyte 
proliferation. Prior published research and experience in the Surgery Branch focused 
our efforts in two areas:  the molecular requirements comprising “costimulation” and 


11-C-0163 
10
a vehicle for presenting surface-bound signals.  Costimulatory signals in T cell 
expansion and activation are critical; however, optimal signals for expanding CD8+ 
memory cells without driving them to exhaustion are not known.  Good candidates 
include members of the B7 family (CD80, CD86, B7-H1) or TNF family (4-1BBL, 
OX40L, CD70).  Several cellular and inert vehicles for presenting costimulation to 
lymphocytes usually referred to as “artificial antigen presenting cells” have been 
studied preclinically and clinically.  These include inert magnetic beads14; insect 
cells15; mouse fibroblast cells16; and the human erythromyeloid cell line K56217;18.  
Literature review and empirical observations with all these costimulatory molecules 
and presentation vehicles led us to pursue 4-1BBL and the K562 cell line for clinical 
applications. 
 K562 is a cell line that grows in suspension and can be indefinitely 
propagated in culture, and is easy to manipulate genetically because it is readily 
transduced by retroviruses in vitro.  The K562 cell is also well suited for use as an 
immune stimulator because it is deficient in HLA class I and II expression 
minimizing endogenous anti-K562 responses, but endogenously expresses high levels 
of the lymphocyte adhesion molecules ICAM-1 (CD54), and LFA-1 (CD58) so it 
optimizes engineered immune activity19.  These K562 attributes have been exploited 
by many investigators for artificial antigen presentation in preclinical studies17-20.  
Additionally, several clinical studies have been approved using K562 derived cell 
lines as an accessory reagent for cellular therapy manufacturing (Butler, Zhang, 
unpublished communication) or directly injected into pateints as a GM-CSF secreting 
vaccine, GVAX21;22.  These physical and immunological attributes and the extensive 
preclinical and clinical experience with the K562 cell line make it an ideal cellular 
vehicle for optimizing TIL proliferation.  
T cell proliferation is dependent on T cell receptor (TCR) engagement, 
gamma chain cytokine signaling (e.g. interleukin-2), and optimal costimulation.  In 
mouse, the CD28/CD80 interaction is optimal for immune activation and T cell 
function.  However, the CD28 does not have a dominant impact on human CD8+ T 
cells.  Recent studies have implicated 4-1BB to be a more potent stimulatory pathway 
for CD8 cells in vitro than CD2823.  4-1BBL expression has been reported “in trans”, 
meaning that expression of 4-1BBL and antigen do not have to exist on the same cell 
for antigen-specific costimulation to occur24.   The high activity and relative 
specificity of 4-1BBL for CD8+ lymphocytes made this costimulatory molecule a 
strong candidate for further investigation in promoting melanoma TIL expansion.   
 
1.2.3. Construction of Engineered Cells with Costimulatory Enhancement
(ECCE) using K562 with CD64 and 4-1BBL and Their Use in Establishing 
Young TIL Cultures 
 
 
Engineered cells with costimulatory enhancement (ECCE) for young TIL 
cultures were based on K562 obtained from the American Type Culture Collection 
(ATCC). The K562 line was verified to be mycoplasma and microbial pathogen free, 
and was adapted to rapid growth in GMP approved media.  K562 cells were first 
retrovirally transduced to express CD64 (the high affinity Fc receptor), then 
transduced with a retrovirus containing the 4-1BBL (CD137L) gene.     


11-C-0163 
11
 
A bulk K562.CD64.4-1BBL line was initially tested for its ability to stimulate 
proliferation of lymphocytes from melanoma tumors with low levels of T cell 
infiltration (<20% lymphocytes) .  Cryopreserved tumors with minimal lymphocyte 
infiltrates were selected, and then thawed TIL cultures were initiated in the absence or 
presence of the K562.CD64.4-1BBL cell line .  Lymphocytes proliferated in six of 21 
samples (29%) in standard conditions, and in 14 samples with costimulation 
enhancement.  These results were confirmed using poorly infiltrated “fresh” 
melanoma tumors.  Four of 12 samples cultured in standard conditions resulted in TIL 
proliferation, while all 12 matched samples demonstrated rapid lymphocyte 
proliferation with the K562.CD64.4-1BBL line. These experiments demonstrate a 
highly significant (p=0.0002) improvement in establishment of ECCE TIL (26 of 33 
samples, 79%) compared to standard TIL (10 of 33 samples 30%, Table 4).  4-1BBL 
expression was essential, with 0/9 TIL established when K562 cells were added 
without 4-1BBL compared to 9/9 TIL established using K562 with 4-1BBL 
expression (p=0.0001, Table 4).  These studies demonstrated the K562.CD64.4-1BBL 
line can be added to poorly infiltrated tumors to improve TIL generation. 
Experiments were performed with the 7F11 ECCE clone (see below) with similar 
results, indicating that ECCE young TIL cultures suitable for therapy may be 
generated for some patients using this approach who would otherwise be ineligible 
for treatment due to lack of a suitable lymphocyte culture.   
To investigate whether ECCE young TIL may have different attributes or 
composition than standard TIL, six tumors were used to generate standard TIL and 
ECCE TIL in parallel cultures, and phenotype and function were compared.  Time to 
generation of TIL was reduced in ECCE TIL cultures compared to standard TIL 
(Figure 4, p=0.004).  The composition of standard TIL and matched ECCE TIL were 
investigated by FACS analysis, and ECCE TIL were found to contain a significantly 
increased fraction of CD8+ lymphocytes (p=0.03).  Importantly, anti-tumor function 
exhibited by ECCE TIL in response to an autologous tumor cell lines was statistically 
similar to standard TIL (p=0.23, Table 5).  These results suggest that CD8+ enhanced, 
functionally equivalent ECCE young TIL can be generated more quickly than 
standard TIL, even from well infiltrated tumors.  The therapeutic efficacy of ECCE 
young TIL cultures cannot be predicted a priori, while a (preliminary) therapeutic 
response rate for standard young TIL cultures has been established.   
 
 
1.2.4. Validating 7F11 Engineered Cells for Costimulation Enhancment
(ECCE) Rapid Expansions of TIL at clinical scale
The number of cells infused for TIL treatments may be a critical determinant 
of patient response.  In our studies, the number of CD8+ cells infused was correlated 
with objective response in prior TIL trials.  Similarly, in a recent trial with young TIL 
following non-myeloablation in melanoma patients, cell number infused was 
correlated with objective response24 The rapid expansion protocol (REP) using 
irradiated peripheral blood mononuclear cell (PBMC) “feeders,” OKT3 and IL-2 is 
required for expansion of TIL to large numbers for patient infusion, but the REP is 
often limited by the number of feeder cells available.  K562.CD64.4-1BBL cells were 


11-C-0163 
12
investigated as a substitute for PBMC in the REP.  Initial experiments demonstrated 
that TIL expanded poorly when K562.CD64.4-1BBL were loaded with OKT3 and 
used alone as stimulators.  However, the K562.CD64.4-1BBL cells functioned well 
when added to PBMC to partially replace PBMC in REPS (Figure 5).  The expression 
of 4-1BBL was required for optimal ECCE TIL REP (p=0.03, Figure 6).   
To obtain a GMP compliant ECCE REP protocol, a K562.C64.4-1BBL master 
cell bank was established in the following way.  Multiple clones from the 
K562.CD64.4-1BBL cell line were isolated and characterized.  The clone 7F11 
(K562.CD64.41BBL-7F11) expressed uniformly high levels of CD64 and 4-1BBL 
(Figure 7), and was further expanded for GMP production and generation of a master 
cell bank (MCB). Full characterization of the MCB including safety information will 
be submitted with an investigational new drug (IND) application.     
  
The 7F11 ECCE line was tested at clinical scale for its ability to replace 
feeder cells in a REP.  Optimal TIL expansion at clinical scale requires a 200:1 ratio 
of PBMC feeder cells:TIL responders.  The addition of 7F11 reduced the requirement 
for PBMC feeders to an optimal ratio of 50:10:1 PBMC feeders:K7F11:TIL 
responders.  These optimal ratios  generated similar numbers of expanded TIL in 
clinical scale REPs (Figure 8).  The composition of REPed cells was determined by 
FACS analysis, and the 7F11 and PBMC expanded TIL had a similar fraction of 
CD8+ cells, but ECCE REP TIL were comprised of significantly fewer CD4+ cells 
and more natural killer (NK) cells (CD3-CD56+, Figure 9).  Although NK cells do 
not have an unambiguously defined role in ACT therapy, they have been implicated 
in reducing metastatic disease in lung metastases models in mice and in eliminating 
HLA loss tumor variants in vitro.  Similarly, reducing CD4+ cells in TIL may 
improve the therapies, although the contribution of the CD4+ compartment is 
complex.  The presence of NK cells at modest numbers and the reduction of CD4+ 
cells from TIL may improve the efficacy of the TIL product.  Media composition and 
other culture conditions are currently being evaluated for differential expansion of T 
cells and NK cells during clinical scale TIL expansions with 7F11.  
 
 
1.2.5. Safety Considerations
Two principal safety concerns arise from TIL cells expanded with the use of 
7F11 accessory cells.  First, the 7F11 may themselves survive the expansion process 
and cause toxicity when infused.  Second, the TIL may acquire new attributes through 
exposure to the 7F11 cells including their 4-1BBL costimulation.   
 
The K562 cell line, the progenitor to 7F11, is well characterized for clinical 
use. Irradiated K562 has been administered directly to patients in GVAX vaccine 
trials21;22 and has also been used as an accessory component in the production of 
autologous cells that have been used for patient therapy (M Butler, Dana Farber 
Cancer Center, personal communications).  These clinical experiences have not 
resulted in evidence of adverse reactions.   
The ECCE used as an ancillary manufacturing reagent are incapable of 
proliferation due to irradiation.  To insure lack of ECCE proliferation, each 
production batch of 7F11 cells is examined for proliferation after 100Gy irradiation, 
the dose that is used in TIL manufacture.  The proliferation of 7F11 following 
irradiation is assessed by tritiated thymidine incorporation (Figure 10). The 


11-C-0163 
13
proliferation assay evaluates live 7F11 at various concentrations spiked into irradiated 
7F11 cells.  Live 7F11 can be reliably detected at concentrations down to and 
including 1 live cell per 105 irradiated cells (detection limit < 0.001%).  100Gy 
irradiated 7F11, the irradiation dose used for 7F11 in clinical TIL production, was 
sufficient to completely inhibit the proliferation of 7F11, and no viable 7F11 was 
detected with 100Gy irradiation. During the REP,  7F11 is eliminated from the final 
product in the same manner as allogeneic feeder cells, through non-specific killing 
mechanisms of highly activated lymphocytes.  However, to insure that 7F11 are not 
infused into patients, additional safety testing will be undertaken.  Each production 
batch of 7F11 will undergo extensive testing and characterization with a detailed, 
batch-specific COA prior to approval for use in ECCE TIL manufacturing protocols.  
The tritium incorporation assay will be one of the tests used in the lot release criteria 
for 7F11. 7F11 production lots will by irradiated and fully characterized prior to 
release for use in production.  In addition, each batch of patient cells will include a 
batch specific release assay to evaluate the elimination of 7F11 cells from the final 
product.  The assay will evaluate the expression of 4-1BBL (CD137) which is 
expressed by 7F11 but not by the infused TIL.  Infused cells will have to demonstrate 
CD137 expression <0.5% prior to release for infusion.   
 
TIL expanded with 7F11 could acquire attributes mediating toxicity upon 
transfer.  This seems unlikely, as no phenotypic or functional characteristics of ECCE 
TIL were observed suggesting that they have superior inflammatory attributes 
compared to conventional TIL.  Over 300 autologous TIL infusions have been safely 
administered in the Surgery Branch to doses as high as 3x1011 cells.   
 
2.0 Eligibility Assessment and Enrollment  
 
 
2.1. 
Eligibility Criteria
 
2.1.1. 
Inclusion Criteria
a.
Measurable metastatic melanoma with at least one lesion that is resectable for 
TIL generation.  
b.
Patients with 3 or less brain metastases are eligible.  Note:  If lesions are 
symptomatic or greater than or equal to 1 cm each, these lesions must have 
been treated and stable for 3 months for the patient to be eligible. 
c. 
Greater than or equal to 18 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 from the time 
of enrollment on this study and for up to four months after receiving the 
preparative regimen. 
i. 
Serology: 
1. Seronegative for HIV antibody.  (The experimental treatment being 
evaluated in this protocol depends on an intact immune system.  Patients 


11-C-0163 
14
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 seronegative for hepatitis C 
antibody. If hepatitis C antibody test is positive, then patient must be 
tested for the presence of antigen by RT-PCR and be HCV RNA negative.  
3. Women of child-bearing potential must have a negative pregnancy test 
because of the potentially dangerous effects of the preparative 
chemotherapy on the fetus. 
j. 
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. 
k. 
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. 
l. 
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).  Patients may have undergone minor surgical procedures within 
the past 3 weeks, as long as all toxicities have recovered to grade 1 or less or 
as specified in the eligibility criteria in Section 2.1.1.   
m.
Six weeks must have elapsed since any prior anti-CTLA4 antibody therapy to 
allow antibody levels to decline. 
n.
Patients who have previously received any anti-CTLA4 antibody and have 
documented GI toxicity must have a normal colonoscopy with normal colonic 
biopsies. 
 
 
 
2.1.2. 
Exclusion Criteria 
a.
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. 
b.
Systemic steroid therapy required. 
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 


11-C-0163 
15
have decreased immune competence may be less responsive to the 
experimental treatment and more susceptible to its toxicities). 
f.
History of severe immediate hypersensitivity reaction to any of the agents 
used in this study. 
g.
History of coronary revascularization or ischemic symptoms 
h.
Any patient known to have an LVEF less than or equal to 45%. 
i.
Documented LVEF of less than or equal to 45% tested in patients with: 
x
Clinically significant atrial and/or ventricular arrhythmias including 
but not limited to: atrial fibrillation, ventricular tachycardia, second or 
third degree heart block 
x
Age  60 years old 
j. 
Documented FEV1 less than or equal to 60% predicted tested in patients with: 
x
A prolonged history of cigarette smoking  
x
Symptoms of respiratory dysfunction 
 
 
2.2. 
Research Eligibility Evaluation  
Within 4 weeks prior to starting the chemotherapy regimen:  
a. 
Complete physical examination, including height, weight and vital signs, and 
eye exam noting in detail the exact size and location of any lesions that exist. 
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. 
Cardiac (MUGA or echocardiogram, stress thallium) and pulmonary 
evaluation (PFTs) if required as indicated in Section 2.1. Note: cardiac 
evaluation may be performed up to 6 months prior to treatment. 
f. 
HIV antibody titer and HbsAG determination, anti HCV, anti CMV antibody 
titer, HSV serology, and EBV panel 
g. 
Medical history (may be conducted at any point prior to this time). 
 
Within 14 days prior to starting the chemotherapy regimen: 
h. 
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) 
i. 
CBC with differential and platelet count 
j. 
PT/PTT 
k. 
Urinalysis and culture, if indicated  
Within 7 days prior to starting the chemotherapy regimen:  
l. 
ȕ-HCG pregnancy test on all women of child-bearing potential 
 
2.3. 
Patient Registration


11-C-0163 
16
 
Patients will be registered on protocol 03-C-0277 (Cell Harvest and 
Preparation for Surgery Branch Adoptive Cell Therapy Protocols) prior to tumor 
resection for TIL generation, 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. Patients will sign the consent 
document for this protocol prior to treatment 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:
3.1.1.1. 
Cell Preparation
 
Patients with evaluable metastatic melanoma who have lesions suitable for 
initiating standard and ECCE TIL that can be resected with minimum morbidity will 
undergo resection of tumor.  TIL will be obtained while enrolled on the Surgery 
Branch protocol 03-C-0277, “Cell Harvest and Preparation for Surgery Branch 
Adoptive Cell Therapy Protocols”.  Separate tumor biopsies may be performed to 
obtain TIL for subsequent lymphocyte cultures. TIL will be grown and expanded for 
this trial according to standard operating procedures submitted in the IND.  Cell 
preparation and patient cohort enrollment will follow the schematic shown in Figure 
11. Briefly, a single cell suspension will be prepared from the resected tumor.   
“Standard” young TIL will be initiated in 3000 IU/ml interleukin (IL)-2 and “ECCE 
young TIL” will be initiated with 100Gy irradiated 7F11 cells plus 3000 IU/ml IL-2.   
TIL cultures will be monitored regularly.  If standard TIL proliferate to at least 5x107 
lymphocytes within 20 days, then this standard TIL culture will be used 
preferentially, and the patient will be eligible for enrollment in Cohort 1.   Clinical 
evaluation of patients in this cohort will establish for the first time the value of ECCE 
replacement of feeder cells in REPs for standard young TIL that have an established 
record of therapeutic benefit.  If standard TIL fail to grow within 20 days then the 
ECCE young TIL will be evaluated for growth and patients who have ECCE young 
TIL available will be eligible for enrollment in Cohort 2. Clinical evaluation of 
patients in this cohort will establish for the first time whether ECCE young TIL are 
therapeutically beneficial.    
 
All TIL will undergo ECCE REP using anti-CD3 (OKT3), IL-2, feeder cells 
and 100Gy irradiated 7F11.  Five to seven days later the cells will be tested for 
potency by interferon-gamma release as specified in the Certificate of Analysis 
shown in Appendix 1.  Volunteers will also undergo apheresis to obtain mononuclear 
cells to be used as feeder cells in cell culture.  The procedures used are the same as 
those in all prior ACT protocols and are in routine use in the Department of 
Transfusion Medicine in the Clinical Center.  Separate consents will be obtained from 
all blood and apheresis volunteers.   
 
It is anticipated that TIL that meet the COA will not be achievable in 
approximately 5% of patients who undergo resection.  These patients may undergo a 
second resection to grow TIL, if another suitable lesion exists, and be treated in the 
same cohort as initially assigned.   


11-C-0163 
17
3.1.1.2. Cell Administration
 
Once cells exceed the potency requirement and are projected to exceed the 
minimum number specified in the COA, (approximately 7 days after the REP 
procedure has been initiated) the patient will receive the lymphocyte depleting 
preparative regimen consisting of fludarabine and cyclophosphamide, followed by 
infusion of up to 1x1011 ECCE REP lymphocytes (minimum of 1x109) and the 
administration of high-dose aldesleukin.  
 
Approximately 4-6 weeks after completion of aldesleukin administration, 
patients will undergo a complete tumor evaluation and evaluation of toxicity and 
immunologic parameters. This will comprise one course of therapy.  Patients will 
receive no other experimental agents while on this protocol. For both cohorts, cells 
will be administered in a volume of 100-200 ml.   
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 
study not attributable to the chemotherapy preparative regimen or IL-2 (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. 
 
 
3.2. 
Drug Administration
(see table 3.2) 
3.2.1. 
Preparative Regimen with Cyclophosphamide and Fludarabine
Day -7 and –6 (Times are offered as examples and may be changed as long as a 
similar time relationship between drug administration is maintained) 
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) 
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.  
 
Furosemide 10-20 mg iv. 
 
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 6.  


11-C-0163 
18
 
1 pm  
Begin to monitor potassium level every 12 hours until hydration is stopped.  
KCl will be adjusted to maintain serum potassium levels in the normal range. 
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 6.  
 
Day -5: 
Stop IV hydration (24 hours after last cyclophosphamide dose)  
If urine output <1.5 ml/kg/hr give additional 20 mg furosemide iv. 
If body weight >2 kg over pre cyclophosphamide value give additional 
furosemide 20 mg iv. 
 
 
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 6.  
 
3.2.2. 
Cell Infusion and Aldesleukin Administration
Cells are delivered to the patient care unit by a staff member from the Cell Production 
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
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).  Cells 
will be administered by an infusion of up to 1x1011 lymphocytes (minimum of 
1x109).  For cell numbers less than 1 x 1010 cells, the volume will be 100 mls; and 
for cell numbers greater than or equal to 1 x1010 cells, the volume will be 200 mls. 
x
Aldesleukin 720,000 IU/kg IV (based on total body weight) over 15 minute 
every eight hours (+/- 1hr) 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 may 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.  
Aldesleukin 720,000 IU/kg IV (based on total body weight) over 15 minute every 
eight hours for up to 5 days. 
 
 
Table 3.2 
 
Day 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
01 
1 
2 
3 
4 
Therapy
 
 
 
 
 
 
 
 
 
 
 
 
Cyclophosphamide 
60 mg/kg 
X 
X 
 
 
 
 
 
 
 
 
 
 


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19
Fludarabine 
25 mg/m2 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 
ECCE TIL Cells 
 
 
 
 
 
 
 
X 
 
 
 
 
Aldesleukin 
 
 
 
 
 
 
 
X2
X 
X 
X 
X 
Filgrastim3 
5 mcg/kg/day 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
TMP/SMX4 
160mg/800mg 
(example) 
 
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 ANC is greater than 1000/mm3 
 
3.2.3. 
Infection Prophylaxis
3.2.3.1. 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) P.O. daily three times a week on non-consecutive days, 
beginning on day -7.  
 
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 
prior to admission and monthly for at least 6 months and until the CD4 count is 
greater than 200 for 2 consecutive measurements. 
 
3.2.3.2. 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 which is continued until 
absolute neutrophil count is greater than 1000/mm3.  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. 
3.2.3.3. Fungal Prophylaxis (Fluconazole)
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. 
3.2.3.4. Empiric Antibiotics 
Patients will start on broad-spectrum antibiotics, either a 3rd or 4th generation 
cephalosporin or a quinolone for fever of 38.3oC once or two temperatures of 38.0oC 


11-C-0163 
20
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. 
 
3.2.4. 
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.  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. 
 
3.2.5 
Aldesleukin: Intravenous Administration
Aldesleukin will be administered at a dose of 720,000 IU/kg (based on total 
body weight) as an intravenous bolus over a 15 minute period every eight hours (+/- 
1hr) beginning on the day of cell infusion and continuing for up to 5 days (maximum 
15 doses). Doses may be delayed or skipped depending on patient tolerance if this is 
determined to be clinically indicated in the best clinical judgment of the attending 
physician.  Doses will be skipped if patients reach Grade 3 or 4 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 
Appendix 2.  Toxicities will be managed as outlined in Appendix 3.  If these 
toxicities can be easily reversed within 24 hours by supportive measures then 
additional doses may be given. Additional instances may arise when in the clinical 
judgment of the attending physician, based on the extensive clinical experience in the 
Surgery Branch with aldesleukin, when doses of aldesleukin may be skipped.  If 
greater than 2 doses of aldesleukin are skipped, aldesleukin administration will be 
stopped. Aldesleukin will be administered as an inpatient and will be purchased by 
the NIH Clinical Pharmacy Department from commercial sources.  
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 
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, 


11-C-0163 
21
ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, 
Total CK, Uric Acid) 
x
Urinalysis 
x
Review of systems and physical exam at least 3 times/week 
x
Vital signs as per routine 
3.3.3. 
After Cell Infusion:
x
Vital signs will be monitored hourly for  3 hours,  and then routinely (every 4 
hours) unless otherwise clinically indicated. 
x
CMV PCR assay will be assessed if clinically indicated (e.g. unexplained fevers, 
pulmonary changes). 
 
3.3.4 
During and after Aldesleukin administration (until hospital discharge)– Every 1-
2 days
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
Review of systems and physical exam at least 3 times/week 
x
Vital signs as per routine 
 
3.3.5. 
Additional research evaluations:
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 (while the 
patient is hospitalized): 
x
5 CPT tubes (10 ml each) 
x
1 SST tube (10 ml) 
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. 
 
3.3.6.2.  Biopsies 
x
Biopsies of tumor tissue or lymph nodes may be performed but are not required 
during the course of therapy.  A maximum of three biopsies will be performed. 
These biopsies will only be performed if they are superficial and extracavitary.  
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. 
 


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22
3.3.6.3.  Immunological Testing: 
x
Apheresis will 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 cryopreserved for immunological monitoring of cell 
function.   
x
Lymphocytes will be tested directly and following in vitro culture using some or 
all of the following tests. Direct immunological monitoring will consist of 
quantifying T cells reactive with targets 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 
ECCE TIL 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. 
 
 
3.4. 
Post Study Evaluation (Follow-up)
 
Patients will be evaluated 4 to 6 weeks after the initial treatment regimen (defined as 
the last day of aldesleukin administration).  
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), complete blood count and thyroid panel 
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 will be performed.  Subsequently, 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
If the patient has SD or tumor shrinkage, repeat complete evaluations will be 
performed monthly for approximately 3-4 months, and then every 3-4 months 
until off study criteria are met. 
 
3.5. 
Off Treatment Criteria


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23
Patients will be taken off treatment (and followed until progression of disease) for the 
following 
x
Grade 3 or greater autoimmunity that involves vital organs (heart, 
kidneys, brain, eye, liver, colon, adrenal gland, lungs). 
x
If a patient experiences a grade 3 or greater toxicity due to cell 
infusion (reaction to cellular product or infusion reaction) the patient 
will receive no further treatment. 
 
 
3.6. 
Off Study Criteria
Patients will be taken off study for the following: 
x
The patient voluntarily withdraws 
x
There is significant patient noncompliance 
x
Progressive disease, unless: 
o
the patient is being followed for a serious adverse event related 
to the research.  The event must resolve to less than or equal to grade 2 
or baseline prior to removing from the study. 
x
The patient develops a second malignancy. 
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. 
 
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. 
Response Criteria 
Clinical response will be determined using RECIST version 1.0 
 
5.1.1. 
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. 


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24
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. 
 
5.1.2. 
 
Evaluation of non-target lesions2
x
Complete Response (CR): Disappearance of all non-target lesions and 
normalization of tumor marker level. 
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.” 
 
 
5.1.3. 
 
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 
 
5.1.4. 
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 


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25
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
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.  
 
 
5.2. 
Toxicity Criteria
This study will utilize the CTCAE version 3.0 for toxicity and adverse event 
reporting.  A copy of the CTCAE v3.0 can be downloaded from the CTEP home page 
(http://ctep.cancer.gov). 
 
Over 150 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 cells can 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 the 68 patients treated with young TIL using the non-myeloablative 
chemotherapy regimen, there was one treatment related death due to overwhelming 
sepsis. 
 
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. 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.  High-dose aldesleukin: a variety of 
side effects have been associated with high-dose aldesleukin administration in our 


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26
experience and a listing of these side effects in 652 patients who received 1,039 
treatment courses are listed in Appendix 2. 
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.    
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.  
 


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27
5.3. Statistical Section 
The primary objective of this study is to determine if the transfer of ECCE young TIL 
in conjunction with aldesleukin after lymphodepletion will be associated with a modest 
response rate (CR+PR) in patients with metastatic melanoma.  
 
Cohort 1
In cohort 1, success with this regimen would allow more rapid and reliable treatment 
of individual patients, and improve procedures for manufacturing TIL so they are more easily 
adopted in other centers. In an ongoing clinical trial (07-C-0176),  two cohorts with standard 
young TIL expanded using conventional feeder cells demonstrated response rates of 21% (5 
of 24 patients) and 35% (6 of 17 patients) for an overall response with unselected young TIL 
in patients who received nonmyeloablative conditioning of 27 % (11 of 41 patients). Accrual 
to this cohort will take place using an optimal two-stage phase II trial design (Simon R, 
Controlled Clinical Trials 10:1-10, 1989), in order to rule out an unacceptably low 10% 
clinical response rate (PR+CR; p0=0.10) in favor of a modest response rate of 30% 
(p1=0.30). With alpha=0.05 (probability of accepting a poor treatment=0.05) and beta = 0.10 
(probability of rejecting a good treatment=0.10), cohort 1 will initially enroll 18 evaluable 
patients and if 0-2 of the 18 have a clinical response, then no further patients will be accrued. 
If 3 or more the first 18 have a response, then accrual would continue until a total of 35 
patients have 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 there are 3-6 
responses in 35 patients, this would be an uninterestingly low response rate, while if there 
were 7 or more responses in 35 patients, then this would be sufficiently interesting to warrant 
further study in later trials.  Under the null hypothesis (10% response rate), the probability of 
early termination is 73%.   
Cohort 2
 
Patients assigned to Cohort 2 would not have any other option for TIL therapy.  For 
these patients, a modest response rate in a relatively limited number of patients would 
warrant subsequent study.  Accrual to this cohort will take place using an optimal two-stage 
phase II trial design, in order to rule out an unacceptably low 5% clinical response rate 
(PR+CR; p0=0.05) in favor of a modest response rate of 25% (p1=0.25). With alpha=0.10 
(probability of accepting a poor treatment=0.10) and beta = 0.10 (probability of rejecting a 
good treatment=0.10), cohort 2 will initially enroll 9 evaluable patients and if 0 of the 9 have 
a clinical response, then no further patients will be accrued. If 1 or more of the first 9 patients 
has a response, then accrual would continue until a total of 24 patients have been enrolled. As 
it may take several weeks to determine if a patient has experienced a clinical response, a 
temporary pause in the accrual to the trial may be necessary to ensure that enrollment to the 
second stage is warranted. If there are 1-2 responses in 24 patients in this cohort, this would 
be an uninterestingly low response rate, while if there were 3 or more responses in 24 
patients, then this would be sufficiently interesting to warrant further study in later trials. 
Under the null hypothesis (5% response rate), the probability of early termination in the trial 
is 63%.   
 


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28
Secondary objectives of this trial include evaluating the safety of TIL that have been 
manufactured using ECCE accessory cells with enhanced costimulation, and the persistence 
of these ECCE TIL.  Accrual of a substantial number of patients in each cohort will also 
assist in achieving these secondary aims.   
 
It is anticipated that 2-5 eligible patients may enroll on this trial in a month. Thus, 
with a requirement of up to 59 patients, this study should be able to complete its accrual 
within two to three years. In order to allow for the possibility of a limited number of 
inevaluable patients, the accrual ceiling will be set at 63 patients. 
 
5.4. 
Clinical Trial Monitoring Plan
A detailed description of the clinical trial monitoring plan has been included 
in the initial IND submission as required.  Briefly, Harris Technical Services 
Corporation provides study auditing/monitoring services under contract with the 
Center for Cancer Research, NCI.  The number of patient records monitored is based 
on actual accrual.  Below is a tabular summary of monitoring evaluations: 
 
 
Time of year
 
 
  
Evaluation 
# of records monitore  
%data verification 
1st quarter 
(Jan.- March)
 
 
Compare CRFs to 
source documentation, 
verify subject 
registration with Drug 
Accountability Records 
3-4 study patients 
100% 
2nd quarter 
(April – June) 
Compare CRFs to 
source documentation, 
verify subject 
registration with Drug 
Accountability Records 
3-4 study patients 
 
100% 
3rd quarter 
(July – Sept.) 
Compare CRFs to 
source documentation, 
verify subject 
registration with Drug 
Accountability Records 
3-4 study patients 
 
100% 
4th quarter 
(Oct. – Dec.)
 
 
Compare CRFs to 
source documentation, 
verify subject 
registration with Drug 
Accountability Records 
3-4 study patients 
 
100% 
            *Based on expected accrual, 25% of enrolled patients will be monitored. 
 
5.5. 
Data and Safety Monitoring Plan 
 


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29
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 3.3.5.  
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. 
Handling of Tissue Specimens for Research Purposes 
Blood or 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 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.  Serum samples will be 
sent to the Clinical Pharmacology Program (CPP) for storage.  Samples will be barcoded and 
stored on site or offsite at NCI Frederick Central Repository Services (Fisher Bioservices) in 
Frederick, MD.  Data is entered and stored securely in the Patient Sample Data Management 
System (PSDMS) utilized by the CPP, and data will be updated to the Surgery Branch central 
computer database weekly.  All samples (serum,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. 
 
6.0 Human Subjects Protections
 
6.1. Rationale for Patient Selection 
The patients to be entered in this protocol have metastatic melanoma 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 


11-C-0163 
30
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 
ECCE TILs in patients who do not have standard TIL available for treatment.  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 
If 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.   
 
 
 


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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, 
‘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. 


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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)
A protocol deviation is any change, divergence, or departure from the study design or 
procedures of a research protocol that is under the investigator’s control and that has not been 
approved by the IRB. 
7.1.8 Protocol Violation (NIH Definition)
Any change, divergence, or departure from the study procedures in an IRB-approved 
research protocol that has a major impact on the subject’s rights, safety, or well-being and/or 
the completeness, accuracy or reliability of the study data.
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 Routine Data Reporting:
All information surrounding patient screening, diagnosis, tumor harvest, treatment, and 
follow up will be recorded in the patient medical record. Data will be reported via the NCI 
SB Immunotherapy reporting system.   
7.3 Expected Adverse Events and Protocol-Specific Expedited Adverse Event Reporting 
Exclusions:
Since the patient is receiving multiple agents which include commercially available agents 
either alone or in combination with investigational agents (aldesleukin, fludarabine, 
cyclophosphamide and supportive medications), Grade 1 and 2 adverse events ‘unrelated’ or 
‘unlikely related’ to the investigational agents, and ‘possibly’, ‘probably’ or ‘definitely’ 
related to the commercially available agents as specified in the package inserts, or Appendix 
2 or 3, do not require reporting/recording.  For additional information about expected 
toxicities, refer to Section 8. 


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7.3.1 Aldesleukin:
Expected toxicities of aldesleukin are listed in the product label and in Appendix 3 and 4.  No 
expedited reporting of expected grade 3 toxicities or grade 4 laboratory toxicities clearly 
related to aldesleukin, expected, easily managed, and reversed to less than grade 3 within 48 
hours is required.  Aldesleukin toxicities that must be reported in an expedited fashion 
include: need for endotracheal intubation, renal dialysis, coma, myocardial infarction, 
myocarditis, bowel perforation, sustained ventricular tachycardia, death or any grade 4 
toxicity that is not listed in Appendix 2 or 3. 
7.3.2 Chemotherapy Preparative Regimen:
Expected toxicities of cyclophosphamide and fludarabine are listed in the product label and 
will not be reported via expedited reporting system, including Grade 4 myelosuppression. 
7.3.3 Adoptive Cell therapy:
Expected toxicities that may occur with cell infusion include fever, chills, headaches, 
malaise, shortness of breath (10% of patients have required intubation either for airway 
protection or dyspnea), renal toxicity and transient rash.  It is possible that patients may 
develop autoimmune reactions after receiving cells (e.g. vitiligo or uveitis). 
7.4 NCI IRB Reporting Requirements
7.4.1 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 days of notification of the event. Follow-
up reports can be submitted to the IRB after the NCI-IRB review.  Information may be sent 
via the NCI iRIS Application: https://iris.nci.nih.gov/iMedris/ 
 
7.4.2 NCI-IRB Requirements for PI Reporting of Adverse Events at Continuing review
The protocol PI will report to the NCI-IRB:  
x
All Grade 2 unexpected events that are possibly, probably or definitely related to 
the research;  
x
All Grade 3 and 4 events that are possibly, probably or definitely related to the 
research;  
x
All Grade 5 events regardless of attribution;  
x
All Serious Events regardless of attribution.  


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NOTE: Grade 1 events are not required to be reported.  
7.4.3 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. 
 
7.5 
Serious Adverse Event Reporting to the FDA:
During the duration of this trial, the principal investigator will assume responsibility for 
reporting any serious and unexpected suspected adverse reaction to the U.S. Food and Drug 
Administration (FDA) with appropriate deadlines defined by the regulations. 
 
FDA will be informed by phone or fax of any unexpected fatal or life threatening suspected 
adverse reaction as soon as possible but in no event, later than 7 calendar days after the 
sponsor’s initial receipt of the information. 
 
FDA will be informed of any other unexpected and serious adverse event, by written 
notification within 15 calendar days after the sponsor’s initial receipt of the information. 
All adverse events, including serious adverse events, will be reported in the annual report to 
the FDA as indicated in 21CFR312.33. 
 
8.0  
Pharmaceutical Information
8.1 
Interleukin-2 (Aldesleukin, Proleukin, Recombinant Human Interleukin 2)
How Supplied: Interleukin-2 (aldesleukin) is manufactured by the Chiron 
Corporation, Emeryville, CA, and will be purchased by the NIH Clinical Pharmacy 
Department from commercial sources.   
Formulation/Reconstitution: Aldesleukin, NSC #373364, is provided as single-use 
vials containing 22 million IU (1.3 mg) IL-2 as a sterile, white to off-white  lyophilized 
cake plus 50 mg mannitol and 0.18 mg sodium dodecyl sulfate, buffered with approximately 
0.17 mg monobasic and 0.89 mg dibasic sodium phosphate to a pH of 7.5 (range 7.2 to 7.8).  
The vial is reconstituted with 1.2 mL of Sterile Water for Injection, USP, and the resultant 
concentration is 18 million IU/ml or 1.1 mg/mL.  Diluent should be directed against the side 
of the vial to avoid excess foaming.  Swirl contents gently until completely dissolved.  Do 
not shake.  Since vials contain no preservative, reconstituted solution should be used with 24 
hours. 
Storage: Intact vials are stored in the refrigerator (20 - 80C) protected from light.  
Each vial bears an expiration date. 
Dilution/Stability: Reconstituted aldesleukin should be further diluted with 50 mL of 
5% Human Serum Albumin (HSA).  The HSA should be added to the diluent prior to the 
addition of RIL-2.  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 – 
300C. 


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Administration:  The dosage will be calculated based on total body weight.  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 5 and 6.  Grade III toxicities common to aldesleukin include diarrhea, nausea, 
vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or constitutional 
symptoms and laboratory changes as detailed in Appendix 2.  Additional grade III and IV 
toxicities seen with aldesleukin are detailed in Appendix 3.   
 
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 2-80C; 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, ribonucleotide 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 6. 
 
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 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 


11-C-0163 
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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 2-80C.   
 
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 6. 
 
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 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.  
 


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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 6.  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 (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 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 within 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)


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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.  
Valacyclovir 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
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 
Cell Preparation  
The procedure for growing and expanding the autologous young TIL and the Certificate of 
Analysis are similar to those approved by the Food and Drug Administration and used at the 
NCI in protocols 04-C-0181, 04-C-0251, and 07-C-0003 with slight changes to reflect 
improved techniques, including a potency assay consisting of cytokine release (IFN > 200 
pg/mL) following OKT3 stimulation.  This product will be provided for investigational use 
only under the investigator-sponsor IND 14265.  The Certificate of Analysis is in Appendix 1 
and the Standard Operating Procedures for the growth of TIL and TIL expansion in the REP 
procedures are included in  IND 14265. 
 


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8.10 
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 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. 
 
8.11  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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cytotoxic T cells specific for both dominant and subdominant viral epitopes for adoptive 
therapy. J. Immunol. 183:2837-2850. 
 17.  Suhoski,M.M., T.N.Golovina, N.A.Aqui, V.C.Tai, A.Varela-Rohena, M.C.Milone, 
R.G.Carroll, J.L.Riley, and C.H.June. 2007. Engineering artificial antigen-presenting cells to 
express a diverse array of co-stimulatory molecules. Mol. Ther. 15:981-988. 


11-C-0163 
43
 18.  Zhang,H., K.M.Snyder, M.M.Suhoski, M.V.Maus, V.Kapoor, C.H.June, and C.L.Mackall. 
2007. 4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes 
for adoptive immunotherapy. J. Immunol. 179:4910-4918. 
 19.  Maus,M.V., A.K.Thomas, D.G.Leonard, D.Allman, K.Addya, K.Schlienger, J.L.Riley, and 
C.H.June. 2002. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes 
by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat.
Biotechnol. 20:143-148. 
 20.  Butler,M.O., J.S.Lee, S.Ansen, D.Neuberg, F.S.Hodi, A.P.Murray, L.Drury, A.Berezovskaya, 
R.C.Mulligan, L.M.Nadler, and N.Hirano. 2007. Long-lived antitumor CD8+ lymphocytes for 
adoptive therapy generated using an artificial antigen-presenting cell. Clin. Cancer Res. 
13:1857-1867. 
 21.  Borrello,I.M., H.I.Levitsky, W.Stock, D.Sher, L.Qin, D.J.DeAngelo, E.P.Alyea, R.M.Stone, 
L.E.Damon, C.A.Linker, D.J.Maslyar, and K.M.Hege. 2009. Granulocyte-macrophage colony-
stimulating factor (GM-CSF)-secreting cellular immunotherapy in combination with 
autologous stem cell transplantation (ASCT) as postremission therapy for acute myeloid 
leukemia (AML). Blood 114:1736-1745. 
 22.  Smith,B.D., Y.L.Kasamon, J.Kowalski, C.Gocke, K.Murphy, C.B.Miller, E.Garrett-Mayer, 
H.L.Tsai, L.Qin, C.Chia, B.Biedrzycki, T.C.Harding, G.H.Tu, R.Jones, K.Hege, and 
H.I.Levitsky. 2010. K562/GM-CSF immunotherapy reduces tumor burden in chronic myeloid 
leukemia patients with residual disease on imatinib mesylate. Clin. Cancer Res. 16:338-347. 


11-C-0163 
44
 23.  Stephan,M.T., V.Ponomarev, R.J.Brentjens, A.H.Chang, K.V.Dobrenkov, G.Heller, and 
M.Sadelain. 2007. T cell-encoded CD80 and 4-1BBL induce auto- and transcostimulation, 
resulting in potent tumor rejection. Nat. Med. 13:1440-1449. 
 24.  Besser,M.J., R.Shapira-Frommer, A.J.Treves, D.Zippel, O.Itzhaki, L.Hershkovitz, D.Levy, 
A.Kubi, E.Hovav, N.Chermoshniuk, B.Shalmon, I.Hardan, R.Catane, G.Markel, S.Apter, 
A.Ben-Nun, I.Kuchuk, A.Shimoni, A.Nagler, and J.Schachter. 2010. Clinical responses in a 
phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in 
metastatic melanoma patients. Clin. Cancer Res. 16:2646-2655. 
 


11-C-0163 
45
 
10.0 
Tables, Figures, and Appendices
 
Table 1 
Cell Transfer Therapy (updated 8/1/10) 
Treatment            Total            
PR
CR
OR (%)
number of patients (duration in months)
No TBI
43
16
5
21 (49%)
(84,   36,   29,   28,        (82+,  81+,  79+,
14,   12,   11,     7,
78+,  64+)
7,     7,     7,     4,     
4,     2,     2,     2)     
200 TBI
25
8
5
13 (52%)
(14,     9,     6,     6,       (68+,  64+,  60+,
5,     4,     3,     3)        57+,  54+) 
1200TBI
25
8
10
18(72%)
(21,   13,     7,     6,        (48+,  45+,   44+, 
6,     5,     3,     2)         44+,  39+,   38+,   
38+,  38+,   37+,
19)
(52 responding patients: 43 had prior IL-2; 23 had prior IL-2 + chemotherapy)
(20 complete responses: 19 ongoing at 37 to 82 months)
 
 


46 
 
Table 2:  Transfusions and grade 3 and 4 non-hematologic toxicities associated with 
NMA plus TBI lymphodepleting preparative regimens. 
 
200cGy TBI 
1200cGy TBI 
Total patients 
25 
25 
Transfusions administered (+SD) 
 
 
Platelets (6-10 units per transfusion) 
3.8 (+3.4) 
8.1 (+ 4.4)  
Packed RBCs 
4.0 (+3.7) 
6.2 (+ 4.0) 
 
 
 
Infection related toxicities 
 
 
CMV infection 
1 
1 
Herpes zoster 
1 
2 
Positive blood cultures 
2 
4 
 
 
 
Other toxicities 
 
 
Intubated for somnolence 
1 
4 
Pulmonary hypertension 
1 
0 
Febrile neutropenia 
12 
16 
Jugular venous thrombosis 
1 
0 
Autoimmune uveitis and hearing loss (transient) 
0 
1 
Thrombotic microangiopathy 
0 
4 
Death (bowel-perforation sepsis) 
1 
0 


47 
 
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


48 
 
 
 
Table 4.  Establishment of TIL is improved with ECCE and dependent on 4-1BBL.   
A) Standard TIL expansion from single cell enzymatic tumor cell suspensions was 
compared with Engineered Cellular Costimulation Enhanced (ECCE) TIL growth in 33 
samples.   
B) Subsequently, ECCE TIL growth using 4-1BBL or without 4-1BBL was compared in 
9 samples.  .   
 
Samples 
evaluated 
Standard TIL 
growth 
ECCE TIL 
growth 
P value*
Cryopreserved
21 
6 
14 
0.03 
Fresh
12 
4 
12 
0.001 
Total
33 
10 (30%) 
26 (79%) 
 
 
Samples 
No 4-1BBL 
With 4-1BBL 
 
Total
9 
0 (0%) 
9 (100%) 
.0001 
*Fisher’s exact test
A 
B 


 
 
49
 
 
 
Table 5. TIL and ECCE TIL both demonstrate specific  tumor recognition.  Lymphocyte 
cultures derived from the tumors using standard methods (TIL) or with addition of K562 
expressing 4-1BBL (ECCE TIL) were tested for specific tumor recognition by interferon-
gamma (IFN-g) secretion after overnight coculture.   
 
IFN-g (pg/ml)  
  Allogeneic tumor
Autologous tumor 
Patient 
HLA   TIL
ECCE TIL
TIL 
ECCE TIL 
3018-1a
02,33   
26 
35 
217 
502 
3289
29,32   
55 
58 
789 
410 
3282-1,2,3
02,24   
7 
19 
341 
363 
3084
02,24   
0 
12 
1760
6040 
3281
03,11   
0 
0 
522 
2290 


 
 
50
TABLE 6: 
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.


51 
 
Figure 1 


 
 
52
Figure 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The fraction of lymphocytes in an initial single cell suspension of disaggregated tumor 
predicts TIL growth.  Single cell disaggregated tumor samples from 121 sequential 
patients were evaluated by trypan blue staining and microscopic evaluation for the 
presence of lymphocytes immediately after processing.  The cell suspensions were plated 
for initiation of TIL cultures and subsequently scored for “no growth” or establishment of 
TIL suitable for treatment (“Rx TIL”).   Poorly infiltrated tumors rarely resulted in the 
growth of TIL cultures suitable for therapy.   
 


 
 
53
 
Figure 3 
A 
 
 
 
 
 
B 
 
 
 
 
 
 
 
 
 
 
No impact of genotype (autologous vs. allogeneic) of PBMC on response or expansion.   
Patients treated with CD8+ enriched young TIL were evaluated.  A) The fraction of 
autologous PBMC used as feeder cells in the REP is plotted compared to whether the 
product mediated an objective clinical response (OR) or no response (NR).  There was no 
difference in clinical outcome compared to genotype of the feeder cells.  B) Expansion of 
TIL in the REPis plotted compared to the fraction of autologous PBMC used as feeder 
cells. (some REPS with 100% autologous feeders were jiggered above the 100% mark for 
visibility).  There was no correlation between genotype of feeders and proliferation in the 
REP 
 
 
 
 
 
 
 
 


 
 
54
Figure 4 
 
 
 
TIL cultures are rapidly established with inclusion of Engineered Cellular Costimulation 
Enhancement (ECCE).  Single cell tumor suspensions were plated in the absence (TIL) or 
presence (ECCE TIL) of K562 expressing 4-1BBL.  Lymphocyte growth was monitored 
daily and the day noted when confluent growth was first reached.     
 


55 
 
 
Figure 5 
 
 
 
 
Expansion of TIL is directly dependent on feeder cell number in REP.  1x105 
responding TIL were stimulated with OKT3 and IL-2 in the presence of different 
numbers of PBMC without (REP) or with (ECCE REP) 1x106 Engineered Cellular 
Costimulation Enhancement.  Calculated fold expansion was determined 14 days 
later.   
 
 


 
 
56
Figure 6 
 
 
 
ECCE REP with K562 cells is dependent on expression of 4-1BBL.  TIL were 
expanded by ECCE REP with OKT3, IL-2, reduced feeders cells, and K562 that were 
transduced to express 4-1BBL (With 4-1BBL) or were not transduced with 4-1BBL 
(No 4-1BBL).  The calculated cell number after 14 days of expansion is plotted and 
the two tailed p value for a paired T test is shown (p=0.03).   
 
 
 
 


 
 
57
 Figure 7. 
 
 
A 
 
 
 
 
B 
 
 
 
 
 
 
 
 
 
Clone 7F11 was generated for GMP production of a master cell bank suitable for 
ECCE TIL manufacture.  K562 cells were transduced with a retrovirus encoding the 
CD64 gene then with the 4-1BBL gene.  Cells were cloned by limiting dilution.  
Clone 7F11 was expanded and evaluated by FACS for surface expression of the 
transduced genes. A) 4-1BBL expression by K562 (open histogram) and 7F11 (filled 
histogram).  B) CD64 expression by K562 (open histogram) and 7F11 (filled 
histogram).   





 
 
58
Figure 8 
 
 
 
TIL showed similar proliferation when expanded by conventional REP and ECCE 
REP at clinical scale.  Eight different TIL samples were expanded at clinical scale 
using the standard REP set up with OKT3, IL-2 and 200 fold excess PBMC feeder 
cells.  Each REP was compared to an ECCE REP that was expanded with OKT3, IL-
2, 50 fold excess PBMC feeder cells, and 10fold excess 100Gy irradiated 7F11.  The 
expansions were not significantly different on day 14 (paired T test).   
 
 
 
 
 
 
 
 
 


 
 
59
Figure 9. 
 
 
 
ECCE REP generates similar CD8+ cell frequency to REP, but significantly more NK 
cells in expanded TIL. ECCE REP TIL generated using 7F11 was compared to REP 
TIL generated with PBMC only.  A) CD8+ cell frequency was measured by FACS 
and found to be similar (NS= not significant).  B) NK cell frequency (%CD3-CD56+) 
was significantly higher in the ECCE REP samples.      


 
 
60
Figure 10. 
 
 
Safety of irradiated 7F11 Engineered Cells for Costimulatory Enhancement by 
demonstrating lack of proliferation.  1x105 7F11 cells (100gy gamma-irradiated) were 
plated per well in triplicate wells (circles) in 96 well plates.  In parallel, non-irradiated 
7F11 was added at an average of one cell per 1x105 100gy irradiated 7F11 cells prior 
to plating.  At the indicated times, samples were labeled with tritiated thymidine and 
proliferation was measured by thymidine incorporation.  Live cell controls were 
clearly detected at a sensitivity of 1 cell per 1x105 total cells.  No viable cells were 
detected in 7F11 ECCE cells during 14 days comprising the time of a clinical REP.   
 
 


 
 
61
Figure 11:   Schematic of Young TIL manufactured with ECCE 7F11 
 
 


 
 
62
Appendix 1:  
 
 
Certificate of Analysis:
Engineered Cells for Costimulation Enhanced (ECCE) TIL 
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 
Between 1 x 109 and  
1 x 1011 cells 
 
 
Identity4 
FACS 
> 70 % CD3+ after 
ECCE REP  
 
 
Absence of 
ECCE4 
FACS 
<0.5% CD137L+ after 
ECCE REP 
 
 
Activity2 
IFNgamma release  
>200 pg/ml, and greater 
than 2x background 
 
 
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 
 
 
Presence of 
tumor cells2 
Cytopathology 
No tumor cells per 200 
cells examined 
 
 
1 Performed on sample of the final product immediately prior to infusion.  Results are available at the time of infusion. 
2 Performed 2-10 days prior to infusion.  Results are available at the time of infusion. 
3  Sample collected from the final product prior to infusion.  Results will not be available before cells are infused into the patient. 
4 Performed 2-4 days prior to infusion.  Results are available at the time of infusion but may not be definitive. 
 
 
Prepared by: 
_________________________________________ 
Date:  ____________________ 
QC sign-off: 
__________________________________________ 
Date:  ____________________ 
James C. Yang, M.D. or designee 


 
 
63
Appendix 2: 
 
ADVERSE EVENTS OCCURRING IN 10% OF PATIENTS TREATED WITH ALDESLEUKIN 
(n=525)1 
 
Body System  
% Patients  
 
Body System  
   % Patients 
Body as a Whole  
 
 
 
Metabolic and Nutritional Disorders
Chills   
 
 
52  
 
Bilirubinemia   
 
40 
Fever   
 
 
29  
 
Creatinine increase   
33 
Malaise  
 
 
27  
 
Peripheral edema  
 
28 
Asthenia  
 
 
23 
 
 SGOT increase  
 
23 
Infection  
 
 
13  
 
Weight gain   
 
16 
Pain   
 
 
12  
 
Edema  
 
 
15 
Abdominal pain  
 
11  
 
Acidosis  
 
 
12 
Abdomen enlarged  
 
10  
 
Hypomagnesemia  
 
12 
Cardiovascular  
 
 
 
Hypocalcemia  
 
11 
Hypotension   
 
71  
 
Alkaline phosphatase incr 
10 
Tachycardia   
 
23 
 
Nervous
Vasodilation   
 
13  
 
Confusion  
 
 
34 
Supraventricular tachycardia  12  
 
Somnolence   
 
22 
Cardiovascular disordera  
11  
 
Anxiety  
 
 
12 
Arrhythmia  
 
 
10  
 
Dizziness  
 
 
11 
Digestive  
 
 
 
 
Respiratory
Diarrhea  
 
 
67  
 
Dyspnea  
 
 
43 
Vomiting  
 
 
50  
 
Lung disorderb  
 
24 
Nausea  
 
 
35  
 
Respiratory disorderc   
11 
Stomatitis  
 
 
22  
 
Cough increase  
 
11 
Anorexia  
 
 
20  
 
Rhinitis  
 
 
10 
Nausea and vomiting   
19  
 
Skin and Appendages
Hemic and Lymphatic  
 
 
Rash   
 
 
42 
Thrombocytopenia  
 
37 
 
 Pruritus  
 
 
24 
Anemia  
 
 
29  
 
Exfoliative dermatitis   
18 
Leukopenia 
 
 
16  
 
Urogenital
Oliguria  
 
 
63 
a Cardiovascular disorder: fluctuations in blood pressure, asymptomatic ECG changes, CHF. 
b Lung disorder: physical findings associated with pulmonary congestion, rales, rhonchi. 
c Respiratory disorder: ARDS, CXR infiltrates, unspecified pulmonary changes. 
1Source: Proleukin® Prescribing Information – June 2007 


64 
 
Appendix 3
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 
No 


 
 
65
supportive measures 
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. 
 
 

ICF_001.pdf: 
 
 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
   MEDICAL RECORD 
               •  Adult Patient or     •  Parent, for Minor Patient 
 
 
INSTITUTE: 
National Cancer Institute 
 
 
STUDY NUMBER: 
11-C-0163 
PRINCIPAL INVESTIGATOR:  Steven A. Rosenberg, M.D., Ph.D. 
 
 
STUDY TITLE: 
Phase II Study of Lymphocytes Generated with Engineered Cells for Costimulation Enhancement 
in Patients with Metastatic Melanoma Following Lymphodepletion 
 
 
Continuing Review Approved by the IRB on 07/23/12 
Amendment Approved by the IRB on 07/09/12 (C) 
 
 
 
Date Posted to Web:  09/06/12 
Standard 
 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL 
 
RESEARCH STUDY 
 
•   Adult Patient or    •   Parent, for Minor Patient 
 
NIH-2514-1 (7-09) 
 
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. 
 
Why is this study being done?  
The purpose of this study is to determine whether we can treat melanoma by taking blood cells called lymphocytes from 
patients' tumors, and grow them in the laboratory in large numbers to then give the cells back to the patient.  These cells 
are called Young Tumor Infiltrating Lymphocytes, or Young TIL and these Young TIL were prepared in the presence of 
another type of  cell called Engineered Cells for Costimulation Enhancement (ECCE) that we hope will help us make the 
Young TIL cells grow easier and faster.  We will test whether this experimental regimen can cause your tumor to shrink, 
as well as test the safety of this regiment and the effects on the immune system.  Before giving the cells back to you, we 
will suppress (make it less able to fight) your immune system with two chemotherapy agents in order to create room for 
your cells and to prevent the immune system cells from working against the cells we give to you.  After giving you the 
cells, we will give you aldesleukin (IL-2) for up to 5 days to help keep the cells alive.   
 


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  2  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
 
Why are you being asked to take part in this study? 
Your doctor has told you that you have been diagnosed with melanoma.  The only treatment available at this time that 
has been shown to cure melanoma is aldesleukin.  We invite you to take part in this research study that is trying to find 
effective treatments for patients with melanoma. 
 
How many people will take part in this Study?  
As many as 63 patients may participate in this study. If you decide to participate, you will undergo a series of tests to 
make sure you are eligible, including blood tests and scans as described below. 
 
Birth Control 
If you are a woman who is breast feeding or pregnant, you may not take part in the study because we don’t know how 
this medicine would affect your baby or your unborn child. If you are a woman who can become pregnant, or are the 
partner of a woman who can become pregnant, you will need to practice an effective form of birth control before starting 
study treatment, during study treatment, and for four months after you finish study treatment.  If you think that you or 
your partner is pregnant, you should tell your study doctor or nurse at once.   
Effective forms of birth control include: 
• 
Abstinence  
• 
intrauterine device (IUD) 
• 
hormonal [birth control pills, injections, or implants]  
• 
tubal ligation 
• 
vasectomy 
 
Description of Research Study 
 
The cell therapy has 5 stages outlined below: 
   
Stage 
Timeframe 
Location 
Comments & Instructions 
Work up 
1-2 weeks 
Inpatient and 
outpatient   
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 
Cells and IL-2 
(Day 0) 
1-5 days 
Inpatient and 
possibly ICU 
Receive Young TIL, 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 
unless disease 
progression 
Outpatient 
Return to clinic for physical exam, review of side 
effects, labs, scans every 1-4 months for the first 
year then every 3-4 months. 


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  3  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
 
What will happen if you take part in this research study?   
Cell harvest and growth 
You will first have some tumors removed from your body so we can grow the TIL cells contained in the tumor in the 
laboratory.  We will be obtaining the cells from a biopsy of your tumor or during surgery.  Based on our previous 
experience growing Young TIL without the help of ECCE cells, at approximately 20% of the time we are unable to 
successfully grow the cells needed for this procedure.  However, we have used ECCE cells in laboratory experiments and 
have shown that these ECCE cells help TIL have a better chance at growing.  For this study, after obtain the cells from 
your tumor, we will try to grow them using two different methods.  If your cells grow well using the original method to 
generate Young TIL, these are the TIL we will use to grow more cells for therapy.  If your cells do not grow this way, we 
will check to see if your Young TIL are growing in the presence of ECCE cells.  If your cells do not grow with either 
method, you will not be able to receive the cell infusion, you will be taken off this study, and we will look at alternative 
treatments for you or return you to the care of your referring physician.  We usually know after about 2-4 weeks whether 
the cells will grow well enough to be used as an experimental treatment on this protocol.  At the time we determine that 
your cells are not growing, we will inform you and discuss your options with you.  
 
The procedure for converting your normal cells into cells that are able to recognize your tumor has been studied in the 
laboratory and although these cells can destroy tumor cells in the test tube, it is not clear that they will be able to destroy 
them inside a patient’s body.  We have grown young TIL cells similar to these and treated over 50 patients.  The Young 
TIL cells in this study are different from others that we have given patients because they are grown in the presence of 
special ECCE cells that help stimulate the cells to grow.  In previous studies we have used other cells from the patient’s 
own blood or from a normal volunteer to stimulate the Young TIL to grow, but the process of collecting these extra cells is 
expensive, time-consuming and sometimes does not generate enough cells to help grow the Young TIL. This experimental 
study will test whether growing young TIL cells this way is effective in shrinking your cancer tumor(s).  In our initial 
experience with Young TIL cells, of 45 patients treated and evaluated for response, 11 had some shrinkage.   
 
Work up 
Prior to receiving the experimental treatment you will undergo many tests. These include imaging procedures, heart and 
lung function tests, and laboratory tests.  If you are a woman, you will undergo a pregnancy test.  You will also have a 
large catheter inserted into a vein and leukapheresis (see description below) 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 therapy, 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. 
 


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  4  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
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 Young TIL and after the 
experimental therapy, you will have leukapheresis so that we can test your cells. 
 
Chemotherapy Regimen (Day -7 through Day -1) 
You will be admitted to the hospital.  For the seven days prior to receiving your cells you will be given two chemotherapy 
medicines to suppress your immune system so the cells we give you can work without any interference from the cells in 
your immune system.  There is evidence in animal experiments and human studies that this can increase how effective 
the cells we give are in fighting cancer cells, but it is not known whether this is true for your cells.   
 
The two chemotherapy medicines are called cyclophosphamide and fludarabine.  The cyclophosphamide will be given into 
your IV over 1 hour for two days and the fludarabine will be given into your IV for 30 minutes every day for the five days 
following the cyclophosphamide.  These chemotherapies will not treat the tumor and are given only to see if the 
suppression of the immune system improves the functioning of the experimental cells we will be giving you. 
 
Cell Infusion and IL-2 Regimen (Day 0 to Day 4) 
One to four days after the last dose of chemotherapy, you will be given the TIL.  The TIL will be given in your catheter 
over 20-30 minutes.  Within 24 hours after the TIL infusion, you will be given high dose aldesleukin (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 help the cells live longer in your body.  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 TIL, we will give you 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.  
 
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; however, you may need to stay in the hospital for longer than 10 days before you are well enough to go home. 
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. 
 


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  5  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
Optional Biopsy 
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.  The biopsy to be performed is 
exclusively for research purposes and will not benefit you.  It might help other people in the future.  Even if you sign "yes" 
to have the biopsy you can change your mind at any time.  Please read each sentence below and think about your choice. 
After reading each sentence, circle and initial the answer that is right for you. The decision to participate in this part of the 
research is optional, and no matter what you decide to do, it will not affect your care.  
 
I agree to have the tumor or lymph node biopsy for the research tests in this study.  
 
Yes              No               Initials_________ 
 
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 approximately 4-6 weeks after receiving the Young TIL.  This will probably take 2 days.  You will have 
scans and x-rays to evaluate your tumor and blood tests.  We will also take some blood from you through leukapheresis 
to see the impact of this therapy on the immune system and see if cells we gave you are still alive.  If your tumor shows 
evidence of shrinking, we will ask you to return for evaluation every month for the first 3 months, then every 3-4 months 
for the next 5 years, and yearly thereafter.  If your tumor appears to be growing, we will look for other investigational 
therapies you may be eligible for, or refer you back to the care of your local physician.   
Alternative Approaches or Treatments 
What other choices do I have if I do not take part in this study?  
 
Instead of being in this study, you have these options: 
 
• 
Getting treatment or care for your cancer without being in a study 
o 
One of the few therapies that have been approved by the US Food and Drug Administration (FDA) for the 
treatment of melanoma is interleukin-2 (IL-2), also called aldesleukin.  IL-2 can shrink tumors in 
approximately 16% of patients with metastatic melanoma.   
o 
Chemotherapy, such as dacarbazine or temozolomide, is also used to treat melanoma. 
• 
Taking part in another study 
• 
Getting comfort care, also called palliative care.  This type of care helps reduce pain, tiredness, appetite problems 
and other problems caused by the cancer.  It does not treat the cancer directly.  Instead, it tries to improve how 
you feel.  Comfort care tries to keep you as active and comfortable as possible. 
 
Please talk to your doctor about these and other options. 


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  6  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
Risks or Discomforts of Participation 
The major side effects of this treatment (described in detail below) 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, shortness of breath, and high heart rate caused by the IL-2.  Other side 
effects of IL-2 are listed below. 
 
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.   
 
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 to chew that takes 
away this tingling.  Rarely, people 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, bleeding or bruising where 
the needles are put in your arms. 
 
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. 
 
TIL Cell Infusion  
All TIL cells will be given through your catheter while we keep you in the patient care unit so we can watch you closely.   
Potential risks include:  
 
 
• 
shortness of breath, or  
• 
an 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.  
• 
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.    
 
 
In the first 24 patients treated on the initial Young TIL study, we have observed an increase in the number of patients 
who have experienced some of the side effects of cells and aldesleukin.  One out of three these patients were intubated 
(a tube was placed in the throat to help them breath), and two patients were put on dialysis due to abnormalities in 
kidney function.   


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  7  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
Medications 
The side effects of cyclophosphamide, fludarabine, IL-2 and some of the other medications you will receive are listed in 
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 Virus Lymphoma.  This can 
be fatal (Two patients on other studies in 
the Surgery Branch developed EBV 
lymphoma, and one died as a result of 
this disease.) 
 
Loss of fertility 
 
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 
 
Skin rash, hives, itching  
 
Tremors, dizziness, Confusion, seizures 


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  8  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
not cause any symptoms 
 
Nausea, vomiting, 
diarrhea, constipation  
 
Pain and irritation at place 
of injection 
 
Fatigue  
 
Blood in the urine 
 
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.  
 
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. 
 
Potential Benefits of Participation 
Are there benefits to taking part in this study?  
One aim of this study is to see if this experimental young TIL will cause your tumors to shrink.  We hope that you will get 
personal medical benefit from taking part in this study, but we cannot be certain.  These potential benefits could include 
shrinking of your tumor or lessening of your symptoms, such as pain, that are caused by the cancer.   
 
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 


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  9  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
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.  Your records may be reviewed by NIH organizations and by 
organizations outside the National Institutes of Health, such as 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.  
 
What are the costs of taking part in this study?  
If you choose to take part in the study, the following will apply, in keeping with the NIH policy: 
• 
You will receive study treatment at no charge to you. This may include surgery, medicines, 
laboratory testing, x-rays or scans done at the Clinical Center, National Institutes of Health 
(NIH), or arranged for you by the research team to be done outside the Clinical Center, NIH if 
the study related treatment is not available at the NIH.  
• 
There are limited funds available to cover the cost of some tests and procedures performed 
outside the Clinical Center, NIH.  You may have to pay for these costs even if they are not 
covered by your insurance company. 
• 
Medicines that are not part of the study treatment will not be provided or paid for by the Clinical 
Center, NIH. 
• 
Once you have completed taking part in the study, medical care will no longer be provided by 
the Clinical Center, NIH. 
 
Stopping Therapy  
Your doctor may decide to stop your therapy for the following reasons: 
• 
if he/she believes that it is in your best interest 
• 
if your disease comes back during treatment 
• 
if you have side effects from the treatment that your doctor thinks are too severe 
• 
if new information shows that another treatment would be better for you 
 
In this case, you will be informed of the reason therapy is being stopped. 
 
You can stop taking part in the study at any time. However, if you decide to stop taking part in the study, we would like 
you to talk to the study doctor and your regular doctor first. 
 
 
Conflict of Interest 
 
The National Institutes of Health (NIH) reviews NIH staff researchers at least yearly for conflicts of interest.  The following 
link contains details on this process http://ethics.od.nih.gov/procedures/COI-Protocol-Review-Guide.pdf.  You may ask 
your research team for a copy of the Protocol Review Guide or for more information.  Members of the research team who 
do not work for NIH are expected to follow these guidelines but they do not need to report their personal finances to the 
NIH. 
 


 
 
  
CONTINUATION SHEET for either: 
  MEDICAL RECORD 
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: 
11-C-0163 
CONTINUATION:  page  10  of  12 pages 
 
 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
 
NIH-2514-1 (10-84) 
 
NIH-2514-2 (10-84) 
 
P.A.: 09-25-0099 
 
 
The National Institutes of Health and the research team for this study have developed the Young TIL being used in this 
study.  This means 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. You will not receive any money 
from the development of Young TIL. 
 
Optional Studies  
We would like to keep some of the specimens and data that are 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.   
 
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 data.  Then any specimens that remain 
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 use 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 data in new research not included 
in this consent.  
 
Yes              No               Initials_________ 
 
 


 
  
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
   MEDICAL RECORD 
•  Adult Patient or    •  Parent, for Minor Patient 
 
STUDY NUMBER: 
11-C-0163 
CONTINUATION:  page  11  of  12  pages 
 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL 
 
RESEARCH STUDY (Continuation Sheet) 
 
•  Adult Patient or   •  Parent, for Minor Patient 
 
NIH-2514-1 (7-09) 
 
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 10 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, Telephone: 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.


 
  
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
   MEDICAL RECORD 
•  Adult Patient or    •  Parent, for Minor Patient 
 
STUDY NUMBER: 
11-C-0163 
CONTINUATION:  page  12  of  12  pages 
 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL 
 
RESEARCH STUDY (Continuation Sheet) 
 
•  Adult Patient or   •  Parent, for Minor Patient 
 
NIH-2514-1 (7-09) 
 
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 
 
 
 
Print Name 
Signature of Parent(s)/Guardian 
Date 
 
 
 
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 JULY 23, 2012 THROUGH JANUARY 22, 2013.  
 
 
 
  
 
 
  
 
Signature of Investigator 
Date 
 
 
 
Print Name 
Signature of Witness 
Date 
 
 
 
Print Name