Clinical trial NCT01807182
Conditions: Stage III Cutaneous Melanoma AJCC v7 Stage IIIA Cutaneous Melanoma AJCC v7 Stage IIIB Cutaneous Melanoma AJCC v7 Stage IIIC Cutaneous Melanoma AJCC v7 Stage IV Cutaneous Melanoma AJCC v6 and v7 Metastatic Melanoma
Sponsor: Fred Hutchinson Cancer Center
Start date: 2013-08-20; Completion date: 2021-08-26
Enrollment: 11 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase II trial studies how well tumor-infiltrating lymphocytes (TIL) after combination chemotherapy works in treating patients with melanoma that has spread to other places in the body. Biological therapies, such as TIL, may stimulate the immune system in different ways and stop tumor cells from growing. Drugs used in chemotherapy, such as cyclophosphamide and fludarabine phosphate, work in different ways to stop the growth of tumor cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Giving TIL after combination chemotherapy may kill more tumor cells.

Detailed description:
OUTLINE:

Patients receive cyclophosphamide intravenously (IV) on days -7 to -6 and fludarabine phosphate IV on days -5 to -1. Patients undergo TIL infusion over 30-60 minutes on day 0 and receive aldesleukin IV every 8 hours on days 1-5 for up to a maximum of 14 doses.

After completion of study treatment, patients are followed up at 6, 12, and 24 weeks.

Arms: arm 1: Patients receive cyclophosphamide IV on days -7 to -6 and fludarabine phosphate IV on days -5 to -1. Patients undergo TIL infusion over 30-60 minutes on day 0 and receive aldesleukin IV every 8 hours on days 1-5 for up to a maximum of 14 doses.
Interventions: intervention 1: Aldesleukin intervention 2: Cyclophosphamide intervention 3: Fludarabine Phosphate intervention 4: Laboratory Biomarker Analysis intervention 5: Therapeutic Tumor Infiltrating Lymphocytes
Intervention descriptions: intervention 1: Given IV intervention 2: Given IV intervention 3: Given IV intervention 4: Correlative studies intervention 5: Undergo TIL infusion

Protocol documents:
Prot_SAP_000.pdf: 
  Page 1 
FRED HUTCHINSON CANCER RESEARCH CENTER (FHCRC) 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE (UW) 
DEPARTMENT OF MEDICINE, DIVISION OF ONCOLOGY 
SEATTLE CHILDRENS 
Current Version: 05/10/2021 
Previous Version: 11/17/2016 
PROTOCOL 2643.00 
1. TITLE: Cellular Adoptive Immunotherapy Using Autologous Tumor-Infiltrating
Lymphocytes Following Lymphodepletion with Cyclophosphamide and
Fludarabine For Patients With Metastatic Melanoma
Investigator 
Professional Title 
Telephone Number 
Sylvia Lee, MD (PI) 
Associate Professor, UWMC 
Associate Professor, FHCRC 
(206) 288-2274
Pager: (206) 540-0757
John Thompson, MD 
Professor, UWMC 
(206) 288-2044
Shailender Bhatia, MD 
Associate Professor, UWMC 
(206) 288-6765
Scott Tykodi, MD 
Associate Professor, UWMC 
(206) 288-7763
David Byrd, MD 
Professor, UWMC 
(206) 543-7512
Stanley Riddell, MD 
Professor, FHCRC 
(206) 667-5249
Biostatistician 
Ted Gooley, PhD 
Member, FHCRC 
(206) 667-6533
Pathologist 
Stephen Schmechel, MD 
Associate Professor, UW (206) 543-1140
Martha Kahuga 
Research Nurse 
 
(206) 667-7582
Emergency (24 hour) Phone:  (206) 598-6190 (page on-call oncology provider) 
IRO received 05/16/22
FHCRC IRB Approval  
JUN 09 2022 
Document Released Date


  Page 2 
TABLE OF CONTENTS 
1. TITLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1  
2. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 
3. BACKGROUND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 
3.1 Immunotherapy of Malignant Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 
3.2 Adoptive Cellular Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 
3.3 Rationale  and Proposed Study of TIL Therapy . . . . . . . . . . . . . . . . . . . . . . . .7 
4. OBJECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 
4.1 Primary Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 
4.2 Secondary Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 
5. STUDY DESIGN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 
6. PATIENT SELECTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 
6.1  Step I: Inclusion and Exclusion Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 
6.2  Step II: Inclusion and Exclusion Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . .  10 
7. STUDY AGENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  13 
7.1 Tumor-infiltrating lymphocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  13 
7.2 Cyclophosphamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  13 
7.3 Fludarabine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  13 
7.4 Interleukin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14 
8. INVESTIGATIONAL PLAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14 
8.1 Tumor Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14 
8.2 Conditioning Regimen with Cyclophosphamide and Fludarabine . . . . . . . . 15 
8.3 TIL Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 
8.4 High-dose Interleukin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  15 
8.5 Prophylaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16 
8.6 Specific Guidelines for Patients on Vemurafenib . . . . . . . . . . . . . . . . . . . .  17 
9. SCHEDULE OF EVALUATIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  17 
9.1 General Toxicity Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  17 
9.2 Immunologic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18 
9.3 Efficacy Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18 
10. RE-TREATMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18 
11. MANAGEMENT OF TOXICITIES AND COMPLICATIONS . . . . . . . . . . . . . . . . . .18 
11.1 Evaluation of Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18 
11.2 Regimen-Related Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19 
11.3 Management of Symptoms during TIL Infusion . . . . . . . . . . . . . . . . . . . .  20 


 
 
 
                      Page 3 
 
 
11.4 Potential Toxicities Warranting Ablation of Adoptively Transferred TIL. . .  20 
 
11.5 Concomitant Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  21 
 
11.6 Premature Discontinuation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  21 
 
12. GUIDELINES FOR ADVERSE EVENTS REPORTING. . . . . . . . . . . . . . . . . . . . .  22 
 
12.1 Reporting of Adverse Events (AEs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  22 
 
12.2 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  22 
 
13. STATISTICAL CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  22 
 
14. ADMINISTRATIVE CONSIDERATIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  23 
 
14.1 Institutional Review Board. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  23 
 
14.2 Termination of Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  23 
 
14.3 Consent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
. . . . . . . .  24 
 
15. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  25 
 
 
APPENDICES 
 
APPENDIX A: ECOG/Zubrod Performance Status . . . . . . . . . . . . . . . . . 
. . . . . . . . .28  
 
APPENDIX B: Generation and Characterization of Tumor-Infiltrating Lymphocytes for  
Adoptive Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  29 
 
APPENDIX C: Administration and Toxicity Management Guidelines for High-Dose  
Interleukin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  30 
1. Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  30 
2. Specific Toxicities and Corrective Intervention . . . . . . . . . . . . . . . . . . . . . .  30 
3. Administration Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  32 
4. Medications Used During HDIL-2 Therapy . . . . . . . . . . . . . . . . . . . . . . . . .  33 
5. Additional Management Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  34 
6. Monitoring (IL-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  34 
7. Patient Management Following Completion of HDIL-2 . . . . . . . . . . . . . . .  35 
 
APPENDIX D: Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  36 
 
APPENDIX E: Data and Safety Monitoring Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  37 
 


 
 
 
                      Page 4 
 
2. INTRODUCTION 
 
This protocol proposes to examine the anti-tumor efficacy and in vivo persistence of 
adoptively transferred, autologous, tumor-infiltrating lymphocytes (TIL) following 
lymphodepletion with cyclophosphamide and fludarabine, in patients with metastatic 
melanoma. This study will also assess prognostic markers and safety of the infused 
autologous TIL following lymphodepletion. 
 
3. BACKGROUND 
 
3.1 Immunotherapy of Malignant Melanoma 
 
3.1.1 Introduction 
The incidence of melanoma is rising at a rate greater than any other cancer. Based on 
2005-2009 data, it is estimated that 1 in 50 men and women will be diagnosed with 
melanoma during his or her lifetime1. Metastatic melanoma is particularly devastating, 
with a median survival of less than one year and low response rates to available 
chemotherapy and immunotherapy agents. Melanoma is notorious for affecting young 
men and women in their 30s and 40s, and thus, the loss of productive years is one of 
the most significant among cancers2.  
 
In a landmark year for melanoma therapy, two new drugs were FDA-approved in 2011 
for advanced melanoma. Ipilimumab, an immune checkpoint blocker, offers long-term 
disease control for approximately 20% of patients, but complete responses are rare3. 
Vemurafenib, a BRAF inhibitor, produces rapid disease regression in roughly 50% of 
patients whose tumor carries a v600 mutation of BRAF, but these responses are 
almost always short-lived, with a median duration of only 6.7 months4. While both drugs 
represent huge advances in the melanoma field, neither agent will change the clinical 
outcome of the majority of melanoma patients in a meaningful way. Thus, the need for 
additional treatment options in melanoma remains urgent.  
 
Melanoma has long been recognized as one of the most immunogenic cancers. Of all 
tumor types, melanoma has provided the most fertile ground for the development of 
immunotherapeutic strategies, such as interleukin-2, interferon-alpha, and ipilimumab. 
Over the past 10 to 15 years, there has been growing enthusiasm for adoptive cell 
therapy (ACT), a technical approach in which the patient’s autologous T cells are 
expanded, manipulated ex vivo, and then re-infused into the patient to exert an anti-
tumor response. Clinical trials of adoptive cellular therapy in our lab and others have 
demonstrated dramatic tumor regressions in an increasing number of melanoma 
patients with refractory metastatic disease5-10.  
 
3.2  Adoptive Cellular Therapy 
 
3.2.1 Rationale as a treatment modality 
Adoptive therapy involves the transfer of ex vivo expanded effector cells as a means of 
augmenting the anti-tumor immune response.  In contrast to tumor vaccination 
strategies, adoptive T cell therapy can allow for far greater control over the magnitude of 


 
 
 
                      Page 5 
 
the targeted response by appropriate generation in vitro of the T cells used for 
therapy11,12. Requisite numbers of high affinity effectors can be routinely achieved 
producing frequencies of tumor-specific T cells in the peripheral blood that are several-
fold higher than that possible by current immunization regimens alone.   
 
3.2.2 Adoptive Therapy of Melanoma Using Antigen-Specific CD8+ T Cell Clones 
The Yee Lab has recently reported the results of a Phase I study of adoptive therapy 
using CD8+ T cell clones targeting the tumor-associated antigens, MART1/MelanA and 
gp100, for the treatment of patients with metastatic melanoma13.  Eleven patients with 
bulky, refractory melanoma received cyclophosphamide 4000 mg/m2 across 2 days, 
then 1010 antigen-specific, CD8+ cytotoxic T cell clones, followed by IL-2. Five of ten 
evaluable patients had stable disease at 8 weeks, and one patient had a complete 
remission that has persisted for greater than 3 years. 
 
In another phase I study for patients with refractory metastatic melanoma, Yee and 
colleagues administered 4 infusions of autologous T cell clones (the first without IL-2) 
and subsequent infusions with low-dose IL-2 for 14 days14.  Among 43 infusions at 
doses up to 3.3 x 109 cells/m2 per dose administered to 10 patients, no serious toxicity 
was observed.  They demonstrated that the adoptively transferred T cell clones 
persisted in vivo in response to low-dose IL-2, preferentially localized to tumor sites, and 
mediated an antigen-specific immune response characterized by the elimination of 
antigen-positive tumor cells and regression of individual metastases.  Historically, 
patients with metastatic disease refractory to standard therapy experience an average 
median survival of 4 months.  Among the initial cohort of 10 patients with progressive 
metastatic melanoma receiving T cell therapy in this study, 8 experienced minor, mixed 
or stable responses for periods of 2-21 months (average of 11 months).  
 
3.2.3  Adoptive Therapy using Tumor-Infiltrating Lymphocytes 
 
In 1988, Rosenberg and colleagues at the NIH demonstrated that tumor-infiltrating 
lymphocytes (TIL) derived from resected tumors and expanded in vitro were capable of 
specifically recognizing tumor antigens and mediating tumor regression in melanoma 
patients15. An analysis of 86 melanoma patients treated with TIL followed by high-dose 
IL-2 at the NIH from 1987 to 1992 demonstrated a 34% overall response rate, with 
similar activity in patients with prior IL-2 exposure or no prior IL-216. Five patients (6%) 
had a complete response but only two were durable at 21 and 46 months. After the 
addition of a lymphodepleting regimen, response rates to TIL have reached 50-70% and 
durable complete responses have reached 20-40%.  
 
An important advantage held by TIL over T cell clones is that TIL generation does not 
rely on exogenous peptide and therefore patient eligibility is not limited to the handful of 
HLA types to which the currently available peptides are restricted. In contrast, our trials 
with T cell clones are only open to patients with HLA type A2, which excludes 
approximately 60% of the population. The shorter generation time of 5-6 weeks for TIL 
versus 10-12 weeks for traditional T cell clones, also allows the inclusion of patients 
who have more rapidly growing disease and can’t wait months for treatment. 
Additionally, because TIL can be generated more rapidly and with less manipulation that 


 
 
 
                      Page 6 
 
traditional T cell clones, a TIL product may be comprised of younger T cells that are less 
terminally differentiated and with a greater proliferative capacity.  
 
3.2.4 Addition of Lymphodepletion Prior to Adoptive Cell Therapy 
In a compelling 2008 analysis, Dudley et al. presented the results of three sequential 
trials with increasing intensities of myeloablation prior to TIL infusion17. In the first trial, a 
nonmyeloablative regimen was administered using cyclophosphamide (60 mg/kg for 2 
days) and fludarabine (25 mg/m2 for 5 days) followed by TIL infusion and high-dose IL-
2. The two subsequent TIL trials gave cyclophosphamide and fludarabine at the same 
doses as before, but with the addition of 2 Gy TBI for the second trial and 12 Gy TBI for 
the third trial. Although the trials were conducted sequentially, limiting the reliability of a 
comparison, they noted that the overall response rates and complete response rates 
were progressively higher with the greater intensity of lympho- and myeloablation. For 
the no-TBI, 2Gy-TBI, and 12Gy-TBI trials, the objective response rate was 49%, 52% 
and 72%, respectively, and the complete response rate was 12%, 20% and 40%, 
respectively10. All but one of the CRs have continued beyond 3 years.  
 
While the response rates are impressive, the myeloablative regimens carried significant 
toxicity. The latter two trials required hematopoietic rescue with autologous stem cells 
after TIL infusion due to the intensity of the myeloablative regimen. There was one 
treatment-related death out of 93 patients and one patient with prolonged pulmonary 
hypertension; both were on the 2Gy-TBI trial. Five patients on the 12Gy-TBI arm 
developed long-standing microangiopathic nephropathy, though interestingly, all five 
patients also achieved CRs and recovered from their renal failure. Because the higher 
response rates of the 12Gy-TBI regimen is likely biased by differences in patient 
population and changes in TIL manufacture across time, a prospective randomized trial 
is ongoing in which patients will receive identical TIL cell and conditioning regimens, but 
half will also receive TBI with autologous stem cell support (study identifier 
NCT01319565, clinicaltrials.gov).  
 
The creation of a lymphopenic environment prior to TIL infusion is believed to enhance 
TIL proliferation and activity by reducing the numbers of immunosuppressive regulatory 
T cells and myeloid derived suppressor cells that are otherwise promoted by many 
factors associated with tumor growth, such as TGF-β and apoptosis-inducing receptor-
ligand interactions18,19. It is also widely believed that the elimination of other 
lymphocytes decreases the competition for homeostatic cytokines IL-7 and IL-15, 
providing both physical and biologic “space” for TIL and other potential effectors, such 
as NK cells, to proliferate and survive20.  Total body irradiation contributes to 
lymphodepletion but also appears to increase the function of antigen-presenting cells by 
activating the innate immune system, in part due to bacterial translocation from gut 
mucosal damage, which provides activation signals to antigen-presenting cells through 
their toll-like receptors21. 
 


 
 
 
                      Page 7 
 
3.3 
Rationale and Proposed Study of TIL Therapy for the Treatment of Patients 
with Metastatic Melanoma 
 
TIL therapy in combination with cyclophosphamide and fludarabine conditioning and 
adjuvant high-dose IL-2 has been demonstrated by the NIH and other institutions to 
achieve impressive response rates of 50%, which is superior to all other available 
treatments for metastatic melanoma. Responses have been seen in patients who have 
failed high-dose IL-2, ipilimumab and vemurafenib.  Current adoptive therapy strategies 
at our institution using T cell clones have been limited to the minority of melanoma 
patients who are HLA-A2 positive. In contrast, TIL therapy would be available to 
patients of all HLA types because it does not rely on HLA-restricted, exogenous peptide. 
In addition, the faster generation time makes TIL a more viable option for patients who 
are experiencing disease progression and can’t wait months for T cell clones to grow.  
 
These early successes of TIL therapy have also given rise to important unanswered 
questions regarding the variability in response among patients and the need to identify 
prognostic factors to determine which patients are likely to respond to TIL and thus 
avoid a costly manufacturing process for patients with little chance of benefit. The 
inherent requirement of a tumor biopsy for TIL generation provides a built-in guarantee 
of melanoma tissue for analysis of immunohistochemical features, molecular and 
genetic markers. 
 
We propose a phase II study evaluating the anti-tumor activity, persistence, safety, as 
well as prognostic markers and immunologic correlates for durable benefit from TIL 
therapy following a lymphodepleting conditioning regimen of cyclophosphamide, and 
fludarabine, and adjuvant high-dose IL-2, for patients with metastatic melanoma.  
 
4. OBJECTIVES 
 
4.A. Primary Objectives 
 
1. Examine the anti-tumor efficacy of cellular adoptive immunotherapy in metastatic 
melanoma patients using autologous tumor-infiltrating lymphocytes with a 
lymphodepleting conditioning regimen of cyclophosphamide and fludarabine, and 
followed by adjuvant high-dose IL-2. 
 
4.B. Secondary objectives 
 
1. Determine the in vivo persistence of transferred tumor-infiltrating lymphocytes. 
 
2. Examine the safety of cellular adoptive immunotherapy in melanoma patients 
using autologous tumor-infiltrating lymphocytes, preceded by a lymphodepleting 
conditioning regimen of cyclophosphamide and fludarabine, and followed by 
adjuvant high-dose IL-2. 
 
3. Evaluate for molecular tumor markers and immunohistochemical features that 
correlate with in vivo persistence and anti-tumor efficacy.  


 
 
 
                    Page 8 
 
 
5. STUDY DESIGN 
This will be a single-center, nonrandomized study that will enroll 20 patients with 
metastatic melanoma for the treatment phase (Step II) of this protocol. The protocol 
utilizes a pre-screening stage (Step I) to obtain the 20 treatment-eligible patients for 
whom TIL can be expanded to sufficient numbers. Enrollment on Step I will stop once 
there are 20 enrolled patients for Step II. The actual enrollment number for Step I is 
anticipated to be 80-120 patients based on an expected 50% success rate for TIL 
generation, mitigated by patient attrition between Step I and Step II due to external 
factors such as disease progression or response to other therapy. 
 
Starting on day -7, patients will receive cyclophosphamide 60mg/kg/day intravenously 
for two days, then fludarabine 25mg/m2/day intravenously for five days, followed by a 
single TIL infusion at a dose up to 1.5 x 1011 cells on day 0. Within 24 hours of the TIL 
infusion, high-dose IL-2 will be administered at 600,000 units/kg every 8 hours, up to 14 
doses as tolerated. Patients will then be monitored with clinical visits and blood draws 
on a weekly basis until week 6, then at weeks 8, 12 and 24. Reimaging with CT or 
PET/CT will be performed at week 6, week 12 and week 24 after TIL infusion.  
 
The chemotherapy of cyclophosphamide and fludarabine (days -7 to -1) will be 
administered at the University of Washington Medical Center inpatient oncology unit, or 
if the patient is deemed suitable by the PI, at the Seattle Cancer Care Alliance on the 
outpatient Immunotherapy Service. Treatment of tumor infiltrating lymphocytes (TIL) will 
be given at the UWMC inpatient oncology unit, or if the patient is deemed suitable by 
the PI, at the Seattle Cancer Care Alliance on the outpatient Immunotherapy Service  
IL-2 therapy (days  +1 to +5) will given at the University of Washington Medical Center. 
in the intensive care unit (ICU) to allow close monitoring and evaluation of the patients.  
 
Figure 1. Treatment course 
 
6. PATIENT SELECTION 
Patients will be enrolled through the Seattle Cancer Care Alliance Melanoma Clinic (a 
clinical consortium of the Fred Hutchinson Cancer Research Center, Seattle Children’s 
Hospital and University of Washington). This protocol is designed to treat patients with 
measurable metastatic melanoma. An average of 300 patients with stage IV disease are 
seen on a yearly basis in the melanoma clinic. With focused recruitment, we expect to 
evaluate roughly 50 patients per year for adoptive cell therapy, and expect 50% of these 
will meet eligibility criteria for Step 1. We anticipate we will succeed in expanding TIL for 


 
 
 
                    Page 9 
 
 
60% of the patients enrolled on Step I of the protocol, and thus, the enrollment for the 
targeted of 20 treated patients will be completed within 30-36 months, and treatment 
and analysis of all patients completed by 36-42 months. 
 
Patient evaluation for eligibility and enrollment will occur in a two-step process: Step I 
for pre-screen and TIL generation, and Step II for treatment. Results of tests and/or 
procedures conducted as per standard of care may be used for eligibility determination 
if conducted within an appropriate window prior to screening. 
 
6.1 Step I: Pre-screen and initiation of TIL expansion. 
Patients must fulfill all the following criteria to be eligible for Step I of the study.  
 
 
6.1.1 Step I Inclusion Criteria  
 
a. 
Stage IV melanoma or stage III melanoma that is unlikely to be 
cured by surgery. 
b. 
Male or female subject, 18 years of age or older and able to 
tolerate high-dose cyclophosphamide, fludarabine and high-dose 
IL-2.  
c. 
ECOG performance status of 0-1 (Appendix A)  
d. 
Patients must have an MRI, CT, or PET of the brain within 2 
months before consenting if known history of brain metastasis or if 
clinically indicated. If new lesions are present, PI or designee 
should make final determination regarding enrollment. 
e. 
Patients must have a site of metastatic disease that can be safely 
resected or biopsied for tissue sufficient for TIL harvest. 
 
 
 
6.1.2 Step I Exclusion Criteria 
 
a. 
Men or women of reproductive ability who are unwilling to use 
effective contraception or abstinence for 4 months after treatment. 
b. 
Calculated creatinine clearance (eGFR) < 60ml/min. EGFR values 
can be determined by either MDRD or Cockcroft-Gault equation 
based on the investigator’s discretion. 
c. 
Significant hepatic dysfunction (AST/ALT > 3x upper limit of 
normal; total bilirubin > 2.0 mg/dl, except in patients with Gilbert’s 
Syndrome whose total bilirubin must not exceed 3.0 mg/dl) 
deemed by investigator to be irreversible. 
d. 
Clinically significant pulmonary dysfunction (FEV1<65% predicted, 
FVC <65% of predicted, DLCO (corrected for Hgb) <50% 
predicted). Pulmonary function tests (PFTs) within 4 months prior 
to consent for Step I will be required for patients with underlying 
risk factors such as smoking history >10 pack years, or a history of 
pre-existing symptomatic lung disease (not including melanoma 
metastases to the lung).  
e.  
Pre-existing known cardiovascular abnormalities as defined by any 
one of the following:  
 
• congestive heart failure,  


 
 
 
                    Page 10 
 
 
• clinically significant hypotension,  
• cardiac ischemia, or symptoms of coronary artery disease, 
• presence of cardiac arrhythmias on EKG requiring drug therapy, 
• ejection fraction < 45% (echocardiogram or MUGA), although 
any patient with an ejection fraction between 45-49% must 
receive clearance by a cardiologist to be eligible for Step II of 
the trial  
f. 
Clinically significant autoimmune disorders or conditions of 
immunosuppression. Patients with AIDS or HIV-1 associated 
complex or known to be HIV antibody seropositive or known to be 
recently PCR+ for hepatitis B or C are not eligible for this study. 
The severely depressed or altered immune system found in these 
patients and the possibility of premature death would compromise 
study objectives.  
g. 
Patients with active systemic infection requiring intravenous 
antibiotics. 
h. 
Clinically significant psychiatric disease which, in the opinion of the 
PI or sub-I, would render immunotherapy and its potential 
sequelae unsafe or compliance with procedural requirements 
unlikely. 
 
6.1.3 Procedures for Step I 
All patients must sign an informed consent form for Step I. Patients will be screened for 
Hepatitis B surface antigen, hepatitis B core antibody, hepatitis B surface antibody, 
hepatitis C virus antibody, HIV 1/2 Antibody, and standard test for syphilis (STS) within 
30 days of signing informed consent.  
 
TIL expansion will be attempted on approximately 80-120 patients to obtain the 20 
patients who continue on to receive treatment on Step II of this protocol. While TIL are 
being grown, patients may be treated with other therapies. However, patients must not 
have received other therapies within 4 weeks prior to lymphodepletion, with the 
exception of commercially available molecularly targeted therapies (e.g., vemurafenib), 
which are not allowed within 7 days prior to lymphodepletion. 
 
6.2 Step II: Treatment with TIL. 
All patients must complete screening procedures for Step I to be eligible for Step II. 
Patients must fulfill all of the following criteria to be eligible for Step II of the study. 
Patients must sign an informed consent form for Step II before receiving treatment with 
TIL therapy on this protocol. 
 
 
 
6.2.1 Step II Inclusion Criteria  
 
a. 
Patients must have measurable metastatic melanoma. 
b. 
Male or female subject, 18 years of age or older and able to 
tolerate high-dose cyclophosphamide, fludarabine, and high-dose 
IL-2.  
c. 
ECOG performance status of 0-1 (Appendix A). 


 
 
 
                    Page 11 
 
 
d. 
Patients must have brain imaging by MRI, CT or PET within 30 
days prior to lymphodepletion. Patients may have asymptomatic 
brain lesions that are ≤1cm each. Lesions that are >1cm that have 
been irradiated and in the opinion of the investigator no longer 
represents active disease will also be allowed.   
e. 
A functional cardiac test (e.g., stress treadmill, stress thallium, 
MUGA, dobutamine echocardiogram) to rule out cardiac ischemia 
within 4 months prior to lymphodepletion is required for all patients.  
f.  
PFTs are required of all patients within 4 months prior to 
lymphodepletion. FEV1 and FVC must be ≥65% predicted and 
DLCO must be ≥50% predicted. 
g.  
Patients must have their tumor sent for BRAF mutational analysis. 
h. 
Patients must have adequate TIL (at least 40 x 106 cells at the pre-
expansion stage). 
 
 
 
6.2.2 Step II Exclusion Criteria 
 
a. 
Pregnant women, nursing mothers, men or women of reproductive 
ability who are unwilling to use effective contraception or 
abstinence. Women of childbearing potential must have a negative 
pregnancy test within 14 days prior to entry. Patients of both 
genders must practice birth control during treatment and for four 
months after treatment. 
b. 
Calculated creatinine clearance (eGFR) < 60ml/min. EGFR values 
can be determined by either MDRD or Cockcroft-Gault equation 
based on the investigator’s discretion. 
c. 
Significant hepatic dysfunction (AST/ALT > 3x upper limit of 
normal; total bilirubin > 2.0 mg/dl, except in patients with Gilbert’s 
Syndrome whose total bilirubin must not exceed 3.0 mg/dl). 
d. 
Clinically significant pulmonary dysfunction (FEV1<65% predicted 
or FVC <65% of predicted, DLCO (corrected for Hgb) <50% 
predicted).  
e. 
Pre-existing known cardiovascular abnormalities as defined by any 
one of the following:  
 
• congestive heart failure,  
• clinically significant hypotension,  
• cardiac ischemia, or symptoms of coronary artery disease, 
• presence of cardiac arrhythmias on EKG requiring drug therapy, 
• ejection fraction < 45%, although any patient with an ejection 
fraction between 45-49% must receive clearance by a 
cardiologist to be eligible for Step II of this trial. 
f.  
Absolute neutrophil count less than 1000/mm3. 
g.  
Platelet count less than 100,000/mm3. 
h. 
Hemoglobin less than 10.0g/dl. 
i. 
Untreated central nervous system metastases that are either 
symptomatic or greater than 1 cm at time of therapy. Lesions that 
are >1cm that have been irradiated and in the opinion of the PI or 
sub-I no longer represent active disease may be allowed. 


 
 
 
                    Page 12 
 
 
j. 
Patients with systemic infections requiring active therapy within 72 
hours of lymphodepletion. 
k. 
Systemic cancer therapy (standard or experimental), including 
cytotoxic chemotherapy, IL-2, or checkpoint blocking agents (e.g., 
CTLA-4 or PD1/PD-L1 inhibitors) received less than 4 weeks prior 
to lymphodepletion, with the exception of targeted therapies (see 
next). 
l. 
Commercially available, molecularly targeted therapies (e.g., 
dabrafenib, trametinib, vemurafenib, imatinib) taken within 7 days 
prior to lymphodepletion.    
m. 
Clinically significant autoimmune disorders or conditions of 
immunosuppression. Patients with AIDS or HIV-1 associated 
complex or known to HIV antibody seropositive or known to be 
recently PCR+ for hepatitis B or C virus are not eligible for this 
study. Virology testing will be done within 6 months of T cell 
infusion. The severely depressed or altered immune system found 
in these patients and the possibility of premature death would 
compromise study objectives. 
n. 
Prior treatment with systemic steroids within 4 weeks prior to  
 
lymphodepletion (except for physiologic replacement doses,  
 
for adrenal insufficiency, premedication for contrast allergies for  
 
scans, and for drug fever related to targeted therapy).  
o. 
Any other significant medical or psychological conditions that would 
make the patient unsuitable candidate for cell therapy at the 
discretion of the PI. 
 
 
6.2.3 Procedures for Step II  
 
  
A complete history and physical examination noting in detail the exact size  
 
 
and location of any lesions appreciable by exam will be performed within 4  
 
 
weeks prior to the initiation of chemotherapy.  
 
  
Blood work including a CBC, differential, complete metabolic panel with  
 
 
LDH, PT/PTT/INR, and a urinalysis (micro) will be performed within 14  
 
 
days prior to initiation of chemotherapy.  
 
  
A pregnancy test (urine or serum) on all women of childbearing  
 
potential will be performed within  14 days prior to initiation of  
 
chemotherapy.  
 
  
Baseline radiographic studies must be obtained within 30 days prior to  
 
 
chemotherapy (CT or PET/CT of chest, abdomen, and pelvis; MRI, CT or  
 
 
PET/CT of brain). If a patient has no history of prior brain metastases and  
 
 
no symptoms concerning for brain disease, brain imaging can be  
 
 
performed within 60 days prior to chemotherapy. 
 
  
Pulmonary function tests must be completed within 4 months of  
 
 
lymphodepletion. 
 
  
An electrocardiogram must be completed within 2 weeks prior to  
 
 
lymphodepletion. 
 
  
Functional cardiac  test is required, within 4 months prior to 
lymphodepletion. 
  
If prior virology studies for Step I were completed more than 6 months  


 
 
 
                    Page 13 
 
 
 
prior to lymphodepletion, these tests must be repeated: Hepatitis B  
 
surface antigen, hepatitis B surface antibody, hepatitis B core antibody, 
hepatitis C virus antibody, HIV 1/2 Antibody, standard test for syphilis 
(STS), CMV antibody, and EBV panel.  
  
HSV and VZV serologies within 2 months prior to lymphodepletion. 
  
HLA typing must be drawn at any time prior to lymphodepletion. 
 
Tumor must have been sent for BRAF mutational analysis at any time  
 
prior to enrollment on Step II. 
  
A research blood draw up to 80cc in ACD tubes for immunologic studies 
at any time prior to lymphodepletion, and at least three weeks after prior 
cytoreductive chemotherapy. 
 
7. 
STUDY AGENTS 
 
7.1 
Tumor-infiltrating lymphocytes (TIL) 
All infused TIL products will be autologous lymphocytes derived from excisional or 
incisional tumor biopsies of metastatic melanoma. Methods employed to generate and 
select TIL for infusion are outlined in Appendix B. In brief, tumor fragments are cultured 
in media containing IL-2 until sufficient lymphocytes grow out. The lymphocytes that 
demonstrate the fastest growth are maintained, and further selected based on CD8 
expression and recognition of autologous tumor by ELISA.  The selected TIL cultures 
are pooled for a 2-week rapid expansion. Prior to infusion they are tested for sterility. 
MD Anderson has found that TIL infusions up to 1.5 x 1011 cells are well tolerated. 
 
7.2  Cyclophosphamide  
The effectiveness of immunotherapy appears to be enhanced when combined with 
conventional cytotoxic agents, and among these, cyclophosphamide has been the most 
extensively studied 22. Cyclophosphamide is an alkylating agent that prevents cell 
division by cross-linking DNA strands. 
 
In clinical studies where no other chemotherapy drugs were administered, 
cyclophosphamide alone at doses of 30 – 120 mg/kg (1000 to 4000 mg/m2) has a 
predictable hematologic profile with a period of neutropenia and leukopenia beginning 
8-10 days after treatment lasting 7-10 days followed by a relatively rapid recovery. 
Thrombocytopenia may be more prolonged, but recovery to normal levels is usually 
complete.  The dose limiting toxicity from cyclophosphamide is not hematopoietic, but 
cardiac at doses up to 8000 mg/m2- when given as an IV bolus23. Cardiac toxicities 
from cyclophosphamide include arrhythmias, potentially irreversible cardiomyopathy 
and pericarditis.  
 
Sterile hemorrhagic cystitis occurs in approximately 20% of patients, ranging from 
microscopic hematuria to extensive cystitis with bladder fibrosis. Mesna (sodium 2-
mercaptoethanesulphonate) is a synthetic sulfhydryl compound that can interact with 
the urotoxic metabolites of cyclophosphamide to decrease the incidence and severity of 
hemorrhagic cystitis.  
 
7.3 
Fludarabine 


 
 
 
                    Page 14 
 
 
Fludarabine is a purine analog that inhibits DNA synthesis by impairment of 
ribonucleotide reductase and DNA polymerase alpha. Fludarabine induces lymphopenia 
in greater than 30% of patients treated with a standard 5-day course of fludarabine at 25 
mg/m2 24.  Although fludarabine is an effective agent against hematologic malignancies, 
such as CLL, low-grade non-Hodgkin’s lymphoma, and Waldenström's 
macroglobulinemia, it has no anti-tumor effect on solid tumors such as melanoma.  
 
Lymphopenia is characterized by a CD4 > CD8 T cell depletion that results in 
lymphocyte counts falling to 30-40% of baseline or less that is sustained for periods of 
several weeks to months and is associated with T cell dysfunction-related infections 
25,26. The risk of lymphopenia, the severity and frequency of infectious complications 
observed in patients receiving multiple courses of fludarabine is much lower when 
patients receive a single five-day course of fludarabine27. Approximately 25% of patients 
receiving a 5-day course of treatment will develop an infection that is generally 
treatable. Patients receiving only a 3-day course of fludarabine exhibit a significantly 
lower incidence of infection per treatment course (14%) without a significant decrease in 
anti-lymphoproliferative effect28. Other common toxicities of fludarabine (< 10%) include 
edema, fever, chills, rash, pain, thrombocytopenia with a nadir of 16 days, malaise, 
fatigue, anorexia, nausea/vomiting, gastrointestinal bleeding, weakness, visual 
disturbances, sleep disorders, cough, dyspnea, and infection. 
 
7.4 
Interleukin-2  
The major physiologic role of IL-2 is to promote the activation and proliferation of T and 
NK cells in an autocrine and paracrine manner 29. It is secreted primarily by T cells in 
response to antigenic stimuli. Exposure of NK cells to IL-2 results in proliferation, 
enhanced cytolytic activity and secretion of other cytokines. B cells also express 
intermediate affinity IL-2 receptors and can secrete IL-2 in cooperation with other 
cytokines, resulting in B cell proliferation and differentiation30.  
 
Initial work by Rosenberg’s group at the NIH found that adoptively transferred IL-2-
activated peripheral blood mononuclear cells with the phenotype and functional 
characteristics of activated NK cells, supported with concomitant administration of IL-2 
in high doses, resulted in significant tumor regression in patients selected for normal 
organ function and good performance status. Further investigation of these encouraging 
results suggested that therapeutic benefit could be seen in a subset of patients treated 
with high doses of IL-2 alone. This led to clinical trials that demonstrated high-dose IL-2 
induces objective clinical responses in 15–20% of patients with advanced melanoma 
and durable complete responses in 5–7% of these patients 31,32. The NIH group has 
been consistently administering a cycle of high-dose IL-2 after TIL infusion to stimulate 
the in vivo expansion of the TIL product. MD Anderson has been administering two 
cycles of high-dose IL-2 following their TIL infusions, spaced 21-days apart, and have 
reported in personal communication, that these are well tolerated. 
 
The toxicity profile of IL-2 is largely associated with a capillary leak syndrome, which is 
characterized by hypotension, tachycardia and peripheral edema secondary to third 
space fluid accumulation. In addition to hypotension, IL-2 may also induce pulmonary 
edema, cardiac arrhythmias, myocarditis, reversible renal and hepatic dysfunction, 
pruritus, electrolyte abnormalities, thrombocytopenia, anemia and coagulopathy. IL-2 


 
 
 
                    Page 15 
 
 
can also cause constitutional symptoms such as fever, chill and fatigue, gastrointestinal 
side effects such as nausea, vomiting, anorexia, transaminase elevation, cholestasis 
and diarrhea 33. Rarely, IL-2 may induce confusion, disorientation or visual 
hallucinations. Although early studies with IL-2 reported a 2% mortality rate, generally 
related to gram-positive sepsis, current IL-2 centers that routinely use prophylactic 
antibiotics report no mortality 34-36. In experienced centers such as University of 
Washington, IL-2-related toxicity can usually be easily managed and all side effects are 
reversible upon cessation of treatment.  
 
8.  INVESTIGATIONAL PLAN 
 
8.1  
Tumor Acquisition and TIL Generation 
Once patients meet screening criteria for Step I, we will obtain a tumor specimen by a 
biopsy or surgery of a safely accessible melanoma lesion, ideally 2 cm3. Whenever 
possible, this tumor will be acquired from a biopsy or surgery that is already being 
performed for medical reasons (e.g. to confirm metastatic disease or for palliation). Part 
of this tumor will be sent to pathology for diagnostic purposes, as well as additional 
immunohistochemical analysis to assess for prognostic features. If there is enough 
tissue, part of this tumor will be flash frozen and stored to enable future analysis.  TIL 
will be generated, tested, selected and expanded across the next 5-6 weeks. If a 
patient is not ready for treatment, the TIL will be cryopreserved prior to the 2-week 
expansion phase. A portion of the TIL product pre- and post-expansion may also be 
removed for research purposes (e.g. immunophenotyping, gene expression analysis.) 
 
8.2.  Conditioning Regimen with Cyclophosphamide and Fludarabine 


 
 
 
                    Page 16 
 
 
Patients will be admitted to the oncology inpatient service at the University of 
Washington Medical Center, or at the discretion of the PI, the outpatient 
Immunotherapy Service at Seattle Cancer Care Alliance on day -7. A PICC line will be 
placed, largely in anticipation of high-dose IL-2 
but also for the administration of chemotherapy 
and TIL. If the PICC team is unavailable on 
admit, chemotherapy may be started using a 
peripheral IV and then switched to the PICC line 
once it is placed. 
 
On day -7 and day -6, cyclophosphamide (Cy) 
at 120 mg/kg will be administered intravenously 
in split doses of 60 mg/kg/day x 2 days. 
Standard Practice Policy guidelines, including 
IV hydration and Mesna therapy for 
uroprotection, will be followed.  
 
Fludarabine will be administered intravenously 
for 5 days from day -5 through day -1, at a dose 
of 25mg/m2/day, and completed at least 24 
hours prior to T cell infusion. 
 
8.3  
TIL infusion 
On day 0, patients will receive the TIL infusion 
at the University of Washington Medical Center 
or at the discretion of the PI, the outpatient 
Immunotherapy Service at Seattle Cancer Care 
Alliance. Patients will receive up to 1.5 x 1011 
cells, depending on the number that is 
generated in the laboratory. The remainder will 
be cryopreserved for research purposes, but 
will not be infused at a later date. The TIL will 
be suspended in a 500cc bag of normal saline 
and infused intravenously over 45 minutes (+/- 
15 minutes) by a peripheral IV, a PICC line or central catheter. The infusion bag will be 
gently mixed, approximately every 15 minutes. Patients will have vital signs obtained 
pre-infusion, every 15 minutes during the infusion, at the end of the infusion and hourly 
for two hours following the infusion.  
 
8.4  
High-dose Interleukin 2 
Within 24 hours of the TIL infusion, the patient will be transferred to the hematology-
oncology intensive care unit at UWMC and started on high-dose interleukin 2 at 
600,000 IU/kg as an IV bolus every eight hours up to a maximum of 14 doses, as 
tolerated, based on the High-Dose Interleukin-2 Administration and Toxicity 
Management Guidelines currently used at the University of Washington (Appendix C). 
Once the patient develops toxicities, the PI or designated surrogate will make a clinical 
decision when to stop interleukin-2. This decision will be based on the Toxicity 
Management Guidelines.  
Figure 2. Investigational Plan 


 
 
 
                    Page 17 
 
 
 
After completion of high-dose interleukin-2, standard supportive care will be provided 
including weaning off vasopressor support, discontinuing parenteral fluids, and 
monitoring. When the patient demonstrates hemodynamic stability and adequate 
pulmonary reserve, the patient will be discharged from the hospital, typically with 3-4 
days of oral diuretics and potassium as indicated.  
 
8.5 
Prophylaxis 
For all prophylactic medications below, dose adjustments and substitutions may be 
made if clinically warranted (e.g. drug allergy, renal impairment). 
 
8.5.1  Bacterial Prophylaxis  
Patients will receive levofloxacin 500 mg daily, starting on day +0 or once ANC 
falls below 500/mm3, whichever is sooner. Levofloxacin will be continued until 
ANC recovers to greater than 500/mm3.  
 
8.5.2  Pneumocystis Jiroveci Pneumonia (PCP) Prophylaxis 
Patients will receive the fixed combination of trimethoprim (TMP) and 
sulfamethoxazole (SMX) as double strength (DS) tablet [DS tabs = TMP 160 
mg/tab and SMX 800 mg/tab ] po bid on two consecutive days a week.  
TMP/SMX-DS will be taken by patients beginning on Day -7 and continued for 6 
months after lymphodepletion.  Patients with sulfa allergies may instead take 
dapsone 100mg po daily or atovaquone 1500mg po daily, starting one week 
prior to lymphodepletion until 6 months after lymphodepletion.   
 
8.5.3  Herpes Simplex Virus and Varicella Zoster Virus Prophylaxis 
Patients with a positive HSV and/or positive VZV serology will be administered 
valacyclovir 500 mg po bid, starting on day+0, for 6 months.  If the patient 
cannot take oral medications, he will be given acyclovir 5 mg/kg IVPB every 8 
hours, which is continued until absolute neutrophil count is greater than 1000/ml.  
 
8.5.4  Fungal Prophylaxis  
Patients will begin Fluconazole 200 mg po daily, starting on Day+0 or once ANC 
falls below 500/mm3, whichever is sooner. Fluconazole will be continued for 2 
months after lymphodepletion.  
 
8.5.5  Empiric Antibiotics  
Patients who develop fevers ≥38.5 C with an ANC less than 500/mm3 will be 
treated on broad-spectrum antibiotics per standard UW practice for neutropenic 
fever as appropriate to the patient’s signs and symptoms of infection.  
Aminoglycosides should be avoided unless clear evidence of sepsis.  
 
8.5.6  Hematologic Support  
In order to reduce neutropenia following chemotherapy and T cell infusion, G-
CSF will be given at 5 μg/kg/day daily subcutaneously from Day +1 until 
neutrophil counts reach >500/mm3x 3 days or >5000/mm3 x 1 day. Using daily 
CBC’s as a guide, the patient will also receive platelets and packed red blood 
cells (PRBC’s) as needed.  Attempts will be made to keep hemoglobin >8.0 g/dl, 


 
 
 
                    Page 18 
 
 
and platelets >20,000/ml.    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.  Irradiated blood and blood products should 
be used. 
 
8.6 
Specific Guidelines for Patients on BRAF and MEK inhibition 
Patients are not allowed to receive other anti-cancer treatments while on this protocol 
with the exception of BRAF and MEK inhibitors. Patients who are responding to 
targeted therapies such as vemurafenib and dabrafenib represent a very different 
patient category. It is well established that the responses to vemurafenib and 
dabrafenib are short-lived, with a 6-7 month median duration of response.4 It often 
takes patients 1-2 months to reach best response, and this response remains stable 
until they ultimately experience disease progression. However, waiting for these 
patients to progress on BRAF/MEK inhibition or requiring they discontinue BRAF/MEK 
inhibitors for an extended period is not ideal, because patients in these two situations 
often experience rapid disease progression that would preclude subsequent 
immunotherapy that could offer more durable disease control.  
 
Therefore, for those patients who are currently on BRAF and MEK inhibitors and are 
experiencing a response that has stabilized (two consecutive scans spaced at least 
one month apart that show no further regression), concurrent treatment on BRAF/MEK 
inhibitors will be allowed with the following guidelines: 
 
• Patients must be off BRAF/MEK inhibitors for the 7 days prior to 
lymphodepletion. 
• Patients may resume BRAF/MEK inhibitors no sooner than 7 days after 
the completion of IL-2, as long as the PI or her surrogate deems this safe 
based on the patient’s clinical status at that time. 
• If the patient achieves a complete response after T cell therapy that lasts 
at least 6 months, the BRAF/MEK inhibitors can be discontinued and a 
follow-up scan performed in 2-3 months. 
• If the patient achieves a partial response or stable disease after T cell 
therapy, that is unchanged for at least 12 months and PET-negative, the 
BRAF/MEK inhibitors can be discontinued and a follow-up scan 
performed in 2-3 months. 
 
9.  SCHEDULE OF EVALUATIONS  
 
9.1     General Toxicity Assessment 
Patients will have vital signs taken, a physical exam, a comprehensive chemistry panel 
and a complete blood count and differential at each planned visit, daily while the patient 
is in the hospital/clinic, weekly until 4 weeks after the T cell infusion, and then on weeks 
6, 8, 12 and 24 (Appendix D). Of note, the dates listed on the study calendar are 
approximate as many patients may reside out of the area and cannot always follow the 
time points as dictated by the protocol—however, patients must remain close to the 
study center until blood counts have recovered sufficiently to be at minimal risk of 


 
 
 
                    Page 19 
 
 
infection or bleeding. All adverse events will be recorded and graded according to the 
NCI CTCAE version 4.0. 
 
 
9.2  
Immunologic Studies  
For evaluation of peripheral blood cells following T cell infusion, 60 ml of blood into 
yellow top tubes (containing ACD solution) should be drawn within 60 days after 
signing of step I consent, prior to receiving chemotherapy on day -7, prior to infusion on 
day 0, and then day +1, +3, +7, +14, +21 +28, +35, +42, +49, +56 (+/- 3 days), and 
then week 12 and week 24, as well as any follow up visits when feasible. These dates 
are approximate as many of our patients reside out of the area and their oncologists 
and laboratories cannot always follow the time points as dictated by the protocol. 
Samples will be used to evaluate the duration of in vivo persistence, phenotype and 
function of infused TIL. Strategies for following in vivo persistence include deep 
sequencing of the T cell receptor CDR3 regions to follow frequencies of T cell 
populations, tetramer staining and ELISPOT analysis using melanoma peptide pools. 
 
9.3 
    Efficacy Assessment  
Radiographic imaging (CT, PET/CT or MRI) and clinical assessment of residual 
disease will be compared with pre-infusion assessment. This assessment will be 
performed at 6 weeks, 12 weeks and 24 weeks after T cell infusion, and for continued 
responders, every 3-6 months for the next two years, at the discretion of the primary 
provider.  
 
Clinical response will be determined at 6 weeks, 12 weeks and 24 weeks based on 
RECIST version 1.137 definitions for CR, PR, SD and PD. 
 
10. RE-TREATMENT 
 
After TIL therapy, if a patient experiences disease control (complete response, partial 
response or stable disease) lasting 6 months, and then shows evidence of disease 
progression, and he experienced less than Grade III toxicity (except for the common 
expected toxicities listed below), the patient may be re-evaluated using the same 
screening procedures for re-treatment with TIL. The retreatment plan would be 
unchanged, with the same cyclophosphamide and fludarabine lymphodepletion 
regimen, TIL infusion and adjuvant high-dose IL-2.  The patient would need to be re-
consented and meet all eligibility criteria, as before. The patient would need to undergo 
a repeat tumor biopsy to harvest TIL, unless there are sufficient TIL cryopreserved from 
the prior TIL culture for expansion (at least 40 x 106 cells). 
 
11. MANAGEMENT OF TOXICITIES AND COMPLICATIONS 
 
 
11.1 Evaluation of Toxicity 
Toxicity grading will be evaluated according to guidelines in NCI Common Toxicity 
Criteria version 4.038. The full text of the NCI CTCAE is available online at: 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html. Toxicities will be monitored on a daily 
basis beginning at day -7 and continuing until discharge from the hospital following IL-2 
infusion and then on subsequent follow up visits through day +42. The principal 


 
 
 
                    Page 20 
 
 
investigator will be responsible for monitoring the data and toxicities to identify trends, 
and for revising the protocol as needed to maintain safety.  
 
A data safety and monitoring board () will be formed and meet after the treatment of the 
5th patient, the 15th patient or in the event of 2 or more unexpected, serious (CTCAE 
grade 3 or greater), treatment-related toxicities or at least annually whichever is sooner. 
If the rate of unexpected, serious, treatment-related toxicities is observed in ≥ 33% of 
enrolled subjects in a cohort of 6 or more subjects, this will trigger the suspension of 
the study pending further review by the DSMB and FDA. Furthermore, any occurrence 
of unexpected grade 4 or greater toxicity will prompt the investigator to conduct a 
thorough investigation of available safety data to justify to the DSMB a decision to 
continue enrolling new subjects into the study. 
 
 Serious adverse events (Appendix E) will be reported to the IRB and the FDA, in 
accordance with regulations detailed in the IND package. 
 
11.2 Regimen-Related Toxicity 
If the patient develops an unexpected and serious toxicity attributable to the study 
regimen, the patient will not receive additional study treatment and a course of 
corticosteroids will be given if clinically indicated (see Section 11.3). A serious toxicity is 
defined as a non pre-existing Grade 3 or higher toxicity that develops after the start of 
treatment with the following exceptions: 
 
Common and expected symptoms of chemotherapy and T cell infusion that are 
considered exceptions to criteria for discontinuation include: 
 
A. For Cyclophosphamide at 60 mg/kg/day x 2 days and fludarabine 25mg/m2/day x 5 
days, expected toxicities are:  
i. Non-life-threatening infections or febrile neutropenia (i.e. absence of 
hemodynamic compromise requiring vasopressor support) 
ii. WBC < 1000 (Grade 4 toxicity) for less than 4 weeks  
iii. ANC < 500 (Grade 4 toxicity) for less than 3 weeks 
iv. Lymphocytes < 500  (Grade 3 toxicity) for less than 3 weeks 
v. Platelets < 50,000 (Grade 3 or 4 toxicity) for less than 4 weeks 
vi. Hyperbilirubinemia and transaminase elevation (Grade 3 toxicity) that 
resolves within 3 weeks 
vii. Nausea, vomiting or diarrhea (Grade 3 toxicity) that resolves within 3 
weeks 
viii. Electrolyte and renal abnormality (Grade 3 toxicity) that resolves to 
Grade 1 or baseline within 3 weeks 
 
B.  Expected toxicities attributable to T cell infusions and considered exceptions to 
criteria for discontinuation include: 
i. 
Cytokine Release Syndrome (CRS) Grade 3 or less, including but 
not limited to asthenia, flu-like symptoms, myalgia, lymphopenia 
and rigors 
ii.       Skin rash/Erythroderma (Grade 3 toxicity)  


 
 
 
                    Page 21 
 
 
iii. 
Hypoxemia requiring continuous oxygen, but not mechanical 
ventilation or intubation < 72 h 
iv.  
Fever that resolves within 72h after cessation of IL-2 
v.  
(Lymphocytes < 500) (Grade 3 or 4 toxicity) that resolves within 4 
weeks to baseline levels (pre-therapy) 
 
C. Expected toxicities attributable to high-dose IL-2:  
A variety of side effects and toxicities have been associated with high-dose IL-2  
administration; a list of these along with guidelines for their management, which  
should be adjusted for the patient’s clinical condition, is listed in Appendix C. 
 
11.3 Management of Symptoms during TIL Infusion 
Mild transient symptoms have been observed with LAK and TIL cell infusions, and with 
infusions of antigen-specific T cell clones. These symptoms occur during the first 24 
hours following T cell infusion. The management of these symptoms is outlined below. 
a. 
Myalgia, fever and chills will be managed with acetaminophen 650 
mg p.o. q 4-6 hrs along with assessment of possible infection.   
b. 
Headaches may be managed with acetaminophen and mild 
opiates following a neurologic examination. 
c. 
Nausea, vomiting will be treated with a non-steroidal anti-emetic of 
choice.  
d. 
Hypotension will initially be managed by intravenous fluid 
administration and further measures as dictated by standard 
medical practice. 
e. 
Hypoxemia will initially be managed with supplemental oxygen and 
further measures as dictated by standard medical practice. 
 
11.4 Potential Toxicities Warranting Ablation of Adoptively Transferred TIL 
Ablation of transferred T cells by corticosteroid administration will be warranted by any 
grade 3 or greater toxicity (NCI Common Toxicity Criteria version 4.038) occurring in 
any other organ system following administration of antigen-specific cytotoxic T cells not 
attributable to underlying metastatic melanoma and excluding exceptions associated  
with known toxicities of cyclophosphamide, fludarabine, T cells and IL-2 .   
 
 
 
T-cell Ablation with Corticosteroids 
a. 
All patients will be hospitalized for the first 48 hours for monitoring.  
The following guidelines are provided for steroid dose and taper 
but will be adjusted to patient’s clinical situation.   
 
Day 1 
Intravenous Solu-Medrol at 2 
mg/kg  
Day 2 
Intravenous Solu-Medrol at 2 
mg/kg 
Day 3-4 
Prednisone at 30 mg po b.i.d. 
Day 5-6 
Prednisone at 15 mg po b.i.d. 
Day 7-8 
Prednisone at 10 mg po b.i.d. 
Day 9-10 
Prednisone at 10 mg po q.d 
Day11-12 
Prednisone at  5  mg po q.d. 


 
 
 
                    Page 22 
 
 
 
b. 
Blood will be drawn for later analysis of the in vivo frequency of 
melanoma antigen-specific T cells will be assayed immediately 
prior to and 48 hours after the start of steroid therapy.  
 
11.5 Concomitant Therapy 
11.5.1 Infections occurring despite antibiotic prophylaxis should be treated according to 
the standard of care. 
 
11.5.2 The following agents are not allowed while on study: systemic corticosteroids 
(except as outlined for management of toxicity of TIL or physiologic doses for adrenal 
insufficiency), chemotherapy that is not part of the treatment plan, other 
immunomodulatory agents, or any investigational agents, including unapproved BRAF 
inhibitors. Patients who are responding to vemurafenib (a BRAF inhibitor) at the time of 
enrollment on this trial may be allowed to resume vemurafenib ≥7 days after IL-2 is 
completed (refer to Section 8.6 for conditions and guidelines for patients on 
vemurafenib).  
 
 11.6 Premature Discontinuation 
Subjects who do not complete the study procedures will be considered to have 
prematurely discontinued the study.  The reasons for premature discontinuation (for 
example, voluntary withdrawal, toxicity, death) must be recorded on the case report 
form.  A subject may re-enter the study after premature discontinuation only by 
approval of the Principal Investigator.  If possible, final study evaluations should be 
completed at the time of discontinuation.  Potential reasons for premature 
discontinuation include: 
 
11.7.1 
The development of a life-threatening infection. 
 
11.7.2  
Judgment of the principal investigator that the patient is too ill to 
continue. 
 
11.7.3     
Patient noncompliance with study therapy and/or clinic 
appointments. 
 
11.7.4 
Pregnancy. 
 
11.7.5     
Voluntary withdrawal; a patient may remove himself/herself from the 
study at any time without prejudice. 
 
11.7.6     
Significant and rapid progression of melanoma requiring alternative   
systemic therapy. 
 
11.7.7    
Grade III or IV toxicity judged to be possibly or probably related to 
study therapy according to criteria and exceptions as described 
above. 


 
 
 
                    Page 23 
 
 
  
11.7.8    
Termination of the study by the principal investigator, the DSMB, 
Institutional Review Office or the Food and Drug Administration.  In 
this case, patients will be informed of the reasons and will continue 
to receive best available therapy of their disease. 
 
12. GUIDELINES FOR ADVERSE EVENTS REPORTING 
 
12.1 Reporting of Adverse Events (AEs) 
 
All unexpected and serious adverse events which may be due to study treatment or 
intervention must be reported to the FHCRC Institutional Review Office (IRO) per their 
current reporting requirements.  
 
AEs of grade 3 or greater (per IRB policy 2.6. section 1.a.) will be collected through 4 
weeks after the T cell infusion. Beginning 4 weeks after the T cell infusion, only study-
related toxicities will be collected. 
  
All serious adverse events that are unexpected and related to study treatment will be 
reported to the FDA on a MedWatch 3500 reporting form that will be submitted to the 
FDA within 15 calendar days. The reports will include the date and time of onset, 
severity and duration of the event, the relationship to study treatment, the treatment 
given, and the eventual outcome. 
 
12.2 Definitions 
 
Definitions associated with reportable events can be found on the FHCRC IRO extranet 
website. (Relevant FHCRC policies include, but are not limited the documents listed 
[Table 1]. Please also refer to the FHCRC IRO website.) 
 
Table 1. FHCRC IRB policies for reportable events 
 
IRB Policy 2.6 
Adverse Events and Other 
Unanticipated Problems 
Involving Risks to Subjects 
or Others 
http://extranet.fhcrc.org/EN/sections/ir
o/irb/ae.html 
IRB Policy 1.9 
Noncompliance with the 
Office of the Director’s 
Human Research 
Protection Program Policy 
http://extranet.fhcrc.org/EN/sections/ir
o/irb/ae.html 
IRB Policy 1.1 
Reporting Obligations for 
Principal Investigators 
http://extranet.fhcrc.org/EN/sections/ir
o/irb/policy/index.html 
IRB Policy 2.2 
Continuing Review 
http://extranet.fhcrc.org/EN/sections/ir
o/irb/policy/index.html 
IRB Policy 
1.13 
Investigational New Drugs 
(IND), Biologics and 
http://extranet.fhcrc.org/EN/sections/ir
o/irb/policy/index.html 


 
 
 
                    Page 24 
 
 
Investigational Device 
Exemptions (IDE) 
 
 
13. 
STATISTICAL CONSIDERATIONS  
 
The primary endpoint of this study will be clinical response, to be assessed at 6, 12 and 
24 weeks, using RECIST 1.1 definitions for CR, PR, SD and PD. Formal guidelines will 
not be used to assess the potential efficacy of this treatment in terms of response rate.  
However, given data from FDA-approved therapies where response rates have ranged 
from 7% to 16%, we previously set a response rate of 20% for this trial as an informal 
benchmark that would support the clinical utility of TIL as an additional treatment for 
melanoma. The original version of this protocol had a targeted accrual of 40 patients 
with a goal of showing a statistically significantly better response rate than the 
benchmark of 20%.  Accrual to this study, however, has been lower than hoped for and 
we therefore are modifying the study with a targeted accrual of 20 patients.  In addition, 
with the introduction in recent years of the immune checkpoint inhibitors, we have 
modified our benchmark from 20% to a new benchmark of 35%, to be comparable with 
the response rates seen from the PD-1 immune checkpoint inhibitors from trials that 
included both treatment-naïve and previously treated melanoma patients, which reflects 
the patient groups eligible for this trial.39-41  As a result of each of these changes, we will 
no longer require a statistically significant improvement over the benchmark to deem 
this treatment as worthy of further study.  Instead, we shall consider the current 
treatment as worthy of further study if the observed response rate exceeds 35%.  If the 
true response rate is 40%, then the probability of an observed response rate in excess 
of 35% among 20 patients (7 or more responses among 20) is 0.75; if the true response 
rate is 50%, the probability is 0.94.  As with the original version, secondary endpoints 
include in vivo persistence of adoptively transferred T cells following TIL infusion and 
safety of the treatment protocol. Various markers will be examined for their association 
with response, among these % expression of central memory T cell markers, CD62L, 
CD27, and CD28.  Logistic regression will be used to assess these correlations.   
 
The original version of this protocol specified that if there are no responses observed 
after the first 20 patients are treated, the study will be suspended pending review by the 
DSMB. There have been 4 responses seen among the first 7 treated, so this 
modification will not have any stopping rules for futility.  
 
 
14. 
ADMINISTRATIVE CONSIDERATIONS 
14.1 
Institutional Review Board 
In accordance with federal regulations (21 CFR 312.66), an Institutional Review Board 
(IRB) that complies with regulations in 21 CFR 56 must review and approve this 
protocol and the informed consent form prior to initiation of the study. 
 
14.2  
Termination of Study 
 
The study will be stopped if any of the following events occur: 
 


 
 
 
                    Page 25 
 
 
• All 20 patients have completed treatment. 
 
• Stopping rules for toxicity have been met. Accrual will be put on hold pending 
discussion with the DSMB regarding whether a change in study design is 
warranted. 
 
• The PI reserves the right to terminate the study at any time. The FDA may also 
terminate the study. 
14.3  
Consent 
The Principal Investigator or her associate must explain verbally and in writing the 
nature, duration, and purpose of the study and possible consequences of treatment.  
Patients must also be informed that they may withdraw from the study at any time and 
for any reason without jeopardizing their future treatment.  In accordance with federal 
regulations (21 CFR 312), all patients must sign the IRB-approved consent form in the 
presence of a witness.  Prior to the start of the study, a copy of the IRB-approved 
consent form must be submitted to the Sponsor. 
 
 
 


 
 
 
                    Page 26 
 
 
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Gattinoni L, Finkelstein SE, Klebanoff CA, et al. Removal of homeostatic cytokine 
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Paulos CM, Wrzesinski C, Kaiser A, et al. Microbial translocation augments the 
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Voss SD, Hank JA, Nobis CA, Fisch P, Sosman JA, Sondel PM. Serum levels of 
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Schwartzentruber DJ. Guidelines for the safe administration of high-dose 
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Atkins MB, Lotze MT, Dutcher JP, et al. High-dose recombinant interleukin 2 
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Weber JS, D'Angelo SP, Minor D, et al. Nivolumab versus chemotherapy in 
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                    Page 29 
 
 
APPENDIX A 
 
ECOG / Zubrod Performance Status 
 
0 
Fully active, able to carry on all pre-disease performance without 
restriction 
 
1 
Restricted in physically strenuous activity but ambulatory and able 
to carry out work of a light or sedentary nature, e.g. light house 
work, office work 
 
2 
Ambulatory and capable of all self-care but unable to carry out any 
work activities. Up and about more than 50% of waking hours 
 
3 
Capable of only limited self-care, confined to bed or chair more 
than 50% of waking hours 
 
4 
Completely disabled. Cannot carry on any self-care. Totally 
confined to bed or chair 
 
5 
Dead 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
                    Page 30 
 
 
APPENDIX B  
 
B.1 
 GENERATION AND CHARACTERIZATION OF TUMOR-INFILTRATING 
LYMPHOCYTES FOR ADOPTIVE IMMUNOTHERAPY 
 
Autologous tumor-infiltrating lymphocytes will be derived from a tumor biopsy from the 
patient with metastatic melanoma. The specimen will first go to the pathologist who will 
assist in distributing parts of the tumor to pathology for diagnostic purposes, to our lab 
for TIL generation, and cryopreservation for banking of remaining tissue. 
 
The tumor sample obtained for the generation of TIL will be dissected into a goal of 24 
fragments, 1-3mm3 in size, which will then be placed in 24-well plates with culture media 
containing IL-2. The remaining tumor will be processed into a single cell suspension and 
then cryopreserved for later use as autologous tumor target cells for TIL testing.  
 
The fragments are cultured in the wells, and inspected every few days for growth and 
evidence of contamination. Once the wells reach confluence, they are resuspended, 
counted and split into wells at a concentration of 1-1.5x106 cells/ml. TIL fragments must 
reach a minimum of 40 x 106 cells within 5 weeks of culture. The cells are tested for 
sterility, endotoxin and mycoplasma. The cells are analyzed for viability, cell number, 
phenotype, and may be assayed for autologous tumor recognition based on interferon-
gamma release after co-culture with the thawed autologous tumor targets, if autologous 
tumor can be reserved at the time of initial tumor processing. Five or six wells are then 
selected based on the wells that demonstrate the best growth, phenotype (e.g., the 
highest CD3+ and CD8+ fractions), and if autologous tumors is available for testing, the 
most selective autologous tumor recognition by IFN-γ release assay. If the patient is 
ready for treatment, a rapid expansion is performed on the fresh, selected cells. If there 
are excess cells, a vial of 10 x 106 cells from each of the selected cultures is 
cryopreserved as a backup. If the patient is not ready for treatment, two vials of 10 x 106 
cells are cryopreserved from each of the selected cultures, one for the future rapid 
expansion and one for the backup.  
 
The requisite cell number to begin the expansion phase is 40 x 106 cells. The selected 
cells are expanded in Grex flasks in CTL media with anti-CD3 monoclonal antibody and 
gamma-irradiated feeder cells prepared from allogeneic peripheral blood mononuclear 
cells obtained from donors with a negative screen for infectious diseases. On day 7 of 
the rapid expansion, a cell count and visual inspection is performed. The patient begins 
the conditioning regimen on day -7, while the cells undergo the last 7 days of rapid 
expansion. Sterility testing is then repeated for bacteria, fungal and mycobacteria, as 
well as gram stain and endotoxin staining, prior to infusion into the patient. 


 
 
 
                    Page 31 
 
 
APPENDIX C 
ADMINISTRATION AND TOXICITY MANAGEMENT GUIDELINES FOR HIGH-DOSE 
INTERLEUKIN-2  
 
(Adapted from University of Washington High-Dose IL-2 Administration and Toxicity 
Management Guidelines) 
 
1) OVERVIEW 
HDIL-2 is administered in 8-hour intervals as tolerated to a maximum of 14 doses. 
Doses that are skipped for management of toxicities are not compensated by 
lengthening the cycle, are not reduced, and are generally intended to be given at 
approximately 8-hour intervals.  As a general rule, if a patient has severe enough 
toxicity to withhold 2 or more consecutive doses, the IL-2 will be discontinued. 
 
2) SPECIFIC TOXICITIES AND CORRECTIVE INTERVENTION 
 
A. Hemodynamics 
 
 Hypotension 
 
• IV Fluids 
 
Upon admission for HDIL-2 treatment, patients should be started on maintenance 
intravenous fluids in anticipation of ensuing capillary leak syndrome, vasodilation 
and decreased systemic vascular resistance.  Colloid has no advantage over 
crystalloid, even when the albumin falls, and D5LR at 100cc/hr is recommended. 
Bolus IV fluids and increase in the continuous IV rate may be of value for initial 
management of hypotension to keep MAP > 60, attempting to limit the cumulative 
positive fluid balance by the end of 5 days to no more than 10% of the patient’s 
dry weight by the end of each 5-day cycle of HDIL-2.  This is accomplished by 
the judicious balance of fluids and vasopressor agents, depending on the 
patient’s hemodynamic status, renal function, acid-base balance and pulmonary 
reserve.     
 
• Vasopressor agents 
 
If hypotension persists despite judicious fluid resuscitation, vasopressor support 
with an α-sympathomimetic agent (generally phenylephrine) is indicated. Titration 
of phenylephrine should be between 0.5 and 2mcg/kg/min to keep the systolic 
blood pressure over 80-90 mm Hg (MAP > 60). Since the BP nadir tends to occur 
4-6 hours after each dose of HDIL-2, a “reserve” should be available in the 
vasopressor dose, so that additional doses of HDIL-2 should be given only if the 
phenylephrine dose can be weaned to below about 1 mcg/kg/minute before the 
next IL-2 dose. 
   


 
 
 
                    Page 32 
 
 
B. Renal 
 
 Oliguria 
 
Oliguria is universal and is due to the combination of hypotension/hypoperfusion 
and decreased intravascular volume.  It may be amplified in uninephric patients 
and patients on vasopressors.  Oliguria per se is not an indication for fluids or 
diuresis; a bladder scan or urinary catheter may be useful to better assess the 
renal status if the patient is anuric, acidotic or has other reasons for urinary 
retention. 
 
C. Pulmonary 
 
 Hypoxia 
 
The need for supplemental oxygen will vary among patients depending on their 
underlying lung function and disease. Supplemental oxygen should be used to 
keep the patient’s oxygen saturation > 90-92%, and intravenous volume should 
be limited in patients whose oxygenation is difficult to maintain and in those with 
significant crackles and/or chest radiographic findings of pulmonary edema or 
significant pleural effusion. 
 
 Cardiogenic Pulmonary Edema 
 
Cardiogenic pulmonary edema is extremely rare in patients receiving HDIL-2 
since patients are well screened and HDIL-2 rarely causes global 
cardiomyopathy. Any patient with sudden pulmonary decompensation should be 
evaluated for causes of cardiac origin.  
 
D. Cardiac 
 
 Ischemia 
 
Cardiac ischemia is also rare, but occasional focal myocarditis may occur, and 
arrhythmias or evidence of ischemia on EKG may mandate discontinuation of 
HDIL-2. 
 
 Cardiac Arrhythmias 
 
Atrial cardiac arrhythmias during HDIL-2 treatment are uncommon, but 
occasionally supraventricular tachycardia and atrial fibrillation may be seen. 
Careful assessment should be performed for supervening causes such as 
ischemia, myocarditis, pericardial effusion or pulmonary events. Treatment 
should include antiarrhythmic medications, and does not preclude continuation of 
HDIL-2.  Serious ventricular arrhythmias are rare and warrant discontinuation of 
HDIL-2 and resumption only if a minor, reversible/treatable cause was found. 
  


 
 
 
                    Page 33 
 
 
 
Edema 
 
Although patients often experience moderate amounts of peripheral edema at the 
end of each cycle, it is rare that they will experience severe symptoms from this 
extravascular fluid accumulation. Patients should be aggressively diuresed at the 
end of each cycle once they are hemodynamically stable. 
 
E. Fever 
 
Blood cultures will be drawn for all patients with fever (temperature > 38.5 C) 
and antibiotic prophylaxis will be given according to SPP guidelines associated 
with IL-2 therapy and neutropenia.  
 
3) ADMINISTRATION GUIDELINES 
Organ-by-organ guidelines for IL-2 dosing must be individualized and considered in the 
context of the whole patient rather than in isolation.  Each dose should be given or 
withheld after an assessment of the patient by the RN and house officer, as supervised 
by the PI or designated surrogate.  
 
System 
Generally Safe to Give 
HDIL-2  
(one or more present) 
Consider Holding HDIL-
2 Dose 
(Especially if >1-2 criteria 
present) 
Unsafe to Given 
HDIL-2 
(any criterion present) 
Cardiac 
Sinus tachycardia up to 
130 bpm, occasional 
ventricular ectopy 
Sustained sinus tachycardia 
after correction of 
contributing factor such as 
fever, chills, hypotension 
or significant ectopy 
Ischemic ECG changes, 
atrial fibrillation, SVT, 
ongoing VT, elevated 
CPK/troponin 
Dermatologic 
Rash 
Urticaria 
Moist desquamation, 
bullae 
Gastrointestinal 
Mild diarrhea controlled 
with antimotility agents, 
isolated bilirubin 
elevation 
Severe nausea, emesis,  
marked elevation/rise of 
transaminases 
Ileus/abdominal 
distension, severe pain, 
intractable emesis 
Hemodynamic 
Phenylephrine support 
up to 1mcg/kg/min 
Phenylephrine support up 
to 1.5mcg/kg/min – must 
be stable or titrating down 
before next dose 
Phenylephrine support 
>1.5mcg/kg/min and or 
rising before next dose 
Hemorrhagic/ 
Thrombotic 
Thrombocytopenia 
Clinically significant 
bleeding requiring platelet 
transfusions 
Life threatening 
bleeding or clot 
Infection 
UTI, viral URI or 
localized HSV 
Bacterial infection 
contributing to 
hemodynamic compromise 
Infection requiring 
surgical intervention, or 
unresponsive to 
antibiotics 
Metabolic 
Asymptomatic and 
correctable: 
hypokalemia, 
hypocalcemia, 
hyperglycemia, 
Symptomatic electrolyte or 
metabolic disturbance  
Arrhythmia or other life 
threatening consequence 
or metabolic disturbance 


 
 
 
                    Page 34 
 
 
hypophosphatemia, 
hypomagnesemia,  
Neurologic 
Mild somnolence, vivid 
dreams 
Mild confusion, 
hallucinations, patient 
aware 
Moderate – severe 
confusion, 
hallucinations, patient 
unaware or combative, 
seizures 
Pulmonary 
Mild dyspnea, hypoxia 
responsive to 4L or less 
of oxygen  
Moderate dyspnea, hypoxia 
requiring more than 40% 
oxygen, significant 
crackles, significant 
effusion not requiring 
treatment  
Severe dyspnea, 
hypoxia on oxygen, 
intubation, severe 
pulmonary edema, 
effusions requiring 
thoracentesis 
Renal 
Creatinine up to 3, 
Oliguria <100cc/in 8 
hours,  correctable 
acidosis 
Creatinine > 3, anuria or < 
100cc/8 hours, persistent 
acidosis 
Persistent anuria, 
dialysis for metabolic or 
renal indications 
 
4) MEDICATIONS USED DURING HDIL-2 THERAPY 
 
1) Standard supportive care 
 
In order to minimize the toxicities of HDIL-2 treatment the following medications should 
be prescribed:    
 
 
Medications 
Indications 
Acetaminophen 650 mg PO q4h 
Fever, myalgia 
NSAID (Example: Naproxen 500 or 750 mg PO BID)  
Fever, myalgia 
Proton pump inhibitor (Pantoprozole 40 mg PO/IV daily, or equivalent) 
Gastritis 
5HT3 blocker (Ex: Ondansetron 8 mg PO/IV daily, or prior to each dose of IL-2 as 
needed) 
Nausea, 
emesis 
Broad spectrum antibiotic, usually Levofloxacin 500 mg PO/IV daily 
Infection 
 
2) Symptom management 
 
For management of toxicity related symptoms, the following should be prescribed on an 
as needed basis:  
 
 
Medications 
Indications 
5HT3 blocker, as above, if not given prophylactically 
Nausea 
Benzodiazepine, usually Lorazepam 0.5 mg IV or 1 mg 
PO q4h 
Mild nausea, anxiety 
Butyrophenone, usually Haloperidol 1-5 mg IV q1h 
Agitation, hallucination 
Loperamide or Atropine-Diphenoxylate 1-2 tablets PO 
q4h, maximum 6 tablets/24h 
Diarrhea 
Meperidine 25 mg IV  or morphine sulfate 1-2 mg, repeat 
up to twice at 10 minute intervals 
Chills 


 
 
 
                    Page 35 
 
 
Tucks topically 
Perianal discomfort 
Diphenhydramine 12.5-25 mg IV or 25-50 mg PO q4h 
Pruritus 
Hydroxyzine 10-25 mg PO q4h 
Pruritus if paradoxical reaction to 
diphenhydramine 
Aveeno bath, lotion 
Severe pruritus 
Lubriderm-based lotion 
Pruritus, skin irritation 
“Magic” mouthwash (Diphenhydramine/Aluminum 
hydroxide/Lidocaine) 
Mouth irritation 
 
Pseudoephedrine 30 mg PO q4h 
Nasal congestion 
Zolpidem or temazepam 
Sleep 
Phenylephrine 25 mg/250 mL concentration,                        
Titrate to BPs 80-90, range 0.1-2 mcg/kg/min 
Hypotension 
Magnesium sulfate  
Hypomagnesemia 
Potassium or sodium phosphate   
Hypophosphatemia 
Potassium chloride  
Hypokalemia 
 
5) ADDITIONAL MANAGEMENT GUIDELINES 
 
Symptom/sign 
Intervention 
Anemia 
Transfuse to keep Hct 27-30 
Arrhythmia 
Correct other factors, R/O ischemia, effusions; arrhythmia 
specific treatment; Consider resuming IL-2 on or off anti-
arrhythmic treatment 
Acidosis 
Keep bicarb level >20 
Edema, threatening 
compartment syndromes 
Elevation, attention to neuropathic symptoms 
Fever pattern atypical (late 
spike) 
Culture, look for sources of bacterial infection 
Hypoalbuminemia 
No specific treatment 
Hypocalcemia 
Correct for low albumin, replace to normal values 
Infection documented 
 
Continuation of IL-2 depends on severity, interventions needed 
Other electrolyte 
disturbances 
Look for contributors, replace as needed   
 
 
6) MONITORING 
 
1) Monitoring guidelines: ICU routine  
 
2) Labs for IL-2 monitoring: According to UWMC standard of care for high-dose IL-2 
 
 


 
 
 
                    Page 36 
 
 
VIII. PATIENT MANAGEMENT FOLLOWING COMPLETION OF HDIL-2 
 
Since the hemodynamic effects of HDIL-2 begin to wane within hours of the last 
infusion, it is usually sufficient to wean the patient off pressors as rapidly as tolerated, 
followed by or accompanied by (depending on the patient’s particular hemodynamic 
needs and volume status) prompt discontinuation of parenteral fluids and then active 
diuresis.  Patients should have sufficient pulmonary reserve at the time of discharge to 
tolerate the possibility of mobilizing a few liters back into the vascular space (which can 
result in pulmonary edema) AND should be given at least 3-4 days of oral diuretics, with 
K as indicated, to return them to near dry weight (which is likely to be about 2-3 kg 
below their prior weight).  Patients can stop the diuretics on their own when their weight 
normalizes and/or edema resolves. All patients experience desquamation and dry, 
pruritic skin, which is managed symptomatically with lubricants and anti-pruritics 
(avoiding steroid). 
 


 
 
 
                    Page 37 
 
 
APPENDIX D  
MONITORING SCHEDULE 
Date* 
Event 
History 
and PE 
Comprehensive 
Panel 
CBC 
Research 
Lab 
Tumor 
biopsy 
Radiographic 
evaluation 
Additional screening tests 
Step 1 Screen 
Evaluation for TIL 
generation 
x 
x 
x 
x 
x 
 
 Brain imaging <2mo; PFTs for high 
risk pts, Virology panel** 
Step 2 Screen 
Evaluation for TIL 
infusion 
x 
x 
x 
x 
 
x 
Brain imaging <30 days; cardiac 
test; PFTs; EKG 
 
Day -7 to 
Day -14 
Admit to UWMC or 
SCCA 
 
x 
x 
x 
x 
 
 
 
 
Day -7 
Lymphodepletion: Cy 
 
 
x*** 
 
 
 
 
 
 
 
 
 
 
 
Day -6 
Lymphodepletion: Cy 
x 
x 
 
 
 
 
 
Day -5 
Lymphodepletion: Flu 
x 
x 
 
 
 
 
 
Day -4 
Lymphodepletion: Flu 
x 
x 
 
 
 
 
 
Day -3 
Lymphodepletion: Flu 
x 
x 
 
 
 
 
 
Day -2 
Lymphodepletion: Flu 
x 
x 
 
 
 
 
 
Day -1 
Lymphodepletion: Flu 
x 
x 
 
 
 
 
 
Day 0 
TIL infusion 
x 
x 
x 
x 
 
 
 
 
Day  +1 
Transfer to ICU; 
IL-2 600,000 U/m2 q8 up 
to 14 doses, as tolerated 
x 
x 
x 
x 
 
 
 
 
Day  +2 
 x 
x 
x 
 
 
 
  
 
Day +3 
x 
x 
x 
x 
 
 
 
 
Day +4 
x 
x 
x 
 
 
 
 
 
Day +5 
x 
x 
x 
 
 
 
 
Week 1 
Day +7 
 
x 
x 
x 
x 
 
 
 
Week 2 
Day +14 
 
x 
x 
x 
x 
 
 
 
Week 3 
Day +21 
 
 
x 
x 
x 
 
 
 
Week  4 
Day +28 
 
x 
x 
x 
x 
 
 
 
Week 5 
Day +35 
 
 
x 
x 
x 
 
 
 
Week 6 
Day +42 
 
x 
x 
x 
x 
 
x 
 
Week  7 
Day +49 
 
 
x 
x 
x 
 
   
   
Week 8 
Day +56 
 
x 
x 
x 
x 
 
 
 
Week 12 
Day +84 
 
x 
x 
x 
x 
 
x 
 
Week 24 
Day+168 
 
x 
x 
x 
x 
 
x 
 


 
 
 
                    Page 38 
 
 
* The dates listed on the study calendar above are approximate as many of our patients reside out of the area and their oncologists and laboratories 
cannot always follow the time points as dictated by the protocol.  
**Patient’s will be screened for Hepatitis B surface antigen, Hepatitis B core antibody, Hepatitis C virus antibody, HIV 1 /2 antibody, standard test for 
syphilis (STS), CMV antibody and EBV panel within 30 days of signing Step 1 consent. 
*** H & Ps during lymphodepletion will be performed according to institutional guidelines/practice.


 
 
 
                    Page 39 
 
 
 
APPENDIX E 
DATA AND SAFETY MONITORING PLAN  
 
Primary Monitoring 
 
The principal investigator (PI) will be responsible for monitoring the data and toxicities to 
identify trends, and for revising the protocol as needed to maintain safety. The PI will be 
responsible for ensuring that the protocol is conducted as approved by the Scientific 
Review Committee and Institutional Review Board, that the monitoring plan is being 
followed, and that all adverse events are reported according to protocol guidelines.  
 
 
Data and Safety Monitoring Board 
 
A dedicated data and safety monitoring board (DSMB) will be formed to review the 
conduct of the trial to date and assess the safety and toxicity of the study intervention. 
At each meeting, all grade III or greater toxicities as defined by version 4.0 of NCI 
Common Toxicity Criteria will be reviewed. The DSMB will determine if the study should 
be prematurely discontinued due to excessive toxicity or if a change in study design is 
warranted. 
 
 
DSMB Meeting Schedule 
 
The DSMB will meet after the treatment of the 5th patient, the 15th patient or if there are 
≥2 unexpected, serious, treatment-related toxicities (see Section 11.2 for definitions) or 
at least annually whichever is sooner.  
 
As described in Section 11, if the rate of unexpected, serious, treatment-related 
toxicities is observed in ≥ 33% of enrolled subjects in a cohort of 6 or more subjects, 
this will trigger the suspension of the study pending further review by the DSMB and 
FDA. Furthermore, any occurrence of unexpected grade 4 or greater toxicity will prompt 
the investigator to conduct a thorough investigation of available safety data to justify to 
the DSMB a decision to continue enrolling new subjects into the study. 
 
DSMB Reporting 
 
The CTSO provides staff to assist with minutes for the DSMB meeting. A report from the 
DSMB is submitted to the FHCRC IRB and Regulatory Affairs Manager.  The FHCRC 
also has a Protocol Data Monitoring Committee (PDMC) that reviews the progress of 
the protocol with respect to the monitoring plan at the time of each annual renewal.  As 
with initial review, annual IRB review and approval is also required.