A Prospective Randomized Study of Cell Transfer Therapy for Metastatic Melanoma Using Short-Term Cultured Tumor-Infiltrating Lymphocytes Plus IL-2 Following Either a Non-Myeloablative Lymphocyte Depleting Chemotherapy Regimen Alone or in Conjunction With 12Gy Total Body Irradiation (TBI)
Phase: PHASE2
Conditions: Metastatic Melanoma, Skin Cancer
Sponsor: National Cancer Institute (NCI)

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
1
Abbreviated Title: 1200 TBI TIL Randomized Study 
NIH Protocol Number: 11C0123  
NCT Number: NCT01319565
Version Date: 04/15/2022
PROTOCOL TITLE
A Prospective Randomized Study of Cell Transfer Therapy for Metastatic Melanoma Using 
Short-Term Cultured Tumor-Infiltrating Lymphocytes Plus IL-2 Following Either a Non-
Myeloablative Lymphocyte Depleting Chemotherapy Regimen Alone or in Conjunction with 
12Gy Total Body Irradiation (TBI) 
NIH Principal Investigator:
Steven A. Rosenberg, M.D., Ph.D. 
Chief of Surgery, Surgery Branch, CCR, NCI
Building 10, CRC, Room 3-3940
9000 Rockville Pike, Bethesda, MD 20892
 Email: Steven.Rosenberg@nih.gov
Investigational Agents:
Drug Name:
Young TIL
IND Number:
14265
Sponsor
Center for Cancer Research
Manufacturer
Surgery Branch Cell Production Facility
Commercial Agents: Cyclophosphamide, Fludarabine, and Aldesleukin

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
2
PRÉCIS
Background:

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes can 
mediate the regression of bulky metastatic melanoma when administered along with 
high-dose aldesleukin (IL-2) following a non-myeloablative lymphodepleting 
chemotherapy preparative regimen consisting of cyclophosphamide and fludarabine.

In a series of consecutive trials using this chemotherapy preparative regimen alone or 
with 2 Gy or 12 Gy total body irradiation (TBI) objective response rates using RECIST 
criteria were 49%, 52%, and 72%, respectively. Complete regression rates in these three 
consecutive trials were 12%, 20%, and 40%, respectively—strongly suggesting that the 
addition of TBI could improve the complete regression rate. Of the 20 complete 
regressions seen in this trial, 19 are on-going at 37 to 82 months.

Because of the complexity of developing selected TIL for use in adoptive transfer, we 
have recently developed a simplified method for producing TIL that is more applicable to 
use in outside institutions. Utilizing “young TIL” cells (sometimes with CD8 
purification) in 105 patients, the objective response rate was 34% with a 6.6 % incidence 
of complete regressions. All patients in this trial received the cyclophosphamide 
fludarabine regimen alone.

Because of the strong suggestion that the addition of TBI to the chemotherapy regimen 
could increase durable, complete regression rates in patients with metastatic melanoma, 
we are now attempting to definitively determine whether the addition of TBI to the 
chemotherapy preparative regimen can improve complete response rates, and overall 
survival in patients receiving “young TIL”. 
Objectives:

To determine, in a prospective randomized trial, the complete response rate and survival 
of patients with metastatic melanoma receiving ACT using young TIL plus aldesleukin 
treatment following either a chemotherapy preparative regimen alone, or the same 
chemotherapy preparative regimen plus TBI.
Eligibility:
Patients who are 18 years or older must have:

Evaluable metastatic melanoma;

Metastatic melanoma lesion suitable for surgical resection for the preparation of TIL;

No contraindications to high-dose aldesleukin administration or total body irradiation;

No concurrent major medical illnesses or any form of immunodeficiency
Design:

Patients with metastatic melanoma will have lesions resected and after TIL growth is 
established patients with will be prospectively randomized to receive ACT with young 
TIL plus aldesleukin following either a non-myeloablative chemotherapy preparative 
regimen or this same regimen plus TBI. 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
3
TABLE OF CONTENTS 
PRÉCIS...........................................................................................................................................2
TABLE OF CONTENTS ..............................................................................................................3
STATEMENT OF COMPLIANCE.............................................................................................7
1
INTRODUCTION .............................................................................................................7
1.1
Study Objectives..............................................................................................................7
1.1.1
Primary Objective........................................................................................................7
1.1.2
Secondary Objectives ..................................................................................................7
1.2
Background and Rationale...............................................................................................7
2
ELIGIBILITY ASSESSMENT AND ENROLLMENT...............................................11
2.1
Eligibility Criteria..........................................................................................................11
2.1.1
Inclusion Criteria .......................................................................................................11
2.1.2
Exclusion Criteria ......................................................................................................12
2.2
Research Eligibility Evaluation .....................................................................................12
2.2.1
Within 2 Months Prior to Randomization .................................................................12
2.2.2
Within 3 Weeks Prior to Randomization AND Treatment........................................13
2.2.3
Within 7 Days Prior to Treatment .............................................................................13
2.3
Participant Registration and Status Update Procedures...............................................13
3
STUDY IMPLEMENTATION.......................................................................................14
3.1
Study Design..................................................................................................................14
3.2
Protocol Stopping Rules ................................................................................................14
3.3
Autologous Stem Cell Collection ..................................................................................15
3.4
Drug Administration......................................................................................................15
3.4.1
Preparative Regimen..................................................................................................15
3.4.2
Cell Infusion and Aldesleukin Administration..........................................................16
3.5
Study Calendar...............................................................................................................17
3.6
Total Body Irradiation (TBI) .........................................................................................17
3.6.1
Energy........................................................................................................................17
3.6.2
Timing........................................................................................................................18
3.6.3
Technique ..................................................................................................................18
3.6.4
Dose/Fractionation.....................................................................................................18
3.6.5
Dose Modifications....................................................................................................18
3.6.6
Toxicities ...................................................................................................................18
3.7
Infection Prophylaxis.....................................................................................................18
3.7.1
Pneumocystis Jirovecii Pneumonia ...........................................................................18
3.7.2
Herpes Virus Prophylaxis..........................................................................................18
3.7.3
Fungal Prophylaxis (Fluconazole).............................................................................19
3.7.4
Infection Prophylaxis for Patients in Cohort 2 (TBI Cohort)....................................19
3.7.5
Empiric Antibiotics....................................................................................................19

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
4
3.8
Blood Product Support ..................................................................................................19
3.9
Aldesleukin: Intravenous Administration......................................................................19
3.10
On Study Evaluation......................................................................................................19
3.10.1
Prior to Starting the Preparative Regimen.............................................................19
3.10.2
During the Preparative Regimen (Daily)...............................................................20
3.10.3
After Cell Infusion.................................................................................................20
3.10.4
During and After Aldesleukin Administration until Hospital Discharge ..............20
3.10.5
Severe Immune Reactions .....................................................................................20
3.11
Post Treatment Evaluation.............................................................................................20
3.11.1
Routine Follow up .................................................................................................20
3.12
Criteria for Removal from Protocol Therapy and Off-Study Criteria ...........................21
3.12.1
Criteria for Removal from Protocol Therapy ........................................................21
3.12.2
Off-Study Criteria..................................................................................................22
4
CONCOMITANT MEDICATIONS/MEASURES.......................................................22
5
CORRELATIVE STUDIES FOR RESEARCH...........................................................22
5.1
Samples Sent to Figg Lab Blood Processing Core (BPC).............................................23
5.2
Samples Sent to Surgery Branch Cell Production Facility (SB-CPF)...........................23
5.3
Prior to Start of Chemotherapy Administration.............................................................23
5.4
Prior to Cell Infusion .....................................................................................................23
5.5
Post Cell Infusion Evaluations.......................................................................................23
5.6
Follow-up.......................................................................................................................23
5.7
Immunological Testing..................................................................................................23
5.8
Sample Storage, Tracking and Disposition for Surgery Branch Cell Production Facility 
(SB-CPF) ...................................................................................................................................24
5.9
Sample Storage, Tracking and Disposition for Figg Lab (BPC)...................................24
5.9.1
Sample Data Collection .............................................................................................24
5.9.2
Sample Storage and Destruction................................................................................25
5.10
Additional Research Evaluations...................................................................................25
6
DATA COLLECTION AND EVALUATION..............................................................27
6.1
Data Collection ..............................................................................................................27
6.1.1
Exclusions to Routine Adverse Event Recording......................................................27
6.2
Data Sharing Plans.........................................................................................................27
6.2.1
Human Data Sharing Plan .........................................................................................27
6.2.2
Genomic Data Sharing Plan.......................................................................................28
6.3
Response Criteria...........................................................................................................28
6.3.1
Evaluation of Target Lesions.....................................................................................28
6.3.2
Evaluation of Non-Target Lesions.............................................................................28
6.3.3
Evaluation of Best Overall Response ........................................................................28
6.3.4
Confirmatory Measurement/Duration of Response...................................................29

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
5
6.3.5
Image Acquisition Plan..............................................................................................29
6.4
Toxicity Criteria.............................................................................................................29
7
NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN 30
7.1
Definitions .....................................................................................................................30
7.2
OHSRP Office of Compliance and Training/IRB Reporting ........................................31
7.2.1
Expedited Reporting ..................................................................................................31
7.2.2
IRB Requirements for PI Reporting at Continuing Review ......................................31
7.3
NIH Required Data and Safety Monitoring Plan...........................................................31
7.3.1
Principal Investigator/Research Team.......................................................................31
7.3.1
Data Safety Monitoring Board (DSMB)....................................................................31
8
SPONSOR PROTOCOL/ SAFETY MONITORING PLAN ......................................31
8.1
Definitions .....................................................................................................................31
8.1.1
Adverse Event............................................................................................................31
8.1.2
Serious Adverse Event...............................................................................................31
8.1.3
Life-Threatening........................................................................................................32
8.1.4
Severity......................................................................................................................32
8.1.5
Relationship to Study Product ...................................................................................32
8.2
Assessment of Safety Events.........................................................................................32
8.3
Reporting of Serious Adverse Events............................................................................33
8.4
Reporting Pregnancy .....................................................................................................33
8.5
Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND .................34
8.6
Sponsor Protocol Deviation Reporting..........................................................................34
9
Clinical Monitoring .........................................................................................................34
10
STATISTICAL CONSIDERATIONS...........................................................................35
11
COLLABORATIVE AGREEMENTS ..........................................................................36
12
HUMAN SUBJECTS PROTECTIONS ........................................................................37
12.1
Rationale for Patient Selection ......................................................................................37
12.2
Participation of Children................................................................................................37
12.3
Participation of Subjects Unable to Give Consent ........................................................38
12.4
Evaluation of Benefits and Risks...................................................................................38
12.5
Consent Document.........................................................................................................38
13
REGULATORY AND OPERATIONAL CONSIDERATIONS.................................39
13.1
Study Discontinuation And Closure ..............................................................................39
13.2
Quality Assurance and Quality Control.........................................................................39
13.3
Conflict Of Interest Policy.............................................................................................39
13.4
Confidentiality And Privacy..........................................................................................40
14
PHARMACEUTICAL INFORMATION .....................................................................40
14.1
Aldesleukin (Interleukin-2, Proleukin, Recombinant Human Interleukin 2) ................40

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
6
14.2
Fludarabine ....................................................................................................................41
14.3
Cyclophosphamide ........................................................................................................42
14.4
Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891) ..........43
14.5
Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim, Neupogen) .43
14.6
Trimethoprim and Sulfamethoxazole Double Strength (TMP / SMX DS) ...................43
14.6.1
Aerosolized Pentamidine in Place of TMP/SMX DS............................................44
14.7
Herpes Virus Prophylaxis..............................................................................................44
14.7.1
Valacyclovir (Valtrex)...........................................................................................44
14.7.2
Acyclovir ...............................................................................................................44
14.8
Fluconazole....................................................................................................................44
14.9
Cell Preparation .............................................................................................................45
14.10 OKT3 .............................................................................................................................45
14.11 Support Medications......................................................................................................45
15
REFERENCES ................................................................................................................46
16
FIGURES, TABLES & APPENDICES.........................................................................47

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
7
STATEMENT OF COMPLIANCE
The trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the following: 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812). 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study. In addition, all changes to the consent form will be IRB-approved; an 
IRB determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form.
1
INTRODUCTION
1.1
STUDY OBJECTIVES
1.1.1
Primary Objective 

To compare in a prospective randomized trial, the complete response rate and survival in 
patients with metastatic melanoma receiving adoptive cell transfer (ACT) using young 
TIL plus aldesleukin treatment following a preparative regimen consisting of 
lymphodepleting chemotherapy with or without total body irradiation.
1.1.2
Secondary Objectives

To determine response rates and progression free survival in patients in the two treatment 
arms.

To determine the toxicity of these two treatment regimens.
1.2
BACKGROUND AND RATIONALE 
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 treatment 
known to cure some patients with metastatic melanoma. Dacarbazine-based chemotherapy has a 
clinical response rate of up to 20% but almost no 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 ACT therapy 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-

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
8
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 2 or 12 Gy whole body 
irradiation prior to cell infusion and aldesleukin administration. The overall objective response 
rate using RECIST criteria in these 93 patients was 56%. The characteristics of these patients are 
shown in Table 1, and the toxicities shown in Table 2 for the 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 in the 2 Gy trial who had an 
undetected diverticular abscess prior to beginning therapy. In the 12 Gy study, five patients had 
hematuria and elevated creatinine levels. Four of these patients underwent a renal biopsy which 
indicated thrombotic microangiopathy. The consulting nephrologists indicated that they believed 
this toxicity would progress to renal failure and all these patients would eventually need dialysis. 
However, this has not occurred and all 5 patients are doing well.
The objective and complete response rates in these three consecutive trials are shown in Table 4. 
The objective response rates (RECIST) were 49%, 52%, and 72% and the complete response 
rates were 12%, 20%, and 40% in the NMA study, 2 Gy study and 12 Gy study, respectively. Of 
the 20 complete responders in the three trials, 19 are ongoing from 37 to 82 months. These 
represent by far the highest complete response rates seen in patients with metastatic melanoma 
(Table 5). 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 cause doubt concerning the validity of this conclusion (Figure 1 and 
Figure 2). 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.
Despite this high rate of objective responses in previously treated patients, ACT is available only 
in the Surgery Branch, NCI and although two other institutions have begun to explore ACT 
therapy very limited numbers of patients have been treated elsewhere. The generation of TIL is 
time consuming, expensive and labor intensive. In addition, the costs for cell preparation as well 
as patient treatment are not covered by third party payers and these factors have discouraged the 
application of the ACT approach. In the original clinical trials, all patients received highly 
selected TIL, grown from multiple individual cultures with expansion of cultures shown to have 
specific reactivity against the autologous tumor or HLA matched tissue culture lines. This 
requirement substantially increased the difficulty in generating the appropriate reagents 
necessary to treat a large number of patients.
Because of the difficulties in generating highly selected reactive TIL we have put considerable 
effort into the development of techniques for the generation of effective TIL using procedures 
that do not require the extensive laboratory manipulations and testing required to develop highly 
selective TIL. In addition, intensive post-hoc analysis of some TIL cultures revealed tumor 
recognition that was not discovered in time to treat patients. This revealed that some TIL were 
considered “non-reactive” due to inadequacies in current assay methods. 
Furthermore, murine and human evidence suggest that TIL cultured for shorter times (young 
TIL) may have better in vivo activity against tumors than extensively cultured TIL. In the initial 
report of the treatment of melanoma patients with adoptive transfer of TIL, 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 examining 
the role of telomere length support this hypothesis. Differentiation and expansion of T cells is 
associated with telomere shortening7,8 and with altered and loss of cell functions, including 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
9
proliferation9,10. 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)11. TIL which persisted in vivo had longer telomere lengths than TIL 
which did not persist (p < 0.001).
To determine the relationship between telomere length and time in culture, we measured 
telomere lengths of 495 TIL cultures generated from 48 consecutive patients by quantitative 
fluorescent in-situ hybridization (QFISH)12. The TIL were evaluated after eight to 56 days in 
culture. Figure 3 shows the close relationship between the age of TIL in culture (days) and their 
mean telomere length (kb). Though the telomere lengths varied widely, there was a strong 
inverse correlation between time in culture and telomere length of TIL (p < 0.001). This 
observation is consistent with earlier observations that shorter time in culture is associated with 
improved response and improved response is associated with longer telomere lengths. 
The hypothesis that younger TIL have improved in vivo survival and anti-tumor activity is 
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 
cells13. 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 persistence14. In a murine 
model of adoptive immunotherapy, Gattinoni et al demonstrated that the differentiation of 
effector T cells in culture had a severe 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 
cells15.
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” TIL. We studied the 
expression of CD27 and CD28 in young and standard TIL to examine the differences in 
phenotypic expression of these TIL12. 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 
fluorescent-activated cell sorting (FACS). Figure 4 shows representative FACS plots comparing 
the phenotypic expression of one young TIL culture with its matched standard TIL culture. 
Figure 5 demonstrates the differences in phenotypic expression of CD27 and CD28 by young 
and standard TIL. Young TIL have 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 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.
A potential advantage of the use of this simpler method of producing TIL is that they will be 
more heterogeneous in their reactivity than highly selected TIL which have grown for longer 
periods of time. Another major advantage of the young TIL procedure is that the great majority 
of patients with resectable lesions can be treated. Thus, young TIL can be successfully grown 
from approximately 80% of all patients with resectable lesions. This high rate of cell production 
makes it possible to treat the great majority of patients with resectable lesions that come to the 
Surgery Branch for treatment. 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
10
We have performed a series of clinical trials to evaluate ACT using these young TIL 
preparations. In the first of these consecutive trials, 24 patients were treated with young TIL and 
in a second cohort, 39 patients were treated with young TIL that had been enriched for CD8+ 
cells. Although there was a suggestion that CD8 enriched young TIL gave higher response rates, 
we then performed a prospective randomized trial in which patients were randomized to receive 
either bulk young TIL or CD8 enriched young TIL. This trial has almost completed accrual and 
there is no suggestion of a difference in either overall response rates, complete response rates, or 
toxicities between the young TIL and CD8 enriched young TIL arms. The CD8 enrichment 
procedure can be quite expensive, including the use of the GMP quality Miltenyi separation 
apparatus.  Thus, for future trials we will be using bulk young TIL. Since there were no 
differences between these two cell preparations, combined data of 105 patients that received 
young TIL or CD8 enriched young TIL are presented. In this combined group of 105 patients, all 
of whom received the cyclophosphamide fludarabine alone non-myeloablative preparative 
regimen, the objective response rate was 34.3% with 6.6% complete regressions. There is no 
significant difference in overall or complete response rates in patients receiving young TIL or the 
selected TIL used in previous trials (p2=0.14 and p2=0.33, respectively) in patients receiving the 
same chemotherapy non-myeloablative preparative regimen alone. The toxicity profile is also not 
significantly different than that using highly selected TIL. A post-hoc analysis of the activity of 
young TIL in-vitro revealed that many of these preparations had specific anti-tumor activity 
quite similar to that of the selected TIL used in prior trials. A pilot trial was conducted in 23 
patients using young TIL and aldesleukin following cyclophosphamide/fludarabine and a 6 GY 
intermediate dose of TBI. Objective responses were seen in 48% of patients with 13% of patients 
experiencing a complete regression. This complete regression was double the 6.6% complete 
regression rate in patients receiving young TIL and the chemotherapy preparative regimen alone. 
Toxicities were similar to that in the trials without TBI.
Interestingly, 2 additional groups have now reported their results utilizing the 
cyclophosphamide/fludarabine non-myeloablative preparative regimen followed by young TIL 
and IL-2 administration. Besser et al., treated 31 patients with an objective response rate of 
48.4% and a complete regression rate of 12.9 %. Joseph, et al., reported at the last meeting of the 
International Society for the Biologic Therapy of Cancer on 25 patients treated with young TIL 
with an objective response rate of 52% with 4% complete regressions. Thus, the conglomerate 
data available concerning the use of young TIL and the chemotherapy alone preparative regimen 
in 161 patients revealed an objective response rate of 39.8 % with a 7.5 % incidence of complete 
regressions.
In summary, this data reveals that the results of the use of young TIL is quite similar to that of 
the previous trials with selected TIL utilizing the cyclophosphamide fludarabine preparative 
regimen. In our previously published trials using selected TIL, the complete regression rate using 
the chemotherapy preparative regimen alone was 11.6% compared to 40% when 12Gy TBI was 
(p2=0.01). Thus, in the current trial we will attempt to definitively answer the question as to 
whether the 12 Gy TBI does indeed improve results by prospectively randomizing patients to 
receive TIL plus IL-2 following either the chemotherapy preparative regimen alone or the 
chemotherapy/12 Gy TBI regimen.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
11
2
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. The lesion must be of at least 1 cm in diameter that can be surgically removed 
with minimal morbidity (defined as any operation for which expected hospitalization ≤ 7 
days).
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 and less than or equal to 66 years of age.
d. Willing to practice birth control during treatment and for four months after receiving all 
protocol related therapy.
e.
Life expectancy of greater than three months
f.
Willing to sign a durable power of attorney.
g. Able to understand and sign the Informed Consent Document
h. Clinical performance status of ECOG 0 or 1.
i.
Hematology:
o
Absolute neutrophil count greater than 1000/mm3 
o
Hemoglobin greater than 8.0 g/dl
o
Platelet count greater than 100,000/mm3
j.
Serology: 
o
Seronegative for HIV antibody. (The experimental treatment being evaluated in this 
protocol depends on an intact immune system. Patients who are HIV seropositive can 
have decreased immune competence and thus be less responsive to the experimental 
treatment and more susceptible to its toxicities.)
o
Seronegative for hepatitis B antigen, or hepatitis C antibody or antigen.
k. Chemistry:
o
Serum ALT/AST less than three times the upper limit of normal.
o
Calculated creatinine clearance (eGFR) > 50 mL/min.
o
Total bilirubin less than or equal to 2 mg/dl, except in patients with Gilbert’s 
Syndrome who must have a total bilirubin less than 3 mg/dl.
l.
More than four weeks must have elapsed since any prior systemic therapy at the time of 
randomization, and patients’ toxicities must have recovered to a grade 1 or less (except 
for alopecia or vitiligo). Patients must have stable or progressing disease after prior 
treatment. 
Note: 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.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
12
m. Six weeks must have elapsed since any prior anti-CTLA4 antibody therapy to allow 
antibody levels to decline.
Note: Patients who have previously received ipilimumab or tremelimumab, anti- PD1 or 
anti-PD-L1 antibodies, and have documented GI toxicity must have a normal 
colonoscopy with normal colonic biopsies.
2.1.2
Exclusion Criteria 
a.
Prior cell transfer therapy which included a non-myeloablative or myeloablative 
chemotherapy regimen.
b. Women of child-bearing potential who are pregnant or breastfeeding because of the 
potentially dangerous effects of the preparative chemotherapy on the fetus or infant.
c.
Systemic steroid therapy requirement.
d. Active systemic infections, coagulation disorders or other active major medical illnesses 
of the cardiovascular, respiratory or immune system, as evidenced by a positive stress 
thallium or comparable test, myocardial infarction, cardiac arrhythmias, obstructive or 
restrictive pulmonary disease.
e.
Any form of primary immunodeficiency (such as Severe Combined Immunodeficiency 
Disease and AIDS). 
f.
Opportunistic infections (The experimental treatment being evaluated in this protocol 
depends on an intact immune system. Patients who have decreased immune competence 
may be less responsive to the experimental treatment and more susceptible to its 
toxicities.)
g. History of severe immediate hypersensitivity reaction to any of the agents used in this 
study.
h. History of coronary revascularization or ischemic symptoms.
i.
Any patient known to have an LVEF less than or equal to 45%.
j.
In patients > 60 years old, documented LVEF of less than or equal to 45%.
k. Documented FEV1 less than or equal to 60% predicted tested in patients with:
o
A prolonged history of cigarette smoking (20 pk/year of smoking within the past 2 
years).
o
Symptoms of respiratory dysfunction
l.
Prior radiation therapy that, in the judgment of the radiation oncologist, precludes the 
administration of total body irradiation.
2.2
RESEARCH ELIGIBILITY EVALUATION
2.2.1
Within 2 Months Prior to Randomization 
a.
HIV antibody titer and HbsAG determination, anti HCV (may be performed within 3 
months of chemotherapy start date)
b. Anti CMV antibody titer, HSV serology, and EBV panel. (may be performed within 3 
months of chemotherapy start; patients who are known to be positive for any of the above 
do not need to be retested. 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
13
c.
Verification that HLA typing is completed (testing is permitted to be conducted at any 
time prior to this point).
d. Stress cardiac (MUGA or echocardiogram) in all patients > 60 years old regardless of 
history, and in patients meeting requirements noted in Section 2.1.2i. 
e.
Pulmonary evaluation (PFTs) for patients meeting requirements in Section 2.1.2k.
f.
Colonoscopy for patients meeting requirements noted in Section 2.1.1n. 
2.2.2
Within 3 Weeks Prior to Randomization AND Treatment
Note: The following evaluations should be repeated for safety if treatment will not begin within 
3 week of randomization.
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. (Note: Patient history 
may be obtained within 8 weeks)
b. Chest x-ray
c.
EKG
d. Baseline CT of the chest, abdomen and pelvis, and brain MRI to evaluate the status of 
disease. Additional scans and x-rays may be performed if clinically indicated based on 
patients’ signs and symptoms.
e.
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]and thyroid blood 
chemistry panel.
f.
CBC, differential, PT/PTT, platelet count 
g. Urinalysis and culture, if indicated 
h. Calculated creatinine clearance
2.2.3
Within 7 Days Prior to Treatment 
a) β-HCG pregnancy test (serum or urine) on all women of child-bearing potential.
b) ECOG
2.3
PARTICIPANT REGISTRATION AND STATUS UPDATE PROCEDURES
When approximately 5x108 cells are available, patients will enter the study, be registered, and 
then stratified by M1a versus M1b and M1c. Following stratification, patient will be randomized 
to receive the non-myeloablative chemotherapy regimen of cyclophosphamide and fludarabine, 
young TIL and high dose aldesleukin (arm 1) or the same regimen plus 12 Gy total body 
irradiation (TBI). Randomization will be conducted a 1:1 fashion using variable block sizes. 
Registration and status updates (e.g., when a participant is taken off protocol therapy and when a 
participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant 
Registration & Status Updates found here.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
14
3
STUDY IMPLEMENTATION
3.1
STUDY DESIGN
Patients with evaluable metastatic melanoma will undergo resection of tumor under protocol 03-
C-0277. Patients with evaluable metastatic melanoma who have TIL successfully growing from 
a metastatic lesion will be prospectively randomized to arm 1 or arm 2 as described in Section 
2.3. TIL will be grown and expanded for this trial according to standard operating procedures 
submitted in the IND. Volunteers will also undergo apheresis to obtain mononuclear cells to be 
used as feeder cells in cell culture according to protocol 03-C-0277. 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 apheresis 
volunteers. TIL cultures will be monitored regularly and when approximately 5x108 cells are 
available the cells will undergo a rapid expansion. Prior to starting the rapid expansion, patients 
will be randomized. Patients randomized to TBI (Arm 2) will undergo collection of CD34+ 
hematopoietic stem cells as indicated in 03-C-0277 prior to beginning the preparative regimen. 
Prior to the start of the preparative regimen (approximately one week prior to infusion) TIL will 
be assessed for potency by interferon gamma release as specified in the Certificate of Analysis 
shown in Appendix 1. 
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, followed by infusion of 
between 1x109 to 2x1011 lymphocytes and the administration of the FDA approved standard 
regimen of aldesleukin at 720,000 IU/kg every eight hours to tolerance starting within 24 hours 
of cell infusion. Patients will be evaluated for response approximately 4-6 weeks following the 
completion of aldesleukin. Patients will receive one course of treatment.  The start date of the 
course will be the start date of the chemotherapy; the end date will be the date of the first post-
treatment evaluation. 
Patients who are randomized to the TBI arm for whom the MINIMUM dose of CD34+ cells 
cannot be obtained will cross over to the chemotherapy preparative regimen without TBI arm 
(arm 1).
3.2
PROTOCOL STOPPING RULES 
The study may be halted if any of the following conditions are met:
1. If 1 or more treatment related death occurs in either arm, we will promptly discuss this 
the protocol with the IRB and the FDA but we may not immediately stop accrual. 
2. The study will be halted pending discussions with the FDA and IRB if the following 
conditions are met: 
o
Two or more patients remain neutropenic (grade 3 or 4 ANC) 4 weeks following 
treatment. 
o
Two or more patients remain thrombocytopenic (grade 3 or 4 platelets) 8 weeks 
following treatment.
o
If 2 or more patients have a greater than or equal to grade 3 creatinine level at 3 
months’ post treatment.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
15
3. The NCI DSMB futility evaluation determines that the trial is unlikely to find an effect at 
the 0.025 two-tailed significance level with continued accrual, based on the fraction 
surviving at one year.
4. The NCI DSMB efficacy evaluation determines that the p-value at the interim point 
(based on approximately 56 total patients enrolled and followed for 6 months) is <0.0054, 
then the trial will stop accrual for better than expected efficacy. 
3.3
AUTOLOGOUS STEM CELL COLLECTION
Prior to beginning the preparative regimen, patients randomized to the TBI (Arm 2) will have 
stem cells collected and stored for re-infusion after the myeloablation and cell therapy as per 
protocol 03-C-0277. Patients will receive filgrastim at 8 mcg/kg dose BID (total = 16 
mcg/kg/day) by subcutaneous injection for 5 days. The morning dose of filgrastim will be given 
at approximately 7:00 am each day, except for the last morning dose which will be at 5 am. 
Eleven hours prior to the apheresis procedure, a single dose of plerixafor at 240 µg/kg may be 
administered by SQ injection to improve stem cell mobilization. Stem cells will be collected by 
apheresis. 
Autologous apheresis will start on the fifth day of filgrastim administration, and filgrastim and 
plerixafor administration and apheresis will be repeated one more day if necessary to achieve a 
sufficient dose of CD34+ cells after ex vivo processing (Miltenyi ClinicMACs). For each daily 
apheresis a blood volume of 15- 25 liters of blood will be processed, using ACD-A 
anticoagulation, peripheral or central venous access, and calcium replacement as needed, per 
standard operating procedure of the NIH Clinical Center DTM. Sufficient CD34+ cell doses after 
Miltenyi ClinicMACs positive selection are defined as a TARGET of > 4 x 106/kg recipient 
weight, and a MINIMUM of ≥ 2 x 106/kg recipient weight. Patients who do not reach the 
MINIMUM dose of selected CD34+ cells after two apheresis collections may be considered for a 
second cycle of filgrastim mobilization and apheresis collections. 
If the MINIMUM dose of CD34+ cells has not been reached after 2 cycles of mobilization and 
collection, the patient in the TBI arm will receive the chemotherapy preparative regimen without 
TBI. 
CD34+ cells will be processed and cryopreserved according to DTM policy and procedure until 
needed for cell infusion.
3.4
DRUG ADMINISTRATION
Treatment schedule will be according to the following schedule (see Section 3.5). Only patients 
in arm 2 will receive 12Gy TBI. (Times are offered as examples and may be changed as long as a 
similar time relationship between administrations of the drugs is maintained. Study medication 
start times for drugs given once daily may be within 2 hours of the scheduled time. All other 
medications may be given ± one hour of the scheduled time; the length of administration may be 
± 15 minutes. Administration of diuretics, electrolyte monitoring and replacement, and hydration 
should all be performed as clinically indicated – the times noted below are offered only as 
examples.)
3.4.1
Preparative Regimen
The following will comprise a course of therapy for Day -7 through Day 2:
Day -7 and -6 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
16
1 am: Hydrate: Begin hydration with 0.9% Sodium Chloride Injection containing 10 meq/L of 
potassium chloride at 2.6 mL/kg/hr. (starting 11 hours’ pre-cyclophosphamide and continue 
hydration until 24 hours after last cyclophosphamide infusion). At any time during the 
preparative regimen, if the urine output <1.5 mL/kg/hr or if body weight >2 kg over pre-
cyclophosphamide value, furosemide 10-20 mg IV maybe administered. Serum potassium should 
be monitored and treated as indicated following administration of furosemide.
11 am: Ondansetron (0.15 mg/kg/dose [rounded to the nearest even mg dose between 8 mg and 
16 mg based on patient weight] IV q 8 hours X 3 days) will be given for nausea.
12 pm (NOON): Cyclophosphamide 60 mg/kg/day X 2 days IV in 250 mL D5W with mesna 15 
mg/kg/day over 1 hr X 2 days. If the patient is obese (BMI > 35) drug dosage will be calculated 
using practical weight as described in Appendix 2.
1 pm: Begin mesna infusion at 3 mg/kg/hour intravenously diluted in a suitable diluent (see 
Section 14) over 23 hours after each cyclophosphamide dose. If the patient is obese (BMI > 35) 
drug dosage will be calculated using practical weight as described in Appendix 2. 
Day -7 to Day-3
Fludarabine 25 mg/m2/day IVPB daily over 15-30 minutes for 5 days. 
If the patient is obese (BMI > 35) drug dosage will be calculated using practical weight as 
described in Appendix 2. (The fludarabine will be started approximately 1-2 hours after the 
cyclophosphamide and mesna on Days -7 and -6). To allow as much time between the last dose 
of fludarabine and the administration of the cell treatment, the timing of the fludarabine 
administration can be moved to an hour earlier each day.)
For patients randomized to TBI (Arm 2) the following will be added to the preparative 
regimen:
Day –3 to Day -1
Prior to TBI, patients will receive a single dose of IV ondansetron (ondansetron 0.15 mg/kg IV 
[rounded to the nearest even mg dose between 8 mg and 16 mg based on patient weight] x 1 dose 
pre-TBI).
Patients will receive 2Gy of TBI twice a day for 3 days (total dose 12 Gy) using a linear 
accelerator in Radiation Oncology. Lungs will be shielded to a mean dose of 6 Gy.
Day-2 (Arm 2 only)
For infection prophylaxis, levofloxacin will be administered at a dose of 500mg PO once daily 
starting on day -2 and will continue daily as clinically indicated in the best clinical judgment of 
the attending physician.
3.4.2
Cell Infusion and Aldesleukin Administration
Day 0 (one to three days after the last dose of agent in the preparative regimen):

Autologous young TIL infusion will be administered intravenously over 20 to 30 minutes 
(minimum 1 X 109 and up to a maximum of 2 X 1011 lymphocytes). 

Within 24 hours of cell infusion administration of aldesleukin will be initiated 
Day 1-4 (Day 0 is the day of cell infusion):

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
17

Aldesleukin 720,000 IU/kg IV (based on total body weight) over 15 minute 
approximately every eight hours (± 1 hour) for up to 5 days (maximum 15 doses).

For patients in cohort 2 only (TBI cohort): 
o
On day 1, the cryopreserved autologous CD34+ selected stem cell product will be 
thawed and administered intravenously immediately. The minimum dose will be > 2 
x 106 CD34+ cells per kg. 

For all patients: 
o
Beginning on day 1 or 2: Filgrastim will be started at 5 mcg/kg/day daily 
subcutaneously until neutrophil count >1x109/l X 3 days or > 5x109/L. The maximum 
filgrastim dose will be 300 mcg per day.
3.5
STUDY CALENDAR
Day
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
Therapy
Cyclophosphamide
60 mg/kg
X
X
Fludarabine
25 mg/m2
X
X
X
X
X
TBI - 2Gy BID 
(cohort 2 only)
X
X
X
Young TIL Cells1
X
Aldesleukin2
X
X
X
X
X
CD34+ Cells (Cohort 
2 only)
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
X
X
Valacyclovir po or 
Acyclovir IV6
X
X
X
X
X
X
X
1One to three days after the last dose of agent in the preparative regimen
2Initiate within approximately 24 hours after cell infusion
3Continue until neutrophils count > 1X109/L X 3 days or > 5x109/L.
4The TMP/SMX schedule should be adjusted to QD three times per week (Monday, Wednesday, Friday) 
and continue for at least six months and until CD4 > 200 X 2
5Continue until ANC > 1000/mm3
6In patients positive for HSV continue until CD4 > 200 X 2
3.6
TOTAL BODY IRRADIATION (TBI)
3.6.1
Energy
All patients should be treated with a linear accelerator using energies higher than 4MV.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
18
3.6.2
Timing
It is anticipated that TBI will be delivered on day -3, -2, -1.
3.6.3
Technique
TBI will be delivered with lateral fields using extended SSD/SAD values of 200-500cm 
(depending on machine/vault size) and partial transmission lung blocks. Patients will then 
receive AP/PA Mediastinal Boost fields using full blocks in the lung area.
3.6.4
Dose/Fractionation
Patients will be treated with TBI to a total dose of 1200 cGy delivered in 200 cGy fractions bid, 
at least 6 hours apart (six total treatments in three days). The mediastinal boost fields will be 
delivered at a dose of 100cGy per fraction for a total dose of 600cGy, also at least six hours 
apart.
3.6.5
Dose Modifications 
Occasionally, the total dose/technique of TBI may require modifications due to patient factors 
(unexpected or severe (grade 4-5) adverse events, serious medical illnesses not conducive to 
stable patient transfer, patient refusal, etc) or treatment factors (linear accelerator machine 
offline, etc.) Modifications to the radiation treatment will be at the discretion of the treating 
radiation oncologist and will be discussed with the Principal Investigator or Study Chairperson. 
3.6.6
Toxicities
Toxicities associated with TBI include mucositis, infections, cataracts, pulmonary insufficiency, 
liver toxicity and secondary late malignancies. Treatment-related mortality in series receiving 
comparable doses are approximately 2 to 5%.
3.7
INFECTION PROPHYLAXIS
Note: Other anti-infective agents may be substituted at the discretion of the treating investigator.  
3.7.1
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 the first Monday, Wednesday, or Friday on 
or after the first dose of chemotherapy. 
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 
continued monthly until CD4 count is above 200 and for at least 6 months’ post chemotherapy.
3.7.2
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. 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.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
19
Prophylaxis for Pneumocystis and Herpes will continue for 6 months’ post chemotherapy. If the 
CD4 count is less than 200 at 6 months’ post chemotherapy, prophylaxis will continue until the 
CD4 count is greater than 200 X 2.
3.7.3
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.
3.7.4
Infection Prophylaxis for Patients in Cohort 2 (TBI Cohort)
Patients who receive the 1200 TBI regimen are myeloablated and consequently more susceptible 
to infection. Therefore, patients in cohort 2 only will receive levofloxacin as follows: 500mg PO 
once daily starting on day -2. Dosing will continue daily as clinically indicated in the best 
clinical judgment of the attending physician.
3.7.5
Empiric Antibiotics 
Patients will start on broad-spectrum antibiotics, either a 3rd or 4th generation cephalosporin or a 
quinolone for fever of 38.3°C once or two temperatures of 38.0°C or above at least one hour 
apart, AND an ANC <500/mm3. Aminoglycosides should be avoided unless clear evidence of 
sepsis. Infectious disease consultation will be obtained for all patients with unexplained fever or 
any infectious complications.
3.8
BLOOD PRODUCT SUPPORT 
Using daily CBC’s as a guide, the patient will receive platelets and packed red blood cells 
(PRBC’s) as needed. Attempts will be made to keep Hb >8.0 gm/dl, and plts >20,000/mm3. All 
blood products with the exception of the stem cell product will be irradiated. Leukocyte filters 
will be utilized for all blood and platelet transfusions to decrease sensitization to transfused 
WBC’s and decrease the risk of CMV infection.
3.9
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 5-minute period every eight hours eight hours (± 1 hour) beginning on 
the day of cell infusion and continuing for up to 5 days (maximum 15 doses). 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 3. Toxicities will be managed as outlined in Appendix 4. If these toxicities 
can be easily reversed within 24 hours by supportive measures then additional doses may be 
given. If greater than 2 doses of aldesleukin are skipped, aldesleukin administration will be 
stopped. In addition, dosing may be held or stopped at the discretion of the treating investigator. 
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.
3.10 ON STUDY EVALUATION
3.10.1 Prior to Starting the Preparative Regimen

Apheresis as indicated

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
20

Within 21 days prior to starting the preparative regimen, patients will have a complete 
blood count, electrolytes, BUN, creatinine, liver function tests and serum chemistries 
performed. If any results are beyond the criteria established for eligibility, the patient will 
not proceed until the abnormalities can be resolved. 
3.10.2 During the Preparative Regimen (Daily)

Complete Blood Count

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]

Urinalysis 

CMV antigen assay will be assessed if clinically indicated (e.g. unexplained fevers, 
pulmonary changes). 

Review of systems and physical exam at least 3 times/week

Vital signs as per routine
3.10.3 After Cell Infusion

Vital signs will be monitored hourly (± 15 minutes) for four hours and then routinely 
(every 4 -6 hours) unless otherwise clinically indicated
3.10.4 During and After Aldesleukin Administration until Hospital Discharge
Every 1-2 days

Complete Blood Count

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].

Review of systems and physical exam at least 3 times/week

Vital signs as per routine
3.10.5 Severe Immune Reactions
Cytokine levels may be assayed on patients who experience rapidly escalating grade 3 or 4 
events assessed as being likely related to the cell product. If the cytokine levels are greatly 
elevated, and the patient is decompensating, aggressive supportive measures will be instituted as 
appropriate until cytokine levels are on a significant downward trend.
3.11 POST TREATMENT EVALUATION
3.11.1 Routine Follow up
The initial post treatment evaluation will be conducted approximately 6 weeks (± 2 weeks) 
following administration of the cell product.
Patients who experience stable disease, a partial response, or a complete response or have 
unresolved toxicities will be evaluated as noted below:

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
21

Week 12 (± 2 weeks) 

Every 3 months (± 1 month) x3

Every 6 months (± 1 month) x 5 years

As per PI discretion for subsequent years
Note: Patients may be seen more frequently as clinically indicated.
3.11.1.1 At each scheduled evaluation patients will undergo:

Physical examination 

Chem 20 equivalent: 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

Thyroid panel as clinically indicated

Toxicity assessment, including a review of systems. 

CT of the chest, abdomen and pelvis as clinically indicated. If clinically indicated, other 
scans or x-rays may be performed, e.g. brain MRI, bone scan.

Visual symptoms will be evaluated and if changes have occurred from baseline, i.e. 
changes in visual acuity, an ophthalmologic consult will be performed.

A 5 liter apheresis may be performed at the first follow up visit, if the patient is unable to 
undergo pheresis, approximately 96 mL of blood may be obtained.  Subsequently, 
approximately 60 mL of blood will be obtained at follow up visits for at least 3 months. 
Peripheral blood mononuclear cells will be cryopreserved so that immunologic testing 
may be performed.  

Patients who develop renal failure will undergo a nephrology consultation and a renal 
biopsy, if indicated.
3.11.1.2 Patients who develop progressive disease or are unable or unwilling to return for follow 
up evaluations will be followed via phone or e-mail contact. The following information 
may be requested from the patient:

Summary of treatment received, including adverse events, since the previous contact

Estimation of performance status

Request for imaging studies, physical exam and laboratory reports to be sent to the PI
3.12 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF-STUDY CRITERIA
Prior to removal from protocol therapy, effort must be made to have all subjects complete a 
safety visit approximately 30 days following the last dose of study therapy.
3.12.1 Criteria for Removal from Protocol Therapy
Patients will be taken off treatment (and followed for survival) for the following:

Completion of treatment

Grade 3 or greater autoimmunity that involves vital organs (heart, kidneys, brain, eye, liver, 
colon, adrenal gland, lungs). 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
22

If a patient experiences grade 3 or 4 toxicity due to cell infusion as referred to under Section 
6.4 occurring within 24 hours post cell infusion that does not reverse to grade 2 or less within 
8 hours with 2 doses of 650 mg p.o. of acetaminophen or two dose of 50 mg po of 
diphenhydramine the patient will receive no further cells but may continue to receive IL-2. 

Grade 2 or greater allergic reaction including bronchospasm or generalized urticaria 

Grade 3 or greater toxicity due to cytokines (as referred to under Appendix 4) that does not 
decrease to grade 2 or less within 96 hours of management according to study site SOP. 

Patients with progressive disease who elect not to undergo further active treatment.

If the PI determines that continued treatment is not in the best interest of the patient.
3.12.2 Off-Study Criteria
Patients will be taken off-study for the following:

The patient voluntarily withdraws

There is significant noncompliance

General or specific changes in the patient’s condition render continued participation the 
patient unacceptable for further follow up in the judgment of the investigator.

Death
Note: Once a subject is taken off study, no further data can be collected.
Note: Patients who are taken off study for study closure may be followed on Protocol 09-C-
0161 - “Follow up Protocol for Subjects Previously Enrolled in NCI Surgery Branch Studies.”
4
CONCOMITANT MEDICATIONS/MEASURES
Patients will have blood transfusions in order to maintain a minimum hemoglogin of greater than 
8.0 g/dL. Concomitant medications to control side effects of therapy will be given. Meperidine 
(25-50 mg) will be given intravenously if severe chills develops. Other supportive therapy will 
be given as required and may include acetaminophen (650 mg q4h), indomethacin (50-75 mg 
q6h) and ranitidine (150 mg q12h). Patients who require transfusions will receive irradiated 
blood products. Additional antiemetic therapy will be administered for breakthrough nausea and 
vomiting. Patients will receive supportive care as indicated for aldesleukin toxicities as listed in 
Appendix 4. 
5
CORRELATIVE STUDIES FOR RESEARCH 
Blood and tissue are tracked at the patient level and can be linked to all protocols on which the 
patient has been enrolled. Samples will be used to support the specific objectives listed in the 
treatment protocol(s), e.g., immunologic monitoring, cytokine levels, persistence, as well as to 
support long-term research efforts within the Surgery Branch and with collaborators as specified 
in our companion protocol, 03-C-0277 (Cell Harvest and Preparation for Surgery Branch 
Adoptive Cell Therapy Protocols).
Samples will be ordered in CRIS and tracked through the Clinical Trial Data Management 
system. Should a CRIS screen not be available, the CRIS downtime procedures will be followed. 
Samples will not be sent outside NIH without appropriate approvals and/or agreements, if 
required.
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. Tubes/media 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
24
studies. Immunological monitoring will consist of quantifying T cells reactive with HLA-
matched tumor cells using established techniques such as intracellular FACS, cytokine 
release assays, and Elispot assays. Immunological assays will be standardized by the 
inclusion of 1) pre-infusion PBMC and 2) an aliquot of the T cells cryopreserved at the 
time of infusion. TCR gene usage may be quantitated in samples using conventional 
sequencing techniques of the T cell receptor variable regimen of the beta chain.

A variety of tests including evaluation of specific lysis and cytokine release, intracellular 
FACS of cytokine production, ELISA-spot assays, and lymphocyte subset analysis may 
be used to evaluate the immunological correlates of treatment. In general, differences of 2 
to 3 fold in these assays are indicative of true biologic differences. 

Samples of all infused cell products will be cryopreserved, and extensive retrospective 
analysis of infused cell phenotype and function will be performed to attempt to find in 
vitro characteristics of the infused cells which correlate with in vivo antitumor activity. 
Analyses of TIL samples will include evaluation of the activity, specificity, and telomere 
length of the infused TIL.
5.8
SAMPLE STORAGE, TRACKING AND DISPOSITION FOR SURGERY BRANCH CELL 
PRODUCTION FACILITY (SB-CPF)
Blood and tissue collected during the course of this study will follow the Cell Tracking and 
Labeling System established by the SB CPF. 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 Blood Processing Core (BPC) for storage. 
Samples will be barcoded and stored on site or offsite at NCI Frederick Central Repository 
Services in Frederick, MD. All samples (blood or tissue) are entered into a central computer 
database with identification and storage location, and this database is backed up every night.
If, at any time, a patient withdraws from the study and does not wish for their existing samples to 
be utilized, the individual must provide a written request. Following receipt of this request, the 
samples will be destroyed.
The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will 
be as per the requirements in Section 7.2.
5.9
SAMPLE STORAGE, TRACKING AND DISPOSITION FOR FIGG LAB (BPC)
5.9.1
Sample Data Collection
All samples sent to the BPC will be barcoded, with data entered and stored in the Labmatrix 
utilized by the BPC, and data will be updated to the Surgery Branch central computer database 
weekly. This is a secure program, with access to Labmatrix limited to defined Figg lab 
personnel, who are issued individual user accounts. Installation of Labmatrix is limited to 
computers specified by Dr. Figg. These computers all have a password restricted login screen. 
Labmatrix  creates a unique barcode ID for every sample and sample box, which cannot be 
traced back to patients without Labmatrix  access. The data recorded for each sample includes 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
25
the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material 
type, as well as box and freezer location. Patient demographics associated with the clinical center 
patient number are provided in the system. For each sample, there are notes associated with the 
processing method (delay in sample processing, storage conditions on the ward, etc.).
5.9.2
Sample Storage and Destruction
Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80°C 
according to stability requirements. These freezers are located onsite in the BPC and offsite at 
NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are 
required to be accompanied by laboratory staff at all times. 
Access to stored clinical samples is restricted. Samples will be stored until requested by a 
researcher named on the protocol. All requests are monitored and tracked in Labmatrix . All 
researchers are required to sign a form stating that the samples are only to be used for research 
purposes associated with this trial (as per the IRB approved protocol) and that any unused 
samples must be returned to the BPC. It is the responsibility of the NCI Principal Investigator to 
ensure that the samples requested are being used in a manner consistent with IRB approval.
Following completion of this study, samples will remain in storage as detailed above. Access to 
these samples will only be granted following IRB approval of an additional protocol, granting 
the rights to use the material. 
If, at any time, a patient withdraws from the study and does not wish for their existing samples to 
be utilized, the individual must provide a written request. Following receipt of this request, the 
samples will be destroyed.
Sample barcodes are linked to patient demographics and limited clinical information. This 
information will only be provided to investigators listed on this protocol, via registered use of the 
Labmatrix. It is critical that the sample remains linked to patient information such as race, age, 
dates of diagnosis and death, and histological information about the tumor, in order to correlate 
genotype with these variables.
The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will 
be as per the requirements in Section 7.2.
Note: Blood and tissue collected during the course of this study will be stored, tracked, and 
disposed of as specified in our companion protocol, 03-C-0277 (Cell Harvest and Preparation for 
Surgery Branch Adoptive Cell Therapy Protocols).
5.10 ADDITIONAL RESEARCH EVALUATIONS
To determine predictors of renal toxicity and possible biodosimeters of radiation dose, the 
following samples will be collected, and then delivered to Dr. Deborah Citrin, M.D., Radiation 
Oncology Branch, NCI.

For patients in Arm 2 (receiving TBI)

Prior to the first dose of radiation (Day -3):
3 tubes of blood consisting of 1 red top tube (4 mL), 1 purple top tube (6 mL), and 
1 CPT tube (10 mL) 
Urine specimen collected in blue cup (20-30 mL) 

 Prior to the second dose of radiation (Day -3, 6 hrs. after the 1st radiation dose)

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
26
3 tubes of blood consisting of 1 red top tube (4 mL), 1 purple top tube (6 mL), and 
1 CPT tube (10 mL)

Prior to the last dose of radiation on day -1
Urine specimen collected in blue cup (20-30 mL)

If renal toxicity occurs:
1 tube of blood consisting of 1 purple top tube (6 mL), 
Urine specimen collected in blue cup (20-30 mL)

Six months (± 1 month) following the last dose of treatment
1 tube of blood consisting of 1 purple red tube (4 mL), 
Urine specimen collected in blue cup (20-30 mL)

For patients in Arm 1 (not receiving TBI)

On Day -3
3 tubes of blood consisting of 1 red top tube (4 mL), 1 purple top tube (6 mL), and 
1 CPT tube (10 mL) 
Urine specimen collected in blue cup (20-30 mL)

If renal toxicity occurs:
1 tube of blood consisting of 1 purple top tube (6 mL), 
Urine specimen collected in blue cup (20-30 mL)

Six months (± 1 month) following the last dose of treatment
1 tube of blood consisting of 1 purple top tube (6 mL), 
Urine specimen collected in blue cup (20-30 mL)
With approval of amendment C, to analyze metabolites of vitamin A in the serum, the following 
samples may be collected from patients treated on both arms of this study and sent to the Dept. 
of Nutritional Science and Toxicology, University of California at Berkley. One tube of blood (1 
mL) may be collected in a 2.5 mL red top tube at the following times:

On day -8 

Between day +10 and day +14

At follow-up visits
In addition, to assess dendritic cell subsets, 4 tubes of blood (10 mL) may be collected in CPT 
tubes at the following times:

On day -8 

Between day +10 and day +14

At follow-up visits

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
27
6
DATA COLLECTION AND EVALUATION
6.1
DATA COLLECTION
The PI will be responsible for overseeing entry of data into a 21 CFR Part 11-compliant data 
capture system provided by the NCI CCR and ensuring data accuracy, consistency and 
timeliness. The principal investigator, associate investigators/research nurses and/or a contracted 
data manager will assist with the data management efforts. Primary and final analyzed data will 
have identifiers so that research data can be attributed to an individual human subject participant. 
All adverse events (AEs), including clinically significant abnormal findings on laboratory 
evaluations, regardless of severity, will be followed until return to baseline or stabilization of 
event. All events occurring during the treatment phase of the study will be followed until 
resolution to grade 2 or baseline. During the follow up period, only grade 3 and 4 and 
unexpected grade 2 events that are related the treatment will be captured in C3D. Patients will be 
followed for adverse events from registration through 30 days after the last treatment. 
An abnormal laboratory value will be recorded in the database as an AE only if the laboratory 
abnormality is characterized by any of the following:

Results in discontinuation from the study

Is associated with clinical signs or symptoms 

Requires treatment or any other therapeutic intervention

Is associated with death or another serious adverse event, including hospitalization. 

Is judged by the investigator to be of significant clinical impact

If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome.
End of study procedures: Data will be stored according to HHS and FDA regulations, and NIH 
Intramural Records Retention Schedule as applicable. 
Loss or destruction of data: Should we become aware that a major breech in our plan to protect 
subject confidentiality and trial data has occurred, this will be reported expeditiously per 
requirements in Section 7.2.1.
6.1.1
Exclusions to Routine Adverse Event Recording
Patients will be receiving multiple agents which include commercially available agents 
(fludarabine, cyclophosphamide, aldesleukin, and supportive medications) in combination with 
the investigational agents. Therefore, all grade 1 and all expected grade 2 adverse events do not 
require reporting/recording.
6.2
DATA SHARING PLANS
6.2.1
Human Data Sharing Plan
De-identified human data generated for use in future and ongoing research will be shared 
through a NIH-funded or approved repository (ClinicalTrials.gov) and BTRIS. At the completion 
of data analysis, data will be submitted to ClinicalTrials.gov either before publication or at the 
time of publication or shortly thereafter. Data may also be used to support long-term research 
efforts within the Surgery Branch and de-identified data may also be shared with collaborators as 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
28
specified in our companion protocol, 03-C-0277 (Cell Harvest and Preparation for Surgery 
Branch Adoptive Cell Therapy Protocols).
6.2.2
Genomic Data Sharing Plan
The NIH Genomic Data Sharing Policy does not apply to this study.
6.3
RESPONSE CRITERIA
For the purposes of this study, patients should be re-evaluated for response at 6 and 12 weeks (± 
2 weeks), then every 3 months (± 1 month) x3, then every 6 months’ (± 1 month) x 5 years. In 
addition to a baseline scan, confirmatory scans should also be obtained approximately 4 (not less 
than 4) weeks following initial documentation of objective response.
Clinical response will be determined using RECIST version 1.0.
6.3.1
Evaluation of Target Lesions1
Complete Response (CR): Disappearance of all target lesions
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.
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.
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase 
to qualify for PD taking as references the smallest sum LD.
6.3.2
Evaluation of Non-Target Lesions2
Complete Response (CR): Disappearance of all non-target lesions and normalization of 
tumor marker level.
Non-Complete Response: Persistence of one or more non-target lesions
Progression (PD): Appearance of one or more new lesions. Unequivocal progression of 
existing non-target lesions
6.3.3
Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of 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. The time to progression will be 
measured from the date of randomization.
Target Lesions
Non-Target Lesions
New Lesions
Overall Response
CR
CR
No
CR
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.”

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
29
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
6.3.4
Confirmatory Measurement/Duration of Response
Confirmation
To be assigned a status of PR or CR, changes in tumor measurements must be confirmed by 
repeat studies that should be performed at least 4 weeks after the criteria for response are first 
met. In the case of SD, follow-up measurements must have met the SD criteria at least once after 
study entry at a minimum interval of 6-8 weeks.
Duration of Overall Response
The duration of overall response is measured from the time measurement criteria are met for 
CR/PR (whichever is first recorded) until the first date that recurrent or progressive disease is 
objectively documented (taking as reference for progressive disease the smallest measurements 
recorded since the treatment started).
The duration of overall complete response is measured from the time measurement criteria are 
first met for CR until the first date that recurrent disease is objectively documented.
Duration of Stable Disease
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. 
6.3.5
Image Acquisition Plan
Images will be obtained digitally and reviewed by a radiologist in a blinded fashion for the 
determination of tumor response.  
6.4
TOXICITY CRITERIA
Careful evaluation to ascertain the toxicity, immunologic effects and anti-tumor efficacy of the 
treatment regimens will be performed. This study will utilize the CTCAE version 3.0 for toxicity 
and adverse event reporting. A copy of the CTCAE version 3.0 can be downloaded from the 
website http://ctep.cancer.gov. All appropriate treatment areas should have access to a copy of 
the CTCAE version 3.0.
Over 100 patients have been treated in the Surgery Branch, NCI with TIL. Early toxicities 
related specifically to the infusion of the cells (those which are seen immediately following cell 
infusion and prior to aldesleukin administration) are generally mild and include fevers, chills, 
headache, and malaise. Toxicities which occur following administration of aldesleukin but are 
thought to be related to the cells include immune mediated events such as vitiligo, transient 
uveitis, hearing loss and vestibular dysfunction. The use of the non-myeloablative regimen prior 
to cell administration increases the toxicity of this treatment as profound myelosuppression 
occurs in all patients. In 93 patients treated with TIL using the non-myeloablative chemotherapy 
regimen with or without total body irradiation, there was one treatment related death (NMA + 
200 cGy TBI) due to an unexpected but preexisting diverticular abscess. 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
30
The standard approach to the administration of high-dose aldesleukin in all studies is to continue 
dosing until grade 3 or 4 events occur. The most commonly seen grade 4 events are pulmonary 
and renal impairment, and mental status changes. These toxicities may sometimes require 
intubation for protection of the patient’s airway. It is important to note that although these 
patients require significant supportive measures during this period, all toxicities are reversible 
and the overwhelming majority of patients have suffered no long-term sequelae following this 
treatment regimen. However, fatal complications are possible and it is therefore only appropriate 
to carry out this experimental treatment in the context of life threatening metastatic cancer.
To ensure safety using this treatment, the NCI SB will review safety data on all protocols semi-
annually at the time of continuing review. Data will be presented for both the recent 6 month 
period and for the entire length of time the protocol has been open. The toxicity data for review 
will include all toxicities captured on the protocol and will be presented in individual tables as 
follows: 

All toxicities attributed to the cells, 

All incidences of intubation including the duration of and reason for intubation,

All grade 2 unexpected adverse events, and all grade 3 or greater events regardless of 
attribution.  
Toxicities seen on protocols using this non-myeloablative regimen and aldesleukin, that occur 
during the follow up period are rare but have included EBV lymphoma following prolonged 
lymphopenia, herpes zoster infection, and sensory neuropathy likely related to fludarabine. 
The major discomforts of the research are those of nausea and vomiting, mucositis, anorexia, 
diarrhea, fever and malaise. Side effects of common drugs used in this 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, 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, photosensitivity. 

TBI: Mucositis, infections, cataracts, pulmonary insufficiency, liver toxicity, thrombotic 
microangiopathy, and secondary late malignancies. 

High-dose aldesleukin administration: A listing of these side effects in 652 patients who 
received 1,039 treatment courses are listed in Appendix 3.
SAEs will be reported as per section 8.3.
7
NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
7.1
DEFINITIONS
Please refer to definitions provided in Policy 801: Reporting Research Events found here.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
31
7.2
OHSRP OFFICE OF COMPLIANCE AND TRAINING/IRB REPORTING 
7.2.1
Expedited Reporting
Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 
802: Non-Compliance Human Subjects Research found here. Note: Only IND Safety Reports 
that meet the definition of an unanticipated problem will need to be reported per these policies.
7.2.2
IRB Requirements for PI Reporting at Continuing Review
Please refer to the reporting requirements in Policy 801: Reporting Research Events found here.
7.3
NIH REQUIRED DATA AND SAFETY MONITORING PLAN
7.3.1
Principal Investigator/Research Team
The clinical research team will meet on a regular basis when patients are being actively treated 
on the trial to discuss each patient. Decisions about enrollment will be made based on the 
toxicity data from prior patients.
All data will be collected in a timely manner and reviewed by the principal investigator. Adverse 
events will be reported as required above. Any safety concerns, new information that might 
affect either the ethical and or scientific conduct of the trial, or protocol deviations will be 
immediately reported to the IRB using iRIS.
The principal investigator will review adverse event and response data on each patient to ensure 
safety and data accuracy. The principal investigator will personally conduct or supervise the 
investigation and provide appropriate delegation of responsibilities to other members of the 
research staff.
7.3.1
Data Safety Monitoring Board (DSMB)
This protocol requires monitoring by the NCI CCR Data Safety Monitoring Board (DSMB) as 
described in Section 10. Interim outcome results will not be revealed to the investigators of the 
trial; results will be presented to the investigators prior to final accrual to the trial only if the 
DSMB recommends early termination of the trial. (Note: The study statistician is responsible for 
providing the description of how the monitoring will take place, including endpoints to be 
monitored and the frequency or timing of monitoring.)
8
SPONSOR PROTOCOL/ SAFETY MONITORING PLAN
8.1
DEFINITIONS
8.1.1
Adverse Event
An adverse event (AE) is defined as any untoward medical occurrence in a in a patient or clinical 
investigation subject administered a pharmaceutical product and which does not necessarily have 
a causal relationship with this treatment. An adverse event (AE) can therefore be any 
unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or 
disease, temporally associated with the use of a medicinal (investigational) product, whether or 
not related to the medicinal (investigational) product (ICH E6 (R2)).
8.1.2
Serious Adverse Event
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:

Death,

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
32

A life-threatening adverse drug experience (see section 8.1.3)

Inpatient hospitalization or prolongation of existing hospitalization
o
A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery 
arranged prior to the start of the study), a planned hospitalization for pre-existing 
condition, or a procedure required by the protocol, without a serious deterioration in 
health, is not considered a serious adverse event.
o
A hospitalization/admission that is solely driven by non-medical reasons (e.g., 
hospitalization for patient or subject convenience) is not considered a serious adverse 
event. 
o
Emergency room visits or stays in observation units that do not result in admission to 
the hospital would not be considered a serious adverse event. The reason for seeking 
medical care should be evaluated for meeting one of the other serious criteria.

A persistent or significant disability/incapacity or substantial disruption of the ability to 
conduct normal life functions

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.
8.1.3
Life-Threatening
Any adverse event or suspected adverse reaction is considered "life-threatening" if, in the view 
of the investigator or sponsor, its occurrence places the patient or subject at immediate risk of 
death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a 
more severe form, might have caused death. (21CFR312.32)
8.1.4
Severity
The severity of each Adverse Event will be assessed utilizing the CTCAE version 3.0.
8.1.5
Relationship to Study Product
All AEs will have their relationship to study product assessed using the terms:  related or not 
related.  

Related – There is a reasonable possibility that the study product caused the adverse 
event. Reasonable possibility means that there is evidence to suggest a causal relationship 
between the study product and the adverse event.

Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event.
8.2
ASSESSMENT OF SAFETY EVENTS
AE information collected will include event description, date of onset, assessment of severity 
and relationship to study product and alternate etiology (if not related to study product), date of 
resolution of the event, seriousness and outcome. The assessment of severity and relationship to 
the study product will be done only by those with the training and authority to make a diagnosis 
and listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  AEs 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
33
occurring during the collection and reporting period will be documented appropriately regardless 
of relationship.  AEs will be followed through resolution.
SAEs will be:

Assessed for severity and relationship to study product and alternate etiology (if not 
related to study product) by a licensed study physician listed on the Form FDA 1572 as 
the site principal investigator or sub-investigator.

Recorded on the appropriate SAE report form, the medical record and captured in the 
clinical database.

Followed through resolution by a licensed study physician listed on the Form FDA 1572 
as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to section 6.1. All serious adverse events 
recorded from the time of first investigational product administration must be reported to the 
sponsor.
8.3
REPORTING OF SERIOUS ADVERSE EVENTS
Any AE that meets protocol-defined serious criteria or meets the definition of Adverse Event of 
Special Interest that require expedited reporting must be submitted immediately (within 24 hours 
of awareness) to OSRO Safety using the CCR SAE report form.  
All SAE reporting must include the elements described in section 8.2.
SAE reports will be submitted to the Center for Cancer Research (CCR) at: 
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator.  CCR SAE report form 
and instructions can be found at: 
https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions
Following the assessment of the SAE by OSRO, other supporting documentation of the event 
may be requested by the OSRO Safety and should be provided as soon as possible.
8.4
REPORTING PREGNANCY 
All required pregnancy reports/follow-up to OSRO will be submitted to: 
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator.  Forms and instructions 
can be found here: 
https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions
8.4.1.1
Maternal Exposure
If a patient becomes pregnant during the course of the study, the study treatment should be 
discontinued immediately and the pregnancy reported to the Sponsor no later than 24 hours of 
when the Investigator becomes aware of it. The Investigator should notify the Sponsor no later 
than 24 hours of when the outcome of the Pregnancy becomes known.
Pregnancy itself is not regarded as an SAE. However, congenital abnormalities or birth defects 
and spontaneous miscarriages that meet serious criteria (section 8.1.2) should be reported as 
SAEs. 
The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic 
pregnancy, normal birth, or congenital abnormality) should be followed up and documented.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
34
8.4.1.2
Paternal Exposure
Male patients should refrain from fathering a child or donating sperm during the study and for 
120 days after the last dose of aldesleukin or pembrolizumab.
Pregnancy of the patient’s partner is not considered to be an AE. However, the outcome of all 
pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or 
congenital abnormality) occurring from the date of the first dose until 120 days after the last dose 
should, if possible, be followed up and documented.
8.5
REGULATORY REPORTING FOR STUDIES CONDUCTED UNDER CCR-SPONSORED IND
Following notification from the investigator, CCR, the IND sponsor, will report any suspected 
adverse reaction that is both serious and unexpected.  CCR will report an AE as a suspected 
adverse reaction only if there is evidence to suggest a causal relationship between the study 
product and the adverse event.  CCR will notify FDA and all participating investigators (i.e., all 
investigators to whom the sponsor is providing drug under its INDs or under any investigator’s 
IND) in an IND safety report of potential serious risks from clinical trials or any other source, as 
soon as possible, in accordance to 21 CFR Part 312.32. 
All serious events will be reported to the FDA at least annually in a summary format.
8.6
SPONSOR PROTOCOL DEVIATION REPORTING
Protocol Deviation is defined as any non-compliance with the clinical trial Protocol, Manual of 
Operational Procedures (MOP) and other Sponsor approved study related documents, GCP, or 
protocol-specific procedural requirements on the part of the participant, the Investigator, or the 
study site staff inclusive of site personnel performing procedures or providing services in support 
of the clinical trial.
It is the responsibility of the study Staff to document any protocol deviation identified by the 
Staff or the site Monitor in the CCR Protocol Deviation Tracking System (PDTS) online 
application. The entries into the PDTS online application should be timely, complete, and 
maintained per CCR PDTS user requirements. 
In addition, any deviation to the protocol should be documented in the participant’s source 
records and reported to the reviewing IRB per their guidelines. OSRO required protocol 
deviation reporting is consistent with E6(R2) GCP: Integrated Addendum to ICH E6(R1): 4.5 
Compliance with Protocol; 5.18.3 (a), and 5.20 Noncompliance; and ICH E3 16.2.2 Protocol 
deviations.
9
CLINICAL MONITORING 
As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring 
program. The CCR’s program allows for confirmation of: study data, specifically data that could 
affect the interpretation of primary study endpoints; adherence to the protocol, regulations, and 
SOPs; and human subjects’ protection. This is done through independent verification of study 
data with source documentation focusing on:

Informed consent process

Eligibility confirmation

Drug administration and accountability

Adverse events monitoring

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
35

Response assessment.
The monitoring program also extends to multi-site research when the CCR is the coordinating 
center.
This trial will be monitored by personnel employed by a CCR contractor. Monitors are qualified 
by training and experience to monitor the progress of clinical trials. Personnel monitoring this 
study will not be affiliated in any way with the trial conduct.
10 STATISTICAL CONSIDERATIONS
The primary objectives of the trial are to compare in a prospective randomized study both the 
complete response rate and the overall survival of patients with metastatic melanoma receiving 
ACT using young TIL plus aldesleukin treatment following either a chemotherapy preparative 
regimen alone, or the same chemotherapy preparative regimen plus TBI.
The secondary objectives are to determine clinical response rate and progression free survival in 
patients in the two treatment arms, and to determine the toxicity of these two treatment regimens.
In a series of consecutive trials using this chemotherapy preparative regimen alone or with 2 Gy 
or 12 Gy total body irradiation (TBI) objective response rates using RECIST criteria were 49%, 
52%, and 72%, respectively. Complete regression rates in these three consecutive trials were 
12%, 20%, and 40%, respectively— suggesting that the addition of TBI could be associated with 
a greater complete regression rate. Of the 20 complete regressions seen in this trial, 19 are on-
going at 37 to 82 months.
A total of 112 evaluable patients (56 per arm) will be randomized to ACT following a chemo 
preparative regimen + 12 Gy TBI vs. ACT following a chemo preparative regimen alone. The 
patients will be stratified for M1a vs. M1b+M1c disease. 
By enrolling 112 patients, the trial will have the following statistical characteristics.
As there are two primary endpoints, the study will have sufficient patients for each of the two 
main outcomes to be evaluated using a 0.025 alpha level two-tailed test in order to 
conservatively adjust for the effect of two endpoints while maintaining an overall 0.05 alpha 
level test. In practice, a Hochberg adjustment to the p-values may be performed, which is less 
overly stringent than the Bonferroni correction using 0.025 for each test.
With 56 patients per arm, there will be 80% power to detect a difference between an 8% 
complete response rate and a 33% complete response rate using a two-tailed Fisher’s exact test at 
the 0.025 significance level. These rates were selected since each represents a modest decline 
from that identified using a more standard TIL regimen.
With respect to overall survival, it will be assumed that the arm without TBI will be associated 
with an 8-month median survival, since the overall survival using standard TIL without TBI was 
approximately 12 months based on 43 patients. This arm would have an associated hazard rate of 
0.0866, or approximately a 9% death rate per month. The arm including TBI will be estimated to 
have a 16 month median survival time, based on the median survival of approximately 24 
months using standard TIL. This arm would have an associated hazard rate of 0.0433, or 
approximately 4% death rate per month. Assuming a 36-month accrual period and an additional 
24 months of follow-up to evaluate survival after the last patient has been enrolled, 56 patients 
per arm will have 88% power to detect this hazard ratio of 2.00 using a 0.025 two-tailed log rank 
test. The primary analysis with respect to this endpoint will thus be determined with Kaplan-
Meier curves and a log-rank test.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
36
Analysis will be done on an intent-to-treat basis; patients will be included in the primary analysis 
according to the arm on which they have been randomly assigned, whether they are able to 
receive the intended therapy or not. 
Progression free survival will be estimated with a Kaplan-Meier curve and results between the 
curves compared with a log-rank test, as a secondary endpoint. The rates of clinical response 
(PR+CR) will also be estimated and compared as a secondary endpoint, with a two-tailed 
Fisher’s exact test. Finally, toxicities will also be compared using a Cochran-Armitage test for 
those types of toxicities which are experienced by greater than 5 patients on each arm. 
Although accrual is expected to be fairly rapid, and approximately 40 or more patients per year 
are expected to enroll on the trial, the study will be monitored by the NCI/CCR Data Safety and 
Monitoring Board on an annual basis to evaluate the toxicity on the two arms and be sure that the 
toxicity on the TBI arm is not appreciably worse than what is observed on the other arm. In 
addition, at the first DSMB meeting held following the point at which approximately one half of 
the required total subjects have been enrolled (56 total evaluable patients) and potentially 
followed for 6 months, a single evaluation for futility and for better than expected efficacy will 
be undertaken. Note that this may mean that approximately 75-80 patients may have been 
enrolled by the time that 56 have been potentially followed for 6 months. 
The futility evaluation will be performed as follows: based on the overall survival percentage of 
the first 56 patients randomized and potentially followed for 6 months, a conditional power 
analysis will be performed to determine if the trial is unlikely to find an effect at the 0.025 two-
tailed significance level with continued accrual. Using 40% and 60% as the projected probability 
values for 12 months overall survival for the patients who will be accrued on the two arms under 
the alternative, and using the actual observed proportions alive at 12 months from among those 
patients who are randomized and potentially followed for 6 months, the conditional power of the 
trial will be determined assuming accrual of the remaining patients to achieve a total of 112 
patients. If the conditional probability of finding a difference at the 0.025 two-sided level at the 
conclusion of the trial is less than 10%, then it will be reasonable to recommend that no further 
patients will be enrolled. 
The evaluation for better than expected efficacy will be performed as follows: an O’Brien-
Fleming interim evaluation boundary will be used. If the p-value at the interim point (based on 
approximately 56 total patients enrolled and followed for 6 months) is <0.0054, then the trial will 
stop accrual for better than expected efficacy. By using this interim evaluation, the final 
significance level should be <0.0246 instead of 0.025 to declare the result to be significant at the 
0.05 level. 
It is expected that 40 to 50 patients per year can be accrued onto this trial. Allowing for a very 
small number of inevaluable patients, the accrual ceiling will be set at 118 patients. Thus, it is 
anticipated that the trial can complete its accrual in approximately 3 years. 
11 COLLABORATIVE AGREEMENTS
With Amendment C, the NCI Surgery Branch will enter into an agreement (MTA #33284-11) 
with colleague Joseph L. Napoli, Ph.D., Professor and Chairman of Nutritional Science and 
Toxicology, University of California at Berkeley, to analyze metabolites of vitamin A, including 
all trans retinoic acid, retintyl ester, and retinol in serum samples from patients treated on both 
arms of this study. We are interested in analyzing metabolites of vitamin A since using a mouse 
model that closely mimics our current adoptive T cell protocols in patients, we found that host 
conditioning (irradiation) caused an acute vitamin A-deficient state and a loss of a vitamin A-

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
38
12.3 PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT
Adults unable to give consent are excluded from enrolling in the protocol. However re-consent 
may be necessary and there is a possibility, though unlikely, that subjects could become 
decisionally impaired. For this reason and because there is a prospect of direct benefit from 
research participation (Section 12.4), all subjects ≥ age 18 will be offered the opportunity to fill 
in their wishes for research and care, and assign a substitute decision maker on the “NIH 
Advance Directive for Health Care and Medical Research Participation” form so that another 
person can make decisions about their medical care in the event that they become incapacitated 
or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH 
Ability to Consent Assessment Team for evaluation. For those subjects that become 
incapacitated and do not have pre-determined substitute decision maker, the procedures 
described in MEC Policy 87-4 for appointing a surrogate decision maker for adult subjects who 
are (a) decisionally impaired, and (b) who do not have a legal guardian or durable power of 
attorney, will be followed.
12.4 EVALUATION OF BENEFITS AND RISKS
The experimental treatment has a chance to provide clinical benefit though this is unknown. The 
NCI Surgery Branch has extensive experience with ACT following treatment with high dose 
aldesleukin, however this experimental treatment is only available at a very few centers 
throughout the country. Although we have seen responses to this treatment, we do not know 
whether adding total body irradiation to the lymphodepleting chemotherapy regimen will result 
in a higher completer response rate or longer survival. The risks associated with ACT are 
substantial, including, a delay in treatment due to the need to harvest and grow the cells, a 
surgical procedure (possible major) to obtain tumor for the cell product, the possibility that a cell 
product cannot be generated, infection and sepsis due to non-myeloablative chemotherapy, 
intubation, and renal toxicities due to aldesleukin, and death.  Side effect associated with TBI 
include mucositis, infections, cataracts, pulmonary insufficiency, liver toxicity and secondary 
late malignancies. 
The risks in this treatment are further detailed in Section 6.4. The goal is to compare survival and 
complete response rate in patients receiving adoptive cell therapy and aldesleukin with or 
without TBI. 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 of both 
arms is thought to outweigh the potential risks. 
12.5 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.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
39
13 REGULATORY AND OPERATIONAL CONSIDERATIONS
13.1 STUDY DISCONTINUATION AND CLOSURE
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to study participants, 
funding agency and the Investigational New Drug (IND) sponsor and regulatory authorities.  If 
the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly 
inform study participants, the Institutional Review Board (IRB), and sponsor and will provide 
the reason(s) for the termination or suspension.  Study participants will be contacted, as 
applicable, and be informed of changes to study visit schedule.
Circumstances that may warrant termination or suspension include, but are not limited to:

Determination of unexpected, significant, or unacceptable risk to participants

Demonstration of efficacy that would warrant stopping   

Insufficient compliance to protocol requirements

Data that are not sufficiently complete and/or evaluable

Determination that the primary endpoint has been met

Determination of futility
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA).
13.2 QUALITY ASSURANCE AND QUALITY CONTROL
The clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion.  An individualized quality management 
plan will be developed to describe a site’s quality management.
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution.
Following written Standard Operating Procedures (SOPs), the monitors will verify that the 
clinical trial is conducted and data are generated and biological specimens are collected, 
documented (recorded), and reported in compliance with the protocol, International Conference 
on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements 
(e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). 
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local 
and regulatory authorities.
13.3 CONFLICT OF INTEREST POLICY
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons who 
have a role in the design, conduct, analysis, publication, or any aspect of this trial will be 
disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be 
required to have such conflicts managed in a way that is appropriate to their participation in the 
design and conduct of this trial.  The study leadership in conjunction with the National Cancer 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
40
Institute has established policies and procedures for all study group members to disclose all 
conflicts of interest and will establish a mechanism for the management of all reported dualities 
of interest.
13.4 CONFIDENTIALITY AND PRIVACY
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants. 
Therefore, the study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor. 
All research activities will be conducted in as private a setting as possible.
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and 
records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study. The 
clinical study site will permit access to such records.
The study participant’s contact information will be securely stored at the/each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a 
secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or 
sponsor requirements.
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored at the NCI CCR. This will not include the 
participant’s contact or identifying information. Rather, individual participants and their research 
data will be identified by a unique study identification number. The study data entry and study 
management systems used by the clinical site(s) and by NCI CCR research staff will be secured 
and password protected. At the end of the study, all study databases will be archived at the NIH.
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by the National Institutes of Health (NIH).  This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research participation in any 
civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or 
local level. By protecting researchers and institutions from being compelled to disclose 
information that would identify research participants, Certificates of Confidentiality help achieve 
the research objectives and promote participation in studies by helping assure confidentiality and 
privacy to participants.
14 PHARMACEUTICAL INFORMATION
14.1 ALDESLEUKIN (INTERLEUKIN-2, PROLEUKIN, RECOMBINANT HUMAN INTERLEUKIN 2)
How Supplied: Aldesleukin (Interleukin-2) is manufactured by the Novartis Pharmaceuticals 
Corporation, Florham Park, NJ and will be purchased by the NIH Clinical Pharmacy Department 
from commercial sources. 
Formulation/Reconstitution: Aldesleukin 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 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
41
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 (2° - 8°C) 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, 2° – 30°C.
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 3 
and Appendix 4. Grade 3 toxicities common to aldesleukin include diarrhea, nausea, vomiting, 
hypotension, skin changes, anorexia, mucositis, dysphagia, or constitutional symptoms and 
laboratory changes as detailed in Appendix 3. Additional grade 3 and 4 toxicities seen with 
aldesleukin are detailed in Appendix 4. 
14.2 FLUDARABINE
Description: (Please refer to package insert for complete product Information) Fludarabine 
phosphate is a synthetic purine nucleoside that differs from physiologic nucleosides in that the 
sugar moiety is arabinose instead of ribose or deoxyribose. Fludarabine is a purine antagonist 
antimetabolite. 
How Supplied: It will be purchased by the NIH Clinical Pharmacy Department from commercial 
sources. Fludarabine is supplied in a 50 mg vial as a fludarabine phosphate powder in the form of 
a white, lyophilized solid cake. 
Stability: Following reconstitution with 2 mL of sterile water for injection to a concentration of 
25 mg/mL, the solution has a pH of 7.7. The fludarabine powder is stable for at least 18 months 
at 2-8°C; when reconstituted, fludarabine is stable for at least 16 days at room temperature. 
Because no preservative is present, reconstituted fludarabine will typically be administered 
within 8 hours. Specialized references should be consulted for specific compatibility 
information. Fludarabine is dephosphorylated in serum, transported intracellularly and converted 
to the nucleotide fludarabine triphosphate; this 2-fluoro-ara-ATP molecule is thought to be 
required for the drug’s cytotoxic effects. Fludarabine inhibits DNA polymerase, 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 
Appendix 2.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
42
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 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.
14.3 CYCLOPHOSPHAMIDE 
(Refer to FDA-approved package insert for complete product information)
Description: Cyclophosphamide is a nitrogen mustard-derivative alkylating agent. Following 
conversion to active metabolites in the liver, cyclophosphamide functions as an alkyating agent; 
the drug also possesses potent immunosuppressive activity. The serum half-life after IV 
administration ranges from 3-12 hours; the drug and/or its metabolites can be detected in the 
serum for up to 72 hours after administration. 
How Supplied: Cyclophosphamide will be obtained from commercially available sources by the 
Clinical Center Pharmacy Department.  
Stability: Following reconstitution as directed with sterile water for injection, cyclophosphamide 
is stable for 24 hours at room temperature or 6 days when kept at 2-8°C.  
Administration: It will be diluted in 250 mL D5W and infused over one hour. The dose will be 
based on the patient’s body weight. If patient is obese (BMI > 35) drug dosage will be calculated 
using practical weight as described in Appendix 2.
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 

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
43
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. 
14.4 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 Appendix 2. 
Toxicities include nausea, vomiting and diarrhea. 
14.5 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. Filgrastim 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. Filgrastim will be given as a daily subcutaneous injection. The side effects of filgrastim 
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.
14.6 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 the first Monday, Wednesday, or 
Friday on or after the first dose of chemotherapy 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.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
44
14.6.1 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. 
14.7 HERPES VIRUS PROPHYLAXIS
14.7.1  Valacyclovir (Valtrex)
Valacyclovir will be obtained by the Clinical Center Pharmacy Department from commercial 
sources. It will be used orally to prevent the occurrence of herpes virus infections in patients with 
positive HSV serology. It is supplied in 500 mg tablets. 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.   
14.7.2 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. gancyclovir. In renal disease, the dose is adjusted as per product labeling. 
14.8 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.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
45
14.9 CELL PREPARATION 
The procedure for growing and expanding the autologous young CD8+ TIL and the Certificate of 
Analysis are similar to those approved by the Food and Drug Administration and used at the NCI 
in protocol 07-C-0176. This product will be provided for investigational use only under a 
sponsor-investigator IND. The Certificate of Analysis is in Appendix 1 and the Standard 
Operating Procedures for the growth of TIL and the purification of CD8+ cells are included in 
the IND.
14.10 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.
14.11 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.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
46
15 REFERENCES
1.
Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J 
Clin 2007;57:43-66.
2.
Atkins MB, Kunkel L, Sznol M, Rosenberg SA. High-dose recombinant interleukin-2 
therapy in patients with metastatic melanoma: long-term survival update. Cancer J Sci Am 2000;6 
Suppl 1:S11-S4.
3.
Gogas HJ, Kirkwood jM, Sondak VK. Chemotherapy for metastatic melanoma: time for a 
change? Cancer 2007;109:455-64.
4.
Wunderlich JR, Hodes RJ. Principles of tumor immunity:Biology of cellur immune 
responses. Biologic Therapy of Cancer, DeVita, Hellman and Rosenberg, eds 1991:3-21.
5.
Dudley ME, Yang JC, Sherry R, et al. Adoptive cell therapy for patients with metastatic 
melanoma: Evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin 
Oncol 2008;26:5233-9.
6.
Rosenberg SA, Yannelli JR, Yang JC, et al. Treatment of patients with metastatic melanoma 
using autologous tumor-infiltrating lymphocytes and interleukin-2. J Natl Cancer Inst 
1994;86:1159-66.
7.
Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM. Telomere length 
dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 
1998;16:743-7.
8.
Reed JR, Vukmanovic-Stejic M, Fletcher JM, et al. Telomere erosion in memory T cells 
induced by telomerase inhibition at the site of antigenic challenge in vivo. J Exp Med 
2004;199:1433-43.
9.
Blackburn EH. Switching and signaling at the telomere. Cell 2001;106:661-73.
10.
de Lange T. Protection of mammalian telomeres. Oncogene 2002;21:532-40.
11.
Zhou J, Shen X, Hodes RJ, Rosenberg SA, Robbins P. Telomere length of transferred 
lymphocytes correlates with in vivo persistence and tumor regression in melanoma patients 
receiving cell transfer therapy. J Immunol 2005;175:7046-52.
12.
Tran KQ, Zhou J, Durflinger KH, et al. Minimally cultured tumor-infiltrating lymphocytes 
display optimal characteristics for adoptive cell therapy. J Immunother 2008;31:742-51.
13.
Hendriks J, Xiao Y, Borst J. CD27 promotes survival of activated T cells and complements 
CD28 in generation and establishment of the effector T cell pool. J Exp Med 2003;198:1369-80.
14.
Huang J, Kerstann KW, Ahmadzadeh M, et al. Modulation by IL-2 of CD70 and CD27 
expression on CD8+ T cells: importance for the therapeutic effectiveness of cell transfer 
immunotherapy. J Immunol 2006;176:7726-35.
15.
Gattinoni L, Klebanoff CA, Palmer DC, et al. Acquisition of full effector function in vitro 
paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J Clin 
Invest 2005;115:1616-26.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
47
16 FIGURES, TABLES & APPENDICES

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
48
Table 1
Table 1. Characteristics of patients enrolled on lymphodepleting adoptive cell therapy protocols
 NMA 
 TBI-200 
 TBI-1200 
 TOTAL 
 TOTAL 
 Patients 
 43 
 (100%) 
 25 
 (100%) 
 25 
 (100%) 
93
 (100%) 
 Sex 
 Male 
 26 
 ( 60%) 
 17 
 ( 68%) 
 18 
 ( 72%) 
61
(66%)
 
 Female 
 17 
 ( 40%) 
 8 
 ( 32%) 
 7 
 ( 28%) 
32
(34%)
 Age 
 11 - 20 
 2 
 ( 5%) 
 0 
 ( 0%) 
 1 
 ( 4%) 
3
(3%)
 
 21 - 30 
 3 
 ( 7%) 
 3 
 ( 12%) 
 2 
 ( 8%) 
8
(9%)
 
 31 - 40 
 8 
 ( 19%) 
 5 
 ( 20%) 
 6 
 ( 24%) 
19
(20%)
 
 41 - 50 
 16 
 ( 37%) 
 9 
 ( 36%) 
 4 
 ( 16%) 
29
(31%)
 
 51 - 60 
 12 
 ( 28%) 
 8 
 ( 32%) 
 12 
 ( 48%) 
32
(34%)
 
 61 - 70 
 2 
 ( 5%) 
 0 
 ( 0%) 
 0 
 ( 0%) 
2
(2%)
 ECOG 
 0 
 33 
 ( 77%) 
 20 
 ( 80%) 
 19 
 ( 76%) 
72
(77%)
 
 1 
 10 
 ( 23%) 
 5 
 ( 20%) 
 6 
 ( 24%) 
21
(23%)
 Prior Rx 
 None 
 0 
 ( 0%) 
 0 
 ( 0%) 
 0 
 ( 0%) 
0
(0%)
 
 Surgery 
 43 
 (100%) 
 25 
 (100%) 
 25 
 (100%) 
93
(100%)
 
 Chemotherapy 
 22 
 ( 51%) 
 7 
 ( 28%) 
 5 
 ( 20%) 
34
(37%)
 
 Radiotherapy 
 15 
 ( 35%) 
 6 
 ( 24%) 
 2 
 ( 8%) 
23
(25%)
 
 Hormonal 
 1 
 ( 2%) 
 0 
 ( 0%) 
 1 
 ( 4%) 
2
(2%)
 
Immunotherapy 
 42 
 ( 98%) 
 24 
 ( 96%) 
 21 
 ( 84%) 
87
(94%)
 
 Any 2 or more 
 43 
 (100%) 
 25 
 (100%) 
 22 
 ( 88%) 
90
(97%)
 
 Any 3 or more 
 27 
 ( 63%) 
 9 
 ( 36%) 
 6 
 ( 24%) 
42
(45%)

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
49
Table 2
Table 2: Transfusions and grade 3 and 4 non-hematologic toxicities associated with NMA plus 
TBI lymphodepleting preparative regimens.
*Two of these patients have had biopsy evidence of microangiopathy and have stable renal 
function and are ongoing complete responders at 49+ months and 46+ months and are 
living normally. Two other patients were suspected to have this diagnosis. One is an 
ongoing complete responder at 52+ months and one had a partial response lasting 13 
months but died of progressive metastatic melanoma. 
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

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
50
Table 3
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

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
51
Table 4
Table 4: Objective response rates in 3 ACT studies.
Cell Transfer Therapy                            (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: 42 had prior IL-2; 22 had prior IL-2 + chemotherapy)
(20 complete responses: 19 ongoing at 37 to 82 months)

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
52
Table 5
Objective Responses in Patients with Metastatic Melanoma
___________________________________________________
Total             CR
PR                   OR
number of patients (%)
Dacarbazine1
149
4(2.7%)           14(9.4%)       18(12.1%)
Interleukin-22
270         17(6.3%)           26(9.6%)       43(17.9%)
Ipilimumab3
540
3(0.6%)           35(6.4%)         38(7.0%)
PLX 40324
48
3(6.3%)           34(70.8%)       37(77.1%)
Cell Transfer5
93
20(21.5%)         32(34.4%)       52(55.9%)
1)  Middleton et al JCO, 18:158, 2000
2)  Atkins et al JCO, 17:2105, 1999
3)  Hodi et al NEJM, 363:711, 2010
4)  Flaherty et al NEJM, 363:809, 2010
5)  Dudley et at JCO, 26:5233, 2008 (updated 2010)

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
53
Figure 1

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
54
Figure 2

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
56
Figure 4
Figure 4:  Expression of CD4, CD8, CD 27, and CD28 of TIL generated from one tumor specimen. Young TIL 
were 15 days old and standard TIL were 29 days old. Plots of CD27 and CD 28 expressions are gated for CD8+ 
lymphocytes.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
58
Appendix 1
Certificate of Analysis:          
Young TIL
Patient:
Date of preparation of final product:
Unique TIL identifier (tumor and culture number):
Allogeneic PBMC
Donor Name(s):
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 109 and 
2 X 1011
Identity
FACs 
> 80 % CD3+ on REP 
cells
TIL potency2
OKT3-stimulated IFN 
release 
>200 pg/mL per 105 
cells and > 2 times 
background
Microbiological 
studies 
aerobic culture5 
no growth
anaerobic culture5
no growth
gram stain1,3
no micro-organisms 
seen
aerobic culture3,4
no growth
fungal culture3,4
no growth
anaerobic culture3,4
no growth
mycoplasma test2
negative
Endotoxin1 
limulus assay
5 E.U./kg
Presence of 
tumor cells2
Cytopathology
No tumor cells per 200 
cells examined
1 Performed on the final product prior to infusion. Results are available at the time of infusion.
2 Performed 2 - 10 days prior to infusion (test performed prior to final manipulation). Results are available at the 
time of infusion.
3 Performed 2-4 days prior to infusion. Results are available at the time of infusion but may not be definitive.
4 Sample for test collected on the final product prior to infusion. Results will not be available before cells are infused 
into the patient.
5 Sample for test collected on the in process cells prior to the REP. Results will be available before cells are infused 
into the patient.
Prepared by:
_________________________________________
Date: ____________________
QC sign-off:
__________________________________________
Date: ____________________
James C. Yang, M.D. or designee

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
59
Appendix 2
Modification of Dose Calculations* in Patients whose BMI is > 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.

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
60
 Appendix 3
 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 
12 
Somnolence 
22
tachycardia
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

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
61
Appendix 4
Expected Aldesleukin 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 q4h p.r., 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 interspersed with 
activity
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

Abbreviated Title: 1200 TBI TIL Randomized Study
Version Date: 04/15/2022
62
Hypotension
3
Fluid resuscitation
Vasopressor support
If uncontrolled 
despite all 
supportive 
measures
No
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
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 re-treatment.