T Cell Therapy in Combination With Peginterferon for Patients With Metastatic Melanoma
Phase: PHASE1
Conditions: Metastatic Melanoma
Sponsor: Inge Marie Svane

T cell therapy in combination with peginterferon 
for patients with metastatic malignant melanoma 
 
 
 
A pilot study 
 
EudraCT no.: 2014-001420-29 
 
 
 
The clinical trial will be performed according to the present protocol, current Good Clinical Practice 
(GCP) guidelines and requirements from the authorities. The investigator allows direct access to 
source data/documents (including patient records) in the event of monitoring, auditing, and/or 
inspection from the Danish Medicines Agency, the GCP units, or from the health authorities of 
other countries, respectively. 
 
 
 
Sponsor 
 
Inge Marie Svane, Professor, Chief Physician, PhD 
Date: _______________________________ 
Director of the National Center for Cancer Immune Therapy  
Department of Hematology 54P4 and  
Department of Oncology 54B1 
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev, Denmark 
Phone: +45 38 68 21 31 
 
 
Inge Marie Svane: ____________________ 
 
 
Primary investigator 
Troels Holz Borch, physician, PhD student 
Date: _______________________________ 
National Center for Cancer Immune Therapy  
Department of Hematology 54P4 and  
Department of Oncology 54B1 
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev, Denmark 
Phone: +45 38 68 17 56 
   
                  Troels Holz Borch:_______________________ 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
2
Protocol responsible parties 
 
Sponsor 
Inge Marie Svane, Professor, Chief Physician, PhD 
 
Director of the National Center for Cancer Immune Therapy  
Department of Oncology and Hematology 
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev, Denmark 
Phone: +45 38 68 21 31 
 
 
 
Primary investigator 
Troels Holz Borch, physician, PhD student 
 
CCIT-DK, Department of Oncology 
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev, Denmark 
Phone: +45 38 68 17 56 
 
 
 
Sub-investigators 
Rikke Andersen, physician, PhD student 
CCIT-DK, Department of Oncology 
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev, Denmark 
Phone: +45 38 68 93 57 
 
Marco Donia, physician, PhD 
CCIT-DK, Department of Oncology and Hematology  
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev, Denmark 
Phone: +45 38 68 14 56  
 
Collaborators and involved departments 
Per thor Straten and Mads Hald Andersen 
Center management, National Center for Cancer Immune Therapy (CCIT-DK) 
Hematology Laboratory 54P4 
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev, Denmark 
Phone: +45 38 68 38 68 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
3
Department of Oncology 
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 7, 2730 Herlev, Denmark 
Phone: +45 38 68 38 68 
 
Hanne Marie Michelsen 
Clinical Research Unit, Department of Oncology  
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 9, 5th floor, 2730 Herlev, Denmark 
Phone: +45 38 68 38 68 
 
Lisbet Rosenkrantz Hölmich 
Department of Plastic Surgery 
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 5, 2730 Herlev, Denmark 
Phone: +45 36 68 38 68 
 
Helle Westergren Hendel 
Department of Clinical Physiology and Nuclear Medicine  
Copenhagen University Hospital Herlev 
Borgmester Ib Juuls Vej 71, 4th floor, 2730 Herlev 
Tlf.: 36 68 38 68 
 
Monitoring 
 
GCP unit 
GCP unit at Copenhagen University Hospital 
Bispebjerg Hospital, Building 51, 3rd floor 
Bispebjerg Bakke 23 
2400 København NV 
Denmark 
Phone: +45 35 31 38 90 
 
 
 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
4
Table of contents 
 
PROTOCOL RESPONSIBLE PARTIES  ..................................................................................................................... 2 
MONITORING ................................................................................................................................................................. 3 
TABLE OF CONTENTS ................................................................................................................................................. 4 
ABBREVIATIONS ........................................................................................................................................................... 7 
SYNOPSIS ......................................................................................................................................................................... 8 
INDICATION AND TREATMENT ......................................................................................................................................... 8 
RATIONALE ..................................................................................................................................................................... 8 
PURPOSE ......................................................................................................................................................................... 8 
STUDY DESIGN ................................................................................................................................................................ 8 
POPULATION ................................................................................................................................................................... 9 
TOXICITY ........................................................................................................................................................................ 9 
CLINICAL RESPONSE EVALUATION .................................................................................................................................. 9 
IMMUNOLOGICAL RESPONSE EVALUATION ...................................................................................................................... 9 
INTRODUCTION AND RATIONALE ........................................................................................................................ 10 
MALIGNANT MELANOMA .............................................................................................................................................. 10 
TUMOR IMMUNOLOGY................................................................................................................................................... 11 
TUMOR-INFILTRATING LYMPHOCYTES (TILS) ............................................................................................................... 11 
T CELL THERAPY ........................................................................................................................................................... 11 
CCIT-DK experiences with T cell therapy ............................................................................................................... 13 
RATIONALE BEHIND THE SUBSTANCES USED IN THE CLINICAL TRIAL ............................................................................ 14 
Lymphocyte-depleting chemotherapy ....................................................................................................................... 14 
Cyclophosphamide ................................................................................................................................................... 15 
Fludarabine phosphate ............................................................................................................................................ 15 
Interleukin-2 ............................................................................................................................................................. 15 
Interferon alpha ....................................................................................................................................................... 15 
Improvement of T cell therapy with pegylated interferon alpha 2b ......................................................................... 16 
RATIONALE BEHIND THE TREATMENT REGIME USED IN THE CLINICAL TRIAL ................................................................ 17 
PURPOSE AND HYPOTHESES .................................................................................................................................. 17 
PRIMARY ....................................................................................................................................................................... 17 
SECONDARY .................................................................................................................................................................. 17 
STUDY DESIGN ............................................................................................................................................................ 18 
STUDY POPULATION ................................................................................................................................................. 18 
CRITERIA FOR INCLUSION AND EXCLUSION ................................................................................................................... 18 
Inclusion criteria for surgery (Step 1) ..................................................................................................................... 19 
Exclusion criteria for surgery (Step 1) ..................................................................................................................... 20 
Inclusion criteria for treatment (Step 2) .................................................................................................................. 21 
Exclusion criteria for treatment (Step 2).................................................................................................................. 22 
Evaluation before inclusion in the clinical trial ....................................................................................................... 23 
Examination plan associated with the treatment ..................................................................................................... 23 
TREATMENT STRATEGY .......................................................................................................................................... 25 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
5
SUBSTANCES IN THE CLINICAL TRIAL ............................................................................................................................ 27 
Cyclophosphamide ................................................................................................................................................... 27 
Fludarabine phosphate ............................................................................................................................................ 27 
TILs .......................................................................................................................................................................... 27 
Interleukin-2 ............................................................................................................................................................. 27 
Pegylated interferon alpha 2b .................................................................................................................................. 27 
TREATMENT PLAN ......................................................................................................................................................... 28 
SIMULTANEOUS TREATMENT......................................................................................................................................... 28 
Guidelines for supportive care and treatment .......................................................................................................... 28 
Prophylactic treatment ............................................................................................................................................. 28 
Supportive treatment ................................................................................................................................................ 29 
Local radiotherapy ................................................................................................................................................... 30 
TREATMENT DISCONTINUATION .................................................................................................................................... 30 
Normal discontinuation............................................................................................................................................ 30 
Follow-up ................................................................................................................................................................. 30 
Early discontinuation of treatment ........................................................................................................................... 31 
Subsequent treatment ............................................................................................................................................... 32 
PRODUCTION OF TILS .............................................................................................................................................. 32 
REQUISITION OF TUMOR TISSUE .................................................................................................................................... 32 
ESTABLISHING ”YOUNG TIL” CULTURES ...................................................................................................................... 32 
RAPID EXPANSION PROTOCOL (REP) ............................................................................................................................ 33 
ISOLATION OF TUMOR CELLS ........................................................................................................................................ 33 
PHENOTYPE AND CLONOTYPE DETERMINATION ............................................................................................................ 33 
CYTOKINE RELEASE ASSAY .......................................................................................................................................... 33 
GENE-BASED ARRAYS ................................................................................................................................................... 33 
ADVERSE EVENTS, POTENTIAL RISKS, AND PRECAUTIONS ....................................................................... 34 
ADVERSE EVENTS (ADVERSE EVENT = AE) ................................................................................................................... 34 
Graduation of adverse events .................................................................................................................................. 34 
Serious adverse event (Serious Adverse Event = SAE) ............................................................................................ 34 
GUIDELINE FOR POSSIBLE RELATIONSHIPS BETWEEN ADVERSE EVENTS AND TREATMENT ............................................ 35 
Side effects ............................................................................................................................................................... 35 
Reporting of events and side effects  ........................................................................................................................ 35 
KNOWN SIDE EFFECTS ................................................................................................................................................... 36 
Chemotherapy .......................................................................................................................................................... 36 
Cyclophosphamide ................................................................................................................................................... 36 
Fludarabine phosphate ............................................................................................................................................ 42 
TILs .......................................................................................................................................................................... 44 
Interleukin-2 ............................................................................................................................................................. 44 
Peginterferon alpha 2b ............................................................................................................................................ 49 
RISKS AND DISADVANTAGES ASSOCIATED WITH SURGERY AND SAMPLE COLLECTION .................................................. 55 
MONITORING AND PRECAUTIONS .................................................................................................................................. 55 
EFFECT EVALUATION, DATA ANALYSIS, AND MONITORING ..................................................................... 57 
EFFECT EVALUATION .................................................................................................................................................... 57 
Primary effect parameter ......................................................................................................................................... 57 
Secondary effect parameter ..................................................................................................................................... 57 
RESPONSE CRITERIA ...................................................................................................................................................... 57 
RECIST .................................................................................................................................................................... 57 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
6
IMMUNOLOGICAL MONITORING ..................................................................................................................................... 57 
Blood samples for immunological monitoring ......................................................................................................... 57 
Tumor biopsies ......................................................................................................................................................... 58 
STATISTICS .................................................................................................................................................................... 58 
DATA REGISTRATION AND ANALYSIS ............................................................................................................................ 59 
END OF STUDY REPORT ................................................................................................................................................. 59 
AMENDMENTS ............................................................................................................................................................... 60 
ETHICAL CONSIDERATIONS ................................................................................................................................... 60 
RECRUITMENT OF STUDY PARTICIPANTS AND INFORMED CONSENT ............................................................................... 60 
INSURANCE ................................................................................................................................................................... 60 
ETHICAL ASPECTS ......................................................................................................................................................... 61 
RESEARCH BIOBANK ..................................................................................................................................................... 61 
REPORTING TO THE DANISH DATA PROTECTION AGENCY ............................................................................................ 61 
ADMINISTRATIVE ASPECTS AND PUBLICATION ............................................................................................. 62 
PATIENT IDENTIFICATION .............................................................................................................................................. 62 
PUBLICATIONS .............................................................................................................................................................. 62 
ECONOMY ..................................................................................................................................................................... 62 
REFERENCES ............................................................................................................................................................... 63 
APPENDIX 1 ................................................................................................................................................................... 69 
GUIDELINE FOR PROVIDING VERBAL PARTICIPANT INFORMATION ................................................................................. 69 
APPENDIX 2 ................................................................................................................................................................... 71 
PATIENT CHARACTERISTICS AND CLINICAL RESULTS .................................................................................................... 71 
APPENDIX 3 ................................................................................................................................................................... 72 
ANTI-TUMOR RESPONSE IN THE TIL INFUSION PRODUCT ............................................................................................... 72 
APPENDIX 4 ................................................................................................................................................................... 73 
CD8+ AND CD4+ TIL CYTOKINE PRODUCTION IS INCREASED BY STIMULATION WITH AUTOLOGOUS TUMOR PRETREATED 
WITH INTERFERON-ALPHA. ............................................................................................................................................ 73 
APPENDIX 5 ................................................................................................................................................................... 74 
ECOG-FUNCTION STATUS/PERFORMANCE STATUS ....................................................................................................... 74 
APPENDIX 6 ................................................................................................................................................................... 75 
COMMON TERMINOLOGY CRITERIA FOR ADVERSE EVENTS (CTCAE) VERSION 4 ........................................................ 75 
APPENDIX 7 ................................................................................................................................................................... 76 
GUIDELINES FOR MONITORING, DOSE MODIFICATIONS, AND SUPPORTIVE TREATMENT DURING INFUSION OF 
INTERLEUKIN-2 (DECRESCENDO-REGIME) ASSOCIATED WITH T CELL THERAPY ............................................................ 76 
APPENDIX 8 ................................................................................................................................................................... 95 
FEBRILE NEUTROPENIA DURING T CELL THERAPY ......................................................................................................... 95 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
7
Abbreviations 
AE = Adverse Event 
ALAT = Alanine Aminotransferase  
AR = Adverse Reaction 
ASAT = Aspartate Aminotransferase 
CCIT-DK = National Center for Cancer Immune Therapy 
CD4+ Cells = Helper T cells 
CD8+ Cells = Cytotoxic T cells 
CR = complete response 
CTC = Common Toxicity Criteria 
CTCAE = Common Terminology Criteria for Adverse Events 
CTL = Cytotoxic T lymphocytes 
Cy = Cyclophosphamide 
DMSO = Dimethyl Sulfoxide 
eCRF = electronic Case Report Form 
Flu = Fludarabine phosphate 
GM-CSF = granulocyte-macrophage colony-stimulating factor 
HLA = Human Leukocyte Antigen 
IFN-γ = Interferon-gamma 
IFN-α = Interferon alpha 
IL-2 = Interleukin-2 
IMPD = Investigational Medical Product Dossier 
KFE = Klinisk Forsknings Enhed (Clinical Research Unit) 
LDH = Lactate dehydrogenase 
MHC = Major Histocompatibility Complex 
MM = malignant melanoma 
ORR = Overall Response rate 
PD = Progressive Disease 
Peginterferon = pegylated interferon alpha 2b 
PET = Positron Emission Tomography  
PR = Partial Response 
PS = Performance Status, ECOG scale 0-4 
RECIST = Response Evaluation Criteria In Solid Tumors 
REP = Rapid Expansion Protocol 
SAE = Serious Adverse Event 
SAR = Serious Adverse Reaction 
SD = Stable Disease 
SUSAR = Suspected Unexpected Serious Adverse Reaction 
TAA = Tumor Associated Antigens 
TIL = Tumor Infiltrating Lymphocytes 
Treg = Regulatory T cells 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
8
VEK = Videnskabsetisk Komité (The National Committee on Health Research Ethics) 
Synopsis 
 
Indication and treatment 
In this pilot study patients with metastatic malignant melanoma (MM) are treated with T cell 
therapy in combination with pegylated interferon alpha 2b (peginterferon). The treatment includes 
treatment with the immune modulating cytokine peginterferon, high-dose chemotherapy, infusion of 
tumor-infiltrating lymphocytes (TILs) isolated from the patient’s own tumor tissue and 
administration of the immune-stimulating cytokine interleukin-2 (IL-2). 
 
Rationale 
T cell therapy is an experimental immuno-therapeutic treatment, where TILs are isolated from the 
patient’s own tumor tissue, expanded ex vivo into billions of cells and infused into the patient with 
the intention to eliminate the cancer cells. Before TIL infusion a lympho-depleting chemotherapy 
regime consisting of cyclophosphamide and fludarabine is administered with the intention to reduce 
irrelevant T cells and eliminate the regulatory T cells, which are known to be able to inhibit T-cell 
mediated killing of tumor cells. After TIL infusion IL-2 is administered to activate and stimulate 
further proliferation of the infused T cells. In this clinical trial the T cell therapy regime described 
above is combined with peginterferon, which is administered as an injection two days before TIL 
infusion and 7 and 14 days, respectively, after TIL infusion, with the specific purpose to increase 
the tumor cell sensitivity to T cell killing and thereby increase the clinical effect of the T cell 
therapy.  
 
Purpose 
The primary purpose is to evaluate tolerability and safety of the treatment. The secondary goals are 
to characterize the immune response against tumor cells in the tumor as well as in the blood and to 
evaluate the clinical effect of the treatment, determined by the objective response rate (assessed 
according to RECIST 1.1). In addition, survival and progression-free survival is described.  
 
Study design 
The clinical trial is a pilot study. All patients are included and treated at the Department of 
Oncology, Herlev Hospital. The patients are referred from oncology departments at Copenhagen 
University Hospital Herlev, Aarhus University Hospital, and Odense University Hospital. All 
suitable patients will be treated during hospitalization, with one treatment series stretching over 
approximately three weeks. The last dose of peginterferon may be given on an outpatient basis. 
From the removal of tissue from the patient for TIL production until the start-up of the treatment it 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
9
will typically take 4-6 weeks. In some cases, TILs will be cryopreserved in preparation for 
treatment of the patient at a later time point. After the treatment patients will be followed by an 
immunotherapeutic team with outpatient control visits for up to five years. In the case of 
progression, patients will be excluded from the study. The inclusion period for the clinical trial is 
expected to last approximately two years. The inclusion period is expected to last from June 2014 
until December 2016. The clinical trial will be reported to the GCP unit, the Danish Medicines 
Agency, the National Committee on Health Research Ethics, and the Danish Data Protection 
Agency. 
 
Population 
Patients with histologically verified malignant melanoma (MM), which has metastasized, will be 
candidates for the treatment. The patients, who are included in this clinical trial, must have an 
acceptable functional status and acceptable kidney and liver function and be free of competing 
serious conditions. The clinical trial will include 20 patients in total. Only the patients, for whom it 
is possible to grow T cells from the tumor tissue (approximately 90 % of the patients), will be 
offered treatment.  
 
Toxicity 
We have already in an ongoing phase II study shown that T-cell based immunotherapy with 
lymphodepleting chemotherapy, TIL infusion, and intermediary dose IL-2 has acceptable toxicity 
and can be administered safely in an oncological setting. Peginterferon is approved as adjuvant 
treatment for patients with metastatic melanoma and the side effects are known and manageable. 
 
Clinical response evaluation 
Patients are evaluated clinically through outpatient attendance six and 12 weeks after treatment with 
T cells and thereafter approximately every 3rd month. In addition, imaging-based evaluation (CT, 
MR, PET-CT, or PET-MR) will be performed before treatment and thereafter in connection with 
clinical evaluations starting from six weeks after the T cell treatment. Before starting treatment 
CT/MR scans of the cerebrum will be performed to rule out CNS metastases.  
 
Immunological response evaluation 
Project blood samples of 110 ml will be taken at surgery, before TIL infusion, at discharge 
(approximately 10 days after TIL infusion), and thereafter in connection with clinical evaluation 
after the treatment. During hospitalization blood serum samples (10 ml blood sample) are taken on 
day 0 before TIL infusion, two hours after TIL infusion, and thereafter every second day until 
discharge. Immune cells are isolated from the collected blood samples using the LymphoPrepTM 
technique and are stored frozen until analysis. Flow cytometry analyses are performed to assess 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
10
different immune cells (e.g. CD4+ and CD8+ T cells) before treatment start and during the course of 
treatment. 
 
Introduction and rationale 
 
Malignant melanoma 
In Denmark, approximately 2,100 new cases of malignant melanoma (MM) are diagnosed annually 
(2011)1, and the incidence is increasing with approximately 5 % per year. Approximately 5 – 10 % 
of patients have metastases at the time of diagnosis, and an additional approximately 10 – 15 % 
develop metastases later in the course of illness2. Thus, approximately 350-400 new cases of 
metastasizing malignant melanoma can be expected per year in Denmark. MM is a very aggressive 
cancer, and once the disease has metastasized the five-year survival has so far been below 10 %3.  
 
Adjuvant treatment of malignant melanoma 
Interferon alpha 2b (IFN-α) was the first drug, which in a randomized controlled clinical trial 
showed significant effect on the disease-free survival in patients with malignant melanoma. Based 
on the ECOG 1684 study, in 1996 high-dose interferon was approved by the FDA as adjuvant 
treatment for high-risk patients with malignant melanoma4, and in 2004 high-dose IFN- was 
approved by the European Medicines Agency (EMA). In 2011, peginterferon (the pegylated form of 
IFN-α) was approved by the FDA as adjuvant treatment for intermediary and high-risk malignant 
melanoma patients5, even though the ECOG 18991 study, which led to FDA approval, exclusively 
showed increased disease-free survival without effect on the actual survival6.  
Pegylation of IFN-α, through a covalent bond to polyethylene glycol, increases the compound’s 
biological half-life from 3-8 hours to 22-60 hours without changing its tertiary structure or 
biological activity. The advantage of this is that peginterferon can be injected only once weekly, 
whereas standard IFN-α must be injected 3 times weekly7. It is still not clarified, which form, 
dosage, and duration of IFN-α treatment gives the optimal antineoplastic effect with the least 
possible toxicity8,9.  
 
Treatment of disseminated malignant melanoma 
In Denmark, four treatments for metastatic MM are currently approved: temodal, interleukin-2 (IL-
2) + peginterferon, ipilimumab, and vemurafenib. Temodal is a chemotherapeutic, which has been 
shown to have an objective response rate (ORR) of 10-15 % but no effect on survival10. IL-2 is an 
immune-stimulating cytokine, and IFN-α is immune modulating. The combination of IL-2 and IFN-
α is used for patients in good general condition, younger than 70 years of age, and has shown an 
ORR of approximately 15 % with complete response (CR) in less than 5 %11 but there seems to be a 
positive effect on survival. Ipilimumab is one of the new compounds, which in 2012 was approved 
in Denmark for use after first line treatment of metastatic MM. It is a human monoclonal antibody, 
which works by blocking the T-cell surface receptor CTLA4 (Cytotoxic T-Lymphocyte Antigen 4), 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
11
which works like a natural break in the immune system. In a large phase III study with ipilimumab 
an ORR of approximately 10 % was found, as well as a CR in 0.6 %, but approximately 20 % 
achieve stable disease (SD) with significant effect on survival12,13. In 2012, Vemurafenib was also 
approved in Denmark for the approximately 50 % of MM patients, which have an activating 
mutation in the BRAF-gene. Approximately 50 % of the treated patients have an objective response 
to Vemurafenib. In addition, up to 30 % have stable disease (stable disease: SD) but the disease 
recurs among most patients within a median of seven months and the effect on long-term survival 
and recovery is unknown, as long-term follow-up has not been completed yet. The effect of 
Vemurafenib is typically fast-acting but most patients will unfortunately only experience short-
lasting effects of the treatment14.  
There is therefore still a large need for the development of new and more effective treatments for 
metastatic MM. 
 
Tumor immunology  
In the recent years remarkable progress in the understanding of the immune system reaction against 
cancer has been made. Thus, it is today clarified that the immune system reacts against certain 
tumors in vivo, and that an immunological response against cancer cells in some cases is associated 
with a better prognosis15,16. Likewise it has been shown that a patient’s immune response against 
tumor cells can be reinforced by treatment with different immune-stimulating therapies, which in 
some cases appear to be curative17. 
 
Tumor-infiltrating lymphocytes (TILs) 
Tumors are often infiltrated by large numbers of T cells (TILs), which specifically recognizes tumor 
antigens but typically are inactive. The T cells include both cytotoxic T cells (CD8+ T cells), helper 
T cells (CD4+ T cells), and regulatory T cells (Tregs), where the latter inhibit tumor cell killing. The 
T cells consist of different clones with varying degrees of specificity towards tumor antigens, where 
only few clones have a particularly high specificity. The inactive state of T cells in the tumor tissue 
is characterized by abnormal intracellular signaling, apoptosis, and a decreased proliferative ability, 
presumably caused by different immune-suppressing factors in the tumor environment18,19. 
However, it is possible in vitro to multiply and reactivate such TILs for tumor cell killing using 
activating factors such as IL-220,21.  
 
 
T cell therapy 
T cell therapy, also called ”Adoptive T cell Therapy” is an immunotherapeutic cancer treatment, 
which has shown very promising results in particular for malignant melanoma. This type of 
treatment, which uses the patient’s own T cells for tumor cell killing, was developed by the 
American National Institutes of Health (NIH), and in the more recent years several studies have 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
12
been published from other cancer research centers in the US and Europe, where more than 500 
patients have received the treatment so far22–27.  
 
TIL based T cell therapy uses the fact that there is a high number of tumor reactive T cells in the 
tumor tissue compared to peripheral blood28. TILs are isolated from the patient’s own tumor tissue 
(metastasis or primary tumor) and expanded in vitro for 4-6 weeks into billions of T cells, which are 
given back into the patient with the purpose to eliminate the cancer cells. This makes T cell therapy 
a highly specialized and individualized type of cancer immunotherapy.  
 
The actual treatment consists of an initial week of lymphodepleting chemotherapy (cyclophos-
phamide and fludarabine phosphate) with the purpose of eliminating existing Tregs in the patient 
and reducing irrelevant T cells. After a week of chemotherapy T cells (TILs) are infused 
intravenously and shortly thereafter, the patient receives high-dose IL-2 to increase activation and 
expansion of the infused T cells.  
 
 
 
 
Figure 1: Treatment course for T cell therapy 
Shortly after inclusion of the patient, tumor tissue for production of the T cell infusion product is 
removed. The week before the T cells are ready for infusion, treatment with cyclophosphamide (Cy) 
and fludarabine phosphate (Flu) is started, followed by T cell infusion and treatment with IL-2.  
The duration and dosing of the IL-2 treatment varies in different protocols. 
 
T cell therapy has shown very promising results for metastatic MM, where an ORR of 
approximately 50 % has been observed, which has been confirmed in several phase I/II “single-
institution” studies24,25,29–31. In addition, CR has been observed in 20 % of the treated patients, of 
which most are lasting, and the patients remain disease-free for more than seven years after the 
treatment22. TIL-based T cell therapy thereby appears to have the potential to cure a significant 
proportion of patients with metastatic MM. 
 
Experiences from previous clinical trials with more than 300 treated patients have shown that T cell 
therapy despite considerable toxicity is safe for use in patients in good general condition with 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
13
sufficient organ function. The described side effects are reversible, acute side effects consisting of 
known and expected side effects of lymphodepleting chemotherapy and high-dose interleukin-2.  
 
CCIT-DK experiences with T cell therapy 
Clinical trial 
As one of the few places in the world the complicated methods for producing TILs are established 
at the National Center for Cancer Immune Therapy (CCIT-DK), Herlev Hospital32 and has been 
translated into a clinical trial (clinicaltrials.gov identifier: NCT00937625), where we so far have 
treated 26 patients with metastatic MM. All patients were treated with classical lymphodepleting 
chemotherapy with cyclophosphamide and fludarabine phosphate followed by TIL infusion with 
approximately 100x109 cells and subsequent administration of IL-2 (see appendix 2). 
 
In the original “T-cell regime” described by Dudley et al.15 very high doses of IL-2 (720,000 IU/kg 
IV) are given as bolus injections every eighth hours until treatment-limiting toxicity. Since it is 
unknown, whether these very high doses of IL-2 are necessary to maintain T-cell expansion, we 
have at CCIT-DK, Herlev Hospital tested treatment with low to intermediary doses of IL-2 to 
determine, whether the response rates can be maintained, while the toxicity is reduced. 
   
A pilot study where six patients were treated with low-dose IL-2 SC was initiated in the summer of 
2009, and the results of this are now published23. Of the six treated patients, two patients achieved 
CR. 
 
To achieve a higher RR, the IL-2 dose was thereafter increased to an intermediary dose, where IL-2 
is administered after a decrescendo regime33, which is equal to the IL-2 decrescendo regime, which 
in Denmark is used as standard treatment of metastatic MM. 
 
After increasing the dose of IL-2 we have treated an additional 20 patients, of which 18 have 
currently been evaluated with imaging. In total (n = 26), we have seen an ORR of 46 %, which is 
comparable with what has been achieved in other studies with high-dose IL-2. Three patients 
achieved complete response (CR; 49, 13, 27+ months), and eight patients achieved a partial 
response (PR), of which six are still followed with responses lasting from 2-25 months. Five 
patients had stable disease (SD) for 2-4 months, and three patients progressed shortly after the 
treatment.  
 
Further, clearly reduced toxicity has been observed at low to intermediary dose IL-2, and the 
treatment has shown to be manageable in an ordinary oncology department without the need for 
intervention from an intensive care unit. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
14
Translational research 
Further development of the T cell therapy in the form of optimization and expansion to other kinds 
of cancer is a highly prioritized research area at CCIT-DK. Our already established platform for T 
cell therapy for MM has given us a unique opportunity to study interactions between tumors and the 
immune system, and thereby to clarify possible methods for optimization of the T cell therapy. 
 
Several studies have shown that the following characteristics for T cells are important in order to 
achieve a clinical response from T cell therapy: long telomeric length, short expansion time, a 
favorable phenotype of T cells (CD27+, CD28+), a high absolute number of T cells and a high 
number of cytotoxic tumor-reactive T cells in the infusion product34 and increased persistence of the 
T cells in the peripheral blood after infusion22,32,35. On the basis of this we have at CCIT-DK 
changed the primary expansion method from “traditional expansion” to “Young TIL” expansion, 
whereby the first expansion period is shortened from 4-7 weeks to 2-4 weeks. Shorter time in 
culture (Young TIL) gives TILs with a longer telomeric length and more favorable phenotypes 
(CD27+, CD28+) with characteristics such as increased proliferation, increased persistence in vivo 
and higher antitumor activity, which as mentioned is correlated with an increased clinical 
response22,32. By decreasing the time-span during which the T cells are grown in culture, a simpler 
and faster method for production of the TIL infusion product has been achieved and testing of the 
TIL specificity is no longer necessary. This optimization of the TIL production has made it possible 
to produce clinically applicable TIL infusion products from more than 90% of the patients36–39.  
Further, we have during the final expansion phase, the Rapid Expansion Protocol (REP), introduced 
the use of the Wave® bioreactor40, which optimizes the proliferation conditions and has made it 
possible to achieve a higher absolute cell number, as well as a higher number of tumor-reactive T 
cells in the TIL infusion product. Based on these TIL production protocols, developed at CCIT-DK,  
we have standardized and harmonized TIL production methods among three European cancer 
research centers, and a randomized, multicenter TIL based T cell therapy phase III clinical trial 
aiming at approval of T cell therapy as standard treatment for patients with malignant melanoma is 
planned. 
 
Rationale behind the substances used in the clinical trial 
Lymphocyte-depleting chemotherapy 
To maintain an immunological response against the tumor, activating cytokines (the signaling 
molecules IL-2, IL-7, IL-15, IL-21 etc.) must be available for the tumor-specific T cells. Many 
“irrelevant” T cells will competitively reduce the availability of these cytokines for the relevant T 
cells. To create an environment, which facilitates a T-cell mediated anti-tumor response, there must 
in addition to the presence of many tumor-specific T cells with high specificity be made a reduction 
of irrelevant T cells, as well as an elimination of Tregs. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
15
To create such an environment, a combination of two days of cyclophosphamide and five days of 
fludarabine phosphate will be used in the present study. A combination chosen on the basis of 
experiences from previous studies41,42.  
 
Cyclophosphamide 
Cyclophosphamide is an alkylating compound, which works by forming covalent bonds with 
biologically important macromolecules. In particular, bonds are formed with the DNA and in some 
cases cross-binding of the DNA takes place. If the cross-binding is not removed by the cell repair 
systems, the cell division can be prevented. The binding to important proteins in the cell may harm 
important functions and lead to cell death. Cyclophosphamide is used in oncology for e.g. breast 
cancer and in malignant hematological diseases such as myelomatosis43.  
 
Fludarabine phosphate 
Fludarabine phosphate is a prodrug, which is changed into the active triphosphate 2-fluoro-ara-
ATP. The compound is part of the group of anti-metabolites, which inhibits the DNA synthesis, 
and at the same time leads to a reduction of the RNA and protein synthesis. Fludarabine phosphate 
is used for the treatment of malignant hematological diseases e.g. CLL44.  
 
Interleukin-2  
Interleukin-2 (IL-2) after the decrescendo-regime33 is used in Denmark as a standard first-line 
treatment for suitable patients with metastatic malignant melanoma. Low-dose IL-2 is also used for 
treatment of metastatic kidney cancer. IL-2 is normally produced by activated T-lymphocytes and 
stimulates through specific receptors both the antigen specific and the unspecific immune defense45.  
IL-2 will in this experiment be given according to the decrescendo-regime, which is used for standard 
IL-2 treatment of disseminated malignant melanoma.  
 
Interferon alpha 
Interferons, which are pleiotropic cytokines, were first described in 1957 by Isaacs and 
Lindenmann46. They work by binding to surface receptors of both normal and neoplastic cells, 
whereby a complex sequence of intracellular processes is initiated, including induction of different 
proteins, such as the Major Histocompatibility Complex (MHC) molecules. Human interferons are 
classified as , , and . Apart from an upregulation of MHC, interferons have been shown to have 
growth-inhibiting effects, when they are added to tumor cells in vitro, and several other indirect 
immune-modulating effects have recently been described: an increase in tumor-infiltrating cells; 
development of auto-antibodies and manifestation of autoimmunity; inhibition of the angiogenesis; 
reduction of circulating Treg; modulation of the STAT1/STAT3 balance in tumor cells and 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
16
lymphocytes; changes in cytokine concentrations and normalization of T-cell STAT1 signaling 
defects in peripheral circulating lymphocytes47–49. 
 
Interferon alpha 2b (IFN-) is a potent immune modulating cytokine, which in several studies has 
been shown to increase the MHC-dependent antigen presentation in a number of different cell 
types, including melanoma cells50. Through this upregulation of the MHC expression, interferons 
are believed to make malignant cells more immunogenic47. Tumor-associated antigens (TAA), 
which are expressed by malignant cells, can be presented on the surface of the malignant cell by 
MHC class I or II molecules and can thereafter be recognized by cytotoxic CD8+ and CD4+ T cells. 
A specific T-cell response against cancer cells thereby requires that these cells express MHC class I 
or II. Tumor cells can evade a specific immune cell killing by downregulating MHC expression51, 
and the MHC expression is also found downregulated in the majority of malignant melanomas52,53. 
Changes in MHC class I expression on malignant cells are correlated with a poor survival, disease 
progression and limited response to immunotherapy, and generally metastases are characterized by 
a higher prevalence of loss of MHC class I molecules than primary tumors. 
 
Improvement of T cell therapy with pegylated interferon alpha 2b 
At CCIT-DK we have recently characterized the response of a number of TIL products against 
autologous tumors from patients with MM. We have shown that the tumor reactivity of both the 
CD8+ and CD4+ T cells can be improved in most TIL products by pretreatment of the autologous 
tumor cells with IFN- and/or IFN-, respectively (see appendix 4). 
 
We have also previously shown that IFN- pretreatment can induce or upregulate Human Leukocyte 
Antigen (HLA) molecules on the tumor cells and thereby restore or increase the anti-tumor 
reactivity34. Previous studies have as indicated shown that IFN- can increase the MHC-dependent 
antigen presentation in several different cell types, including melanoma cells50. 
 
IFN- has as described been used safely for many years as an adjuvant treatment for patients with 
malignant melanoma in different forms, doses, and regimes and is also used in combination with 
IL-2 for treatment of patients with metastatic melanoma. 
 
Recently, a Dutch research group combined adoptive T cell therapy with interferon alpha54. In this 
phase I/II clinical trial 10 melanoma patients were treated with T cells isolated from peripheral 
blood in combination with subcutaneous injections of IFN- 3 MIU (million units) daily for 12 
weeks. Degree 2 (CTC) side effects were observed in 5/10 patients and no ≥ degree 2 side effects, 
and one CR, one PR, and three patients with SD were observed. 
 
Our experiences and those of others indicate that the combination of T cell therapy with IFN- 
potentially can increase the clinical effect of T cell therapy by increasing the tumor reactivity of the 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
17
infused TILs, which we have previously shown is a critical factor in achieving clinical response34 
(see appendix 3). 
 
 
Rationale behind the treatment regime used in the clinical trial 
 
The T cell therapy regime is structured according to guidelines described by Dudley et al15. A few 
approaches, however, differ from this regime, and these deviations are described below.  
 
As it is unknown whether high-dose IL-2 is necessary to maintain the T-cell expansion after 
infusion, we have chosen to treat the patients with intermediary dose IL-2 (decrescendo regime) 
instead of high-dose bolus infusions, which have been used in earlier studies, and which are 
considerably more toxic22. Our results indicate that it is possible to achieve a response rate of 
approximately 50 % with intermediary dose IL-2, which is comparable with earlier studies, which 
have used high-dose bolus IL-2 (see appendix 2).  
 
By combining the T cell therapy with IFN-, we hope to increase the tumor cell sensitivity to the 
cytotoxic T cells in the TIL infusion product, whereby the clinical effects of the treatment can be 
further increased. Our experiences with T cell therapy indicate that the effects of the treatment 
begin within a few days to weeks after the actual T-cell infusion.  
 
We have among responding patients observed the largest reduction in the tumor size within the first 
couple of months after the treatment. We will therefore administer IFN- shortly before (day -2) 
and in the period immediately after the T-cell infusion (day 7 and 14) to give the T cells the best 
possible conditions for tumor cell killing. We have chosen to use pegylated interferon, since this 
formulation only needs to be given 1x weekly. Peginterferon will be given in a dosage of 3 g/kg 
1x weekly, in a total of three doses. The weekly dose of peginterferon thereby corresponds to the 
dose, which is used in adjuvant treatment of malignant melanoma, where the treatment is given 
once weekly for two years. 
 
Purpose and hypotheses 
Primary 
1) To evaluate tolerability and feasibility of the treatment  
Secondary 
1) To determine whether T cell therapy in combination with peginterferon for patients with 
MM can induce a measurable immune response against tumor cells 
 
2) To describe an objective response using RECIST 1.1 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
18
 
3) To describe survival and progression-free survival 
 
Study design 
The clinical trial is a pilot study for patients with metastatic (inoperable stage III or stage IV) 
malignant melanoma. All patients are included and treated at the Department of Oncology, Herlev 
Hospital. The patients are referred for treatment from oncology departments at Copenhagen 
University Hospital Herlev, Aarhus University Hospital, and Odense University Hospital. 
 
We will include and treat 20 patients and expect that this can be done within two years. We expect 
that the patients can finish the treatment and the first six months of follow-up within approximately 
three years. The patients will be evaluated for objective response six and 12 weeks after infusion of 
T cells, and thereafter every third month. 
 
The inclusion period is expected to begin in June 2014, and the clinical trial is expected to end in  
December 2016. The actual treatment course can vary from patient to patient depending on the 
time-span between surgery and treatment. In most cases the patient may receive treatment 
approximately 4-6 weeks after surgery, and in this case the treatment course from surgery until the 
first imaging-based evaluation (six weeks after treatment) will last approximately 4-5 months. 
 
Study population 
Patients with histopathologically verified metastatic or locally advanced malignant melanoma will 
be candidates for this clinical trial, except for patients whom have previously been treated with T 
cell therapy. As is evident from the exclusion criteria below, the patients must be in good general 
condition, be free of serious competing conditions, and have acceptable organ function. Only the 
patients, where it is possible to grow T cells from the tumor tissue (more than 90 % of the patients), 
will be offered treatment with T cells.   
 
Criteria for inclusion and exclusion 
It typically takes 4-6 weeks from the patients have tumor tissue excised until the T cells are ready 
for treatment. If there is long waiting time for the treatment, the T cells will be cryopreserved after 
the initial expansion (“Young TIL), and the cells can at a later time point be thawed and prepared 
for treatment in two weeks in the final expansion (REP). Therefore, in some cases it takes longer 
time between the time of surgery and the actual treatment, and the patients are therefore included in 
two steps: 
 
Step 1: Inclusion and exclusion criteria for surgery 
Step 2: Inclusion and exclusion criteria for treatment 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
19
 
This two-step inclusion also makes it possible to include patients for surgery (Step 1), even though 
they do not progress after previous treatment for malignant melanoma and therefore do not fulfill 
the criteria for treatment. In these cases, the T cells will be cryopreserved, until the patient 
progresses and fulfills criteria for treatment (Step 2). 
 
If the patient is included for surgery but later do not fulfill the requirements for treatment the 
removed tumor tissue and the blood samples will be used for research. 
 
Inclusion criteria for surgery (Step 1) 
All criteria listed below must be fulfilled in order for patients to be included for surgery. 
 
1. Histologically verified metastatic or locally advanced malignant melanoma, where surgical 
removal of tumor tissue is feasible. 
2. Age: 18 - 70 years 
3. ECOG Performance Status of ≤1 (see appendix 5) 
4. Life expectancy > 3 months  
5. At least one measurable parameter according to RECIST 1.1 criteria 
6. No significant toxicity or side effects (CTC ≤ 1) from any previous treatment 
7. Sufficient organ function, including:  
System 
Laboratory values 
Hematology 
 
ANC (Absolute Neutrophil Count) 
≥ 1,500/µl 
Leukocytes 
≥ normal range 
Thrombocytes 
≥ 100,000/µl and < 700,000/µl 
Hemoglobin 
> 6.0 mmol/l (e.g. after blood transfusion) 
Kidney 
 
S-creatinine  
< 140 mol/l 
Liver 
 
Total serum bilirubin 
≤ 1.5 times the upper normal limit 
ASAT/ALAT 
≤ 2.5 times the upper normal limit  
Alkaline phosphatase 
≤ 5 times the upper normal limit 
LDH 
≤ 5 times the upper normal limit  
Coagulation 
 
PP 
> 40, unless the patient receives therapeutic 
anticoagulation 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
20
INR 
< 1.5, unless the patient receives therapeutic 
anticoagulation 
 
8. Women of child-bearing potential must use safe means of birth control. Likewise, men who 
participate in the clinical trial or female partners of men, who participate in the clinical trial 
must use safe anticonception. This goes from the inclusion until six months after the last 
dose of study drug. Oral contraceptive pills, intrauterine device (IUD), depot injection of 
progestogen, sub-dermal implantation, hormonal vaginal rings, and transdermal patches are 
considered safe contraceptive methods. 
9. Signed informed consent form after oral as well as written information. 
10. Prepared to meet up at the planned control visits and capable of handling toxicity.  
 
Exclusion criteria for surgery (Step 1) 
Patients should be excluded from surgery, if they meet just one of the criteria listed below: 
 
1. Other malignant tumors in the anamnesis with the exception of basal cell carcinoma and 
adequately treated carcinoma in situ colli uteri. Patients treated for other malignant 
conditions can participate, if the patient is without signs of illness for at least five years after 
ended treatment.   
2. Patients with cerebral metastases. Patients with solitary cerebral metastases can be included 
after radical surgery or stereotaxic radiotherapy, if the patient at least 1 month later is 
without signs of cerebral disease activity, clinically as well as by MR scanning. Patients 
with non-previously treated asymptomatic cerebral metastases can be included according to 
the investigator’s discretion. 
3. Patients with ocular malignant melanoma. 
4. In the case of confirmed sensitivity to one of the active compounds or one or more of the 
adjuvant substances.  
5. Serious medical condition e.g. serious asthma/COLD, badly regulated coronary disease, 
Morbus cordis, badly regulated insulin-demanding diabetes mellitus  
6. Acute/chronic infection with e.g. HIV, hepatitis, tuberculosis. 
7. Serious allergy or previous anaphylactic reactions. 
8. Active autoimmune disease e.g. autoimmune neutropenia/thrombocytopenia or hemolytic 
anemia, systemic lupus erythematosus, Sjögren’s syndrome, scleroderma, myasthenia 
gravis, Goodpasture syndrome, Addison’s disease, Hashimoto’s thyroiditis, active Grave’s 
disease. 
9. Pregnant and breast-feeding women. 
10. Concurrent treatment with systemic immunosuppressive medication (including 
prednisolone, methotrexate etc.) excluding planned shorter-lasting treatment, which 
according to the study responsible physician’s assessment can be discontinued before 
planned treatment with T cell therapy. 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
21
11. Concurrent treatment with other experimental drugs. 
12. Patients with active uncontrolled hypercalcemia. 
Inclusion criteria for treatment (Step 2) 
All criteria listed below must be completed in order for the patients to be included for treatment. 
Changes with respect to step 1 are written in bold: 
 
1. Histologically verified metastatic or locally advanced progressing malignant melanoma. 
2. Age: 18 – 70 years 
3. ECOG Performance Status of ≤ 1 (see appendix 5) 
4. Life expectancy > 3 months 
5. At least one measurable parameter according to RECIST 1.1 criteria 
6. No toxicity or side effects (CTC ≤ 1) from any previous treatment 
7. Sufficient organ function, including:  
System 
Laboratory values 
Hematology 
 
ANC (Absolute Neutrophil Count) 
≥ 1,500/µl 
Leukocytes 
≥ normal range 
Thrombocytes 
≥ 100,000/µl and < 700,000/µl 
Hemoglobin 
> 6.0 mmol/l (e.g. after blood transfusion) 
Kidney 
 
S-creatinine 
< 140 mol/l 
Liver 
 
Total serum bilirubin 
≤ 1.5 times the upper normal limit 
ASAT/ALAT 
≤ 2.5 times the upper normal limit  
Alkaline phosphatase 
≤ 5 times the upper normal limit 
LDH 
≤ 5 times the upper normal limit  
Coagulation 
 
PP 
> 40, unless the patient receives therapeutic 
anticoagulation 
INR 
< 1.5, unless the patient receives therapeutic 
anticoagulation 
 
8. Women of child-bearing potential must use safe means of birth control. Likewise, men who 
participate in the clinical trial or female partners of men, who participate in the clinical trial 
must use safe anticonception. This goes from the inclusion until six months after the last 
dose of study drug. Oral contraceptive pills, intrauterine device (IUD), depot injection of 
progestogen, sub-dermal implantation, hormonal vaginal rings, and transdermal patches are 
considered safe contraceptive methods. 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
22
9. Signed informed consent form after verbal as well as written information 
10. Prepared to meet up at the planned controls and capable of handling toxicity. 
 
Exclusion criteria for treatment (Step 2) 
Patients should be excluded, if they meet just one of the criteria listed below: 
Changes with respect to step 1 are written in bold: 
 
1. Other malignant tumors in the anamnesis with exception of basal cell carcinoma and 
adequately treated carcinoma in situ colli uteri. Patients treated for other malignant 
conditions can participate, if the patient is without signs of illness for at least five years after 
ended treatment.  
2. Patients with cerebral metastases. Patients with solitary cerebral metastases can be included 
after radical surgery or stereotaxic radiotherapy, if the patient at least 1 month later is 
without signs of cerebral disease activity, clinical as well as MR scanning. Patients with 
non-previously treated asymptomatic cerebral metastases can be included according to the 
investigator’s opinion. 
3. Patients with ocular malignant melanoma. 
4. In the case of confirmed sensitivity to one of the active compounds or one or more of the 
adjuvant substances. 
5. Serious medical condition e.g. serious asthma/COLD, badly regulated coronary disease, 
Morbus cordis, badly regulated insulin-demanding diabetes mellitus. 
6. S-creatinine clearence < 70 ml/min*. 
7. Acute/chronic infection with e.g. HIV, hepatitis, and/or tuberculosis. 
8. Serious allergy after previous anaphylactic reactions. 
9. Active autoimmune disease, e.g. autoimmune neutropenia/thrombocytopenia or hemolytic 
anemia, systemic lupus erythematosus, Sjögren’s syndrome, scleroderma, myasthenia 
gravis, Goodpasture syndrome, Addison’s disease, Hashimoto’s thyroiditis, active Graves’ 
disease. 
10. Pregnant and breast-feeding women. 
11. Concurrent treatment with systemic immunosuppressant medication (including 
prednisolone, methotrexate etc.) 
12. Concurrent treatment with other experimental substances.  
13. Other concurrent systemic anti-cancer treatment. 
14. Patients with active uncontrolled hypercalcemia. 
 
*In special cases it can be decided to include the patient despite a GFR < 70 ml/min using a reduced 
dosage of chemotherapy. 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
23
Evaluation before inclusion in the clinical trial 
The following tests must be performed within one month prior to treatment start (laboratory tests, 
however, within 1 week): 
 
- 
Anamnesis and objective examination 
- 
Performance Status according to the ECOG Scale  
- 
Electrocardiogram 
- 
Cr-EDTA clearance 
- 
Urine analysis test 
- 
Laboratory tests:  
A) Hematology: hemoglobin, leukocytes, granulocytes, and thrombocytes. 
B) Blood chemistry tests: Sodium, potassium, creatinine, LDH, alkaline phosphatase, 
ASAT, ALAT, bilirubin, ionized calcium, CRP, TSH, Calcium ion, INR, PP 
C) Infections: Hepatitis B, Hepatitis C (IgG), HIV, HTLV-I (IgG), EBV 
- 
Pregnancy test: Women of child-bearing potential must do a pregnancy test. This means 
women, who are not surgically sterilized, post-menopausal, or have used safe birth-control 
for more than 6 months. 
- 
Baseline tumor evaluation: CT, MRI, PET-CT, or PET-MRI scans can be used. 
- 
Revision of check-list for inclusion/exclusion for treatment.  
Examination plan associated with the treatment 
 
Inclusion Step 1 
Surgery 
Inclusion Step 2 
(before 
chemotherapy) 
Before 
peginterferon 
Before 
T-cell infusion  
Before 
peginterferon 
Before discharge 
Before 
peginterferon 
Day 
 
 
-8 
-2 
0 
7 
App. 10 
14 
Performance 
Status 
x 
 
x 
x 
x 
x 
x 
x 
Objective 
examination 
x 
 
x 
x 
 
 
x 
x 
Weight 
x 
 
x 
x 
x 
x 
x 
x 
Adverse 
events (CTC) 
 
 
x 
x 
x 
x 
x 
x 
Screening 
blood samplesa 
x 
 
 
 
 
 
 
 
Immunological 
blood samplesb 
 
x 
x 
 
 
 
x 
 
Miscellaneous 
blood samplesc 
 
 
x 
x 
x 
x 
x 
x 
EKG 
x 
 
x 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
24
Cr-EDTAd 
 
 
x 
 
 
 
 
 
Urine tests 
 
 
x 
 
 
 
 
 
Biopsye 
 
 
x 
 
 
 
 
 
Tumor 
evaluationf 
x 
 
x 
 
 
 
 
 
Examination plan 
a: 
Screening blood samples: T-cell screening (Labka blood sample package): 
hemoglobin, thrombocytes, leukocytes, differential counts, sodium, potassium, 
creatinine, ALAT, ASAT, alkaline phosphatase, bilirubin, LDH, INR, APTT, PP, 
ionized calcium, CRP, TSH, calcium-ion free, Hepatitis B virus s antigen (HBVSAG), 
Hepatitis C virus antibody (HCVAB), Human immunodeficiency virus type 1 and 2 
antibody and antigen (HIVABAG), HTLV type I antibody + HTLV type II antibody 
(IgG) (HTLVIGG), Ebstein-Barr virus antibody (EBV), P-Treponema pallidum 
antibody (TREPONE) 
b: 
Immunological blood samples: HEREKS11. See the section on "Immunological 
monitoring, blood samples”. 
c:  
Miscellaneous blood samples: Hemoglobin, leukocytes, differential counts, 
thrombocytes, creatinine, sodium, potassium, LDH, ASAT, ALAT, bilirubin, alkaline 
phosphatase, albumin, ionized calcium, TSH, CRP, INR, PP.  
d: 
Chrome-EDTA clearance is performed in the week before admission. 
e: 
If possible, and depending on localization and availability, excision-, punch- 
(“stanse”), crude needle- or fine needle biopsy is taken. 
f: 
CT, MRI, PET-CT, or PET-MRI scans can be used. Use of PET/CT scan is advised. 
Scans before chemotherapy should be performed in the week before admission. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
25
Treatment strategy 
The treatment course consists of two treatment steps and a subsequent control course. 
 
 
Figure 2: Overview of the treatment course 
 
Step 1: Screening and inclusion and surgical removal of tumor material with subsequent preparation 
and expansion of TIL cultures in the laboratory. 
 
Step 2: Treatment during admission with chemotherapy, peginterferon, TIL infusion, and IL-2. The 
last dosage of peginterferon is given as outpatient care, or the patient receives a syringe for self-
injection at home. 
 
Control course: Evaluation of treatment effect and follow-up.  
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
26
 
Figure 3: The figure shows a schematic representation of TIL isolation from tumor tissue and 
expansion, as well as T cell therapy in combination with peginterferon. 
Tumor tissue (metastasis or primary tumor) of a minimum size of 2 cm3 is removed from the patient, 
and the tumor tissue is brought to the laboratory under sterile conditions, where it is cut into 
suitable fragments of 1-3 mm3 and placed in cultivation wells with growth medium and IL-2. TILs 
are initially grown for 2-4 weeks until an appropriate cell number of a minimum of 50 x 106 is 
reached. At this time the cells can be cryopreserved for later use or further expanded in the Rapid 
Expansion Protocol (REP), where T cells for 2 weeks are stimulated with anti-CD3 antibody, 
allogenic irradiated PBMCs (peripheral blood mononuclear cells) feeder cells, and IL-2. The 
expanded TILs (now billions of cells) are washed, pooled, and re-infused intravenously into the 
patient. 
Before TIL infusion 1 week of lymphodepleting chemotherapy is given in the form of cyclo-
phosphamide (C) day -7 until day -6 and fludarabine (F) day -5 until day -1 in order to remove all 
existing lymphocytes in the patient to make room for the infused TILs and remove regulatory T cells 
(Treg). Two days before TIL infusion a subcutaneous injection of peginterferon (day -2) is given, 
and after TIL infusion another two doses of peginterferon (day 7 and day 14) is given with the 
purpose of increasing T cell tumor reactivity. Approximately 6 hours after TIL infusion, continuous 
IL-2 infusion according to the decrescendo regime (day 0 until day 5) is started to activate and 
stimulate the infused T cells into further expansion in the patient. 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
27
Substances in the clinical trial 
The substances, which are included in the clinical trial, are cyclophosphamide, fludarabine 
phosphate, interleukin-2, and pegylated interferon alpha 2b. Mixing and storage of the substances is 
performed according to existing standard guidelines in the department. 
 
Cyclophosphamide 
Cyclophosphamide is given as an intravenous infusion for two consecutive days in a dose 
corresponding to 60 mg per kg body weight. The treatment will take place during hospitalization 
and with supplemental hydration and mesna injections. 
 
Fludarabine phosphate 
Fludarabine phosphate is given as an intravenous infusion in a dose corresponding to 25 mg per m2 
body surface given daily for five consecutive days (the day after the last dose of 
cyclophosphamide). The treatment will take place during hospitalization.     
 
TILs 
The tumor-specific T cells are infused intravenously the day after the last dose of fludarabine 
phosphate (day 0). The number of T cells in the product depends on the feasible degree of 
expansion in vitro and is therefore variable, but an infusion will normally consist of approximately 
1010 cells. The treatment will take place during hospitalization. 
 
Interleukin-2 
After a week of chemotherapy and infusion of T cells on day 0, treatment with interleukin-2 as a 
continuous intravenous infusion on day 0-4 is started: 18 MU/m2 over 6 hours, 18 MU/m2 over 12 
hours, 18 MU/m2 over 24 hours, followed by 4.5 MU/m2 over 24 hours for 3 days. 
 
Pegylated interferon alpha 2b 
Pegylated interferon alpha 2b (peginterferon) is administered as a subcutaneous injection at 3 g/kg 
(maximal dose of 300 g) on day -2 (two days before T cell infusion), day 7 and 14. The two first 
injections are given during hospitalization. If the patient is discharged before the peginterferon 
injection on day 14, this injection can be administered on an outpatient basis, or the patient is given 
a syringe and detailed oral and written instructions in how to self-administer the injection at home. 
Peginterferon therapy is labeled according to the current Annex 13 in the GMP rules. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
28
Treatment plan 
Year:       Date: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Week 
-2 
-1 
0 
1 
Treatment day 
-12 -8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
1 
2 
3 
4 
7 
14 
Time 
(indicative 
timepoints) 
12:
00 
14:
00 
18:
00 
24:
00 
12:
00 
12:
00 
12:
00 
12:
00 
Peginterferon 
3 μg/kg SC 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
x 
x 
Patient is 
admitted 1) 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cyclophosphamid
e 60 mg/kg IV 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fludarabine phos-
phate 25 mg/m2 
IV 
 
 
 
 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
TIL IV 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
Pegfilgrastim 
6 mg SC*  
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
IL-2 18 MIU/m2 
IV continuously 
for 6, 12, and 24 
hours 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
 
 
 
 
 
IL-2 4.5 MIU/m2  
IV continuously  
for 24 hours 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
 
 
Treatment plan 
* See the section ”supportive treatment” 
 
 
Simultaneous treatment 
Guidelines for supportive care and treatment 
Is given on standard medical indication according to the assessment of the treatment responsible 
physician and must be specified in the medical record and flow sheets. The following are guidelines 
and other drugs may be used at your discretion. However, the patient may not receive concurrent 
systemic adrenocortical hormone.   
 
Prophylactic treatment 
Prophylactic treatment includes fluid therapy during cyclophosphamide treatment as well as 
supportive treatment with mesna (25 % of cyclophosphamide dose IV 4 times daily on day -7 and -
6) in order to protect the bladder mucosa. During IL-2 treatment fluid therapy is also administered 
in order to prevent electrolyte derangement as well as too low blood pressure, cf. appendix 7. 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
29
In order to prevent opportunistic infections, the following drugs are given: 
 
• Tabl. Sulfamethizole with trimethoprim, 400/80 mg, 1 tabl. daily, day -7 and 6 mo. on. 
• Tabl. Aciclovir, 400 mg x 2 daily. Day 0 and 6 mo. on. 
• Tabl. Diflucan, 100 mg daily. Day 0 and until neutrophilocyte number > 1000/µl. 
 
In order to prevent and relieve nausea during chemotherapy (day -7 to -1) the following is given: 
• Inj. Aloxi 250 μg IV day -7 and -5 
• Tabl. Emend 125 mg day -7, 80 mg day -6, 80 mg day -5 
• Tabl. Motilium 20 mg x 4 
• Tabl. Temesta 1-2 mg max x 4 as needed. 
• Tabl. Pantoloc 40 mg x 1-2 daily. 
 
In order to prevent and relieve nausea during IL-2 (day 0 to 5) the following is given: 
• Tabl. Motilium 20 mg x 4 
• Ondansetron 8 mg x 1 as needed max x 2 
• Imolope 2 mg as needed max x 8 
 
In order to reduce the duration of neutropenia, Pegfilgrastim, which is a human granulocyte-
stimulating factor (G-CSF), is given, 6 mg SC on day 0 after TIL infusion (at 14:00). 
 
Supportive treatment 
During infusion with T cells, the patient may experience chills, which can be relieved with injection 
of Petidin 25 mg subcutaneously as needed max x 4. 
Similarly, light difficulties breathing, possibly with a drop in the oxygen saturation, may be seen, 
which is treated with oxygen through a nasal catheter. 
 
Neutropenia: 
In the case of simultaneous neutropenia and fever will be treated according to the instruction (see 
appendix 8) for “Febrile neutropenia during T cell therapy”.  
 
Diarrhea:  
Diarrhea will immediately be treated with appropriate supportive care and treatment, including 
Loperamide. Loperamide must be discontinued, if there is blood or slime in the feces related to the 
diarrhea. In these circumstances appropriate diagnostic microbiological samples should be collected 
in order to exclude an infectious etiology. The patients should also be instructed in drinking plenty 
of clear fluids to prevent dehydration due to the diarrhea. 
  
Anemia: 
Blood transfusion is given if Hgb ≤ 6,0 mmol/L, or if clinically indicated. Irradiated and filtered 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
30
blood from day -7 and 6 mo. on will be used. 
 
Thrombocytopenia:  
Thrombocyte transfusion is given to patients with thrombocyte numbers < 20/μl, or if clinically 
indicated. 
 
Local radiotherapy 
Local radiotherapy of metastases can be prescribed against bone pain, wounds, or other indication. 
Out of consideration for the patient radiotherapy should be avoided during the 3-week period, 
where the actual treatment will take place. Irradiated areas cannot be used as a parameter for 
assessment of response. If possible, not all evaluable lesions should be included in the irradiated 
area. If this cannot be completed, the patient is not evaluable for response and will leave the study.  
 
Treatment discontinuation 
Normal discontinuation 
The patients will only receive one treatment series consisting of one week of chemotherapy, one 
single infusion of T cells (day 0), 3 subcutaneous injections of peginterferon (on day -2, 7, and 14, 
respectively), and 5 days of IL-2 according to the decrescendo regimen. The patients will be 
followed by the immune team for up to 5 years after treatment or until progression. 
 
Follow-up 
After the treatment the patients will be followed with outpatient control visits at 6 and 12 weeks 
after the treatment and thereafter every 3. month. After 2 years the patients will be followed every 
6. month. The patients will be followed by an immunotherapeutic team for up to 5 years or until 
progression. 
 
At each outpatient control visit an objective examination, toxicity assessment, blood samples 
including immunological blood samples, as well as PET/CT scan will be performed. If the patients 
have available remaining tumor tissue, tumor biopsies will also be taken at the first outpatient 
attendance. 
 
 
 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
31
Follow-up schedule 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1) Blood samples (T cell follow-up): hemoglobin, thrombocytes, leukocytes, differential count, 
sodium, potassium, creatinine, ALAT, ASAT, alkaline phosphatase, bilirubin, LDH, INR, APTT, PP, 
ionized calcium, CRP, TSH, calcium-ion free, immunological blood samples – HEREKS11 (See the 
section on “Immunological monitoring, blood samples”) 
2) See the section on “Immunological monitoring, tumor biopsy”. 
3) CT, MR, PET-CT, or PET-MR scan can be used. Use of PET/CT is recommended. 
 
Early discontinuation of treatment 
Not possible to grow TIL: If it is not possible to grow TILs, the patient cannot be offered treatment. 
 
The patient’s preference: The treatment can be stopped at any time, if the patient wants it. 
 
Medical decision: The treatment can at any time be stopped, if the investigator, for medical reasons, 
finds it to be in the patient’s best interest. 
 
Other treatment: The patients will leave the study, if treatment with other experimental drugs or 
other systemic anticancer treatment is started after the patient has been included for T cell therapy. 
The patient will leave the study, if treatment with adrenocortical hormone is started, unless it is 
based on a vital indication as agreed with the protocol responsible physician. 
 
Year:         
Date: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Week no. 
6 
 
12 
 
24 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Month no. 
1,
5 
 
3 
 
6 
 
9 
 
12 
 
15 
 
18 
 
21 
 
24 
 
27 
 
30 
Objective 
examination
s and 
subjective 
complaints 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
Toxicity 
assessment 
0 
 
0 
 
0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
0 
 
0 
 
0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PS 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
Blood 
samples:  
T cell 
follow-up1) 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
Biopsy 2) 
0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tumor 
evaluation 3) 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 
 
0 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
32
Side effects: If side effects arise in a patient in relation to the treatment to such a degree that the 
study cannot be completed, the treatment is aborted. 
 
Patients, who have stopped with IL-2 treatment early, will still be followed according to the 
protocol. 
 
Patients, who have left the protocol before infusion of tumor-specific T cells, will be replaced by 
new study subjects. They will be followed until cessation of side effects of the received treatment 
but will not be followed with further controls after that. 
 
Subsequent treatment 
If the patients leave the study, they can freely receive other treatment. If the patient develops 
progressive disease, they can freely receive other treatment. 
 
 
Production of TILs 
Requisition of tumor tissue  
Before the patients are operated, they will be informed verbally, and written consent will be 
collected according to procedures described elsewhere. When sufficient tissue has been obtained for 
a possible pathological examination, a biopsy of at least 2 cm3 will be taken from the tumor tissue.  
The biopsy is labelled with date and patient code number, placed in a sterile container, and 
transported to the GMP laboratory 54J7, Herlev Hospital, where the further processing takes place. 
 
Establishing ”Young TIL” cultures 
The T cells are expanded through our newly established method for “Young TILs”32. The tumor 
tissue is cut into fragments of 1-3 mm3 and placed in a culture plate with 24 wells. From each 
fragment a TIL culture is established by either migration of T cells from the tumor tissue or by 
enzymatic processing. The cell density is maintained at approximately 1x106 cells/ml growth 
medium, where the immunostimulatory cytokine IL-2 has been added. IL-2 belongs to the group of 
homeostatic cytokines, characterized by having a beneficial effect on the activation of tumor-
specific T cells and thereby on tumor cell killing. The cell cultures from the different fragments are 
pooled into a combined cell culture. The T cell expansion proceeds in an unselected manner, as a 
polyclonal TIL repertoire directed against multiple epitopes is desired in order to potentially obtain 
a more efficient destruction of the tumor cells in vivo. The establishment of “Young TIL” cultures 
typically takes 2-4 weeks with a success rate of more than 90 %. For any further inquiries, please 
refer to the Investigational Medicinal Product Dossier (IMPD). 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
33
Rapid Expansion Protocol (REP)  
When the TIL cultures have been expanded to approximately 5 x 107 cells, they are transferred to 
further expansion through the Rapid Expansion Protocol (REP), where TILs are grown together 
with irradiated (40 Gy) allogenic PBMCs (peripheral blood mononuclear cells), which serve as 
“feeder cells”, and IL-2 and anti-CD3 antibody, which activate the TILs. A very high number of 
activated tumor-specific T cells with a high activity towards tumor associated antigens (TAA) and 
tumor can be obtained in the course of 14 days. In the end the autologous T cells are concentrated in 
a 400 ml infusion bag for the purpose of intravenous infusion. For any further inquiries, please refer 
to the IMPD. 
 
Isolation of tumor cells 
Tumor cells are isolated from the tumor fragments by enzymatic processing or seeding from the 
tumor fragments and frozen for the purpose of later use for determination of anti-tumor T cell 
activity. 
 
Phenotype and clonotype determination 
In both Young TIL and REP TIL cultures the prevalence of T cell types (e.g. CD4+ and CD8+) and 
T cell stages are characterized (naïve and activated T cells) through flow cytometry. 
 
Cytokine Release Assay 
The TIL cultures are screened for activity towards TAA and autologous tumor by determining the 
production of activating cytokines (INF-γ + TNF-α). The production of activating cytokines is 
quantified using the ELISPOT method, flow cytometry, and combinatorial coding flow cytometry. 
 
Gene-based arrays 
Simultaneous analyses will be performed in order to identify specific tumor gene expression 
signatures55 and mutations in the tumor cells, which lead to the identification of patient-specific 
neo-antigens derived from these mutations56. These analyses can contribute to the identification of 
patients, whom are more likely to respond to the treatment, and further it may contribute to 
optimization of the T cell therapy based on the selection of neo-antigen specific T cells. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
34
Adverse Events, potential risks, and precautions 
Adverse events (Adverse Event = AE) 
Adverse events are unwanted signs, symptoms, or events, which occur during participation in the 
study, regardless of whether they are causally linked to the treatment or not. All adverse events 
must be described in the patient’s medical record and in the electronic Case Report Form (eCRF). 
The severity and effects after the treatment should be recorded for each adverse event. The severity 
of the adverse event and relationship with the administered treatment must be evaluated in 
accordance with the guidelines described below.   
The medical investigator should try to find all clinical and objective reactions from patients in 
treatment and determine their relationship with the experimental drugs. The investigator evaluates 
the relationship between adverse event and treatment using the following guidelines: 
 
Graduation of adverse events 
Severity refers to the intensity of a reaction. 
 
Events are graded according to CTCAE version 4.0 (see appendix 6)57. If this cannot be used, use 
the following scale: 
 
1 = light 
2 = moderate 
3 = severe 
4 = life-threatening 
5 = lethal 
 
Patients, who experience adverse events, will be monitored with relevant clinical assessments and 
laboratory testing according to the recommendations by the treatment responsible physician. All 
adverse events must be followed until satisfactory restitution or stabilization. Results from the 
follow-up must be recorded in the patient’s medical record and in the eCRF. 
Abnormal laboratory test results should not be recorded in the eCRF, unless these events either 
caused a clinical event, led to the discontinuation of the treatment, or fulfill the criteria for an SAE 
(see below). 
 
Serious adverse event (Serious Adverse Event = SAE) 
A serious adverse event must be reported to the sponsor within 24 hours and is defined as an event, 
which indicates a significant risk, contraindication, side effect, or precaution, and includes events, 
which: 
- result in death or are life-threatening 
- lead to hospitalization or prolong existing hospitalization 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
35
- result in considerable or continuing disability or incapacity for work 
- lead to an inborn anomaly or malformation 
- is a significant medical event. 
 
Guideline for possible relationships between adverse events and treatment 
0  
Not related – no temporal relationship, other etiologies very likely to be the cause. 
 
1  
Possibly related – less evident temporal relationship, other etiologies are also possible.  
 
2  
Probably related – clear temporal relationship with improvement upon discontinuation of 
treatment, and not reasonably explained by the patient’s known clinical condition. 
 
3 
Related – clear temporal relationship with laboratory confirmation or a positive re-treatment 
test. 
 
If the event is determined to be causally related to the experimental treatment, it is classified as a 
side effect (Adverse Reaction = AR) or as a serious side effect (Serious Adverse Reaction – SAR). 
 
Side effects 
A side effect may be expected, if it is described in the IMPD (appendix 12) or relevant “Summary 
of Product Characteristics” (SPC), or unexpected, if character or seriousness does not agree with the 
product information in these documents. 
If the side effect is unexpected, fulfill the criteria for an SAE, and is determined to be related to the 
experimental treatment, it is classified as a serious unexpected side effect (Suspected Unexpected 
Serious Adverse Reaction = SUSAR).  
 
Reporting of events and side effects 
The investigator reports SAEs, SARs, and SUSARs to the sponsor within 24 hours. 
The sponsor reports SUSARs to the Danish Medicines Agency within 7 days, if they are deemed 
life-threatening or are fatal, and otherwise within 15 days. Consequences for the study must be 
reported. 
The sponsor submits a list annually, which sums up possible SAEs and SUSARs, as well as a report 
of the safety of the study subjects to the Danish Medicines Agency and the National Committee on 
Health Research Ethics (NVEK) (reporting to the NVEK can also be done by the investigator). 
 
At the conclusion of the study, the sponsor submits a final report to the Danish Medicines Agency, 
where all events and side effects (SAEs, SARs, SUSARs) will be described. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
36
The following should not be reported: 
- deaths where the death is due to progression of the malignant disease  
- hospitalizations or prolongation of existing hospitalization, which are caused by the 
cancer: 
▪ weight loss 
▪ fatigue 
▪ electrolyte disturbances 
▪ pain management 
▪ anxiety 
▪ admission for palliative care 
▪ hospice stay or terminal care 
▪ progression of the basic disease 
- hospitalizations or prolongation of existing hospitalizations, if the only reason for the 
hospitalization or the prolongation is the following: 
▪ fluid or nausea treatment 
▪ blood transfusion 
▪ thrombocyte transfusion 
▪ hospitalization due to febrile leukopenia/neutropenia 
▪ administration of examination procedure 
▪ insertion of a permanent intravenous catheter. 
 
These events should be recorded in the eCRF. 
 
Known side effects 
 
Chemotherapy 
The side effects of chemotherapy described below are all general side effects of the two drugs, 
which are seen, when the drugs are given as primary treatment of oncological and hematological 
diseases. For such indications the treatments will often be given over several treatment series. In 
this study, the treatment will only be given in one session, and a milder side effect profile is 
therefore expected. 
 
Cyclophosphamide 
In patients, who receive cyclophosphamide, the dose-limiting toxicities consist of myelosuppression 
(neutropenia, thrombocytopenia, and anemia) and urotoxicity (cystitis, hematuria, and hemorrhagic 
cystitis). Sufficient treatment with mesna and hydration can markedly reduce the frequency and 
severity of bladder toxicity. 
Other commonly presenting side effects are alopecia, nausea, and vomiting.  

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
37
 
Patients, who receive cyclophosphamide treatment, can experience the following side effects: 
Infections and parasitic diseases  
Common (  1/100 to <1/10)  
   
Uncommon (  1/1,000 to <1/100)  
   
Very rare (<1/10,000)  
   
Infections  
   
Pneumonia, sepsis  
   
Septic shock  
Benign, malignant, and unspecified tumors (incl. 
cysts and polyps)  
Rare (  1/10,000 to <1/1,000)  
   
   
   
Very rare (<1/10,000)  
   
Unknown (cannot be estimated based on available 
data)  
   
   
   
Secondary tumors, bladder cancer, 
myelodysplastic changes, urinary tract 
cancer, acute leukemia.  
   
Tumor lysis syndrome.  
   
Lymphoma, sarcoma, kidney carcinoma, 
renal pelvic cancer, thyroid cancer, 
carcinogenic effect in offspring, 
progression of underlying malignancy. 
Blood and lymphatic system  
Very common (  1/10)  
   
 
Common (  1/100 to <1/10)  
   
Uncommon (  1/1,000 to <1/100)  
   
Very rare (<1/10,000)  
   
   
Unknown (cannot be estimated based on available 
data)  
   
   
Myelosuppression, leukopenia, 
neutropenia.  
   
Neutropenic fever.  
   
Thrombocytopenia, anemia.  
   
Hemolytic uremic syndrome, disseminated 
intravascular coagulation.  
   
Pancytopenia, agranulocytosis, 
granulocytopenia, lymphopenia, reduced 
hemoglobin.  
Immune system  
Very common (  1/10)  
   
Uncommon (  1/1,000 to <1/100)  
   
   
Very rare (<1/10,000)   
  
   
Immunosuppression.  
   
Anaphylactic reactions, hypersensitivity 
reactions.  
   
Anaphylactic shock.  
The endocrine system  
Very rare (<1/10,000)  
   
   
   
Vasopressin hypersecretion 
(SIADH/Schwartz-Bartter syndrome).  
   

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
38
Unknown (cannot be estimated based on available 
data)    
   
Water intoxication.  
Metabolism and nutrition  
Uncommon (  1/1,000 to <1/100)  
   
Rare (  1/10,000 to <1/1,000)  
   
Very rare (<1/10,000)  
   
Unknown (cannot be estimated based on available 
data)  
   
   
Anorexia.  
   
Dehydration.  
   
Water retention, hyponatremia.  
   
Increased blood glucose, decreased blood 
glucose.  
Mental disturbances  
Very rare (<1/10,000)  
 
   
Confusion.  
Nervous system  
Uncommon (  1/1,000 to <1/100)  
   
 
Rare (  1/10,000 to <1/1,000)  
   
Very rare (<1/10,000)  
   
   
Unknown (cannot be estimated based on available 
data)  
   
Peripheral neuropathy, polyneuropathy, 
neuralgia.  
   
Dizziness.  
   
Cramps, paresthesia, taste disturbances, 
hepatic encephalopathy.  
   
Encephalopathy, posterior reversible 
leukoencephalopathy syndrome, 
myelopathy, dysesthesia, hypoesthesia, 
tremor, hypogeusia, parosmia.  
Eyes  
Rare (  1/10,000 to <1/1,000)  
   
Very rare (<1/10,000)  
   
   
   
Unknown (cannot be estimated based on available 
data)  
   
Blurred vision.  
   
Visual disturbances, conjunctivitis and 
*eye edema in connection with 
hypersensitivity.  
 
   
Increased lacrimation.  
Ear and labyrinth  
Uncommon (  1/1,000 to <1/100)  
   
Unknown (cannot be estimated based on available 
data)  
   
Deafness.  
   
Hearing impairment, tinnitus.  
Heart  
Uncommon (  1/1,000 to <1/100)  
   
   
   
Cardiomyopathy, **heart failure, 
tachycardia, myocarditis.  
   

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
39
Rare (  1/10,000 to <1/1,000)  
   
   
Very rare (<1/10,000)  
   
   
   
Unknown (cannot be estimated based on available 
data)  
   
Arrhythmia (e.g. ventricular arrhythmia, 
supraventricular arrhythmia).  
   
Atrial fibrillation, ventricular fibrillation, 
angina pectoris, myocardial infarction, 
**cardiac arrest, pericarditis.  
   
Ventricular tachycardia, cardiogenic shock, 
pericardial effusion, hemorrhagic 
myocardium, left-sided ventricular failure, 
bradycardia, palpitation, EKG QT 
prolongation, reduced ejection fraction.  
Vascular diseases  
Rare (  1/10,000 to <1/1,000)  
   
Very rare (<1/10,000)  
   
   
Unknown (cannot be estimated based on available 
data)  
   
   
Hemorrhage.  
   
Thromboembolia, hypertension, 
hypotension.  
   
Pulmonary embolism, venous thrombosis, 
vasculitis, peripheral ischemia, flushing.  
Airways, thorax, and mediastinum  
Very rare (<1/10,000)  
   
   
   
   
Unknown (cannot be estimated based on available 
data)  
   
Bronchospasm, dyspnea, cough, interstitial 
pneumonia, chronic interstitial pulmonary 
fibrosis, pulmonary edema, pleural 
exudate, shock lung (ARDS), hypoxia.  
   
Pulmonary veno-occlusive disease, 
obliterative bronchiolitis, interstitial 
pneumonia, allergic alveolar diseases, 
pneumonia, unspecified lung diseases, 
nasal congestion, nasal discomfort, pain in 
the throat, rhinitis, sneezing.  
Gastrointestinal tract  
Very rare (<1/10,000)  
   
   
   
 
Unknown (cannot be estimated based on available 
data)  
   
Ascites, hemorrhagic enterocolitis, acute 
pancreatitis, mucosal ulcerations, 
stomatitis, diarrhea, vomiting, congestion, 
nausea.  
   
Gastrointestinal bleeding, abdominal pain, 
abdominal discomfort, colitis, enteritis, 
cecitis, inflammation of the parotid gland.  
Liver and biliary tract  
Rare (  1/10,000 to <1/1,000)  
   
Very rare (<1/10,000)  
   
   
Liver function disturbances, hepatitis.  
   
Hepatic veno-occlusive disease, enlarged 
liver, icterus, activation of viral hepatitis.  

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
40
   
Unknown (cannot be estimated based on available 
data)  
   
   
   
Cholestatic hepatitis, cytolytic hepatitis, 
cholestasis, liver toxicity with liver failure, 
increased bilirubin in the blood, abnormal 
liver function, increase in liver enzymes 
(ALAT, ASAT, alkaline phosphatase, and 
gamma-glutamyl transferase).  
Skin and subcutaneous tissue  
Very common (  1/10)  
   
Uncommon (  1/1,000 to <1/100)  
   
Rare (  1/10,000 to <1/1,000)  
   
Very rare (<1/10,000)  
   
   
   
   
   
Unknown (cannot be estimated based on available 
data)  
   
   
   
Alopecia.  
   
Total alopecia.  
   
Exanthem, dermatitis.  
   
Stevens-Johnson syndrome, toxic 
epidermal necrolysis, pruritus, erythema of 
the irradiated area, serious skin reactions, 
discoloration of palms, finger nails, and 
soles of the feet, toxic skin inflammation.  
   
Palmar-plantar erythrodysesthesia (hand-
foot syndrome), erythema multiforme, 
urticaria, blisters, erythema, swelling of the 
face, hyperhidrosis.  
Bones, joints, muscles, and connective tissue  
Very rare (<1/10,000)  
   
Unknown (cannot be estimated based on available 
data)  
   
   
Rhabdomyolysis, cramps.  
   
Scleroderma, muscle cramps, myalgia, 
arthralgia.  
Kidneys and urinary tracts  
Very common (  1/10)  
   
Common (  1/100 til <1/10)  
   
Very rare (<1/10,000)  
   
   
   
   
   
Unknown (cannot be estimated based on available 
data)  
   
   
Cystitis, microhematuria.  
   
Hemorrhagic cystitis, macrohematuria.  
   
Suburethral bleeding, edema of the bladder 
wall, kidney failure, reduced kidney 
function. Interstitial inflammation, fibrosis, 
and sclerosis of the bladder. Increased 
creatinine in the blood.  
   
Tubular necrosis, tubular disturbances, 
toxic nephropathy, hemorrhagic urethritis, 
ulcerative cystitis, bladder contractions, 
nephrogenic diabetes insipidus, atypical 
epithelial cells in the urinary bladder, 
increased urea in the blood.  

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
41
Pregnancy, puerperium, and the perinatal 
period  
Unknown (cannot be estimated based on available 
data)  
   
   
Premature birth 
The reproductive system and breast  
Common (  1/100 to <1/10)  
   
Uncommon (  1/1,000 to <1/100)  
   
   
Rare (  1/10,000 to <1/1,000)  
   
   
 
Unknown (cannot be estimated based on available 
data)  
   
   
Reduced spermatogenesis.  
   
Ovulation disturbances, reduced levels of 
female sex hormones.  
   
Oligospermia***, azoospermia***, 
irreversible ovulation disturbances, 
amenorrhea***.  
   
Infertility, ovarian failure, oligomenorrhea, 
testicular atrophy, reduced estrogen in the 
blood, increased gonadotropin in the blood.  
Inborn, familial, and genetic diseases 
 
Unknown (cannot be estimated based on available 
data)  
   
   
   
Fetal death, fetal malformation, delayed 
fetal development, fetal toxicity.  
General symptoms and reactions at the 
administration site  
Very common (  1/10)  
   
Common (  1/100 to <1/10)  
   
   
Rare (  1/10,000 to <1/1,000)  
   
Very rare (<1/10,000)  
   
   
   
   
Unknown (cannot be estimated based on available 
data)  
   
   
   
Fever.  
   
Chills, asthenic conditions (e.g. fatigue, 
weakness, discomfort), mucositis.  
   
Chest pain.  
   
Multiple organ failure, headache, pain. 
Reactions at the administration site, e.g. 
phlebitis, thrombosis, necrosis, pain, 
swelling, erythema.  
   
Pyrexia, edema, influenza-like illness.  
Examinations 
Uncommon (  1/1,000 to <1/100)  
   
   
   
ECG changes, reduced left ventricle 
ejection fraction (LVEF), increased LD, 
increased C-reactive protein.  

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
42
Source: The Danish Medicines Agency SPC, cyclophosphamide58 
The text is abbreviated compared to the approved SPC. The full SPC is available at www.produktresume.dk. 
 
Fludarabine phosphate 
The most frequent side effects are myelosuppression (neutropenia, thrombocytopenia, and anemia),  
infections including pneumonia, cough, fever, fatigue, malaise, nausea, vomiting, and diarrhea.  
Other commonly presenting side effects are chills, edemas, discomfort, peripheral neuropathy, 
visual disturbances, anorexia, mucositis, stomatitis, and skin rash. Serious opportunistic infections 
have arisen among patients treated with fludarabine phosphate. Deaths caused by serious side 
effects have been reported. 
 
Patients, who receive fludarabine phosphate, may experience the following side effects: 
   
Very rare (<1/10,000)  
   
Weight increase  
Infections and parasitic diseases 
Very common (  1/10)  
 
 
 
 
 
 
Rare (  1/10,000 to <1/1,000)  
 
 
 
Infections/opportunistic infections (as 
latent virus reactivation, such as e.g. 
progressive multifocal leukoencephalo-
pathy, Herpes zoster virus, Epstein-Barr-
virus) 
Pneumonia 
 
Lymphoproliferative diseases (EBV-
associated) 
Benign, malignant, and uspecified tumors (incl. 
cysts and polyps)  
Common (  1/100 to <1/10)  
 
 
 
Myelodysplastic syndrome and acute 
myeloid leukemia (mainly associated with 
previous, concurrent, or later treatment 
with alkylating compounds, 
topoisomerase-inhibitors, or radiotherapy) 
 
Blood and lymphatic system 
Very common (  1/10)  
 
Common (  1/100 to <1/10)  
 
 
Neutropenia, anemia, thrombocytopenia 
 
Myelosuppression 
 
The immune system 
Uncommon (  1/1,000 to <1/100)  
 
 
Autoimmune diseases (including 
autoimmune hemolytic anemia, Evans 
syndrome, thrombocytopenic purpura, 
acquired hemophilia, pemphigus) 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
43
Metabolism and nutrition 
Common (  1/100 to <1/10)  
 
Uncommon (  1/1,000 to <1/100)  
 
 
Anorexia 
 
Tumor lysis syndrome (including kidney 
failure, metabolic acidosis, hyperkalemia, 
hypocalcemia, hyperuricemia, hematuria, 
urinary stones, hyperphosphatemia) 
 
The nervous system 
Common (  1/100 to <1/10)  
 
Uncommon (  1/1,000 to <1/100)  
 
Rare (  1/10,000 to <1/1,000)  
 
Unknown 
 
 
Peripheral neuropathy 
 
Confusion 
 
Coma, seizures, agitation 
 
Cerebral bleeding 
 
Eyes 
Common (  1/100 to <1/10)  
 
Rare (  1/10,000 to <1/1,000)  
 
 
Visual disturbances 
 
Blindness, optic neuritis, optic neuropathy 
Heart  
Rare (  1/10,000 to <1/1,000)  
 
 
Cardiac arrest, arrhythmia 
 
Airways, thorax, and mediastinum  
Very common (  1/10)  
 
Uncommon (  1/1,000 to <1/100)  
 
 
Unknown 
 
 
Cough 
 
Pulmonary toxicity (including pulmonary 
fibrosis, pneumonitis, dyspnea) 
 
Pulmonary bleeding 
 
Gastrointestinal tract 
Very common (  1/10)  
 
Common (  1/100 to <1/10)  
 
Uncommon (  1/1,000 to <1/100)  
 
 
Vomiting, diarrhea, nausea 
 
Stomatitis 
 
Gastrointestinal bleeding, abnormal 
pancreatic enzymes  
Liver and biliary tract 
Uncommon (  1/1,000 to <1/100)  
 
 
Abnormal liver enzymes 
Skin and subcutaneous tissues 
Common (  1/100 to <1/10)  
 
Rash 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
44
Source: The Danish Medicines Agency SPC, fludarabine phosphate59 
The text is abbreviated compared to the approved SPC. The full SPC is available at www.produktresume.dk. 
 
TILs 
No serious side effects of TIL infusion are expected. The patients can briefly experience fever, 
chills, and light dyspnea, and a small drop in saturation has been observed a few times. In addition, 
autoimmune reactions, including vitiligo and uveitis60, can be seen. 
Theoretically, there is a risk for the development of allergic reactions/anaphylactic shock. 
According to the literature this has not been seen yet. 
 
Interleukin-2  
Frequency and severity of side effects associated with interleukin-2 have in general proven to be 
dependent on route of administration, dose, and dosing interval. Most side effects are self-limiting and 
will go away within 1-2 days of treatment discontinuation. 
 
See also the enclosed ”Vejledning til monitorering, dosismodifikationer og understøttende 
behandling under infusion af høj-dosis Interleukin-2” (”Guidelines for monitoring, dose 
modifications, and supportive treatment during infusion of high-dose interleukin-2”) (appendix 7) 
 
The following side effects were reported from clinical studies and from post-marketing experience 
with interleukin-2: 
 
Rare (  1/10,000 to <1/1,000)  
 
 
Skin cancer, toxic epidermal necrolysis, 
(Lyell-type), Stevens-Johnson-syndrome 
Kidneys and urinary tract 
Unknown 
 
 
Hemorrhagic cystitis 
General symptoms and reactions at the 
administration site 
Very common (  1/10)  
 
Common (  1/100 to <1/10)  
 
 
 
Fever, fatigue, lethargy 
 
Edema, mucositis, chills, discomfort 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
45
Infections and parasitic diseases 
Common (≥1/100 to <1/10) 
 
 
Respiratory infection. 
 
Blood and lymphatic system (see further 
information below the table) 
Very common (≥1/10) 
 
Common (≥1/100 to <1/10) 
 
 
Uncommon (≥1/1,000 to <1/100) 
 
 
Rare (≥1/10,000 to <1/1,000) 
 
 
 
Anemia, thrombocytopenia. 
 
Leukopenia, coagulopathy including 
disseminated intravascular coagulation, 
eosinophilia. 
 
Neutropenia  
 
Agranulocytosis, aplastic anemia, 
hemolytic anemia, neutropenic fever. 
The immune system  
Uncommon (≥1/1,000 to <1/100) 
 
Rare (≥1/10,000 to <1/1,000) 
 
Hypersensitivity reactions. 
Anaphylaxis. 
The endocrine system  
Very common (≥1/10) 
 
Common (≥1/100 to <1/10) 
 
 
Hypothyroidism 
 
Hyperthyroidism. 
Metabolism and nutrition   
Very common (≥1/10) 
 
Common (≥1/100 to <1/10) 
 
 
 
Uncommon (≥1/1,000 to <1/100) 
 
Rare (≥1/10,000 to <1/1,000) 
 
Anorexia. 
 
Acidosis, hyperglycemia, hypocalcemia, 
hypercalcemia, hyperkalemia, 
dehydration. 
 
Hypoglycemia. 
 
Diabetes mellitus 
 
Mental disturbances  
Very common (≥1/10) 
 
Common (≥1/100 to <1/10): 
 
Anxiety, confusion, depression, insomnia. 
 
Irritability, agitation, hallucinations. 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
46
The nervous system  
Very common (≥1/10) 
 
 
Common (≥1/100 to <1/10) 
 
 
 
Uncommon (≥1/1,000 to <1/100) 
 
 
Unknown (cannot be estimated based on available 
data) 
 
Dizziness, headache, paresthesia, 
somnolence. 
 
Neuropathy, syncope, speech 
disturbances, lost sense of taste, 
drowsiness. 
 
Coma, cramps, paralysis, muscle 
weakness 
 
Intracranial/cerebral bleeding, 
cerebrovascular event, leukoencephalo-
pathy (see further information below the 
table). 
Eyes  
Common (≥1/100 to <1/10) 
 
Rare (≥1/10,000 to <1/1,000) 
 
 
Conjunctivitis. 
 
Optic nerve disease including optic 
neuritis. 
Heart  
Very common (≥1/10) 
 
 
Common (≥1/100 to <1/10) 
 
 
 
 
 
Uncommon (≥1/1,000 to <1/100) 
 
 
Rare (≥1/10,000 to <1/1,000) 
 
Unknown 
 
 
Tachycardia, arrhythmia, chest pain. 
Arrhythmia, cyanosis. 
 
Cyanosis, transient ECG changes, 
myocardial ischemia, palpitations, 
cardiovascular disease including heart 
failure. 
Ventricular hypokinesis. 
 
Myocarditis, cardiomyopathy, cardiac 
arrest, pericardial exudate.   
 
Ventricular hypokinesis. 
 
Cardiac tamponade 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
47
Vascular diseases  
Very common (≥1/10) 
 
Common (≥1/100 to <1/10) 
 
Uncommon (≥1/1,000 to <1/100) 
 
 
Hypotension. 
 
Phlebitis, hypertension. 
 
Thrombosis, thrombophlebitis, bleeding. 
 
Airways, thorax, and mediastinum   
Very common (≥1/10) 
 
Common (≥1/100 to <1/10) 
 
 
 
Rare (≥1/10,000 to <1/1,000) 
 
Dyspnea, cough. 
 
Pulmonary edema, pleural exudate, 
hypoxia, hemoptysis, epistaxis, nasal 
congestion. rhinitis. 
 
Pulmonary embolism, adult respiratory 
distress syndrome. 
Gastrointestinal tract   
Very common (≥1/10) 
 
 
Common (≥1/100 to <1/10) 
 
 
 
 
Uncommon (≥1/1,000 to <1/100) 
 
 
 
Rare (≥1/10,000 to <1/1,000) 
 
 
Nausea with or without vomiting, diarrhea, 
stomatitis. 
 
Dysphagia, dyspepsia, constipation, 
gastrointestinal bleeding including rectal 
bleeding, hematemesis, ascites, cheilitis, 
gastritis. 
 
Pancreatitis, intestinal obstruction, 
gastrointestinal perforation including 
necrosis/gangrene. 
 
Activation of latent Crohn’s disease, 
pancreatitis, intestinal obstruction. 
Liver and biliary tract 
Common (≥1/100 to <1/10) 
 
 
 
Rare (≥1/10,000 to <1/1,000) 
 
 
Elevation of liver transaminases, increase 
in alkaline phosphatase, increase in lactate 
dehydrogenase, hyperbilirubinemia, 
hepatomegaly or hepatosplenomegaly. 
 
Cholecystitis, liver failure with lethal 
outcome. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
48
Skin and subcutaneous tissues  
Very common (≥1/10) 
 
 
Common (≥1/100 to <1/10) 
 
Uncommon (≥1/1,000 to <1/100) 
 
Rare (≥1/10,000 to <1/1,000) 
 
 
Erythema/rash, skin exfoliation, pruritus, 
sweating. 
 
Alopecia, urticaria. 
 
Vitiligo, Quincke’s Edema. 
 
Vesiculobullous rash, Stevens-Johnson’s 
syndrome. 
Bones, joints, muscles, and connective tissue  
Common (≥1/100 to <1/10) 
 
Uncommon (≥1/1,000 to <1/100) 
 
 
Myalgia, arthralgia. 
 
Myopathy, myositis. 
Kidneys and urinary tract  
Very common (≥1/10) 
 
 
Common (≥1/100 to <1/10) 
 
 
Oliguria, increased serum urea, increased 
serum creatinine. 
 
Hematuria, kidney failure, anuria. 
General symptoms and reactions at the 
administration site  
Very common (≥1/10) 
 
 
 
 
 
 
Common (≥1/100 to <1/10) 
 
 
Rare (≥1/10,000 to <1/1,000) 
 
 
 
Reaction at the injection site*, pain at the 
injection site*, inflammation at the 
injection site*, fever with or without 
chills, discomfort and fatigue, pain, 
edema, weight increase, weight loss  
Necrosis at the injection site. 
 
Mucositis, lump at the injection site, 
hypothermia. 
 
Necrosis at the injection site. 
Source: The Danish Medicines Agency SPC, proleukin61  
The text is abbreviated compared to the approved SPC. The complete SPC is available at www.produktresume.dk. 
 
Comments: 
* The frequency of reactions, pain, and inflammation at the injection site is lower than what is seen 
with continuous intravenous infusion.  
 
Leukoencephalopathy 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
49
In the literature rare cases of leukoencephalopathy in connection with Proleukin have been reported, 
in particular among patients treated for HIV infection. In some cases, other risk factors such as 
opportunistic infections, co-administration of interferons, as well as several series of chemotherapy, 
which may predispose the patients to leukoencephalopathy, were present. 
 
Capillary leak syndrome 
Cardiac arrhythmia (supraventricular and ventricular), angina pectoris, myocardial infarction, 
respiratory insufficiency demanding intubation, gastrointestinal bleeding or infarction, renal 
insufficiency, edema, and mental state changes can be linked with capillary “leak” syndrome. The 
frequency and severity of the capillary “leak” syndrome is lower after subcutaneous administration 
than for continuous intravenous infusion. 
 
Serious manifestations of eosinophilia 
During treatment most patients develop lymphocytopenia and eosinophilia with reactive 
lymphocytosis within 24-48 hours after cessation of treatment. These conditions are not considered 
to be side effects and may be attributed to the Proleukin mechanism of anti-tumor activity. 
 
Cerebral vasculitis 
Cerebral vasculitis, both isolated and in combination with other manifestations, has been reported. 
Cutaneous and leukocytoclastic hypersensitivity vasculitis have been reported. Some of these cases 
respond to treatment with corticosteroids. 
 
Side effects associated with concurrent treatment with interferon alfa 
The following side effects have been reported as rare with respect to parallel treatment with 
interferon alfa: Extracapillary IgA glomerulonephritis, oculo-bulbar myasthenia gravis, 
inflammatory arthritis, thyroiditis, bullous pemphigoid, rhabdomyolysis, as well as Stevens-
Johnson’s syndrome. Serious rhabdomyolysis and myocardial damage, including myocardial 
infarction, myocarditis, and ventricular hypokinesis, appear to be increased among patients treated 
with Proleukin (intravenously) and interferon alfa simultaneously. 
 
Bacterial infection 
Bacterial infection or worsening of bacterial infection, including septicemia, bacterial endocarditis, 
septic thrombophlebitis, peritonitis, pneumonia, and local infection surrounding the catheter site has 
been reported, mainly in association with intravenous administration. 
 
Peginterferon alpha 2b 
The following treatment-related side effects were reported among adults in clinical trials or through 
post-marketing surveillance of patients with chronic hepatitis C treated with peginterferon alfa-2b, 
including PegIntron monotherapy or PegIntron/ribavirin. 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
50
Infections and parasitic diseases 
Very common (≥ 1/10) 
 
Common (≥ 1/100 to < 1/10) 
 
 
 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
Viral infection*, pharyngitis *  
 
Bacterial infection (including sepsis), 
fungal infection, influenza, upper 
respiratory tract infection, bronchitis, 
herpes simplex, sinusitis, otitis media, 
rhinitis  
 
Infection at the injection site, lower 
respiratory tract infection  
 
Blood and lymphatic system  
Very common (≥ 1/10) 
 
Common (≥ 1/100 to < 1/10) 
 
Very rare (< 1/10,000) 
 
 
Unknown (cannot be estimated based on available 
data) 
 
Anemia, neutropenia  
 
Hemolytic anemia, leukopenia, 
thrombocytopenia, lymphadenopathy  
 
Aplastic anemia  
 
Pure red cell aplasia  
 
The immune system  
Uncommon (≥ 1/1,000 to < 1/100) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
Unknown (cannot be estimated based on available 
data) 
 
Hypersensitivity towards the drug  
 
Sarcoidosis  
 
Acute allergic reactions including 
angioedema, anaphylaxis and 
anaphylactic reactions including 
anaphylactic shock, idiopathic 
thrombocytopenic purpura, thrombotic 
thrombocytopenic purpura, systemic 
lupus erythematosus  
 
The endocrine system 
Common (≥ 1/100 to < 1/10) 
 
 
Hypothyroidism, hyperthyroidism  
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
51
Metabolism and nutrition  
Very common (≥ 1/10) 
 
Common (≥ 1/100 to < 1/10) 
 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
 
Anorexia  
 
Hypocalcemia, hyperuricemia, 
dehydration, increased appetite  
 
Diabetes mellitus, hypertriglyceridemia  
 
Diabetic ketoacidosis  
 
Mental disturbances  
Very common (≥ 1/10) 
 
 
Common (≥ 1/100 to < 1/10) 
 
 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
Unknown (cannot be estimated based on available 
data) 
 
Depression, anxiety*, emotional lability*, 
decreased concentration, insomnia  
 
Aggression, agitation, anger, mood 
changes, abnormal behavior, nervosity, 
sleep disturbances, decreased libido, 
apathy, abnormal dreams, crying  
 
Suicide, suicide attempt, suicidal ideation, 
psychosis, hallucinations, panic attacks  
 
Bipolar conditions  
 
Homicidal ideation, mania  
 
The nervous system  
Very common (≥ 1/10) 
 
Common (≥ 1/100 to < 1/10) 
 
 
 
 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
Very rare (< 1/10,000) 
 
 
Unknown (cannot be estimated based on available 
data) 
 
Headache, dizziness  
 
Amnesia, failing memory, syncope, 
migraine, ataxia, confusion, neuralgia, 
paresthesia, hypoesthesia, hyperesthesia, 
hypertonia, drowsiness, attention 
disturbances, tremor, dysgeusia 
 
Neuropathy, peripheral neuropathy  
 
Seizures  
 
Cerebrovascular bleeding, 
cerebrovascular ischemia, encephalopathy  
 
Facial paralysis, mononeuropathies  
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
52
Eyes  
Common (≥ 1/100 to < 1/10) 
 
 
 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
 
 
 
 
Unknown (cannot be estimated based on available 
data) 
 
Visual disturbances, blurred vision, 
photophobia, conjunctivitis, eye irritation, 
lacrimation disorder, eye pain, dry eyes 
 
 
Retinal exudates  
 
Loss of visual acuity or field of vision,  
retinal hemorrhage, retinopathy, 
retinal artery occlusion, retinal vein 
occlusion, optic neuritis, papilledema, 
macular edema 
  
 
Serous retinal detachment  
 
Ear and labyrinth  
Common (≥ 1/100 to < 1/10) 
 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
 
Reduced hearing/hearing loss, tinnitus, 
dizziness 
 
Ear pain  
Heart  
Very common (≥ 1/10) 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
 
Very rare (< 1/10,000) 
 
Unknown (cannot be estimated based on available 
data) 
 
Palpitations, tachycardia  
 
Myocardial infarction  
 
Congestive heart failure, cardiomyopathy,  
arrhythmia, pericarditis  
 
Cardiac ischemia  
 
Pericardial effusion  
 
Vascular diseases  
Common (≥ 1/100 to < 1/10) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
 
Hypotension, hypertension, flushing 
 
Vasculitis  
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
53
Airways, thorax, and mediastinum  
Very common (≥ 1/10) 
 
Common (≥ 1/100 to < 1/10) 
 
 
 
 
Very rare (< 1/10,000) 
 
 
Dyspnea*, cough*  
 
Dysphonia, epistaxis, breathing problems, 
clogged airways, sinus congestion, nasal 
congestion, rhinorrhea, increased upper 
airway secretion, pharyngolaryngeal pain 
 
Interstitial lung disease 
Gastrointestinal tract  
Very common (≥ 1/10) 
 
 
Common (≥ 1/100 to < 1/10) 
 
 
 
 
 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
Very rare (< 1/10,000) 
 
 
Vomiting*, nausea, abdominal pain, 
diarrhea, dry mouth*  
 
Dyspepsia, gastroesophageal reflux, 
stomatitis, mouth sores, glossalgia, 
gingival bleeding, constipation, 
flatulence, hemorrhoids, cheilitis, 
abdominal distension, gingivitis, glossitis, 
tooth disorder 
 
Pancreatitis, mouth pain  
 
Ischemic colitis  
 
Colitis ulcerosa 
 
Liver and biliary tract  
Common (≥ 1/100 to < 1/10) 
 
 
Hyperbilirubinemia, hepatomegaly  
 
Skin and subcutaneous tissues  
Very common (≥ 1/10) 
 
Common (≥ 1/100 to < 1/10) 
 
 
 
 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
Very rare (< 1/10,000) 
 
 
 
Alopecia, pruritus*, dry skin*, rash*  
 
Psoriasis, photosensitivity reaction, 
maculopapular rash, dermatitis, 
erythematous rash, eczema, night sweats, 
hyperhidrosis, acne, furunculosis, 
erythema, urticaria, abnormal hair 
structure, nail disorder  
 
Cutaneous sarcoidosis  
 
Stevens-Johnson syndrome, toxic 
epidermal necrolysis, erythema 
multiforme  
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
54
Bones, joints, muscles, and connective tissue  
Very common (≥ 1/10) 
 
Common (≥ 1/100 to < 1/10) 
 
 
Uncommon (≥ 1/1,000 to < 1/100) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
 
Myalgia, arthralgia, musculoskeletal pain  
 
Arthritis, back pain, muscle spasms, pain 
in the extremities 
 
Bone pain, muscle weakness  
 
Rhabdomyolysis, myositis, rheumatoid 
arthritis  
Kidneys and urinary tract  
Common (≥ 1/100 to < 1/10) 
 
Rare (≥ 1/10,000 to < 1/1,000) 
 
Changes in urinary frequency, polyuria, 
abnormal urine 
 
Kidney failure, reduced kidney function  
 
The reproductive system and breasts  
Common (≥ 1/100 to < 1/10) 
 
 
Amenorrhea, chest pain, menorrhagia, 
menstruation irregularities, ovarian 
disorders, vaginal problems, sexual 
dysfunction, prostatitis, erectile 
dysfunction  
 
General symptoms and reactions at the 
administration site  
Very common (≥ 1/10) 
 
 
 
 
Common (≥ 1/100 to < 1/10) 
 
 
 
Rare (≥ 1/10,000 to < 1/1,000): 
 
 
 
Reactions at the injection site*, 
inflammation at the injection site, fatigue,  
asthenia, irritability, chills, fever, 
influenza-like symptoms, pain  
 
Chest pain, chest discomfort, pain at the 
injection site, discomfort, facial edema, 
peripheral edema, abnormal sensation, 
thirst  
 
Necrosis at the injection site  
Examination  
Very common (≥ 1/10) 
 
 
Weight loss  
 
Source: European Medicines Agency (EMA) SPC: PegIntron (EMA7).  
The text is abbreviated compared to the approved SPC. The full SPC is available at the EMA home 
page: www.ema.europa.eu/ema/ 
 
Comments: 
*These side effects were common (≥ 1/100 to < 1/10) in clinical trials among patients treated with 
PegIntron monotherapy. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
55
Risks and disadvantages associated with surgery and sample collection 
 
Risks associated with the removal of tumor tissue 
Prior to the inclusion it will be assessed, whether it is possible to remove part of the patient’s own 
tumor tissue, either from the primary tumor or from a metastasis, through a smaller surgical 
procedure. The operation will be performed by physicians at the Department of Plastic Surgery at 
Herlev Hospital or by physicians from other surgical specialties, if necessary. Use of 
subcutaneous/cutaneous metastases or lymph nodes is prioritized. If readily accessible tumor tissue 
is not available, or if the removal is associated with serious risks for the patient, the patient cannot 
participate in the study. 
 
Risks associated with biopsy collection 
In association with biopsy collection there will be a small risk of infection and/or bleeding. Also, 
there may be discomfort such as pain or ecchymosis in the biopsy area.  
 
Risks associated with blood sampling 
In association with blood sampling the patient may experience discomfort such as pain or 
ecchymosis at the puncture site. The blood samples will also be associated with frequent visits at 
the hospital. 
 
Monitoring and precautions 
 
Hematological parameters 
Close hematological surveillance of blood counts is indicated for all patients during the treatment. 
Leukocyte count, thrombocyte count, and hemoglobin values will be controlled at fixed intervals. 
Measurements will be made prior to commencing chemotherapy, Peg-Intron, and IL-2, and daily 
during treatment until the neutrophilocyte number is > 500/l and the leukocyte number is > 
1000/l. Chemotherapy will not be administered to patients with leukocyte numbers below 500/l 
and/or a thrombocyte number below 50,000/l before commencing chemotherapy. 
 
Kidney and urinary tract function 
Before treatment start any obstruction of the efferent urinary tract, cystitis, or infection will be 
resolved. The patients will be treated with mesna and plenty of fluids to reduce the frequency and 
severity of bladder toxicity. If, during the treatment with cyclophosphamide, cystitis associated with 
micro- or macrohematuria is detected, the treatment will be stopped. The urine will be controlled 
for the presence of microscopic hematuria with a urine analysis test prior to commencing treatment 
with cyclophosphamide.   
 
Cardiotoxicity 

 
 
 
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56
Cardiotoxicity is especially seen in association with administration of high doses of 
cyclophosphamide (120-240 mg/kg body weight). An electrocardiogram will be taken prior to 
treatment. Patients with known heart disease cannot be included in the study. If the patient 
experiences symptoms from the coronary circulation (e.g. chest pain, shortness of breath) the 
necessary examination-procedures will be performed. 
  
Infertility 
Treatment of men can increase the risk of irreversible infertility, and they will therefore be informed 
about the possibility of freezing down semen before treatment start. There is also a risk of fertility 
problems among women. 
 
Live vaccines 
Vaccination with live vaccines must be avoided during and immediately after treatment with 
chemotherapy due to the immunosuppressive effect. 
 
Interactions: 
Cyclophosphamide inhibits the cholinesterase activity and thereby reinforces the effect of 
depolarizing muscle relaxants, such as suxamethoniumchloride. This may result in longer-lasting 
apnea in association with anesthesia. The anesthesiologist must be informed, if a patient has 
received treatment with cyclophosphamide within 10 days prior to treatment with 
suxamethoniumchloride. The combination should be avoided. 
 
Patients should not eat grapefruit or drink grapefruit juice, as grapefruit contains a substance, which 
may impair the activation of cyclophosphamide and thereby its effects. 
 
Transfusion-related graft-versus-host reactions have been observed among patients treated with 
fludarabine phosphate after transfusion with non-irradiated/non-filtered blood. Therefore, patients 
who require blood transfusion and whom receive or have received treatment with fludarabine 
phosphate within ½ a year, may only receive irradiated or filtered blood. An agreement has been 
made with Blodbanken (the “blood bank”) at Herlev Hospital, that only irradiated blood will be 
ordered for these patients for half a year after treatment. All blood within the Capital Region of 
Denmark is filtered.  
 
Results from a multi-dose study, which evaluated CYP substrates among chronic hepatitis C 
patients, who received pegylated interferon (PegIntron, 1.5 g/kg) once weekly for 4 weeks, 
showed an increased activity of CYP2D6 and CYP2C8/9. Changes in the activity of CYP1A2, 
CYP3A4, or N-acetyltransferase were not observed. 
 
Caution should be shown when co-administering peginterferon alfa-2b with drugs, which are 
metabolized by CYP2D6 and CYP2C8/9, especially drugs with a narrow therapeutic window, such 
as warfarin and phenytoin (CYP2C9) and flecainide (CYP2D6). 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
57
Effect evaluation, data analysis, and monitoring 
Effect evaluation 
Primary effect parameter 
Paraclinical evaluation: to evaluate the immunological effects of the treatment, the patients will be 
followed by continuous in vitro analyses of the specific T-cell reactivity against tumor antigens. 
The immunological response towards tumor antigens before and after the treatment will be 
compared. These analyses will be performed on blood samples and tumor biopsies. 
Secondary effect parameter  
Clinical evaluation: The clinical effects of the treatment will be assessed using the objective 
response rate according to RECIST 1.1, survival, and progression-free survival. 
 
Response criteria 
RECIST 
Clinical evaluation will be made according to the RECIST 1.1 Guidelines62:  
 
Complete response (CR): All lesions disappear. 
Partial response (PR): Defined as ≥ 30 % reduction of the sum of all measurable lesions’ longest 
diameter. 
Stable disease (SD): Defined as < 30 % reduction of the sum of all measurable lesions’ longest 
diameter, or < 20 % increase in the sum of all measurable lesions’ longest diameters. 
Progressive Disease (PD): Defined as > 20 % increase in the sum of all measurable lesions’ longest 
diameter or the presence of new lesions. 
 
Complete and partial response must be verified by examination at the earliest 4 weeks after the 
response has been documented. 
 
Immunological monitoring 
Blood samples for immunological monitoring 
Blood samples of 100 ml heparinized blood are taken for immunological monitoring, as well as 10 
ml blood in dry glasses to freeze down serum; at surgery, before treatment start (at “baseline”), at 
discharge (approximately 1 week after T-cell infusion), and then 6 and 12 weeks after infusion of T 
cells (see schedule for examination plan and for follow-up). Thereafter, blood samples for 
immunological monitoring will be taken, when the patients show up for evaluation every third 
month, and until the patient leaves the study. Further, additional serum samples (10 ml blood 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
58
sample) are taken on day 0 before TIL-infusion, 2 hours after TIL-infusion, and then every second 
day until discharge. 
This means, that a total of 500 ml blood will be taken for research in the period from the time of 
surgery until the patient reports to the outpatient clinic to receive the results of the first scan after 
the treatment (typically for a period of 3-4 months). These blood samples are taken in order to 
assess the effect of the treatment on the immune system for research purposes. The volume of 
blood, which will be taken for the study, does not surpass what the body itself can produce between 
each sampling. Research blood samples will not be taken, if the hemoglobin levels are not 
acceptable (> 6 mmol/l). 
 
Mononuclear cells from peripheral blood (PBMCs) are isolated using Lymphoprep/Leucosep 
density gradient technique. The mononuclear cells are washed and resuspended in a freezing 
medium consisting of 90 % heat inactivated human AB serum and 10 % DMSO. The cells are 
frozen at -150 oC until analysis. A panel of relevant immunological assays for testing of antigen-
specific immune reactivity will be applied, including measurement of cytokine production 
(multimer fluorescence staining, ELISPOT, ELISA), and proliferative and cytotoxic potential. 
 
Tumor biopsies 
Biopsy sampling from available tumor lesions or involved lymph nodes is planned. Depending on 
the localization and availability, excision-, punch-, crude needle-, or fine needle biopsies are taken. 
The biopsies are requested at the evaluation, 6 weeks after finished treatment, and in the case of 
progression, if possible (see the schedule for the examination plan and the schedule for follow-up). 
If the implicated areas are not readily accessible, the biopsy sampling will, if possible, take place 
under the guidance of ultrasound in sterile conditions at the ultrasound department at Herlev 
Hospital. The biopsy sampling will take place on an outpatient basis, and the biopsy size will here 
be approximately 5 mm3. 
 
Biopsies are examined for their content of immune cells. Further, TILs will be isolated from the 
lesions and analyzed for clonotype and specificity. 
 
Statistics 
 
This is a non-blinded, non-comparative study. Only descriptive statistics will be used, where the 
immunological and clinical response rate will be determined. Descriptive statistics will also be used 
to sum up response duration and patient characteristics. The study is designed as a pilot study and 
primarily aims at determining the safety and toxicity of the treatment. It is therefore not possible to 
formally determine a required sample size, which will allow determination of primary as well as 
secondary and tertiary end points. Based on experiences from previous clinical trials, inclusion of 
20 patients63 is deemed appropriate. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
59
Data registration and analysis 
The patient receives a patient number upon entry in the study in order to ensure patient anonymity. 
However, clinical personnel and selected individuals in the laboratory will be granted access to 
personal information, as this information is necessary to make sure the patient receives the correct 
treatment. The study responsible physician has access to the patient medical records to be able to 
request information about your illness, as this information shall be compared with the project 
specific analyses, which are made on cancerous tissue and blood samples. 
 
All relevant data are registered in an eCRF (electronic Case Report Form), which is created together 
with the KFE. The principal investigator is responsible for eCRF creation and typing of data into 
the eCRF, when the study treatment has been completed. The eCRFs will be reported to the 
sponsor. Sponsor and principal investigator are responsible for data analysis for all included 
patients. Patient data and eCRF will be stored for 5 years according to existing guidelines for the 
storage of person sensitive information. A final report is compiled in collaboration between the 
members of the project group. 
 
The analysis will include overviews of: 
• Toxicity (side effect registration) 
• Immunological response  
• Clinical effect parameters 
 
Upon termination of the study, person-referable data and any remaining samples will be coded. 
All patients, who receive T-cell infusion, will be included in the statistical analysis. Patients, who 
due to one of the following causes are excluded, will not be included in the statistical analysis:  
• Does not have enough tissue to produce TILs 
• Where TILs cannot be produced in the laboratory 
• Where inclusion criteria at step 2 before starting chemotherapy are not fulfilled 
• Withdraw their consent 
• Has started another treatment 
 
End of study report 
Sponsor will inform the Danish Medicines Agency that the study has been concluded within 90 
days. The study is deemed finalized at the last visit of the last patient. If the study is finished 
prematurely, the Danish Medicines Agency will also be informed with an explanation of the early 
ending. 
 
Within one year of the conclusion of the study, sponsor will submit a final study report with the 
study results including publications based on the study to the Danish Medicines Agency and the 
National Committee on Health Research Ethics. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
60
 
Amendments 
Permission to make substantial changes to the protocol should be applied for from the Danish 
Medicines Agency and the National Committee on Health Research Ethics, and these changes 
cannot be implemented until approval has been received.  
Changes in the protocol are considered substantial (substantial amendments), when they relate to 
• The safety of the study participants 
• The interpretation of the scientific documentation  
• The completion or management of the study 
• Quality and safety of the study drugs 
• Other substantial changes 
 
In the case of changes relating to  
• extension relative to the date stated in the original approval 
• new centers/changing of centers 
• changing of principal investigator or sponsor 
• when the study ends 
• other small changes apart from typographical errors 
the Danish Medicines Agency and the National Committee on Health Research Ethics will be 
informed, even though these authorities do not have to approve these changes. 
 
Ethical considerations 
Recruitment of study participants and informed consent  
Patients with metastatic or locally advanced MM will be referred to the clinical trial from the 
oncology or plastic surgery department at Herlev Hospital or from one of the other oncological 
centers in Denmark, which treat patients with MM (Odense University Hospital, Aarhus University 
Hospital). Information about the study will be provided at scientific meetings for physicians at the 
involved departments. Referral of patients for the study is directed to the urogynecological (UG-
team) visitation office, Department of Oncology at Herlev Hospital. 
 
All patients will be informed about the study cf. appendix 1. 
 
Insurance 
The patients, who participate in the study, are covered by the hospital liability insurance. 
 
 

 
 
 
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melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
61
Ethical aspects 
Malignant melanoma has through several years shown increasing incidence and has a bad 
prognosis, when the illness is disseminated. Metastasizing MM can rarely be cured by either 
surgery, radiotherapy, chemotherapy, or immunotherapy, and the need for new treatment modalities 
is therefore considerable. 
The purpose of this study is to improve the survival of patients with MM in the long term. Based on 
the current knowledge and the lack of treatment alternatives, there are no unacceptable risks or 
disadvantages associated with this study. 
 
Participation is voluntary and preceded by both verbal as well as written information, and the 
treatment will be stopped in the event of unacceptable side effects, or at any time point if the patient 
so wishes. If the patient does not want treatment according to the protocol, the patient will receive 
treatment according to the usual guidelines of the department. The study is therefore deemed 
ethically sound. 
 
The study follows the guidelines of the Declaration of Helsinki, and the study responsible physician 
will obtain permission from the National Committee on Health Research Ethics and the Danish 
Medicines Agency. 
 
Research biobank 
In relation to the present study, blood samples (110 ml/blood sample) and tumor biopsies will be 
taken and stored coded at -150 oC in a research biobank at CCIT in room PA102, until all analyses 
linked to the study have been made or for a maximum of 15 years, after which any remaining 
material will be safely destroyed. 
The samples may only be used for other research studies within other areas of research, if approved 
following a new application to the National Committee on Health Research Ethics. The National 
Committee on Health Research Ethics may according to the circumstances waive the requirement 
for consent in such future research projects. 
 
The analyses will primarily be performed at CCIT, Department of Oncology, Herlev Hospital. 
Some special analyses of tumor tissue and blood samples may be performed at a foreign research 
institution, which we will make a specific collaboration with, and an independent Data Processor 
Agreement will be made. All patient information will in such cases be provided in coded form. If 
tissue and blood samples are sent abroad, they will be covered by the national legislation of the 
country, they are sent to. If the patient withdraws his or her informed consent, the biological 
material will be destroyed, if the patient does not want the material to be used. 
 
Reporting to the Danish Data Protection Agency 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
62
The clinical trial is reported to the Danish Data Protection Agency. The law on treatment of 
personal data will be followed. Data pertaining to study subjects will be protected according to the 
law on treatment of personal data and the Health Act Section 3 regarding the legal position of 
patients. 
 
Administrative aspects and publication 
Patient identification 
After entry into the study the patient will receive a number. This number will be used to identify the 
patient and will be used on electronic Case Report Forms (eCRFs). Data and patient material will be 
handled in a coded form and confidentially. The number will be awarded sequentially according to 
entry in the protocol and is not based on the patient’s initials or birthdate. 
 
Publications 
The investigators are Inge Marie Svane, Rikke Andersen, Troels Holz Borch, and Marco Donia. 
Provided that the Vancouver rules, moreover, are fulfilled, the members of the project group will 
have shared copyright for the obtained results. Positive as well as negative results will be published 
in international scientific journals. Manuscripts will be prepared in collaboration between the 
investigators and the other members of the project group, with the investigators being primarily 
responsible for the preparation. The investigators will be co-authors on publications based on this 
study.  
The order of authors will be determined according to the contribution of each author. The use of 
data from the study, both written and verbal, such as for e.g. conference participation, teaching, or 
the like can only take place after approval from the investigators. The investigators are obliged to 
publish results from the study and are naturally also interested in the results being disseminated and 
implemented in the everyday clinical practice. Publication is expected to be completed during 2017. 
 
Economy 
The study is initiated by the National Center for Cancer Immune Therapy (CCIT-DK) in 
collaboration with the Department of Oncology, Herlev Hospital, and is financed in part by these 
two departments, by the Capital Region of Denmark’s research fund, and through operating and 
salary funds from private research foundations, which we continuously apply for. 
 
The Committees on Health Research Ethics will be informed, if/when further funding is obtained. 
None of the physicians at the participating departments have any financial interest in the study, and 
there are no financial gains related to the study for the departments or their personnel. There are no 
financial connections between the funding bodies and the investigators. 
The clinical trial is part of the study responsible physician Rikke Andersen’s PhD project. 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
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melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
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melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
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Donia, M. et al. Methods to Improve Adoptive T-Cell Therapy for Melanoma: IFN-γ 
Enhances Anticancer Responses of Cell Products for Infusion. J. Invest. Dermatol. (2012). 
doi:10.1038/jid.2012.336 
35. 
Tran, K. Q. et al. Minimally cultured tumor-infiltrating lymphocytes display optimal 
characteristics for adoptive cell therapy. J. Immunother. 31, 742–51 (2008). 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
66
36. 
Besser, M. J. et al. Minimally cultured or selected autologous tumor-infiltrating lymphocytes 
after a lympho-depleting chemotherapy regimen in metastatic melanoma patients. J. 
Immunother. 32, 415–23 (2009). 
37. 
Dudley, M. E. et al. CD8+ enriched “young” tumor infiltrating lymphocytes can mediate 
regression of metastatic melanoma. Clin. cancer Res. 16, 6122–31 (2010). 
38. 
Goff, S. L. et al. Tumor infiltrating lymphocyte therapy for metastatic melanoma: analysis of 
tumors resected for TIL. J. Immunother. 33, 840–7 (2010). 
39. 
Joseph, R. W. et al. Impact of clinical and pathologic features on tumor-infiltrating 
lymphocyte expansion from surgically excised melanoma metastases for adoptive T-cell 
therapy. Clin. cancer Res. 17, 4882–91 (2011). 
40. 
Somerville, R. P. T., Devillier, L., Parkhurst, M. R., Rosenberg, S. a & Dudley, M. E. 
Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the 
WAVE® bioreactor. J. Transl. Med. 10, 69 (2012). 
41. 
Gattinoni, L. et al. Removal of homeostatic cytokine sinks by lymphodepletion enhances the 
efficacy of adoptively transferred tumor-specific CD8+ T cells. J. Exp. Med. 202, 907–12 
(2005). 
42. 
Antony, P. a et al. CD8+ T cell immunity against a tumor/self-antigen is augmented by 
CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J. Immunol. 174, 
2591–601 (2005). 
43. 
Lægemiddelstyrelsen. Produktresume Cyclophosphamid. (2011). 
44. 
Lægemiddelstyrelsen. Produktresume Fludarabinphosphat. (2011). 
45. 
Nelson, B. H. & Willerford, D. M. Biology of the interleukin-2 receptor. Adv. Immunol. 70, 
1–81 (1998). 
46. 
Isaacs A, L. J. Virus interference. I. The interferon. Procedings R. Soc. London 147, 258–267 
(1957). 
47. 
Ascierto, P. a & Kirkwood, J. M. Adjuvant therapy of melanoma with interferon: lessons of 
the past decade. J. Transl. Med. 6, 62 (2008). 
48. 
Mozzillo, N. & Ascierto, P. Reduction of circulating regulatory T cells by intravenous high-
dose interferon alfa-2b treatment in melanoma patients. Clin. Exp. Metastasis 29, 801–5 
(2012). 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
67
49. 
Wang, W. et al. Impact of IFNalpha2b upon pSTAT3 and the MEK/ERK MAPK pathway in 
melanoma. Cancer Immunol. Immunother. 57, 1315–21 (2008). 
50. 
Weber, J. S. & Rosenberg, S. A. Modulation of Murine Tumor Major Histocompatibility 
Antigens by Cytokines in Vivo and in Vitro Modulation of Murine Tumor Major 
Histocompatibility Antigens by Cytokines in Vivo and in Vitro. 5818–5824 (1988). 
51. 
Garrido, F. & Algarra, I. MHC antigens and tumor escape from immune surveillance. Adv. 
Cancer Res. 83, 117–58 (2001). 
52. 
Anichini, A. et al. Association of antigen-processing machinery and HLA antigen phenotype 
of melanoma cells with survival in American Joint Committee on Cancer stage III and IV 
melanoma patients. Cancer Res. 66, 6405–11 (2006). 
53. 
Mendez, R. et al. HLA and melanoma: multiple alterations in HLA class I and II expression 
in human melanoma cell lines from ESTDAB cell bank. Cancer Immunol. Immunother. 58, 
1507–15 (2009). 
54. 
Verdegaal, E. M. E. et al. Successful treatment of metastatic melanoma by adoptive transfer 
of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with 
low-dose interferon-alpha. Cancer Immunol. Immunother. 60, 953–63 (2011). 
55. 
Jönsson, G. et al. Gene expression profiling-based identification of molecular subtypes in 
stage IV melanomas with different clinical outcome. Clin. cancer Res. 16, 3356–67 (2010). 
56. 
Robbins, P. F. et al. Mining exomic sequencing data to identify mutated antigens recognized 
by adoptively transferred tumor-reactive T cells. Nat. Med. 19, 747–52 (2013). 
57. 
U.S. Department of health and human services, National Institutes of Heallth, N. C. I. 
Common Terminology Criteria for Adverse Events ( CTCAE ) Version 4.0. 2009, (2010). 
58. 
Sundhedsstyrelsen. Sundhedstyrrelsens produktresume Cyclophosphamid. (2014). 
59. 
Sundhedstyrelsen. Sundhedsstyrelsens produktresume Fludarabinphosphat. (2013). 
60. 
Dudley, M. E. et al. Cancer regression and autoimmunity in patients after clonal repopulation 
with antitumor lymphocytes. Science (80-. ). 298, 850–4 (2002). 
61. 
Sundhedstyrelsen. Sundhedstyrelsens produktresume Interleukin-2. (2013). 
62. 
Eisenhauer, E. a et al. New response evaluation criteria in solid tumours: revised RECIST 
guideline (version 1.1). Eur. J. Cancer 45, 228–47 (2009). 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
68
63. 
Julious, S. a. Sample size of 12 per group rule of thumb for a pilot study. Pharm. Stat. 4, 
287–291 (2005).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
69
Appendix 1 
 
Guideline for providing verbal participant information  
The verbal information regarding the project ”T cell therapy in combination with interferon- for 
patients with metastasized malignant melanoma” will be provided at the Department of Oncology, 
Herlev Hospital. The information session will be carried out by the study responsible physicians. In 
connection with the verbal information, written information material will be handed out. The session 
will take place in compliance with the guidelines from the Committee on Health Research Ethics. 
 
Before the information session 
• a time and a place for the session must be booked 
• it should be stated that it constitutes an invitation to participate in a health science research 
project 
• the right to take time to consider the information and the opportunity to bring an assessor to 
the session should be stated 
 
The information session 
• must be carefully planned 
• must take place in an undisturbed setting and without interruptions 
• the participant must be given sufficient time to read the written information, listen to the 
verbal information, and ask questions 
• must include an understandable presentation of the research study without the use of 
technical or emotionally charged terms, and be provided in a considerate manner, adapted to 
the recipient’s individual preconditions regarding age, maturity, experience etc.  
• must contain information about possible predictable risks, side effects, complications, and 
disadvantages, as well as the fact that there may be unpredictable risks and burdens 
associated with participation in a health science research project. 
• must contain information about other treatment options 
• must contain information about the fact that information about health conditions, strictly 
private circumstances, and other confidential information may be passed on to and be 
handled by individuals, who have to carry out a legally required quality control of the study. 
• must contain information about the participants’ rights to renounce knowledge regarding 
their own health  
• the information is provided by the study responsible physician or by the thereto authorized 
person associated with the study 
 
Period of reflection and obtaining consent 
• The period of reflection depends on the nature of the study. As a rule, the period of 
reflection should be at least 24 hours. 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
70
• There should be a clear relationship between information and consent. This entails that the 
consent to study participation as a rule is given soon after the information has been 
provided, yet still taking into consideration the necessary period of reflection. 
 
After the information session, the study subject will be informed, 
• If during the study new information about effect, risks, side effects, complications, or 
disadvantages should arise. 
• If the research project’s experimental design should change considerably with respect to the 
safety of the study subject (pertains to study subjects who actively participate in the study). 
• If, during the completion of the research study, significant information about the health of 
the study subject should arise, unless the study subject unequivocally has expressed that he 
or she does not want this. 
• About the achieved results, as well as about possible consequences for the individual 
participant. This requires, that it is practically possible, and that the study subject wants this. 
• If the study is discontinued, the study subject should be informed about the cause of this. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
71
Appendix 2  
 
 
Patient characteristics and clinical results 
 
 
 
Patient characteristics and clinical results (unpublished) from studies of T cell therapy at 
CCIT/Department of Oncology, Herlev Hospital (clinicaltrials.gov identifier: NCT00937625). 
CR: complete response, PR: partial response, SD: stable disease, PD: progressive disease 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Patient ID
Age
Sex
AJCC 
stage
Previous 
Treatments
Infused Cells 
(x109)
CD8%
CD4%
d%
Infused CD8 
(x109)
IL-2
Response
Survival
Responders (n=11)
MM909.01
60
F
M1c
1 (IL2)
28,7
78,1
19,5
0,03
22,4
Low
CR (49 mo) NED
53 mo+
MM909.11
41
M
M1a
3 (IL2, Ipi, DC)
74,7
97
2,4
0,01
72,5
Low
CR (13 mo) NED
32 mo+
MM909.15
48
F
M1b
1 (IL2)
85,7
31,3
54,9
10,7
26,8
Intermediate
CR (27 mo+)
27 mo+
MM909.17
49
M
M1c
2 (IL2, Ipi)
142,5
24,5
72
2,5
34,9
Intermediate
PR (25 mo+) NED
25 mo+
MM909.20
65
F
M1c
3 (IL2, Ipi, Tem)
82
88,5
10,5
0,04
92,7
Intermediate
PR (12 mo)
19 mo+
MM909.24
56
M
M1a
3 (Ipi, IL2, Vem)
110
67
20
0,05
73,7
Intermediate
PR (15 mo+)
15 mo+
MM909.26
46
M
M1c
1 (Ipi)
131,2
93
5
0,05
122,0
Intermediate
PR (13 mo+)
13 mo+
MM909.31
65
M
M1c
2 (IL2, Ipi)
117
53
47
0,22
62,0
Intermediate
PR (11 mo)
12 mo+
MM909.22
60
F
M1c
2 (IL2, Ipi)
123
21,6
78,1
0,02
26,6
Intermediate
PR (8 mo+)
8 mo+
MM909.36
40
F
M1c
3 (IL2, Ipi, Vem)
120
54,7
43,8
0,6
65,6
Intermediate
PR (7 mo+)
7 mo+
MM909.42
68
F
M1c
2 (IL2, Ipi)
83
8,2
91
0
6,8
Intermediate
PR (2 mo+)
2 mo+
54 ± 4
2,1 ± 0,8
100 ± 33
56 ± 31
40 ± 31
1,3 ± 3
55 ± 35
Non Responders (n=13)
MM909.07
61
M
M1c
3 (IL2, CD137, DC)
12,7
89,2
9,8
0,17
11,3
Low
SD (5mo)
11 mo
MM909.03
62
M
M1c
2 (IL2, DC)
20
91,8
6,9
0,12
18,4
Low
SD (4mo)
11.5 mo
MM909.06
36
M
M1c
1 (IL2)
3,4
52,5
46,8
0,31
1,8
Low
PD
4.6 mo
MM909.02
47
M
M1b
2 (IL2, DC)
17,7
95,2
3,2
0,84
16,9
Low
PD
7 mo
MM909.16
60
M
M1c
2 (IL2, Ipi)
61,5
87,5
11,3
0,17
53,8
Intermediate
SD (4 mo)
25 mo+
MM909.18
51
M
M1c
2 (IL2, Ipi)
199,5
86,2
8,6
3,6
172,0
Intermediate
SD (4 mo)
5.4 mo
MM909.25
25
M
M1b
3 (Ipi, Il2, Ipi)
127
55
35
9,7
69,9
Intermediate
SD (4 mo)
5.5 mo
MM909.34
46
F
M1c
2 (IL2, Ipi)
110
92,2
4
0,93
101,4
Intermediate
SD (4 mo)
5 mo
MM909.37
36
F
M1c
2 (IL2, Ipi)
125
49,9
28,9
16,9
62,4
Intermediate
SD (3 mo)
6 mo+
MM909.40
53
M
M1c
3 (Ipi, IL2, Ipi)
78
27,7
57,8
9,53
21,6
Intermediate
SD (2 mo+)
2 mo+
MM909.14
43
F
M1c
4 (IL2, Ipi, Tem, DC)
85,7
17,3
82,1
0,25
14,8
Intermediate
PD
3 mo
MM909.29
52
F
M1c
4 (IL2, DC, Ipi, Vem)
81,6
37,3
10,1
51
30,4
Intermediate
PD
6 mo
MM909.27
62
F
M1b
3 (Ipi, IL2, Vem)
98
47,5
51,8
0,20
46,6
Intermediate
PD
4 mo
48 ± 8
2,5 ± 0,9
78 ± 56
64 ± 28
27 ± 25
7 ± 14
48 ± 47
Not Evaluated (n=2)
MM909.43
48
F
M1c
2 (IL2, Tem)
99
31,9
67,1
0,17
31,6
Intermediate
MM909.46
50
F
M1c
2 (IL2, Ipi)
75
31
54
13,5
23,3
Intermediate
Patient Characteristics
Treatment Characteristics
Clinical Outcome

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
72
Appendix 3 
 
Anti-tumor response in the TIL infusion product 
 
 
 
 
 
The TIL infusion product is incubated with autologous (arrow) or allogeneic tumor cell lines, after 
which the number of interferon (IFN)-γ and tumor necrosis factor (TNF)-α producing T cells are 
determined using intracellular cytokine staining. The number of reactive T cells, which are infused 
into the patient, is significantly correlated with clinical response. The reactivity of the infusion 
product has been analyzed for the first 21 patients. Donia M et al., J Invest Dermatol 2013, and 
CCIT, unpublished.  
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
73
Appendix 4 
 
CD8+ and CD4+ TIL cytokine production is increased by stimulation with 
autologous tumor pretreated with interferon-alpha. 
 
 
 
 
TILs were stimulated with autologous melanoma tumor cells in vitro for 4 hours (in the presence of 
brefeldin A). The figure shows the frequency of cytokine producing CD8+ (on the left) and CD4+ 
(on the right) TILs after stimulation with autologous tumor cells pretreated with nothing (control), 
Interferon-gamma (IFN-gamma), Interferon-alpha (IFN-alpha), and IFN-gamma + IFN-alpha, 
respectively. 
 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
74
Appendix 5 
ECOG-function status/Performance status 
 
ECOG PERFORMANCE STATUS* 
Grade 
ECOG 
0 
Fully active, no negative effects on activities of daily living (ADL). 
1 
Limited physically strenuous activity, but ambulatory and capable of performing 
light or sedentary work, e.g. light housework, office work. 
2 
Ambulatory and capable of taking care of him-/herself, but unable to do any 
kind of work. Ambulatory more than 50 % of waking hours. 
3 
Capable of taking care of him-/herself to a limited degree, bedridden or sitting 
more than 50 % of waking hours. 
4 
Completely disabled. Unable to take care of him-/herself. Always bedridden or 
sitting in a chair. 
5 
Dead 
* Published in Am. J. Clin. Oncol.: 
Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., McFadden, E.T., Carbone, P.P.: 
Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol 
5:649-655, 1982.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
75
Appendix 6 
Common Terminology Criteria for Adverse Events (CTCAE) version 4 
 
CTCAE57 is a descriptive terminology, which can be used when reporting side effects. The adverse 
events (AEs) are graded according to the following: 
 
• Grade 1: Mild AE 
• Grade 2: Moderate AE 
• Grade 3: Severe AE 
• Grade 4: Life-threatening AE or AE leading to disability  
• Grade 5: Death related to an AE 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
76
Appendix 7 
 
 
Guidelines for monitoring, dose modifications, and supportive treatment 
during infusion of interleukin-2 (decrescendo-regime) associated with T cell 
therapy 
 
 
 
 
 
Guidelines for protocol MM1413: 
T cell therapy in combination with peginterferon for patients with metastatic malignant melanoma 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Clinically responsible: 
Physician, PhD student Rikke Andersen 
Physician, PhD student Troels Holz Borch 
Professor, Chief Physician Inge Marie Svane 
 
Department of Oncology R 
Herlev Hospital 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
77
Table of contents 
 
TABLE OF CONTENTS .................................................................................................................................... 77 
BACKGROUND AND RATIONALE ............................................................................................................... 78 
TREATMENT ..................................................................................................................................................... 78 
DOSE MODIFICATION .................................................................................................................................... 78 
MONITORING DURING IL-2 TREATMENT ................................................................................................ 79 
GUIDELINES FOR SUPPORTIVE TREATMENT ........................................................................................ 80 
INFLUENZA-LIKE SYMPTOMS ............................................................................................................................. 80 
NAUSEA ............................................................................................................................................................ 80 
FLUID BALANCE/HYPOTENSION .......................................................................................................................... 81 
HEMATOPOIESIS ................................................................................................................................................. 84 
COAGULATION ................................................................................................................................................... 85 
SKIN ................................................................................................................................................................... 86 
DERMATOLOGICAL LONG-TERM SIDE EFFECTS ................................................................................................... 87 
THE CENTRAL NERVOUS SYSTEM ....................................................................................................................... 88 
LUNGS ................................................................................................................................................................ 89 
LIVER ................................................................................................................................................................. 90 
HEART................................................................................................................................................................ 90 
GASTROINTESTINAL TRACT ................................................................................................................................ 91 
CHRONIC TOXICITY ............................................................................................................................................ 92 
THYROID GLAND ................................................................................................................................................ 92 
LYMPHADENOPATHY ......................................................................................................................................... 93 
JOINT PAIN ......................................................................................................................................................... 93 
OTHER AUTOIMMUNE DISORDERS ...................................................................................................................... 94 
ACCOMPANYING MEDICATION ........................................................................................................................... 94 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
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Background and rationale 
T-cell based immunotherapy is an experimental treatment regime with tumor-specific T cells, which 
ex vivo have been activated to show a specific reaction towards the cancer cells. This experimental 
type of treatment has been tested on patients with advanced malignant melanoma, and in this setting 
both complete and partial disease regression has been registered, where 50 % of the patients 
achieved measurable disease reduction. 
Tumors are often infiltrated by large amounts of T cells (tumor-infiltrating lymphocytes), which 
specifically recognize tumor antigens but typically are inactive. It is possible in the laboratory to 
expand and reactivate such cytotoxic T cells for tumor cell killing. The patients’ own tumor tissue is 
prepared in the laboratory, so that the cytotoxic T cells with the largest activity towards autologous 
tumor cells are activated and expanded in vitro. To maintain an immunological response towards 
the tumor, activating cytokines must be available to the specific T cells. Many “irrelevant” T cells 
will competitively reduce the availability of these cytokines to the T cells in question. To create an 
environment, which facilitates a T-cell mediated anti-tumor response, it is necessary, in addition to 
the presence of many tumor-specific T cells, to reduce irrelevant T cells and eliminate regulatory T 
cells. 
 
Treatment 
The treatment consists of one week of lymphocyte-depleting chemotherapy with two days of 
cyclophosphamide (60 mg/kg IV) and five days of fludarabine phosphate (25 mg/m2 IV) followed 
by one infusion of tumor-infiltrating lymphocytes (TILs) (approximately 109-1010 cells). 
After one week of chemotherapy and infusion of T cells on day 0, treatment with interleukin 2 is 
commenced, consisting of a continuous intravenous infusion on day 0-2: 18 MU/m2 for 6 hours, 18 
MU/m2 for 12 hours, 18 MU/m2 for 24 hours, followed by 4.5 MU/m2 for 24 hours for 3 days. 
 
Dose modification 
The following treatment guideline only applies to IL-2. 
The listed toxicity gradings are based on CTC criteria (version 4.0). 
 
IL-2 is paused at the following toxicities: 
• Hypotension according to the instructions of this guideline 
• Newly arisen cardial arrhythmia, other than sinus tachycardia.  
• Suspicion of or verified AMI. 
• Agitation or persisting confusion. 
• Bilirubin increase grade III/IV. 
• Sepsis. 
• Resting dyspnea. 
• In the case of coagulation disturbances, defined as PP < 0.2.  

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
79
 
In the case of pausing of IL-2, the patient should be reassessed every second hour, and the treatment 
cannot be resumed, until the toxicity is reduced to grade 0/1. When the IL-2 infusion is resumed, the 
planned dose is administered, so that the time of treatment is prolonged corresponding to the 
treatment pause. If the pause exceeds 24 hours, the treatment is shortened with this exceedance, so 
that the treatment is maximally prolonged with 24 hours. 
 
Dose modification of IL-2 is primarily done with pausing. In the case of the following toxicities, IL-
2 can be resumed at 50 % dose. IL-2 cannot be resumed until the toxicity is reduced to grade 0 or 1. 
• In the case of an increase in creatinine grade III/IV 
• In the case of grade II or III cerebral toxicity. 
 
Contraindications for continued IL-2 treatment: 
• Documented myocardial ischemia or infarction. 
• Grade IV CNS toxicity.  
 
Monitoring during IL-2 treatment 
 (Please refer to the relevant sections in the guidelines for supportive treatment) 
• Vital functions: 
• Pulse/BP every fourth hour. 
• Temperature every eighth hour. 
• Diuresis every 12 hours. 
• Lung stethoscopy every 24 hours. 
• Weight every 24 hours. 
After treatment cessation, pulse/BP is measured for the last time after 4 hours, however BP is 
followed until normalization. 
 
Monitoring of patients with systolic BP below 80 mm Hg, who are not responding to volume 
treatment are followed with: 
• Pulse/BP every second hour. 
• Diuresis every second hour.  
• Lung stethoscopy every second hour. 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
80
Guidelines for supportive treatment 
 
Influenza-like symptoms 
During treatment with cytokines, the so called ”Flu-like Syndrome” usually arises, that is influenza-
like symptoms such as fatigue, joint pain, fever, and chills. These constitutional symptoms are 
triggered by IL-2 and Interferon through a number of secondary cytokines, in particular TNF and 
IL-1. The degree of these constitutional symptoms varies a lot from person to person. This may be 
due to genetic causes, as the TNF production is determined by a genetic polymorphism in the 
HLA region. 
 
The clinical picture 
The degree of influenza-like symptoms varies a lot. Frequently, pronounced fatigue presents, as 
well as routinely fever, where the maximal increase in temperature varies but where the temperature 
may be between 38 and 41 oC. In the case of a rapid increase in temperature, chills usually present. 
These side effects are usually not critical but can, however, be perceived by patients as burdensome. 
 
Treatment principles 
The fever is perceived as an effect and not a side effect of the treatment and therefore shall not be 
treated with antipyretic drugs such as paracetamol and NSAIDs. Only if the temperature surpasses 
41 oC, should treatment with paracetamol be initiated. It is of paramount importance, that the 
patients prior to the treatment are advised about these things, and that the fever is considered an 
important part of the treatment. It should be noted that opioids also have an antipyretic effect, and 
that their use therefore should be carefully considered. 
 
References 
• 
Blay, J.Y et al: Correlation between clinical response to interleukin 2 therapy and sustained production of 
tumor necrosis factor. Cancer Res. 1990. 
• 
Economou, J.S et al: Production of tumor necrosis factor α and interferon γ in interleukin-2-treated melanoma 
patients: correlation with clinical toxicity. Cancer Immunol. Immunother. 1991. 
• 
Edwards, M.J et al: Passive immunization against tumor necrosis factor inhibits interleukin-2-induced 
microvaascular alterations and reduces toxicity. Surg. 1992. 
• 
Hobohm U: Fever therapy revisited. British Journal of Cancer 2005. 
• 
Hoption Cann S.A: Dr William Coley and tumour regression: a place in history or in the future. Postgrad Med 
J 2003. 
 
Nausea 
The pathogenesis for IL-2 triggered nausea is unknown. 
 
The clinical picture 
 

 
 
 
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Nausea and vomiting can arise during IL-2 treatment. It can vary from single bouts of vomiting 
without nausea to nearly permanent severe nausea with frequent vomiting. On the contrary, loss of 
appetite is seen in nearly all patients. 
 
Treatment principles 
General guidelines cannot be given, as there are large individual differences in the occurrence of 
nausea and vomiting. Both centrally as well as peripherally acting antiemetics can be used. 
  
Treatment guideline 
The following drugs can be used in the case of nausea, administered either perorally or IV. 
Prednisolone may not be used due to its immunosuppressive effects. Vogalene should be avoided 
due to the risk of orthostatic hypotension. 
 
• Emend (Aprepitan)  
 
125 mg on day 1, 80 mg on day 2 and 3 
• Motilium (Domperidon) 
 
20 mg up to 4 times pr. day 
• Zofran (Ondansetron) 
 
16 mg for 24 hours 
• Kytril (Granisetron) 
 
1-2 mg/24 hours 
• Temesta (Lorazepam) 
 
1-2 mg  
 
References 
• 
Siegel, J.P et al: Interleukin-2 toxicity. J. Clin. Oncol. 1991 
 
Fluid balance/hypotension 
Interleukin-2 usually leads to fluid retention. In the majority of the older studies with high-dose 
intravenous infusion, the most frequent dose-limiting side effect was a complex disturbance in the 
fluid balance. Interleukin-2 affects the regulation of the fluid balance through the so-called 
“capillary leak syndrome” and through a direct effect on the kidney function. 
 
Reduced renal perfusion 
Independently of the capillary-leak associated hypovolemia, oliguria is seen upon infusion of IL-2.  
It is evoked by an effect on the renal perfusion. IL-2 leads via leukotrienes to a disturbed renal 
vasoregulation. The large preglomerular vessels respond with pronounced vasoconstriction and 
subsequent reduction of the glomerular perfusion. This effect counteracts the physiological 
renovascular autoregulation, which in the case of a reduction in the glomerular flow actually was 
supposed to lead to a dilation of the preglomerular vessels. The pronounced reduction in the 
glomerular flow leads to a reduced function of the glomeruli and thereby oliguria, increasing to 
anuria. 
 
Capillary leak syndrome 

 
 
 
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The underlying pathophysiology of the capillary leak syndrome is an increased permeability of the 
capillaries. There are several molecular mechanisms involved in this: in part an activation of 
endothelial cells via secondary cytokines, and in part a direct toxic effect of activated mononuclear 
cells, in particular on activated endothelial cells. The result is an increased permeability of the 
capillaries and an outflow of liquid and low molecular weight substances into the extravasal space. 
Hereby, interstitial edemas arise in nearly all organs, as well as an intravasal hypovolemia. 
 
The clinical picture 
The symptoms of the affected regulation of the fluid balance is complex. On one hand the capillary 
leak syndrome causes interstitial edemas and intravasal hypovolemia with subsequent arterial 
hypotonia. On the other hand, the rapidly developing renovascular autoregulatory disturbance leads 
to oliguria, fluid retention, and intravasal hypervolemia. Therefore, during infusion with IL-2 a 
reduced renal perfusion with oliguria and intravasal hypervolemia will occur. Later, as a 
consequence of the capillary leakage, an outflow of liquid to the extracellular space, edemas, and 
intravasal hypovolemia with consequent arterial hypotonia will occur (see below).  
 
The following is a schematic attempt to describe interleukin-2’s effect on the fluid balance: 
 
Reduced renal perfusion 
1) Reduced glomerular perfusion 
2) Oliguria 
3) Fluid retention 
4) Intravasal hypervolemia 
5) Edemas 
 
Capillary leak syndrome 
1) Increased capillary permeability 
2) Outflow of water, electrolytes, and albumin 
3) Interstitial edemas 
4) Intravasal hypovolemia 
5) Arterial hypotonia 
 
Edemas can arise in all organs and are partly the cause of some of the organ side effects mentioned 
below. Interstitial pulmonary edema may be particularly problematic. This, however, should not 
arise during adequate treatment. The disturbances in the fluid balance frequently become more 
complicated due to fever, peripheral vasodilation, and diarrhea. 
 
Treatment principles 
To avoid intravasal hypovolemia, sufficient fluids should be administered. Large fluid infusions 
over a short time should, however, be avoided, as a pulmonary edema may arise. A fluid retention 

 
 
 
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of approximately 5-10 % of the body weight may be necessary during a five-day IL-2 infusion to 
prevent intravasal hypovolemia and thereby arterial hypotonia. 
In the case of oliguria, dopamine or loop diuretics, which increase the glomerular perfusion, may be 
used. Both substances should be dosed correctly to maintain an adequate diuresis and may not be 
overdosed, as a too strong diuresis leads to volume depletion and thereby arterial hypotonia. 
NSAID drugs cannot be used simultaneously with the IL-2 treatment due to a risk of aggravation of 
renal insufficiency. 
 
Treatment guideline  
• Concurrent with the IL-2 treatment, infusion of potassium-sodium-glucose infusion fluid at 
100 ml/hour is administered for the entire IL-2 treatment period. 
• In the case of a drop in systolic blood pressure of more than 30 mm Hg or to below 90 mm 
Hg, the infusion speed of potassium-sodium-glucose infusion fluid is increased to 125 
ml/hour. 
• At systolic blood pressure < 80 mm Hg or a clinically affected patient, 2 x 1000 ml NaCl 
infusion is given within 2 hours. 
• If the blood pressure is not restored after the above, the IL-2 infusion is paused, and the 
patient is reassessed again after 2 hours with the aim to resume the IL-2 infusion. 
• If the systolic blood pressure continues to drop, and the patient is clinically shocked, the 
patient should be transferred to the intensive care unit for treatment with pressors. 
 
It is important, that the primary blood pressure, which is measured, and which forms the 
basis for assessment of blood pressure drop during treatment, is measured resting and 
without stress. 
 
Voluven or HES infusion fluid is not used anymore, after two large studies have indicated a 
negative effect on renal function, and the Danish study showed an increased mortality in the HES 
treatment group. 
 
In the case of hyponatremia below 130 mmol/L, NaCl is used instead of KNAG infusion in the 
same volumes. Hypokalemia can be compensated as usual according to the departmental 
instructions.  
 
At diuresis below 360 ml/12 hours, furosemide is administered as a continuous infusion at 120 
mg/24 hours. The infusion product is produced as two times furosemide 60 mg in 250 ml NaCl for 
infusion over 12 hours. The infusion is administered over a minimum of 24 hours and can be 
continued for as long as requested. Intravenous bolus infusion or tablet furosemide cannot be used.  
 
References 
• 
Cotran, R.S et al: Endothelial activation during interleukin-2 immunotherarpy. A possible mechanismen for 
the vascular leak syndrome. J. Immunol. 1987. 
• 
Keilholz, U.et al: Mechanism and clinical managemant of interleukin-2 indced renal toxicity and fluid 

 
 
 
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retention. Int.J. Immunophath. Pharmaol. 1993. 
• 
Ognibene, F.P et al: Interleukin-2 administration causes reversible hemodynamic changes and left ventricular 
dysfunction similar to those seen in septic shock. Chest 1988. 
 
Hematopoiesis 
During treatment with IL-2, a drop in all hematopoietic cell lines (granulocytes, thrombocytes, and 
erythrocytes) in the peripheral blood can be seen. It is not caused by, as in chemotherapy, a toxic 
effect on the hematopoiesis. The bone marrow is not hypoplastic, and nadir is seen already at 1-2 
days after cessation of the IL-2 infusion. The mechanism behind the hematopoietic toxicity is today 
not fully understood, probably it relates to an inhibition of the last steps of differentiation and an 
inhibited release of hematopoietic cells from the bone marrow through secondary cytokines. Unlike 
this, the lymphopenia, which usually presents after a few hours of IL-2 infusion, is a physiological 
reaction, as IL-2 activated lymphocytes in the peripheral blood express several adhesion antigens, 
which lead to adhesion to the capillary endothelium and subsequent extravasation. Upon cessation 
of IL-2 treatment the lymphopenia is reversible and is usually followed by lymphocytosis. These 
lymphocytes correspond to morphologically highly activated lymphatic cells, which can be 
confused with immature cells. After IL-2 infusion a pronounced eosinophilia is often seen, triggered 
by IL-5. The eosinophilia may in extreme cases constitute 50 % of the leukocytes. This 
phenomenon is not completely understood and has according to current knowledge no therapeutic 
or pathophysiological importance. 
 
The clinical picture 
As described above, a pronounced lymphopenia is seen during IL-2 infusion followed by 
lymphocytosis 1-2 days after cessation of treatment. Whether this has a clinical significance is not 
known. Anemia, thrombocytopenia, and granulocytopenia may occur, and among 10-20 % of 
patients, infusion of blood or thrombocytes is indicated. The hematological toxicity is reversible 
within a couple of days after cessation of IL-2 infusion. The granulocytopenia is very rarely serious 
but a functional disturbance of the granulocyte ability for chemotaxis may appear. This may lead to 
a reduced ability to fight bacterial infections, which predominantly is seen among patients with an 
intravenous catheter.  
 
Treatment principles 
Erythrocytes and thrombocytes may be substituted according to standard guidelines. Lympho- and 
granulocytopenia does not require treatment and does not warrant prophylactic antibiotics. All 
clinically uncharacteristic treatment courses should, however, lead to the consideration of bacterial 
infection. 
 
Upon suspicion of bacterial infection (e.g. unusually high fever, unusually pronounced tachycardia, 
disturbances in coagulation, or affected general condition) the patient should be treated as for a 
bacteremia. This is important, as many of the side effects of IL-2 are similar to a bacteremia. If a 
patient is still having a high fever 3 hours after pausing of IL-2, the patient should be treated as 

 
 
 
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having a bacteremia. Nephrotoxic antibiotics should be avoided due to the simultaneous 
nephrotoxicity.  
  
Treatment guideline 
Upon suspicion of bacterial infection: 
• Follow the usual procedures of the department 
• Nephrotoxic antibiotics should be avoided 
 
References 
• 
Clerigue, M et al: Effects of interleukin-2 and interleukin-2-activated cells on in vitro myelopoiesis. Clin. Exp. 
Immunol. 1990. 
• 
Huang, C.M et al: Changes in laboratory results for cancer patients treated with interleukin-2. Clin. Chem. 
1990. 
• 
Klempner, M.S et al: An acquired chemotactic defect in neutrophils from patients receiving interleukin-2 
immunotherapy. N. Engl. J. Med. 1990. 
• 
Wanebeo, H.J et al: Production of and response to interleukin-2 in peripheral blood lymphocytes of cancer 
patients. Cancer. 1986. 
 
Coagulation 
Activated monocytes and endothelial cells can produce and release coagulation promoting 
substances. Of central pathogenetic importance is here the induction of Factor 3, which activates the 
exogenous coagulation cascade. The coagulation disturbance can be even more complex, as the 
malignant condition in itself can lead to hypercoagulopathy. Finally, the synthesis of coagulation 
factors in the liver may be reduced by IL-2. 
 
The clinical picture  
During IL-2 treatment a drop in coagulation factor 2,7,10 (PP) may be seen. Clinically relevant 
coagulation disturbances, such as disseminated intravascular coagulation (DIC) or thromboembolic 
complications, are, however, only seen rarely. In the case of severe coagulation disturbances, the 
possibility of sepsis should always be considered, and the treatment adapted accordingly.  
 
Treatment principles 
To avoid disruption of IL-2 infusion, K-vitamin should be given intravenously at the following PP 
values. 
• At PP < 0.30 10 mg Konakion IV is given, this can be repeated until PP is above 0.30. 
• At PP < 0.20 Interleukin-2 treatment in paused. Konakion 10 mg IV is given, whereafter a new 
blood sample is taken after 2 hours, the injection can be repeated. The Interleukin-2 treatment 
can be resumed at 100 % dose, when PP is above 0.30.  
• In the case of abnormal bleeding tendency, which can be related to abnormal coagulation 
factors, fresh, frozen plasma (type-specific) can be given, contact e.g. the coagulation 
laboratory. 

 
 
 
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Patients treated with blood thinner will continue this treatment during the immunotherapy, and 
intervention is only needed, if PP falls below 0.20. In this case IL-2 is paused, and Konakion 1 mg IV 
may be used until PP is above 0.20, whereafter IL-2 can be resumed.  
 
References 
• 
Andreesen, R et al.: A new approach to adoptive immunotherapy of cancer using tumorcytotoxic macrophages 
grown from peripheral blood monocytes. Cancer Res. 1991. 
• 
Birchfiel, G.R et al: Hypoprothombinemia associates with interleukin-2 therapy; correction with vitamin K. J. 
Immunother. 1992. 
• 
Fleischmann, J.D et al: Fibrinolysis, thrombocytopenia, and coagulation abnormalities complicating high-dose 
interleukin-2 immonotherapy. J. Lab. Clin. Med. 1991. 
 
Skin 
Several of the secondary cytokines, which are induced by IL-2, lead to a generalized dilatation of 
the peripheral vessels, in particular in the skin and mucosa. Histologically numerous activated T 
lymphocytes are found underneath the basement membrane. It is unclear, if these cells represent a 
proliferation of already present subcutaneous lymphocytes, or if they are extravasated from the 
dilated capillaries in the skin. In the skin these activated T cells release additional cytokines, which 
enhance the inflammatory reaction. 
 
The clinical picture 
The most distinctive clinical manifestation is a pronounced erythema, which is seen in nearly all 
patients, who receive high-dose IL-2. The erythema, however, has no clinical significance. 
Inflammatory skin conditions with generalized pruritus and hives can develop. Further, it is known 
that autoimmune skin conditions, in particular psoriasis, are worsened during IL-2 treatment. 
 
Essential fatty acids to relieve skin problems 
Good symptomatic effects on skin dryness and pruritus has been found with plant oil based dietary 
supplements with the essential fatty acids omega-3, omega-6, and omega-9. This should be 
recommended to the patient as basis treatment alongside of skin lotion. The dietary supplements 
described below can be purchased in health food stores and other products can probably be used; 
however, types which contain the three mentioned types of essential fatty acids are recommended. 
The following products have so far been used by patients: “Perfect balance” or “Livets olie” 
(“Life’s oil”). The recommended daily dose is listed on the product. The ingestion of the oil is 
easiest if used together with beverages e.g. juice. For skin areas with annoying itching, the oil can 
additionally be applied directly to the skin a couple of times per day. 
 
Treatment principles 
The generalized erythema does not require treatment. In the case of pruritus and hives, systemic 
antihistamine products may be effective. Drug-induced exanthems, which may go unnoticed during 
IL-2 treatment, pose a particular problem. The same may be the case for reactions to radiocontrast 

 
 
 
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agent. Drug-induced exanthems may necessitate cessation of further IL-2 treatment. A few cases of 
severe exfoliative reactions have been described. It should here be noted that glucocorticoids block  
both side effects, as well as the antitumoral effect. In severe allergic reactions local glucocorticoids 
can be used, while systemic treatment is only considered if vitally indicated, simultaneously with 
cessation of the IL-2 treatment.  
 
Treatment guideline 
Dietary supplements with essential fatty acids should be used as basis treatment along with skin 
lotion, following the above guidelines. In the absence of effect hereof, the following may be used: 
• Itching: Tablet Atarax 25 mg as needed for maximally 5 days. In the absence of effect tablet 
Tavegyl or Zyrtec may be used. 
• Severe itching: Local glucocorticoid creams. 
• Systemic glucocorticoids may only be used if vitally indicated!  
 
References 
• 
Dummer, R et al: The skin: An immunoreactive target organ during interleukin-2 administration? 
Dermatologica  1991. 
• 
Gaspari, A.A et al: Dermatologic changes associated with interleukin-2 administration. JAMA 1987. 
• 
Staunton, M.R et al: Life-threatening bullous skin eruptions during interleukin-2 therapy. J. Nat. Canc.Inst. 
1991. 
 
Dermatological long-term side effects 
Dermatological long-term side effects are common. Most frequently they consist of dryness and 
itching of the skin, which may persist for months after treatment. Drug-induced exanthems, caused 
by the supportive medication, may be particularly long-lasting and annoying. Pre-existing 
autoimmune skin disorders are usually worsened. This is seen in particular for psoriasis and can 
develop into a psoriatic arthritis. One death has been reported of a patient with pemphigus, who 
shortly after IL-2 treatment had a lethal worsening of his/her skin condition. Of particular interest is 
the manifestation of vitiligo among melanoma patients. Vitiligo is an immunological reaction 
directed towards normal melanocytes but also towards melanoma cells due to a large shared antigen 
pool. Vitiligo is not seen during IL-2 treatment of patients with other tumors and is in melanoma 
patients associated with the effects of the immune therapy. Serious skin conditions such as 
pemphigus, lupus, and dermatomyositis are contraindications for IL-2 treatment.  
Good symptomatic effects on skin dryness and itching by plant oil-based dietary supplements with 
the essential fatty acids omega-3, omega-6, and omega-9 has been found. This should be 
recommended for patients as a basis treatment, alongside of moisturizing cream. The recommended 
daily dose is listed on the product. The ingestion of the oil is easiest if used together with beverages 
e.g. juice. For skin areas with annoying itching, the oil can additionally be applied directly to the 
skin a couple of times per day. 
 
Treatment guideline 

 
 
 
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• Skin dryness, itching: Symptomatic treatment (oil baths, moisturizing cream, essential fatty 
acid dietary supplements).  
• Psoriasis: confer with the department of dermatology, treatment with local glucocorticoids is 
permitted.  
 
References 
• 
Richards, J.M. et al: Sequential chemoimmunotherapie in the treatment of metastic melanoma. J. Clin. Oncol.,  
1992. 
• 
Staunton, M.R.et al: Life-threatening bullous skin eruptions during interleukin-2-therapy. J. Nat. Canc. Inst., 
1990. 
 
The central nervous system 
Effects on the central nervous system during high-dose IL-2 treatment are frequent and caused by 
several mechanisms. An interstitial edema may arise as a consequence of the disturbances in the 
fluid balance. Secondary cytokines have a direct as well as an indirect toxic effect on the CNS. The 
mechanism behind this is only partially known. Probably these compounds exert their effects 
through the central neurotransmitter system. Brain metastases represent a special problem, as the 
IL-2 mediated inflammation of brain metastases can lead to a peritumoral edema. This reaction, 
which in peripheral metastases only rarely give rise to problems, can in the brain lead to generalized 
seizures and in rare cases incarceration. 
 
The clinical picture 
Studies have shown that up to 40 % of the patients in high-dose IL-2 treatment have mental side 
effects. Clinically significant side effects, however, are rare but include the full spectrum of 
psychiatric disorders such as depression, hallucinatory psychoses, schizoaffective psychoses, and 
comatose conditions. Most frequent are sleep disturbances.  
 
Treatment principles 
Mild mental changes should not be treated but observed. More severe psychiatric conditions should 
lead to pausing, treatment with systemic glucocorticoids, and usually discontinuation of the 
treatment. It can be difficult to discern an IL-2 triggered depression from the reactive depression, 
which may arise due to the processing of the disease. In case of doubt, a psychiatric consultation 
may be necessary. The patient should prior to the treatment be informed that the treatment may 
trigger a depression, and that a previous endogenous depression may predispose to this. Patients 
with a clinically significant psychiatric condition cannot be offered treatment with IL-2.  
 
Treatment guideline 
In the case of lasting confusion, coma/cramps, severe motor weakness, or focal neurological 
deficits, the treatment is discontinued. Glucocorticoid treatment with Solumedrol 80 mg IV daily is 
initiated immediately and until the symptoms have disappeared. Additional cerebral CT/MRI-
imaging to check for cerebral metastases should be performed, if the symptoms do not decrease 

 
 
 
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within a couple of days. At degree II and III CNS toxicity treatment can after consultation with the 
treatment responsible and careful information of the patient be resumed at 50 % dose. At degree IV 
CNS toxicity, the treatment is discontinued.  
 
References 
• 
Denicoff, K.D et al: The neuropsychiatric effects of treatment with interleukin-2 and lymphokine-activated 
killer cells. Ann. Intern. Med. , 1987. 
• 
Denicoff, K.D et al: The neuroendocrine effects of interleukin-2 treatment. J. Clin. Endocrin. Metab. , 1989. 
 
Lungs 
An interstitial pulmonary edema may develop due to disturbances in the fluid balance. However, 
only rarely does it develop into a fulminant pulmonary edema. The cause is, as in a toxin provoked 
pulmonary edema, an increased capillary permeability. Therefore, no increase in pulmonary or 
central venous pressure is seen. The IL-2 provoked pulmonary edema can appear despite of 
hypovolemia. Unlike a cardiogenic pulmonary edema, the alveolar fluid is rich in albumin, and 
protein-rich pleural exudates may also appear.  
 
The clinical picture 
At high-dose IL-2 the clinical symptoms usually develop in the following order: 
• Clear respiratory sound 
• Light tachypnea increasing to hyperventilation (low pCO2, normal pO2) 
• Moist respiratory sounds 
• Respiratory insufficiency (drop in pO2) 
Existing lung conditions such as asthma bronchiale, chronic obstructive pulmonary disease, and 
restrictive lung disease can make the clinical picture very complex. Patients with these diseases 
should therefore not be treated with high-dose IL-2, and all patients with known lung disease or a 
smoking anamnesis should prior to treatment perform a pulmonary function test and have a forced 
expiratory volume for 1 second (FEV1) > 70 %. 
 
Treatment principles 
In the case of a mild pulmonary edema, the IL-2 infusion can be continued, if the diuresis can be 
increased, i.e. if there is no intravasal hypovolemia present. In this situation the mild pulmonary 
edema can be treated rapidly. A simultaneous intravasal hypovolemia is, however, an indicator for a 
considerable capillary leak syndrome and should lead to a pausing of the IL-2 infusion. In the case 
of resting dyspnea, the IL-2 infusion should be paused.  
 
Treatment guideline 
In the case of resting dyspnea, the following is done: 
• IL-2 injection is paused, and nasal oxygen is administered. 
• If the above is without effect, furosemide, 10-20 mg intravenously, is given. Coincidence of 

 
 
 
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dyspnea and hypotension should be expected, and the attention should therefore be directed 
towards simultaneous treatment thereof.  
• Depending on arterial puncture and clinical condition, high-pressure ventilation may be 
considered. If high-pressure ventilation is indicated, injection of Solumedrol 80 mg IV should 
be tried first, and may be repeated.  
• In the case of need for respiratory treatment/steroid the current treatment series is stopped, and 
the patient is reassessed after 2 weeks. 
 
References 
• 
Keilholz, U et al: Mechanismen and clinical management of interleukin-2 induced renal toxicity and fluid 
retention. Int. J. Immunopath. Pharmacol. , 1993. 
• 
Ognibene, F.P et al: Interleukin-2 administration causes reversible hemodynamic changes and left ventricular 
dysfunction similar to those seen in septic shock. Chest, 1988. 
 
Liver 
Interstitial edema and secondary cytokines probably play a role here, as well as hepatitis-like 
changes due to activated inflammatory cells, endothelium, and Kupffer cells.  
 
The clinical picture 
Frequently, during IL-2 treatment an increase in transaminases without a concurrent drop in liver 
function is seen. Simultaneously, an increase in alkaline phosphatase is seen. A mild liver synthesis 
disturbance is seen among 5-30 % of the patients. In particular, the synthesis of coagulation factors 
and albumin is of clinical importance. In the case of severely reduced liver function, a bacterial 
infection should always be considered. Further, see the section on coagulation. 
  
References 
• 
Fischer, B et al: Interleukin-2 induces profound reversible cholestasis: a detailed analysis in treated cancer 
patients. J. Clin. Oncol., 1989. 
• 
Huang, C.M et al.: Changes in laboratory results for cancer patients reated with interleukin-2. Clin. Chem. 
1990. 
 
Heart 
Arrythmia and myocardial infarction is described but the exact mechanism is unclear. Secondary 
mediators, as well as the interstitial edema associated with fever, may cause arrythmia. The 
triggering factors for acute myocardial infarction are probably pre-existing arteriosclerosis in 
connection with fever, endothelial activation through secondary cytokines, and possibly activation 
of the coagulation system. In addition, an IL-2 triggered myocarditis probably plays a role for the 
development of arrythmia and myocardial infarction (AMI). 
 
The clinical picture 

 
 
 
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All types of arrythmia may occur, including the febrile sinus tachycardia. In refractory sinus 
tachycardia and atrial flutter, hyperthyreosis should be considered in differential diagnostics. It may 
be triggered by an iodine-containing radiocontrast agent or be due to an IL-2 triggered thyroiditis. 
 
Treatment principles 
In clinically significant arrythmia or myocardial ischemia, the IL-2 infusion should be paused until 
restitution from the toxicity, and until the cardiac toxicity is clarified.  
 
Treatment guideline 
Upon suspicion of AMI, IL-2 treatment is stopped, and the treatment is paused, until AMI is 
confirmed or denied. Upon suspicion of AMI, ECG and cardiac enzymes are evaluated. The patient 
is moved to the coronary unit for continuous monitoring with scope. In the case of evidence of 
morbus ischaemicus cordis, the treatment is discontinued. Arrythmias are treated as usual.  
 
References 
• 
Goel, M et al: Reversible cardiomyopathy after high-dose interleukin-2 therapy. J. Immunol.1992 
• 
Kragel, A.H et al.: Myocarditis or acute myocardial infection associated with interleukin-2 therapy for cancer. 
Cancer , 1990 
• 
Lee, R.E. et al.: Cardiorespiratory effects of immunotherapy with interleukin-2. J. Clin.Oncol. , 1989 
• 
Nora, R et al.: Myocardial toxic effects during recombinant interleukin-2 therapy. J.Nat.Canc.Inst., 1989 
• 
Ravaud, A et al.: Cardiomyopathy after acute myocardial infarction after therapy with interleukin-2 and 
tumour infiltrating lymphocytes. Eur. J. Canc. , 1992 
• 
Shulman, K.L et al: Adverse reactions to intravenous contrast media in patients treated with interleukin-2. J. 
Immunother. 1993 
 
Gastrointestinal tract 
Three factors play a role in the gastrointestinal toxicity: Mucosal edema, motility disturbances, and 
a general stress impact. 
 
The clinical picture 
The most frequently affected organs are the ventricle and the colon. Gastritis and ventricular ulcers, 
which may lead to strong bleeding, have been described. Mucosal edemas of the colon prevent 
reabsorption of fluids, and a characteristic diarrhea presents: watery diarrhea, one to five times a 
day. A few cases of ventricular and colon perforation during IL-2 treatment have been described. 
The pathophysiology behind these very rare side effects is unknown. 
 
Treatment principles 
The treatment is in all cases symptomatic. Prophylactic treatment with antacids and H2 blockers is 
not recommended but can be used in ulcer anamnesis or in treatment-induced dyspepsia. The 
diarrhea is difficult to treat. Loperamide may give slower peristalsis, and the fluid reabsorption in 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
92
the colon may be improved to some degree. If larger losses of fluid occur from the gastrointestinal 
tract, these must be equalized in order to avoid intravasal hypovolemia and arterial hypotonia.   
 
Treatment guideline 
In the case of clinical suspicion of fungus, treatment is initiated according to the usual guidelines of 
the department. Also, general good oral hygiene. In the case of diarrhea, more than 3-4 loose stools, 
tablet Imodium 4 mg is administered, followed by Imodium 2 mg at each loose stool. In the case of 
gastritis, treatment with proton-pump inhibitors is used.  
  
References 
• 
Rahman, R et al: Unusual gastrointestinal complications of interleukin-2 therapy. J. Immunother. , 1991. 
• 
Schwartzentruber, D et al: Colonic perforation: An unusual complication of therapy with high-dose 
interleukin-2. Cancer, , 1988. 
• 
Sparano, J.A et al: Symptomatic exacerbation of Crohn disease after treatment with high-dose interleukin-2. 
Ann. Med., 1993. 
 
Chronic toxicity 
Unlike the acute IL-2 toxicity, which disappears shortly after discontinuation of the infusion, a 
number of side effects, which may last from weeks to months, and which may be worsened by 
continued immunotherapy, appear. These side effects consist mainly of autoimmune reactions, 
which are more frequent among patients, who respond to the treatment. It is possible, that this 
association is seen, because the immune system of these patients is more easily “stimulated”. More 
likely, however, it is that these autoimmune phenomena more frequently appear in responding 
patients, because these patients receive a longer-lasting treatment with IL-2. All chronic side effects 
usually disappear by themselves, and they respond well to glucocorticoid treatment. Treatment with 
glucocorticoids, however, should only be used on vital indication, as the effects on the tumor are 
also inhibited.  
 
Thyroid gland 
Autoimmune disorders in the thyroid gland are frequent, and subclinical conditions often become 
clinically manifest during IL-2 treatment. This is more common upon subcutaneous administration 
than by infusion. In a few studies of long-term treatment up to 50 % of participants show thyroid 
function disturbances, both hyper- and hypothyreosis. This, however, is significantly less frequent 
in high-dose IL-2 infusion and presents in between 1 and 20 %. Before, during, and after the 
treatment, thyroid parameters should be controlled (TSH, T3, and T4).  
 
Treatment guideline 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
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In the case of clinical disturbances in thyroid function, the endocrinology department should be 
consulted but the treatment may continue.  
 
References 
• 
Atkins, M.B et al: Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells. N. 
Eng. J. Med. , 1988. 
• 
Reid, I et al.: Thyroid dysfunction can predict response to immunotherapy with interleukin-2 and interferon-
2α.. 
• 
Sauter, N.P et al: Transient thyrotoxicosis and persistent hypothyroidism due to acute autoimmune thyroiditis 
after interleukin-2 and interferon-α therapy for metastatic carcinoma: A case report. Am. J. Med., 1992. 
• 
Wejl, N. I. et al: Hypothyroidism during immunotherapy with interleukin-2 is associated with antithyroid 
antibodies and response to treatment. J. Clin. Oncol. , 1993. 
 
Lymphadenopathy 
During IL-2 treatment a generalized lymphadenopathy with lymph nodes up to 4 cm in diameter is 
frequently seen. These reactive lymph node changes may be misinterpreted as tumor progression. 
Typically, the peripheral lymph nodes are not tender upon pressure. Lymphadenopathy may also be 
seen in a few locations and typically disappears after a couple of weeks.  
 
Treatment suggestions 
No treatment is necessary. The differential diagnosis between lymphadenopathy and progression 
may be difficult but by having a pause of at least 2 weeks between the last treatment and the 
evaluation, misinterpretation is avoided. 
 
Joint pain 
Transient joint pain is seen as a result of unspecific inflammatory reactions. Actual rheumatological 
joint disorders have so far only been observed among patients, who have been treated for a long 
time. Newly arised conditions, as well as exacerbations of existing conditions such as spondylosis, 
psoriatic arthropathy, and rheumatoid arthritis, have been observed. Usually the symptoms decrease 
for weeks to months after treatment discontinuation.  
 
References 
• 
Baron, N.W et al: Scintigraphic findings in patients with shoulder pain caused by interleukin-2. AJT, 1990. 
• 
Scheibenbogen, C et al: Rheumatic disease following immunotherapy. Ann. Rheum. Dis,. 1993. 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
94
Other autoimmune disorders 
In principle, all clinically relevant autoimmune disorders may be induced or worsened by IL-2 
treatment. In animal experimental studies, severe conditions such as rheumatoid arthritis, lupus 
erythematosus, myositis, nephrotic syndrome, and many others can be seen during long-term IL-2 
treatment. Autoantibodies are frequent, but do not all have clinical relevance. Further, potentially 
life-threatening autoimmune conditions represent a contraindication for IL-2 treatment.  
All autoimmune phenomena during IL-2 treatment can be treated with high-dose glucocorticoids 
but at the same time as this the potential therapeutic effect is abolished.  
 
Accompanying medication 
 
Pain treatment 
During the treatment with IL-2 and interferon, analgesics with antipyretic effect should as far as 
possible be avoided, cf. section on influenza-like symptoms. Opioids have, like paracetamol and 
NSAIDs, an antipyretic effect, and use during the immunotherapy should therefore be carefully 
considered.  
Treatment with NSAID simultaneously with IL-2 is contraindicated due to an increased risk of renal 
insufficiency. 
 
Tapering off of antihypertensive medication 
As IL-2 may lead to a considerable drop in blood pressure, in particular in patients in anti-
hypertensive therapy, a tapering off of all antihypertensive medication before treatment initiation is 
recommended. Antihypertensive medication is paused for as long as the treatment lasts. Most 
antihypertensive medications can be readily discontinued but one should pay attention to 2 groups 
of drugs: 
• Beta blockers. Fast tapering may lead to tachycardia and angina pectoris, why tapering 
should be done over 14 days. 
• ACE inhibitors/Angiotensin II-antagonists. May often be readily discontinued. If the 
indication is cardiac insufficiency, the patient should be referred to a cardiologist and the 
ejection fraction should be evaluated, as this should be normal.   
One may prior to treatment ask one’s own general practitioner to supervise the tapering and 
subsequent resumption of the treatment. 
 
 
 
 
 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
95
Appendix 8 
 
Febrile neutropenia during T cell therapy 
There is indication to initiate empirical antibiotic treatment in severely granulocytopenic (< 0.5 
bn/l) patients with fever (≥ 38.5 °C or ≥ 38.0 °C for ≥ 1 hour).  
Examinations at fever onset:  
• 
Culture for bacteria and fungi from:  
o blood (sterile glass vials) 
o urine for D+R 
o throat  
o potential foci of infection (e.g. wounds)  
• 
C-reactive protein, hematology, fluid numbers (in blood) 
• 
X-ray of thorax  
 
Treatment 
Initially  
o Piperacillin/Tazobactam (Tazocin) dose: 4 g/0.5 g every 8. hours IV.  
o Penicillin allergy: Ceftazidim (Fortum) (dose: 2 g / 8. hours IV).  
Initially, Gentamicin is not used in febrile neutropenia during T cell therapy, as the combination of 
gentamicin and IL-2 can exacerbate the nephrotoxicity. In the case of an aggravation of the clinical 
condition, supplemental Gentamicin can be used, however, at maximally 3 doses. 
 
In the case of positive cultures, possibly adjustment of the initial treatment.  
 
Evaluation on day 3  
Temperature dropping, clinical response:  
As a rule, the treatment is continued unchanged for at least another 4 days corresponding to 
a total of 7 days of treatment; however, so that the patient must have been afebrile for at 
least 2 days before discontinuation. In cases of continued signs of infection and in all cases 
of septicemia with Pseudomonas aeruginosa antibiotic treatment, however, is continued 
until the granulocyte number is > 0.5 bn/l. 
Temperature unchanged/increasing, clinical condition unchanged/aggravated:  
Repetition of initial examinations, supplemented with culturing for Aspergillus spp. from the 

 
 
 
Protocol MM1413: T cell therapy in combination with peginterferon for patients with metastatic malignant 
melanoma. Version 1.4 Date: 14.03.2016 EudraCT no. 2014-001420-29. The National Committee on Health 
Research Ethics, case no. 1402193. Danish Medicines Agency, journal no. 2014041626 
96
nasal vestibules. 
 
Necessary considerations: 
• Lung infiltrates: Bronchoalveolar lavage (BAL) with adjustment of treatment 
depending on findings. Possibly ordering of expectorate testing for Legionella-PCR, 
"atypical pneumonia titers" (LAT (Legionella antibody test), MPT (Mycoplasma 
pneumoniae test), and CKT (Chlamydia complement fixation test)), LUT 
(Legionella-antigen urine test), and CT scan of the thorax.  
• Signs of IV catheter infection: Addition of Vancomycin dose: 1 g/twelfth hour IV 
(dose reduction in case of renal insufficiency) 
• Signs of oral candidiasis: At this time decision should be made about indication to 
add empirical systemic antifungal therapy with Amphotericin B-dose or 
Caspofungin-dose: 70 mg IV day 1, thereafter 50 mg IV daily (dose reduction in 
liver insufficiency). 
 
No objective findings: Change of initial treatment to Meropenem-dose: 1 g/eighth hour IV. 
 
Evaluation on day 8 
In the case of continued fever, the examinations from day 3 are repeated, supplemented with: 
 
• Aspergillus galactomannan-antigen  
• CMV-antigen in blood (PCR-method)  
• CT scan of liver and spleen (chronic disseminated candidiasis):  
• Consider systemic antifungal therapy with Amphotericin B as monotherapy or in case of 
simultaneous suspicion of infection with Aspergillus spp.: Caspofungin-dose: 70 mg IV 
day 1, hereafter 50 mg IV daily (dose reduction in liver insufficiency).  
 
Switching from intravenous to peroral antibacterial therapy 
Can be considered in cases, where the granulocyte number is increasing, and the patient has become 
afebrile.  
The treatment is changed to: Penicillin 1 MIU x 3 perorally + Ciprofloxacin-dose: 500 mg x 2 
perorally. 
In the case of penicillin allergy, penicillin is replaced by Roxithromycin-dose: 150 mg x 2 perorally.