Conditions: Metastatic Ovarian Cancer
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 7
Healthy volunteers: no

Brief summary:
Adoptive T cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) has achieved impressive clinical results with durable complete responses in patients with metastatic melanoma. Recently, the investigators have completed a pilot study treating 6 patients with metastatic ovarian cancer. The TILs are isolated from patients own tumor tissue followed by in vitro expansion and activation for around 4-6 weeks. Before TIL infusion the patients receive 1 week of preconditioning chemotherapy with cyclophosphamide and fludarabine. After TIL infusion Interleukin-2 is administered to support T cell activation and proliferation in vivo.

The investigators recent pilot study has shown TIL therapy in patients with metastatic ovarian cancer to be feasible and tolerable. Mainly transient clinical responses where observed and therefore the investigators plan to combine TIL therapy with checkpoint inhibitors to potentially increase the clinical effect.

Detailed description:
Adoptive T cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) has achieved impressive clinical results with durable complete responses in patients with metastatic melanoma. Recently, the investigators have completed a pilot study treating 6 patients with metastatic ovarian cancer. The TILs are isolated from patients own tumor tissue followed by in vitro expansion and activation for around 4-6 weeks. Before TIL infusion the patients receive 1 week of preconditioning chemotherapy with cyclophosphamide and fludarabine. After TIL infusion Interleukin-2 is administered to support T cell activation and proliferation in vivo.

The investigators recent pilot study has shown TIL therapy in patients with metastatic ovarian cancer to be feasible and tolerable. Mainly transient clinical responses where observed and therefore the investigators plan to combine TIL therapy with checkpoint inhibitors to potentially increase the clinical effect.

Objectives:

To evaluate safety and feasibility when treating patients with metastatic ovarian cancer with ACT with TILs in combination with checkpoint inhibitors.

To evaluate treatment related immune responses To evaluate clinical efficacy

Design:

Patients will be screened with a physical exam, medical history, blood samples and ECG.

Patients will be treated with one dose of Ipilimumab 14 days before undergoing surgery to harvest tumor material for TIL production. Patients is admitted on day -8 in order to undergo lymphodepleting chemotherapy with cyclophosphamide and fludara starting day -7. On day -2 patients will start treatment with Nivolumab every 2 weeks for a total of 4 doses to increase the activity of the infused TIL product.

On day 0 patients receive TIL infusion and shortly after starts IL-2 stimulation with a daily subcutaneous dose for a total of 14 days.The patients will followed until progression or up to 5 years.

Arms:
arm 1: All patients receive the same treatment. All patients are treated with one dose of Ipilimumab 14 days prior to surgical removal of tumor tissue for TIL expansion. Hospitalization for TIL treatment is approximately 3 weeks.

The patients are admitted to hospital on day -8 and receive lymphodepleting chemotherapy (cyclophosphamide and fludarabine= on day -7 to day -1. The first of 4 doses of Nivolumab is administered on day -2 and every 2 weeks for at total of 4 doses.

The TILs are infused on day 0 and Interleukin-2 therapy is administered on day 0 to day 13.

Interleukin-2 is administered as a daily low-dose subcutaneous injection for a total for 14 days.

Interventions: intervention 1: Cyclophosphamide intervention 2: Fludarabine intervention 3: TIL infusion intervention 4: Interleukin-2 intervention 5: Ipilimumab intervention 6: Nivolumab
Intervention descriptions: intervention 1: Cyclophosphamide 60 mg/kg is administered i.v. on day -7 and day -6. intervention 2: Fludarabine 25 mg/m2 is administered on day -5 to day -1. intervention 3: The maximum number of expanded TILs are infused over 30-45 minutes on day 0. intervention 4: Interleukin-2 is administered as a daily low-dose subcutaneous injection of 2 MIU for a total of 14 days. intervention 5: One dose of Ipilimumab 3 mg/kg is administered 14 days prior to surgical removal of tumor tissue for TIL expansion. intervention 6: Nivolumab 3 mg/kg is administered on day -2 before TIL infusion and every 2 weeks for a total of 4 doses.

Protocol document:
Prot_SAP_000.pdf: 
T-cell Therapy in Combination with Checkpoint 
Inhibitors for Patients with Advanced Ovarian-, 
Fallopian Tube- and Primary Peritoneal Cancer. 
 
 
 
A phase I/II study 
 
 
EudraCT nr.: 2017-002179-24 
 
 
The study will be conducted as described in this protocol and according to Good Clinical 
Practice (GCP) guidelines and regulatory requirements. The investigator allows direct access 
to data sources/documents (including patient charts) for monitoring, audit and/or inspection 
from the Danish Medicines Agency, GCP-units or other national health authorities. 
 
 
Sponsor 
 
Inge Marie Svane, professor, M.D., ph.d. 
 
Head of Center for Cancer Immune Therapy (CCIT) 
Department of Hematology (54P4) and 
Department of Oncology (54B1) 
Herlev Hospital, University of Copenhagen 
Date: _______________________________ 
Herlev Ringvej 75, 2730 Herlev 
Phone: 38 68 21 31  
 
 
Inge Marie Svane: ____________________ 
 
 
Principal Investigator 
Anders Kverneland, M.D., PhD-student 
 
 
Center for Cancer Immune Therapy (CCIT) 
Department of Hematology (54P4) and 
Department of Oncology (54B1) 
Herlev Hospital, University of Copenhagen 
Date: _______________________________ 
Herlev Ringvej 75, 2730 Herlev 
Phone: 38 68 64 67  
 
Anders Kverneland:______________________ 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
2
Project Managers 
 
Sponsor 
Inge Marie Svane, professor, M.D., PhD 
Head of Center for Cancer Immune Therapy (CCIT) 
Department of Hematology and Oncology 
Herlev Hospital, University of Copenhagen 
Herlev Ringvej 75, 2730 Herlev 
Phone: 38 68 21 31 
 
 
 
Principal Investigator 
Anders Kverneland, M.D., PhD-student 
 
Center for Cancer Immune Therapy (CCIT) 
Department of Hematology and Oncology 
Herlev Hospital, University of Copenhagen 
Herlev Ringvej 75, 2730 Herlev 
Phone: 38 68 64 67 
 
 
Sub-investigators 
Marco Donia, M.D., PhD 
Center for Cancer Immune Therapy (CCIT) 
Department of Hematology and Oncology 
Herlev Hospital, University of Copenhagen 
Herlev Ringvej 75, 2730 Herlev 
Phone: 38 68 14 56 
 
Additional collaborators 
Ôzcan Met, MSc, PhD 
Center for Cancer Immune Therapy (CCIT) 
Department of Hematology and Oncology 
Herlev Hospital, University of Copenhagen 
Herlev Ringvej 75, 2730 Herlev 
Phone: 38 68 92 29 
 
Marie Christine Wulff Westergaard, PhD-student 
Center for Cancer Immune Therapy (CCIT) 
Department of Hematology and Oncology 
Herlev Hospital, University of Copenhagen 
Herlev Ringvej 75, 2730 Herlev 
Phone: 38 68 91 83 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
3
 
Ulla Peen, M.D., PhD 
Herlev Hospital, University of Copenhagen 
Herlev Ringvej 75, 2730 Herlev 
Phone: 38 68 22 87 
 
Trine Juhler-Nøttrup, M.D., PhD 
Department of Oncology 
Rigshospitalet, University of Copenhagen 
Blegdamsvej 9, 2100 Kbh Ø 
 
Monitoring 
 
GCP unit 
GCP-unit at University of Copenhagen 
Bispebjerg Hospital, Bygning 51, 3.sal 
Bispebjerg Bakke 23 
2400 København NV 
Phone: 35 31 38 90 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
4
Table of Contents 
 
PROJECT MANAGERS ...................................................................................................................................................................... 2 
MONITORING .................................................................................................................................................................................... 3 
TABLE OF CONTENTS ..................................................................................................................................................................... 4 
LIST OF ABBREVIATIONS ............................................................................................................................................................. 8 
SYNOPSIS ......................................................................................................................................................................................... 10 
INDICATION AND TREATMENT ........................................................................................................................................................................... 10 
RATIONALE ........................................................................................................................................................................................................... 10 
PURPOSE ................................................................................................................................................................................................................ 10 
STUDY DESIGN ...................................................................................................................................................................................................... 11 
POPULATION ......................................................................................................................................................................................................... 11 
TOXICITY ............................................................................................................................................................................................................... 12 
EVALUATION OF CLINICAL RESPONSE ............................................................................................................................................................... 12 
IMMUNOLOGICAL RESPONSE EVALUATION ...................................................................................................................................................... 12 
INTRODUCTION AND RATIONALE .......................................................................................................................................... 13 
OVARIAN CANCER ................................................................................................................................................................................................ 13 
TUMOR IMMUNOLOGY ......................................................................................................................................................................................... 13 
TUMOR INFILTRATING LYMPHOCYTES (TILS) ................................................................................................................................................ 13 
T-CELL THERAPY .................................................................................................................................................................................................. 13 
CHECKPOINT INHIBITION .................................................................................................................................................................................... 16 
CCITS EXPERIENCE WITH T-CELL THERAPY ................................................................................................................................................... 17 
Clinical trials ................................................................................................................................................................................................ 17 
Translational research ............................................................................................................................................................................ 18 
THE RATIONALE OF THE DRUGS USED IN THE STUDY ..................................................................................................................................... 19 
Lymphocyte depleting chemotherapy ................................................................................................................................................ 19 
Cyclophosphamide ..................................................................................................................................................................................... 19 
Fludarabine phosphate ............................................................................................................................................................................ 20 
Interleukin 2 ................................................................................................................................................................................................. 20 
Checkpoint inhibition in combination with T-cell therapy ........................................................................................................ 20 
Ipilimumab ................................................................................................................................................................................................... 21 
Nivolumab ..................................................................................................................................................................................................... 21 
THE RATIONALE OF THE TREATMENT REGIMEN USED IN THE STUDY ......................................................................................................... 21 
PURPOSE AND HYPOTHESIS ..................................................................................................................................................... 22 
PRIMARY................................................................................................................................................................................................................ 22 
SECONDARY........................................................................................................................................................................................................... 22 
STUDY DESIGN ............................................................................................................................................................................... 22 
STUDY POPULATION ................................................................................................................................................................... 24 
CRITERIA OF IN- AND EXCLUSION ...................................................................................................................................................................... 24 
Criteria of inclusion ................................................................................................................................................................................... 24 
Criteria of exclusion .................................................................................................................................................................................. 25 
EVALUATION BEFORE INCLUSION ...................................................................................................................................................................... 25 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
5
TREATMENT PLAN ............................................................................................................................................................................................... 26 
EXAMINATION PLAN DURING THE COURSE OF TREATMENT .......................................................................................................................... 27 
MEDICINAL PRODUCTS USED IN THE STUDY .................................................................................................................................................... 28 
Ipilimumab ................................................................................................................................................................................................... 28 
Cyclophosphamide ..................................................................................................................................................................................... 29 
Fludarabine phosphate ............................................................................................................................................................................ 29 
Nivolumab ..................................................................................................................................................................................................... 29 
TILs .................................................................................................................................................................................................................. 29 
Interleukin-2 ................................................................................................................................................................................................ 29 
Pegfilgratim ................................................................................................................................................................................................. 30 
GUIDELINES FOR CONCOMITANT TREATMENT ................................................................................................................................................ 30 
END OF TREATMENT ............................................................................................................................................................................................ 32 
Normal end of treatment including follow-up ................................................................................................................................ 32 
Follow-up schedule .................................................................................................................................................................................... 32 
Early termination of treatment ............................................................................................................................................................ 33 
Subsequent treatment .............................................................................................................................................................................. 34 
PRODUCTION OF TILS ................................................................................................................................................................. 34 
ACQUISITION OF TUMOR TISSUE ........................................................................................................................................................................ 34 
ESTABLISHMENT OF ”YOUNG TIL” CULTURES ................................................................................................................................................ 34 
RAPID EXPANSION PROTOCOL (REP) ............................................................................................................................................................. 34 
HANDLING AND TRANSPORTATION OF THE INFUSION PRODUCT ................................................................................................................. 35 
PHENOTYPE- AND CLONOTYPE DETERMINATION ........................................................................................................................................... 35 
ADVERSE EVENTS, POTENTIAL RISKS AND PRECAUTIONS .......................................................................................... 35 
ADVERSE EVENTS ................................................................................................................................................................................................ 35 
Grading of Adverse Events ...................................................................................................................................................................... 35 
Serious Adverse Events ............................................................................................................................................................................ 36 
GUIDELINES FOR ADVERSE EVENTS POSSIBLE RELATION TO THE TREATMENT ......................................................................................... 36 
Adverse Reactions ...................................................................................................................................................................................... 37 
Reporting of Adverse Events and Adverse Reactions ................................................................................................................... 37 
KNOWN ADVERSE REACTIONS .......................................................................................................................................................................... 38 
Chemotherapy ............................................................................................................................................................................................. 38 
Cyclophosphamide ..................................................................................................................................................................................... 38 
Fludarabine phosphate ............................................................................................................................................................................ 38 
Checkpoint inhibition and irAEs .......................................................................................................................................................... 38 
Ipilimumab ................................................................................................................................................................................................... 39 
Nivolumab ..................................................................................................................................................................................................... 39 
TILs .................................................................................................................................................................................................................. 39 
Interleukin-2 ................................................................................................................................................................................................ 39 
RISKS AND DISADVANTAGES REGARDING SURGERY AND TEST SAMPLING .................................................................................................. 40 
Risks associated with removing tumor tissue ................................................................................................................................. 40 
Risks associated with biopsies ............................................................................................................................................................... 40 
Risks associated with blood tests ......................................................................................................................................................... 40 
MONITORING AND PRECAUTIONS ...................................................................................................................................................................... 40 
Hematological parameters .................................................................................................................................................................... 40 
Kidney- and urine infections .................................................................................................................................................................. 40 
Cardiotoxicity .............................................................................................................................................................................................. 40 
Immune-related toxicity .......................................................................................................................................................................... 41 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
6
Infertility........................................................................................................................................................................................................ 41 
Live vaccines ................................................................................................................................................................................................ 41 
Interactions .................................................................................................................................................................................................. 41 
EFFECT EVALUATION, DATA PROCESSING AND MONITORING ................................................................................... 41 
EFFECT EVALUATION ........................................................................................................................................................................................... 41 
Primary effect parameter ....................................................................................................................................................................... 41 
Secondary effect parameters ................................................................................................................................................................. 42 
RESPONSE CRITERIA ........................................................................................................................................................................................... 42 
RECIST ............................................................................................................................................................................................................ 42 
IMMUNOLOGICAL MONITORING ......................................................................................................................................................................... 42 
Blood samples for immunological monitoring ............................................................................................................................... 42 
Tumor biopsies ............................................................................................................................................................................................ 43 
Specimens for Future Biomedical Research .................................................................................................................................... 43 
ADDITIONAL RESEARCH ANALYSES ................................................................................................................................................................... 43 
Isolation of tumor cells ............................................................................................................................................................................ 43 
Tumor-associated lymphocytes (TALs) and chemokines ........................................................................................................... 44 
Cytokine Release Assay ............................................................................................................................................................................ 44 
Gene-based arrays ..................................................................................................................................................................................... 44 
STATISTICS ............................................................................................................................................................................................................ 45 
DATA REGISTRATION AND -ANALYSIS ............................................................................................................................................................... 45 
END OF STUDY REPORT ....................................................................................................................................................................................... 46 
AMENDMENTS ...................................................................................................................................................................................................... 46 
ETHICAL ASPECTS ........................................................................................................................................................................ 47 
RECRUITMENT OF STUDY PATIENTS AND INFORMED CONSENT ................................................................................................................... 47 
INSURANCE ............................................................................................................................................................................................................ 47 
ETHICAL ASPECTS ................................................................................................................................................................................................ 47 
RESEARCH BIOBANK, INCLUDING FUTURE BIOMEDICAL RESEARCH BIOBANK ......................................................................................... 48 
REPORTING TO THE RESEARCH ETHICS COMMITTEE .................................................................................................................................... 49 
ADMINISTRATIVE ASPECTS AND PUBLICATION ............................................................................................................... 49 
PATIENT IDENTIFICATION .................................................................................................................................................................................. 49 
PUBLICATIONS ...................................................................................................................................................................................................... 49 
ECONOMY .............................................................................................................................................................................................................. 49 
REFERENCE LIST ........................................................................................................................................................................... 50 
APPENDIX 1 .................................................................................................................................................................................... 55 
RETNINGSLINJER FOR AFGIVELSE AF MUNDTLIG DELTAGERINFORMATION ............................................................................................... 55 
APPENDIX 2 .................................................................................................................................................................................... 57 
ECOG-FUNKTIONSSTATUS/PERFORMANCE STATUS ..................................................................................................................................... 57 
APPENDIX 3 .................................................................................................................................................................................... 58 
COMMON TERMINOLOGY CRITERIA FOR ADVERSE EVENTS (CTCAE) VERSION 4 .................................................................................. 58 
APPENDIX 4 .................................................................................................................................................................................... 59 
VEJLEDNING TIL MONITORERING, DOSISMODIFIKATIONER OG UNDERSTØTTENDE BEHANDLING UNDER INFUSION AF INTERLEUKIN-
2 (DECRESCENDO-REGIMET) I FORBINDELSE MED T-CELLE TERAPI ........................................................................................................... 59 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
7
APPENDIX 5 .................................................................................................................................................................................... 82 
FEBRIL NEUTROPENI UNDER T-CELLE TERAPI ................................................................................................................................................ 82 
APPENDIX 6 .................................................................................................................................................................................... 90 
VURDERING OG HÅNDTERING AF BIVIRKNINGER RELATERET TIL IMMUNTERAPI MED CHECKPOINT HÆMMERE. ............................... 90 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
8
List of Abbreviations 
ACT = Adoptive Cell Therapy 
AE = Adverse Event 
ALAT = Alanine-Aminotransferase  
AR = Adverse Reaction 
ASAT = Aspartaet-Aminotransferase 
BP = Blood Pressure 
CA 125 = Cancer Antigen 125 
CCIT = Center for Cancer Immune Therapy 
CD3 = Cluster of differentiation 3 
CD4+ Cells = Helper T cells 
CD8+ Cells = Cytotoxic T cells 
CR = Complete Response 
CRP = C-Reactive Protein 
CTC = Common Toxicity Criteria 
CTCAE = Common Terminology Criteria for Adverse Events 
CTLA-4 = Cytotoxic T-lymphocyte-assosiated antigen 4 
Cy = Cyclophosphamide 
DMSO = DiMethyl SulfOxide 
eCRF = Elektronic Case Report Form 
ELISA = Enzyme-Linked ImmunoSorbent Assay 
ELISpot = Enzyme-Linked ImmunoSpot 
EMA = European Medicines Agency 
GM-CSF = Granulocyte-macrophage colony stimulating factor 
HLA = Human Leukocyte Antigen 
IFN-γ = Interferon-gamma 
IMPD = Investigational Medical Product Dossier 
IL-2 = Interleukin-2 
irAE = Immune related adverse events 
KFE = Klinisk forskningsenhed / Clinical Research Unit  
LDH = Lactate dehydrogenase 
MM = Malignant melanoma = skin cancer 
NSCLC = Non small cell lung cancer 
OC = Ovarian cancer 
ORR = Overall Response Rate 
P = Pulse 
PD = Progressive Disease 
PD-1 = Programmed cell death protein 1 
PD-L1 = Programmed cell death-ligand 1 
PD-L2 = Programmed cell death-ligand 2 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
9
PET =Positron Emission Tomography  
PR = Partial Response 
PS = Performance status, ECOG scale 0-4 
RECIST = Response Evaluation Criteria In Solid Tumours 
REP = Rapid Expansion Protocol 
SAE = Serious Adverse Event 
SAR = Serious Adverse Reaction 
SCLC = Small cell lung cancer 
SD = Stable Disease 
SUSAR = Suspected Unexpected Serious Adverse Reaction 
TAA = Tumor Associated Antigens 
TAL = Tumor Associated Lymphocytes 
TIL = Tumor Infiltrating Lymphocytes 
TNF = Tumor Necrosis Factor 
VEK = Videnskabsetisk Komité / Health Research Ethics Committee 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
10 
Synopsis 
Indication and treatment 
Patients with advanced ovarian-, fallopian tube-, and primary peritoneal cancer will be 
treated in this phase I/II study. The treatment involves treatment with the cytotoxic T-
lymphocyte-associated antigen 4 (CTLA-4) antibody Ipilimumab before surgical removal of 
tumor tissue, administration of lymphodepleting chemotherapy followed by infusion of 
tumor-infiltrating lymphocytes (TILs) isolated from removed tumor tissue, subsequent 
treatment with the programmed cell death protein 1 (PD-1) antibody Nivolumab as well as 
subcutaneous injections with the immune-stimulating cytokine, interleukin-2 (IL-2).  
Rationale 
T-cell therapy is an experimental personalized immunotherapy where TILs are isolated from 
the patient’s own tumor tissue, expanded in vitro to billions of cells and then administered to 
the individual patient with the purpose of eliminating the remaining cancer cells. 
Lymphodepleting chemotherapy with cyclophosphamide and fludarabine phosphate is 
administered to the patient before TIL infusion to reduce the number of irrelevant immune 
cells- as well as regulatory T cells known to inhibit T-cell mediated cancer cell killing. IL-2 is 
administered after TIL administration to activate and stimulate further proliferation of the 
infused TILs.  
At Center for Cancer Immune Therapy (CCIT), we have shown that adoptive cell therapy 
(ACT) with TILs and IL-2 stimulation in accordance with the decrescendo regimen for patients 
with advanced ovarian cancer (OC) is feasible and tolerable (Pedersen M et al., manuscript in 
preparation). 
CTLA-4 is involved in the regulation of the early stages of T-cell activation while PD-1 
predominantly potentiates the effector functions of T cells at the tumor site. It is therefore 
hypothesized that targeting both these two receptors will lead to a positive and synergistic 
effect on the efficacy of TILs1. 
Subcutaneous IL-2 in a low dosage is given to reduce the risk of toxicity when combining the 
treatment with a PD1 antibody.  
Purpose 
The primary objective is to evaluate the tolerability and safety of the treatment. The 
secondary objective is to characterize antitumor immune responses (immune monitoring) as 
well as to assess the clinical effect of the treatment by use of the objective response rate 
(using RECIST 1.1). Also, Cancer antigen 125 (CA-125) meassurements will be done. 
Assessment with PERCIST and irRC will be done exploratively. In addition, overall survival 
(OS) and progression-free survival (PFS) will be described, but not included as endpoints. 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
11 
Study design 
The trial is an open label phase I/phase II study.  
Patients will be included and treated at the Department of Oncology at Herlev Hospital. 
Patients can be referred from other oncology or gynecology centers. 
 
All eligible patients will be treated in the outpatient clinic with one dose of Ipilimumab, 2-6 
weeks before surgical removal of tumor tissue for TIL manufacturing. TIL administration, IL-2 
injections and the start of Nivolumab treatment will take place during hospital admission. It is 
anticipated that it will take approximately 4-6 weeks from the tumor tissue is removed for TIL 
manufacturing to the start of treatment. Hospitalization and treatment will span 
approximately 3 weeks with Nivolumab treatment continuing after discharge in the 
outpatient clinic for a total of 4 series. T-cell therapy will be administered only once 
In some cases, TILs will be isolated from tumor tissue in advance and cryo-preserved for later 
treatment. After the end of treatment, the patients will be followed with clinical- and imaging 
controls for up to 5 years in a specialized immunotherapy unit at the Department of Oncology, 
Herlev Hospital. Patients will be excluded upon clinical or radiological progression.  
The inclusion period is expected to be approximately 2 year, starting from July 2017. We 
expect all patients to have finished treatment and 6 months follow-up within three years. 
The study will be monitored by the Good Clinical Practice (GCP)-unit, and reported to the 
Danish Medicines Agency, the Research Ethics Committee and the Danish Data Protection 
Agency. 
Population 
Patients with histologically verified advanced ovarian-, fallopian tube- and primary peritoneal 
cancer will be eligible for treatment if they meet the criteria’s of inclusion, including an 
acceptable performance status, acceptable kidney- and liver function, and the absence of 
major co-morbidities. Initially, 6 patients will be included and treated. The treatment will only 
be completed in patients with successful manufacturing of TILs. If feasible and tolerable, 
additional 6 patients, giving a total of 12 patients, will be treated. 
Prior clinical trials have shown that the success rate of TIL manufacturing from metastatic 
melanoma exceeds 90% 2. To date, we have successfully generated viable TIL cultures from 34 
out of 34 samples (100%) from patients with ovarian cancer who have undergone debulking 
surgery (our internal observation). In our clinical pilot study, we managed to grow TIL 
cultures from 9 out of 10 ovarian cancer tumor samples (90%) (Pedersen M et al., manuscript 
in preparation). 
The actual success rate of TIL manufacturing in this trial may depend on additional factors e.g. 
previous chemotherapy regimens that the patients have received before enrolment, but any 
major difficulties in manufacturing TILs from the majority of patients are not anticipated.  


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
12 
Toxicity 
As mentioned above, we have shown that TIL therapy and decrescendo intermediate doses of 
intravenous IL-2 for patients with advanced OC is feasible and tolerable. We have previously 
conducted a study treating 6 patients with metastatic MM with TILs and low-dose 
subcutaneous IL-2 that showed minimal toxicity compared to high-dose IL-23. Therefore, low 
dose subcutaneous IL-2 is expected to be safe and with minimal toxicity. 
The toxicities associated with Ipilimumab are well-described4. Two clinical studies by the 
same group have treated patients with OC with a vaccine and subsequent CTLA-4 blockade. 
Initially, 2 OC patients were treated with Ipilimumab several years after the vaccine5 and in 
the continued study 9 additional OC patients were treated with Ipilimumab between 1-4 
months after vaccination6. No serious toxicity was seen in the first study, while 2 patients 
experienced grade 3 gastrointestinal toxicities, which resolved after oral corticosteroid 
administration, in the second study. A low level of toxicity is anticipated as the patients will 
only receive 1 dose of Ipilimumab. 
The toxicities associated with Nivolumab are also well-described, and in general are milder 
and observed less frequently than with ipilimumab7. A clinical study treating 20 patients with 
OC with Nivolumab has shown acceptable toxicity in a phase II trial8. 
Evaluation of clinical response 
The patients will be clinically evaluated 6 and 12 weeks after treatment with TILs and every 3 
months hereafter. Evaluation by diagnostic imaging will take place before the treatment, 
before discharge following TIL treatment, and in connection with the clinical evaluations 
starting from 6 weeks after TIL treatment. 
Immunological response evaluation 
Blood samples of 110 ml will be collected at the time of surgery, before TIL infusion, at 
discharge and in connection with the clinical evaluations. Serum samples (10 ml blood 
samples) will be collected during hospitalization at day 0 before TIL infusion, 2 hours after 
TIL infusion and every 2 days hereafter until discharge. Immune cells will be isolated from the 
collected blood samples by standard gradient centrifugation and cryo-preserved for later 
analysis. Flow-cytometric analyses will be used to assess the quantity and function of different 
immune cell subsets (e.g. CD4+, CD8+) before and after treatment at several time points. 
 
 
 
 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
13 
Introduction and rationale 
Ovarian cancer 
Approximately 1900 women are diagnosed with a gynecological malignancy each year in 
Denmark. Of these, approximately 450 have ovarian-, fallopian tube- and primary peritoneal 
cancer. 70-80% have local- or advanced disease at the time of diagnosis and it is the 4th most 
common cause of cancer death among women in Denmark9. Inoperable patients are treated 
with combinational chemotherapy (carboplatine and a taxan). Despite recent progress in 
treatment strategies, patients with advanced disease still have dismal survival rates10.  
Tumor immunology 
Remarkable progress has been made in the understanding of the reactions of the immune 
system against cancer in recent years. It has become clear that the immune system reacts 
against certain tumors in vivo and that immunological response against cancer cells are 
associated with a better prognosis11,12. In addition, it has been shown that a small percentage 
of patients with widely metastatic cancers can be cured with a variety of immune-activating 
approaches, including transfer of T cells13.  
T-cell therapy in patients with OC has only been investigated scarcely and most of the existing 
studies were conducted over 15 years ago. Importantly, none of the studies used current 
protocols of TIL manufacturing or preparative chemotherapy regimens. These studies are 
described in details below (section “T-cell therapy”). 
Immune checkpoint inhibition in patients with OC has been investigated more recently and in 
several trials. The clinical trials with CTLA-4-, PD-1- and PD-L1 antibodies are described in 
more details below (section “Checkpoint inhibition”). 
Tumor infiltrating lymphocytes (TILs) 
Tumors are often infiltrated by large amounts of T-cells (TILs) that specifically recognize 
tumor antigens but typically are inactive, or not sufficiently active, in the tumor 
microenvironment. The inactive state of the T cells in the tumor tissue is characterized by 
abnormal intracellular signaling, apoptosis and reduced proliferative capability, which are 
probably caused by various immune inhibiting factors in the tumor environment14,15. 
However, it is possible to amplify and reactivate such TILs for tumor cell killing in vitro by use 
of activating factors like IL-216,17.  
 
T-cell therapy 
T-cell therapy, which is also called ”Adoptive T-cell Therapy” (ATC), is an immunotherapeutic 
cancer treatment that has shown very promising results, especially in metastatic MM. This 
treatment, which uses the patient’s own T cells for tumor cell killing, was developed at the 
American National Institute of Health and in recent years several studies from research 


 
 
 
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Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
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centers in other countries in both USA and Europe have been published with more than 500 
patients having received the treatment in total18–23.  
 
The treatment is defined as the infusion of T cells isolated from the patient’s own  
tumor tissue after ex vivo activation and several rounds of expansion, and takes advantage of 
the high number of tumor reactive T cells in tumor tissue compared to peripheral blood24. 
 
Briefly, tumor-infiltrating T-lymphocytes are harvested from freshly resected tumor material 
from an individual patient and initially expanded ex vivo over a period of 2-4 weeks by 
growing T cells in high concentrations of the T-cell growth factor IL-2. Upon TIL isolation and 
initial growth, cells are further expanded to around 5x1010 cells in a standard 14 days rapid 
expansion protocol (REP), where TILs are cultured in the presence of allogeneic or autologous 
irradiated peripheral mononuclear blood cells (PBMCs, “feeder cells”), soluble anti-CD3 
antibodies and IL-2. Prior to infusion of the TIL product, patients receive in-hospital 
lymphodepleting chemotherapy for 7 days as a conditioning treatment. This last step has no 
direct impact on tumor growth, but is crucial for subsequent in vivo TIL persistence and 
expansion. Following intravenous administration of the T-cell product, IL-2 is administered to 
support the growth and survival of the infused T cells. The patients remain in hospital for a 
total of 14-21 days (see Figure 1 for an overview of the procedure). When an autologous and 
polyclonal tumor specific T-cell population is infused under these conditions, migration of 
anticancer T cells to the tumor site leads to a broad and patient-specific recognition of both 
defined and undefined antigens expressed on tumor cells leading to tumor cell killing and, 
eventually, tumor regression.  
 
This makes T-cell therapy a highly specialized and individualized form of cancer immune 
therapy. 
 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
15 
 
Figure 1: Course of treatment with T-cell therapy 
Tumor tissue for manufacturing of the T-cell infusion product is removed immediately after 
inclusion. Treatment with Cyclophosphamide (C) and fludarabine phosphate (F) is commenced a 
week before the T cells are ready for infusion and are followed by infusion of the T cells and 
administration of IL-2. Duration and dosage of IL-2 varies among protocols. 
 
T-cell therapy has shown very promising results in metastatic MM with overall response rates 
(ORR) around 50%, which has been confirmed in several phase I/II “single institution” 
studies19–21,25,26.  Complete response (CR) has been observed in about 20% of the treated 
patients of which most CR are of long duration and with patients being free of disease more 
than 7 years after the treatment18. Thus, TIL based T-cell therapy seems to have the potential 
of curing a significant number of the treated patients with metastatic MM. 
 
Previous experiences with more than 500 patients with metastatic MM treated with T-cell 
therapy has shown that the treatment is safe to administer to patients in a good performance 
status and adequate organ function, despite considerable - but temporary - toxicity. The 
observed side effects are reversible and acute and consist of known and expected side effects 
to lymphodepleting chemotherapy and high dose IL-2. 
Of the older studies investigating T-cell therapy for ovarian cancer, one study administered 
TILs in the peritoneal cavity with a low dosage of IL-2 and found manageable toxicity but no 
measurable response27. Two studies administered TILs without simultaneous IL-2 
administration. One of the studies administered 1 series of cyclophosphamide or cisplatine-
based chemotherapy followed by administration of TILs and found CR in 14,3% and 70%, 
respectively, while finding partial response (PR) in 57,1% and 20%, respectively, in very 
small patient groups (7 and 10 patients)28. The other study was performed in an adjuvant 
setting following primary operation and adjuvant chemotherapy. Thirteen patients were 
treated with 5-floururacile and cyclophosphamide or cyclophosphamide and Adriamycin 


 
 
 
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Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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nr. 2017061517. 
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followed by TILs randomized against chemotherapy alone for 11 patients. They found 100% 
and 67,5% 3 years overall survival rate and 82,1% and 54,5% 3 years disease-free survival 
rate, both significant results29. More recently, a pilot study using gene-modified T cells with 
PBMCs derived from leukapheresis were tested for treatment of patients with recurrent 
epithelial OC. The treatmen was shown to be safe, but no clinical responses were observed30.  
A recent study administering T-cell therapy to patients with metastatic ovarian cancer has 
been performed here at CCIT and is mentioned in details below (see “Clinical trials”). 
Checkpoint inhibition 
Immune checkpoint therapy targets regulatory pathways in T cells with the purpose of 
enhancing antitumor immune responses. The regulatory pathways of interest to this study are 
described briefly in the following.  
MHC-molecule presentation of tumor antigen in the context of B7 costimulation by 
professional antigen-presenting cells is necessary for priming of tumor-specific T cells. This 
priming leads to activation, acquisition of effector functions and infiltration of tumor sites. 
Upon T-cell activation, an induction of  CTLA-4 expression leads to an accumulation of CTLA-4 
on the surface of the activated T cells that, at a certain level, can block B7 costimulation and 
thereby subsequently suppress T-cell activation31–33. The majority of Activated T cells that 
infiltrates the tumor microenvironment express PD-1 on their cell surface and many types of 
cancer express its inhibitory ligands, PD-1 and PD-L1, thus inhibiting local anti-tumor 
responses in the T cells effector phase31,33,34.  
Two clinical studies have investigated CTLA-4 blockade in patients with OC. In one study 9 
previously vaccinated cancer patients were treated with the CTLA-4 antibody Ipilimumab (3 
mg/kg). Two of these patients had OC. The vaccine consisted of irradiated, autologous tumor 
cells engineered to secrete GM-CSF. No serious toxicities directly related to the antibody were 
observed and the two OC patients experienced a brief reduction in CA-125 values followed by 
stabilization (total 4 months) and a stabilization (1 month), respectively5. The other study 
from the same group continued treating previously vaccinated cancer patients with the 
difference that CTLA-4 blockade (Ipilimumab 3 mg/kg with subsequent treatments at 2-3 
months intervals as clinically indicated, range 1-16) was given between 1 and 4 months after 
vaccination instead of after several years, as in their earlier study. The same vaccine was used. 
Nine additional OC patients were treated. Two patients experienced grade 3 gastrointestinal 
toxicities which resolved after the administration of oral corticosteroids. One of the two also 
manifested Sweet’s syndrome. The remaining 7 patients only experiences minor 
inflammatory toxicities. Three patients achieved stable disease (SD) (2,4,6+ months) and one 
patients achieved a partial response (PR) (35+ months)6. 
One clinical study has investigated anti-PD-1 antibodies in patients with OC. They treated 20 
patients with platinum-resistant OC with the PD-1 antibody Nivolumab (two 10-patient 
cohorts, the first treated with 1 mg/kg, the second with 3 mg/kg, treatment every 2 weeks for 
up to six cycles with four doses per cycle). Eight of 20 patients (40%) experienced grade 3- or 
4 treatment-related toxicities. Two patients experienced severe adverse events. Six patients 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
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achived SD (4 in the 1 mg/kg group), one achived PR (in the 1 mg/kg group) and two achieved 
CR (both in the 3 mg/kg group)8.  
One clinical study investigated anti-PD-L1 antibodies in patients with OC. They treated a total 
of 207 patients with the anti-PD-L1 antibody BMS-936559 (escalating doses from 0.3 to 10 
mg/kg every 14 days in 6-week cycles for up to 16 cycles). Seventeen of these patients had OC. 
Treatment-related grade 3 or 4 adverse events were observed in 19 of 207 (9%) and serious 
adverse events in 11 of 207 patients (5%). No information about these events was available 
based on the different cancer types. Of the 17 OC patients, 1 achieved PR and 3 achieved SD 
for at least 24 weeks. All were in the 10-mg dose group35.  
A number of other trials are currently enrolling patients, but results are so far unknown 
(source clinicaltrials.gov). 
CCITs experience with T-cell therapy 
Clinical trials 
The complicated methods of manufacturing TILs has been established at the Center for Cancer 
Immune Therapy (CCIT), Herlev Hospital, as one of the few places in the world36. CCIT and the 
Department of Oncology at Herlev Hospital have several years of experience in treating cancer 
patients with T-cell therapy and different regimens of IL-2. 
All patients have been treated with classic lymphodepleting chemotherapy with 
cyclophosphamide and fludarabine phosphate followed by TIL infusion and subsequent 
administration of IL-2. 
In the original “T-cell regimen” described by Dudley et al15 very high dosages of IL-2 (720.000 
IU/kg i.v.) where given as bolus injection every 8 hours until treatment limiting toxicity. It is 
unknown how high a dosage of IL-2 is necessary to maintain T-cell expansion, and 
consequently CCIT has tested the treatment with low and intermediate doses of IL-2 to 
investigate whether clinical efficacy can be maintained while toxicity is decreased. 
Initially, low dosage subcutaneous IL-2 was given to 6 patients in a pilot study initiated in the 
summer of 2009 and the results are now published19.  Two of the six treated patients achieved 
CR and are presently without evidence of disease (NED). 
To achieve a higher response rate (RR), the dosage of IL-2 was then increased to an 
intermediary dosage and administered after the decrescendo-regimen equal to the 
decrescendo regimen used in Denmark as standard treatment of metastatic MM. 
Additionally 25 patients have been treated after the increase in IL-2 dosage. Of the 31 treated 
patients, five achieved CR (48 (NED), 13 (NED), 38+, 22+ and 11+ months) and 7 patients 
achieved partial PR (35+ (NED), 12, 18+, 25+ (NED), 11, 8 and 6+), of which 7 are having 
ongoing responses varying from 6-38 months. Thirteen patients had stable disease (SD) for 4-
6 months and 5 patients progressed immediately after treatment. An ORR of 39% has been 
observed, which is comparable to other studies administering high dosage of IL-2. The 
lymphodepleting chemotherapy induced, as anticipated, myelosuppression with anemia, 
leucopenia and thrombocytopenia and all patients received prophylactic antibiotics and blood 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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nr. 2017061517. 
18 
transfusions. All patients experienced transient grade III-IV toxicities during the 3 weeks of 
hospitalization but recovered quickly after the treatment. The above mentioned results have 
recently been published37. Markedly reduced toxicities have been observed with the low- to 
intermediary dosage of IL-2 and the treatment has shown to be manageable at a regular 
Department of Oncology with limited need for intervention from an Intensive care unit. 
Recently, an international phase III study treating patients with metastatic MM with TILs and 
a high dosage bolus IL-2 regimen (600.000 IU/kg) was initiated at the Department of 
Oncology, Herlev Hospital. So far, toxicity has been acceptable. 
Our recent pilot study with 6 patients has shown that it is feasible and safe to treat patients 
with metastatic ovarian cancer with T-cell therapy and IL-2 according to the decrescendo 
regimen. Only expected and manageable toxicities have been observed and all patients’ 
hematopoietic system recovered without the need for stem cell support. Four patients 
experienced SD for 3 months and the last two patients had SD for 5 months (Pedersen M et al., 
manuscript in preparation).  
Translational research 
Further development of T-cell therapy with optimizing and expansion to other cancer forms 
has a high priority at CCIT. Our already established platform for T-cell therapy for MM gives 
us a unique opportunity to study the interactions between tumor and immune system and 
thereby identify possible methods for optimization of T-cell therapy, as well as extension to 
other tumor histologies.  
 
Several studies has shown that the following T-cell characteristics are important for achieving 
a clinical response after T-cell therapy: long telomeres, short time spent in culture, a favorable 
T-cell phenotype (CD27+, CD28+), a high absolute number of T cells and a high number of 
cytotoxic  tumor-reactive T cells in the infusion product38 as well as an increased persistence 
of T-cell in the peripheral blood after infusion18,36,39. AT CCIT, we have modified the original T-
cell expansion method from ”Standard TIL expansion” to ”Young TIL expansion” based on 
these characteristics and leading to a reduction of the length of cell manufacturing from 4-7 
weeks to 2-4 weeks. A decreased amount of time in culture (Young TIL) provides the TILs 
with longer telomere sequences and more favorable phenotypes (CD27+, CD28+) with the 
ability of increased proliferation, an increased persistence in vivo and a higher antitumor 
activity, all of which are correlated to an increased clinical response18,36. This optimization of 
TIL production has made it possible to produce clinically usable TIL infusion products from 
more than 90% of the patients40–43. Furthermore, during the final expansion phase, the Rapid 
Expansion Protocol (REP), we have introduced the use of the Wave® bioreactor44, which 
optimizes the conditions of proliferation of T cells and has made it possible to achieve a higher 
total number of cells as well as tumor reactive T cells in the TIL infusion product. We have 
standardized and harmonized TIL production methods between 3 European cancer research 
centers based on these TIL production protocols developed at CCIT and have initiated a 
randomized, multicenter TIL-based phase III trial with T-cell therapy versus standard 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
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immunotherapy (clinicaltrials.gov identifier: NCT02278887) with the purpose of the approval 
of T-cell therapy as standard treatment for patients with MM.  
 
Recent studies suggest that TIL based ACT potentially can be used with success in other 
cancer forms including colon, breast, head and neck, kidney, sarcoma and ovarian cancer45–50. 
It has been shown that a high intratumoral presence of T cells is correlated with longer 
survival and functional analysis has shown that tumor infiltrating T cells show in vitro anti-
tumor activity similar to that seen in MM. In a preclinical study, we have successfully 
manufactured TILs from 34/34 samples obtained with primary debulking surgery from 
patients with ovarian cancer. Tumor cell lines have been established from 14/33 tumor 
samples and we have been able to detect an anti-tumor response (either CD8+ or CD4+ T 
cells) in 19 of 31 patients tested against either tumor cell line or tumor digest (Westergaard 
MCW et al., ESMO IO 2014).  Subsequently, in our aforementioned pilot study with T-cell 
therapy in OC, we were able to manufacture TILs from 9/10 patients, establish tumor cell 
lines from 5/10 tumor samples, and detect anti-tumor responses (either CD8+ or CD4+ T 
cells) in 5/6 of the treated patients tested against either tumor cell line or tumor digest 
(Pedersen M et al., manuscript in preparation).  
The rationale of the drugs used in the study 
Lymphocyte depleting chemotherapy 
Activating cytokines (the signal molecules IL-2, IL-7, IL-15, IL-21 etc.) need to be available for 
the tumor specific T cells to sustain a immunological response against tumor tissue. A large 
number of “irrelevant” T cells will decrease the availability of these cytokines for the relevant 
T cells through competition. Thus, a high number of tumor specific T cells with a high 
specificity as well as a reduction of irrelevant T cells and regulatory T cells (Tregs) are needed 
to create an environment that facilitates the T-cell mediated antitumor response. 
This study will use combination chemotherapy with two days treatment with 
cyclophosphamide and 5 days with fludarabine phosphate to create such an environment. 
This combination has been chosen based on earlier studies where it was shown safe and 
effective51,52. 
Cyclophosphamide 
Cyclophosphamide is an alkylating drug that works by creating covalent bindings with 
biologically important macromolecules. Of special interest is the creation of a binding and 
linkage to DNA. Cell division can be prevented if the linkage is not canceled by the cells repair 
systems. The binding to important proteins in the cell can damage important cellular 
functions and lead to cell death. Cyclophosphamide is among others used to treat breast 
cancer and in the treatment of hematological diseases as myelomatosis53.  


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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nr. 2017061517. 
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Fludarabine phosphate 
Fludarabine phosphate is a pro-drug that is converted to the active triphosphate 2-fluoro-ara-
ATP. It is an anti metabolite which inhibits DNA synthesis while simultaneously reducing RNA 
and protein synthesis. Fludarabine phosphate is used in the treatment hematological diseases 
as CLL among others54.  
Interleukin 2 
IL-2 is physiologically produced by activated T-lymphocytes and stimulates the antigen 
specific and non-specific immune system through specific receptors55. 
Intermediate-dose interleukin-2 (IL-2) administered intravenously according to the 
decresendo-regimen56 is used in Denmark as standard treatment for suitable patients with 
metastatic MM. Low-dose subcutaneous IL-2 has previously been administered to 6 patients 
with metastatic MM in a T-cell therapy pilot study with CR in 2 patients and generally low 
occurrence of toxicity3. Moreover, low-dose IL-2 is used for the treatment of metastatic kidney 
cancer.  
In this study, IL-2 will be given in a low-dose subcutaneous setting to reduce the risk of the 
occurrence of adverse events when given in combination with Nivolumab. 
Checkpoint inhibition in combination with T-cell therapy 
As mentioned earlier, a high absolute number of T cells and a high number of cytotoxic tumor-
reactive T cells in the infusion product is important for achieving a clinical response after T-
cell therapy38. Furthermore, CTLA-4 is involved in the regulation of the early stages of T-cell 
activation while PD-1 predominantly regulates T-cell effector functions in the periphery and it 
is therefore hypothezied that targeting of these two receptors will lead to a positive and 
synergistic effect on the TILs1. 
One study has shown an increased intratumoral infiltration with CD8+ cells in posttreatment 
biopsies when treating patients with metastatic malignant melanoma (MM) with a CTLA-4 
antibody57. Another study investigated 40 patients with metastatic MM treated with the 
CTLA-4 antibody Ipilimumab and found that clinical response was associated with an increase 
in absolute lymphocyte count (p = 0.008), absolute T-cell count (p = 0.02) and the absolute 
number of activated T cells in peripheral blood (p = 0.003) after 1 series of treatment58. Other 
recent studies from our laboratory and others have shown that blockade with a CTLA-4 
antibody broadens the intratumor and peripheral T-cell repertoires59 (Bjørn J et al., 
Oncotarget 2017 in press). Our laboratory has also shown that in vitro stimulation of TIL 
cultures from OC patients with a CTLA-4 antibody increases the frequency of CD8+ T cells. We 
were able to generate TIL cultures in 5/5 cultures stimulated with Ipilimumab vs. 3/5 and 4/5 
cultures not stimulated with Ipilimumab. The Ipilimumab stimulated cultures contained an 
increased amount of TILs per fragment in 4/5 cultures and an increased amount of CD8+ TILs 
in 5/5 cultures compared to the unstimulated ones. 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
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Thus, pretreating the patient with a CTLA-4 antibody before tumor harvest may lead to 
increased quality of the TIL product and in vitro stimulation with a CTLA-4 antibody may 
increase the quality of the TIL product even further. 
Studies investigating the expression of programmed cell death-ligand 1 (PD-L1) in OC has 
shown an uncertain correlation to prognosis60,61, but also that a high intratumoral amount of 
CD3+ T cells is positively correlated to prognosis. It is hypothesized that PD-L1 expression is 
upregulated in tumor tissue as a compensatory reaction to an immune response against 
tumor cells and suppresses antitumor CD8+ T cells60–62. 
In addition, we have shown that a significant fraction of tumor-reactive TILs in the infusion 
product express an increased level of PD-1 and that in vitro addition of anti-PD1 leads to 
increased tumor killing by these TILs (unpublished data).   
Therefore, clinical targeting of the PD-1 receptor could potentially decrease the suppression 
of the infused tumor reactive CD8+ T cells.  
Ipilimumab 
Ipilimumab is an anti-CTLA-4 antibody that inhibits a decrease in the activation and 
proliferation of T cells when presented to tumor antigens by professional antigen presenting 
cells in the priming phase, as mentioned earlier. Ipilimumab is approved by the European 
Medicines Agency (EMA) for the treatment of metastatic malignant melanoma and is 
undergoing clinical trials in combination with PD-1/PD-L1 inhibitors in non-small cell lung 
carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer and metastatic hormone-
refractory prostate cancer63. 
Ipilimumab will be administered in the approved dosage of 3 mg/kg for 1 dose 2-6 weeks 
before surgical removal of tumor tissue for TIL production. 
Nivolumab 
Nivolumab is an anti-PD-1 antibody that inhibits suppression of the activated T cells in 
performing their effector functions in targeting the tumor cells in the tumor 
microenvironment in the effector phase, as mentioned earlier. Nivolumab is approved by the 
EMA for the treatment metastatic malignant melanoma, NSCLC, renal cell carcinoma, 
Hodgkin’s lymphoma, head and neck cancer64.  
Nivolumab will be administered in the approved dosage of 3 mg/kg starting 2 days before T-
cell therapy and every 2nd week continuing after discharge in the outpatient clinic for a total of 
4 series. 
The rationale of the treatment regimen used in the study 
The ACT regimen has been determined according to our previous study carried out in MM2 
and OC (unpublished data).  
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
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Purpose and hypothesis 
Primary 
1) To assess tolerability and feasibility of the treatment. 
Secondary 
1) To clarify whether T-cell therapy in combination with checkpoint inhibitors for 
patients with advanced ovarin-, fallopian tube- and primary peritoneal cancer can 
induce a measurable immune response against tumor cells. 
2) To describe objective responses using RECIST 1.1.  
PERCIST and irRC will be used exploratively. 
 
Furthermore, overall survival (OS) and progression-free survival (PFS) will be described. 
 
Study design 
The study is an open label phase I/phase II study for patients with advanced ovarian-, 
fallopian tube- and primary peritoneal cancer. All patients will be included and treated at the 
Department of Oncology, Herlev Hospital. Patients can be referred to treatment from other 
centers in Denmark. 
 
We expect to include and treat 6 patients initially. If feasible and tolerable, additional 6 
patients will be treated giving a total of 12 treated patients.  
We expect to have completed initial patient enrolment and treatment within 2 years and 6 
months follow-up within 3 years.  
 
The course of treatment consists of 3 steps followed by clinical controls and evaluation-scans 
as follow-up. 
 
- 
Step 1: Screening, inclusion, Ipilimumab treatment and surgical removal of tumor 
material followed by production and growth of TILs in the laboratory. 
- 
Step 2: Treatment during hospitalization with chemotherapy, Nivolumab, TIL infusion 
and IL-2 administration. 
- 
Step 3: Continued treatment with Nivolumab every 2nd week in the outpatient clinic for 
a total of 4 series. 
- 
Follow-up: Evaluation of the effect of treatment during follow-up.  
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
23 
 
Figure 2: Schematic presentation of the course of treatment. One dose of Ipilimumab (3 mg/kg) 
is administered 2-6 weeks before tumor resection. Tumor tissue (metastasis or primary tumor) 
of minimum 1 cm3 is surgically removed from the patient and transported to the laboratory 
under sterile conditions where the tumor is separated into suitable fragments of 1-3 mm3 and 
placed in growth wells with a growth media and IL-2. TILs are then initially grown for 2-4 weeks 
until a cell-number of minimum 50 x 106. At this point the cells can be cryo-preserved for later 
use or pass on to the Rapid Expansion Protocol (REP) in which the T cells for 2 weeks are 
stimulated with anti-CD 3 antibody, allogeneic, radiated PBMC (peripheral mononuclear blood 
cells), feeder cells and IL-2. After stimulation, the expanded TILs (now billions) are then washed, 
pooled and re-infused intravenously back in to the patient.  Seven days of lymphodepleting 
chemotherapy is given to the patient before TIL infusion with cyclophosphamide (C) on day -7 to 
day -6 and Fludarabine (F) on day -5 to day -1 with the purpose of removing all existing 
lymphocytes in the patient to make room for the infused TILs and remove regulatory T cells 
(Tregs). Nivolumab (3 mg/kg) is administered 2 days before TIL infusion to boost the activity of 
the reinfused TILs. Subcutaneous IL-2 (2 MIU) injections is started approximately 6 hours after 
TIL infusion (day 0 to day 5) in order to further increase activation and to stimulate the infused 
TILs for further expansion within the patient. Nivolumab treatment is continued in the 
outpatient clinic for a total of 4 series. 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
24 
 
Study population 
Patients with histological verified advanced ovarian-, fallopian tube- or primary peritoneal 
cancer will be candidates for this study. The patients need to have an acceptable performance 
status, no major co morbidities and acceptable organ functions. 
Only patients in which it is possible to grown T cells from their tumor tissue will be offered 
treatment with T cells in the study and only patients receiving treatment will be included in 
the final study population. 
Criteria of in- and exclusion 
Criteria of inclusion 
All of the criteria listed in the following need to be met before patient inclusion. 
 
1. Histological proven advanced ovarian-, fallopian tube or primary peritoneal cancer 
with the possibility of surgical removal of tumor tissue of > 1 cm3. All histologies can be 
included. 
2. Progressive or recurrent resistant disease after platin-based chemotherapy (platinum 
resistant) or progressive or recurrent disease after second line or additional 
chemotherapy. 
3. Age: 18 – 70 years. 
4. ECOG performance status of ≤1 (Appendix 2). 
5. Life expectancy of > 6 months. 
6. At least one measurable parameter in accordance with RECIST 1.1 –criteria’s. 
7. No significant toxicities or side effects (CTC ≤ 1) from previous treatments, except 
sensoric- and motoric neuropathy (CTC ≤ 2) and/or alopecia (CTC ≤ 2).  
8. Sufficient organ function, including:  
o Absolute neutrophil count (ANC) ≥ 1.500 /µl 
o Leucocyte count ≥ normal limit 
o Platelets ≥ 100.000 /µl and <700.000 /µl 
o Hemoglobin ≥ 6,0 mmol/l (regardless of prior transfusion) 
o S-creatinine < 140 
o S-bilirubin ≤ 1,5 times upper normal limit 
o ASAT/ALAT ≤ 2,5 times upper normal limit 
o Alkaline phosphatase ≤ 5 times upper normal limit 
o Lactate dehydrogenase ≤ 5 times upper normal limit 
o Sufficient coagulation: APPT<40 and INR<1,5 
9. Men and women in the fertile age must use effective contraception. This applies from 
inclusion and until 6 months after treatment. Birth control pills, spiral, depot injection 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
25 
with gestagen, subdermal implantation, hormonal vaginal ring and transdermal depot 
patch are all considered safe contraceptives. 
10. Signed statement of consent after receiving oral and written study information 
11. Willingness to participate in the planned controls and capable of handling toxicities. 
Criteria of exclusion 
Patients will be excluded if they meet one of the criteria’s listed below 
 
1. A history of prior malignancies. Patients treated for another malignancy can 
participate if they are without signs of disease for a minimum of 5 years after 
treatment. 
2. Known hypersensitivity to one of the active drugs or one or more of the excipients. 
3. Severe medical conditions, such as severe asthma/COLD, significant cardiac disease, 
poorly regulated insulin dependent diabetes mellitus among others. 
4. Creatinine clearence < 70 ml/min*. 
5. Acutte/chronic infection with HIV, hepatitis, syphilis among others. 
6. Severe allergies or previous anaphylactic reactions. 
7. Active autoimmune disease, such as autoimmune neutropenia/thrombocytopenia or 
hemolytic anemia, systemic lupus erythematosis, Sjögren’s syndrome, sclerodermia, 
myasthenia gravis, Goodpasteur’s disease, Addison’s disease, Hashimotos thyroiditis, 
active Graves disease. 
8. Pregnant women and women breastfeeding. 
9. Simultaneous treatment with systemic immunosuppressive drugs (including 
prednisolone, methotrexate among others)**. 
10. Simultaneous treatment with other experimental drugs. 
11. Simultaneous treatment with other systemic anti-cancer treatments. 
12. Patients with active and uncontrollable hypercalcaemia. 
 
* In selected cases it can be decided to include a patient with a GFR < 70 ml/min with the use 
of a reduced dose of chemotherapy. 
** In selected cases a systemic dose of ≤10 mg prednisolone or a transient planned treatment 
that can be stopped before TIL therapy can be tolerated. 
Evaluation before inclusion 
The following need to be carried out before the start of treatment: 
- 
Medical history and clinical examination 
- 
Performance status in accordance to the ECOG-scale 
- 
Electrocardiogram 
- 
Cr-EDTA clearance 
- 
Urine sample 
- 
Screening blood tests:  


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
26 
o Hematology: Hemoglobin, leukocytes with diff-count and platelets 
o Liver: ASAT, ALAT, albumin, alkaline phosphatase, bilirubin, LDH, total protein, 
INR, APPT. 
o Kidney: sodium, potassium, creatinine, carbamide, magnesium, bicarbonate, 
phosphate, Ca-ion. 
o Other: Glucose, CRP. 
o Chronic infections: HIV, Hepatitis B surface antigen, Hepatitis B surface 
antibody, Hepatitis B core antibody, Hepatitis C antibody, HTLV IgG, EBV, 
Trepone. 
o Endocrine: TSH, T3, T4. 
o Female specific: Estradiol, prolactin. 
o Ovarian Specific: CA-125 
- 
Pregnancy test: Women in the fertile age must take a pregnancy test. This includes 
women who are not surgically sterilized, who are not postmenopausal and who have 
not used safe contraception’s regularly within the last 6 months.  
- 
Baseline tumor evaluation: CT, MR or PET-CT scans can be used. A PET/CT scan is 
preferred.  
- 
Reviewing the checklist for inclusion/exclusion for treatment. 
Treatment plan 
The course of treatment can vary between patients depending on the time period from 
surgery to treatment. In most cases (e.g. TILs undergoing massive expansion and infusion 
without cryopreservation of intermediate products), the patients will receive treatment 
approximately 4-6 weeks after the operation and in these cases the course of treatment from 
Ipilimumab treatment before surgery until the first evaluation with diagnostic imaging (6 
weeks after treatment) will span across approximately 3-4 months. 
 
Surgical removal of tumor tissue for TIL expansion can be repeated if not possible to grow TIL 
after initial surgery if additional accesible tumor tissue is present and based on an individual 
clinical assessment. Presurgical treatment with Ipilumumab is to be administered 2-6 weeks 
prior to surgery and is to be repeated if more than 6 weeks has passed since the initial 
Ipilimumab treatment. 
 
Stem cell harvest by leukapheris will not be performed since we and others have observed 
that the patients’ hematopoietic system recovers after the lymphodepleing chemotherapy 
without the need for stem cell support65–68 (unpublished data). 
 
Location 
 
Hospital 
Outpatient 
Week 
* 
-2 
-1 
0-1 
3 
5 
Day 
* 
-8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
1-11 
12 
13 
27 
41 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
27 
Ipilimumab (3 mg/kg) iv 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pt. is admitted 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cyclophosphamide (60 mg/kg) iv 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
Fludarabine (25 mg/m2) iv 
 
 
 
 
x 
x 
x 
x 
x 
 
 
 
 
 
 
Nivolumab (3 mg/kg) iv 
 
 
 
 
 
 
 
x 
 
 
 
x 
 
x 
x 
TILs iv 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
Pegfilgrastime (6 mg) sc 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
IL-2 (2 MIU) subcut. inj. 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
 
 
* Ipilimumab is administered 2-6 weeks before surgery 
Examination plan during the course of treatment 
The patients are continuously monitored on several parameters before, during and after TIL 
infusion. 
 
Blood samples will be collected before, during and after hospitalizing. After ended treatment, 
the patients will be evaluated for up 5 years at the unit of experimental cancer treatment 
(EFEK) at the Department of Oncology, Herlev Hospital. Patients will be excluded upon clinical 
or radiological progression. 
 
Time point 
Screening 
Before Ipilimumab 
Tumor tissue removal 
<14 days to admission 
At admission 
Daily during hospitalization 
Before Nivolumab, 1st of 4 
At TIL infusion 
At discharge 
Before Nivolumab, 2nd of 4 
Before Nivolumab, 3rd of 4 
Before Nivolumab, 4th of 4 
Week 
 
a 
b 
-4 → -2 
-2 
-1→c 
-1 
0 
c 
2 
3 
5 
Day 
 
a** 
b 
-22 → -
9 
-8 
-7→c 
-5 
0 
c 
12 
26 
40 
Performance Status 
x 
x 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
Clinical examination 
x 
x 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
Weight, BP, P, Temp. 
x 
x 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
Toxicity assessment (CTC) 
 
x 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
Screening blood tests 
x 
 
 
 
 
 
 
 
 
 
 
 
Research blood tests 
 
x 
 
 
x 
x* 
 
x 
x 
 
 
 
Standard blood tests 
 
x 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
Immunological blood tests 
x 
 
 
 
x 
 
 
 
 
 
 
 
Ovarian specific blood tests 
x 
x 
 
 
x 
 
x 
 
x 
x 
x 
x 
ECG 
x 
x 
 
 
x 
 
x 
 
 
x 
 
 
Cr EDTA 
 
 
 
x 
 
 
 
 
 
 
 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
28 
Urine sample 
 
 
 
 
x 
 
 
 
 
 
 
 
Biopsy 
 
 
x 
 
 
 
 
 
 
 
 
 
Tumor Evaluation 
x 
 
 
x 
 
 
 
 
x 
 
 
 
a At least 14 days before tumor tissue removal (b) 
b Day of tumor tissue removal. 
c Depends on recovery period after TIL infusion, approximately at day 11. 
* Every second day untill discharge. 
** Examination and blood tests will only be repeated if clinically indicated or >2 weeks from 
screening 
 
Overlapping samples are only taken once. 
 
Screening blood tests: Se previous section “Evaluation before inclusion”. 
 
Research blood tests: See section “Blood samples for immunological monitoring” under the 
section “Immunological Monitoring”. 
 
Standard blood tests: Hemoglobin, platelets, leukocytes with differential-count, creatinine, 
sodium, potassium, magnesium, phosphate, ASAT, ALAT, bilirubin, alkaline phosphatase, Ca-
ion, CRP, INR, APPT.  
 
Immunological blood tests: Hemoglobin, platelets, leukocytes with differential-count, 
creatinine, sodium, potassium, carbamide, bicarbonate, albumin, urate, ALAT, ASAT, LDH, 
alkaline phosphatase, bilirubin, amylase, glucose, TSH, T4, Ca-ion, cortisol, prolactin, Estradiol. 
 
Ovarian specific blood tests: CA-125. 
 
Tumor evaluation: CT, MR, PET-CT or a PET-MR scan can be used. Before inclusion a current 
scan need to be available for review. A PET/CT scan is preferred. Before hospital admission a 
baseline scan has to be performed. 
Medicinal products used in the study 
The medicinal products used in this study are Ipilimumab, Nivolumab, cyclophosphamide, 
fludarabine phosphate and interleukin-2. Mixing and storage of the products is carried out 
according to existing standard guidelines at the Department of Oncology. 
Ipilimumab 
Ipilimumab is an antibody targeting the CTLA-4 receptor on T cells. The antibody functions as 
a negative regulator by blocking of the suppressive signaling through the receptor and 
thereby potentiating T-cell activity.  
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
29 
Ipilimumab is administered in the outpatient clinic 2-6 weeks before surgical removal of 
tumor tissue for TIL production as an intravenous infusion. The dosage is 3 mg/kg body 
weight and administration takes 90 minutes.  
Cyclophosphamide 
Cyclophosphamide is given as an intravenous infusion for two consecutive days in a dosage of 
60 mg per kg of body weight. The treatment takes place during hospital admission and with 
supplementary hydration and Mesna injections.  
Fludarabine phosphate 
Fludarabine phosphate is given as an intravenous infusion for 5 consecutive days in a dosage 
of 25 mg per m2 body surface (starting the day after the last dosage of cyclophosphamide). 
The treatment takes place during hospital admission.  
Nivolumab 
Nivolumab is an antibody targeting the PD-1 receptor which is expressed on activated T-
lymphocytes. By blocking this receptor, Nivolumab prevents inhibition of T-lymphocyte 
activity through binding of the receptors ligands, PD-L1 and PD-L2.  
 
Nivolumab is administered 2 days before TIL administration as an intravenous infusion over 
60 minuttes in a dosage of 3 mg/kg body weight and every 2 weeks for a total of 4 doses. 
TILs 
The tumor specific T cells are infused intravenously back in to the patient during 
hospitalization on the day after the last dosage of fludarabine phosphate (day 0). The number 
of T cells in the product depends on the possible in vitro degree of expansion and is therefore 
variable, but will normally consist of approximately 1010 cells. See the Investigational Product 
Medicinal Dossier (IMPD) for more information. 
Interleukin-2 
A daily subcutaneous injection with IL-2 (2 MIU) will be administered from day 0 to 14. 
During hospitalization the injection will be given by a trained nurse. If the patient is well 
enough to be discharged within these 14 days, the patient or relatives will be taught to 
perform the remaining injections. Injections for self-administration will be labeled by the 
cytostatica unit, Herlev Hospital, and in accordance with Annex 13 of the GMP rules. 
The supplied amount of IL-2 injections for selfadministration will be registered by use of 
batch number, so that the total amount of administed IL-2 can be determined. Moreover, IL-2 
will be labeled in accordance with current guidelines regarding study drugs. 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
30 
Pegfilgratim 
Is an analog of human granulocyte colony stimulatory factor (G-CSF). It works by stimulating 
the bone marrow into producing white blood cells and increasing the peripheral blood count. 
It is usually given to cancer patients who suffer from low blood counts following 
chemotherapy69.  
Pegfilgrastim is administered as a single dose of 6 mg s.c. at 2 hours after TIL infusion to help 
patients recover from the lymphodepleting chemotherapy by.  
 
Guidelines for concomitant treatment 
Supportive treatment is given on ordinary medical indications estimated by the physician 
responsible for the treatment. Any measures should be specified in the patient chart and flow 
sheet. The following is meant as guidance and other medications can be administered as it is 
seen fit. One exception is systemic corticosteroid which can be administered du to immune 
related adverse events (irAEs) in relation to the treatment with checkpoint inhibitors. 
During lymphodepleting chemotherapy: 
In order to protect the mucosa of the bladder the following is administered: 
• Supplementary fluid therapy during cyclophosphamide treatment. 
• Inj. Mesna (25% of the cyclophosphamide dosage i.v. x 4 daily on day -7 and -6).  
In order to prevent and relieve nausea during chemotherapy (day -7 to -1) the following is 
administered:  
• Inj. Aloxi 250 μg i.v. on day -7 and -5. 
• Tabl. Emend 125 mg on day -7, 80 mg on day -6, 80 mg on day -5. 
• Tabl. Motilium 20 mg x 3. 
• Tabl. Temesta 1-2 mg max x 4 if needed. 
• Tabl. Pantoloc 40 mg x 1-2 daily. 
 
After TIL infusion: 
• Inj. Pegfilgrastime, 6 mg x 1 s.c. on day 0, two hours after TIL infusion. 
• Tbl. Pethidine, 25 mg max x 4, if needed. 
• Oxygen on nasal catheter if needed. 
 
During IL-2 treatment: 
Low-dose subcutaneous IL-2 is expected to be safe and tolerated with minimal toxicity as 
previously described3. If supportive treatment is indicated, it will be as in our prevous trials 
with TIL therapy. 
In order to prevent deranging of electrolytes as well as low blood pressure during IL-2 
treatment the following is administered:  
• Fluid therapy in accordance with appendix 4. 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
31 
In order to prevent and relieve nausea during IL-2 treatment (day 0 to 5) the following is 
administered: 
• Tabl. Motilium 20 mg, if needed max x 3. 
• Ondansetron 8 mg x 1 if needed max x 2. 
• Imolope 2 mg if needed max x 8. 
In case of fever: 
• Tabl. Paracetamol 1000 mg if needed*, max x4. 
*Only if patients develop fever >41○c. 
 
To prevent opportunistic infections:  
• Tabl. Sulfamethizole with Trimethoprime, 400/80 mg, 1 tabl. Daily on day -7 and 6 months 
ahead. 
• Tabl. Aciclovir, 400 mg x 2 daily on day 0 and 6 months ahead. 
• Tabl. Diflucan, 100 mg daily on day 0 and until the neutrophile count is > 1000/µl. 
 
In case of fever: 
If simultaneous neutropenia and fever occurs it will be treated in accordance with local 
guidelines described in appendix 5. 
 
In case of diarrhea: 
• Patients are treated according to local guidelines that include loperamide and fecal 
samples. 
• Loss of fluid and electrolytes are evaluated daily and corrected orally or intravenously. 
 
Immune related adverse events (irAEs) to checkpoint inhibitors:  
• irAEs are to be managed in accordance with local guidelines described in appendix 6.  
 
In case of anemia and thrombocytopenia:  
• Transfusions with blood should be administered if hgb ≤ 6.0 mmol/L or if it is otherwise 
clinically indicated.  
• Transfusion with platelets is indicated if platelets <20/μl or if it is otherwise clinically 
indicated. 
• Radiated and filtered blood is to be administered from day -7 and 6 months ahead if 
indicated. 
 
Others: 
In some cases, e.g. localized bone pain, local radiotherapy can be prescribed.  
o Radiotherapy is preferably to be avoided within the 3 weeks period of hospitalization  
o Radiated areas cannot be used as parameters in the assessment of treatment response.  


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
32 
o If possible, not all evaluable areas should be included in the radiated area. If all 
evaluable areas are treated, the patient is withdrawn from the study to ensure correct 
evaluation. 
End of treatment 
Normal end of treatment including follow-up 
Patients who finish the treatment course will be followed with clinical controls and diagnostic 
imaging before hospital discharge following TIL treatment, at 6 and 12 weeks after treatment 
and every 3 months hereafter. After 2 years the controls will change to every 6 months for a 
total of up to 5 years or until progression.  
 
A clinical examination, blood tests and diagnostic imaging will be performed in connection 
with every evaluation. At the first three evaluations the blood tests will include immunological 
parameters and a toxicity assessment (any late onset adverse events will registered 
throughput the entire follow-up period). If tumor tissue is accessible, a tumor biopsy will be 
performed at the first evaluation and/or after progression. 
 
End of study is defined as 6 months after the last patients’ treatment or after progression. OS 
and PFS will subsequently be followed for up to 5 years. 
Follow-up schedule 
Evaluation, month 
1,5 
3 
6 
9 
12 
15 
18 
21 
24 
30 
36 
42 
48 
54 
60 
Clinical examination 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
BP, P, PS, Weight 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Subjective complaints 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
ECG 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
Toxicity assessment 
(CTC) 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
Immunological blood 
tests 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
Research blood tests 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Evaluation blood 
tests 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Ovarian specific 
blood tests 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Tumor evaluation 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Tumor biopsy* 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*If possible, a biopsy is also perfomed at the time of progression. 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
33 
Research blood tests: See section “Blood samples for immunological monitoring” under the 
section “Immunological Monitoring”. 
 
Evaluation blood test: Hemoglobin, platelets, leukocytes with differential-count, creatinine, 
sodium, potassium, magnesium, phosphate, Ca-ion, ASAT, ALAT, bilirubin, alkaline 
phosphatase, albumin, LDH, CRP, INR, APTT, TSH, T3, T4.  
 
Immunological blood tests:  Hemoglobin, platelets, leukocytes with differential-count, 
creatinine, sodium, potassium, carbamide, bicarbonate, albumin, urate, ALAT, ASAT, LDH, 
alkaline phosphatase, bilirubin, amylase, glucose, TSH, T4, Ca-ion cortisol, prolactin, Estradiol. 
 
Ovarian specific blood tests: CA-125. 
 
Tumor evaluation: CT, MR, PET-CT or a PET-MR scan can be used. A PET/CT scan is preferred. 
 
Tumor biopsy: A tumor biopsy is carried out at 6 weeks evaluation and in case of progression 
if it is accessible and safe to perform. This is further explained in the “Tumor biopsies” section. 
Early termination of treatment 
Several reasons can lead to early termination of the planned treatment course: 
 
Not possible to grow TILs: The patient cannot be offered treatment if it is not possible to grow 
TILs. 
 
Patients own wish: The treatment can be stopped at any time the patient wishes so.  
 
Medical decision: The treatment can be stopped because of medical conditions at any time the 
investigator finds it in the patients’ best interest. 
 
Other treatment: Patients will be excluded at any time a new treatment with an experimental 
drug or other systemic anticancer treatment is initiated after inclusion in this protocol. The 
patient will be excluded if systemic treatment with corticosteroids is initiated unless it is on 
vital indication an in agreement with the physician responsible for the protocol. 
 
Adverse events: Treatment is terminated if adverse events of a degree that makes completion 
of the study impossible do occur. 
 
Patients who stop IL-2 treatment prematurely will still be followed in accordance with the 
protocol. 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
34 
If a patient is excluded before infusion of TILs a replacement subject will be found. They will 
be followed until end of adverse events caused by the treatment, but will not be followed with 
subsequent controls. 
Subsequent treatment 
Patients that are excluded from the study can receive other treatment freely. If patients 
progress they can receive other treatment. 
 
Production of TILs 
Acquisition of tumor tissue 
The patients will receive oral and written information as described elsewhere and a written 
consent will be obtained before surgery. A tumor biopsy will be performed after sufficient 
tissue material for pathological examination has been removed. The biopsy will be labeled 
with date and patient code, placed in a sterile container and transported to the GMP facilities 
54J7 or 54I6 at Herlev Hospital for further processing. See the IMPD for more details. 
If present, ascites fluid will collected during surgery, if possible to do so without increased 
risk to the patient. Ascites fluid will be labeled and transported in the same way as the biopsy, 
Acquisition and examination of ascites fluid will be for research purposes only. See the section 
‘Immunological monotoring’ and ‘Additional research analyses’ for more details. 
Establishment of ”Young TIL” cultures 
T cells are expanded using a recently established method for ”Young TILs”36. The tumor mass 
will be isolated with a scalpel, and cut into small 1-3 mm3 fragments. Fragments (typically 
from 24 to 72 fragments in total) will be placed separately in the wells of a 24 well/plate. A 
TIL culture is established from each fragment by passive migration of T cells from tumor 
tissue in the IL-2 based media. IL-2 belongs to the group of homeostatic cytokines which are 
characterized by having a positive effect on the activation of tumor specific T-cell and thereby 
tumor cell killing. T-cell density is kept at about 1x106 cells/ml growth media containing the 
immune stimulating cytokine IL-2. Cell cultures from the different fragments are pooled to a 
single cell culture. T-cell expansion is performed unselected to produce a polyclonal TIL 
repertoire targeted against multiple epitopes to potentially achieve more effective tumor cell 
destruction in vivo. The establishment of ”Young TIL” cultures usually takes 2-4 weeks with a 
rate of success more than 90%. See the IMPD for more details. 
Rapid Expansion Protocol (REP) 
When the TIL cultures are expanded to approximately 5x107 cells they are either frozen for 
later use or transferred directly for further expansion by use of the Rapid Expansion Protocol 
(REP) in which TIL are grown with radiated (40 Gy) allogeneic PBMCs (peripheral blood 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
35 
mononuclear cells) that work as “feeder cells”, IL-2 and anti-CD3 antibody that activates the 
TILs. In this way it is possible to reach a large number of activated tumor specific T-cell with a 
high level of activity against tumor associated antigens (TAA) and tumor in 14 days. 
Ultimately, the autologous T cells are concentrated in a 400 ml infusion bag for intravenous 
infusion. See the IMPD for further details. 
Handling and transportation of the infusion product 
The infusion bag is labeled with patient ID and a patient specific transport schedule is filled 
out and both are then placed in a secure hatch. A trained physician controls the information 
on the infusion product and transport schedule, signs the latter and transports the infusion 
product to the patient. Before administration, the infusion product is controlled again by the 
treatment staff and matched to the ID of the patient through patient identification. 
Phenotype- and clonotype determination 
The prevalence of T-cell types (e.g. CD4+ and CD8+) as well as characterization of T-cell stages 
in both Young TILs and REP TILs will be determined by use of flow cytometric analysis. See 
the IMPD for more details. 
 
Adverse events, potential risks and precautions 
Adverse Events 
Adverse events (AE) are defined as any undesirable experience occurring to a subject during a 
clinical trial, whether or not considered related to the investigational treatment. All AEs 
reported spontaneously by the subject or observed by the investigator or his staff will be 
recorded and described in the patient chart and the electronic Case Report Form (eCRF). The 
severity and consequences will be recorded for each AE. The severity and relation to the study 
medication will be assessed in accordance with the guidelines described in the following. 
The investigator must attempt to identify all clinical and objective events from patients 
receiving treatment and determine their relation to the study medication. The investigator 
determines the relationship between AEs and treatment using the following guidelines: 
Grading of Adverse Events 
The severity of an AE refers to the intensity of the reaction. 
 
Events are graded using CTCAE version 4.0 (appendix 3)70. The following scale can be used if 
this grading is not applicable:  
 
1 = light 
2 = moderate 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
36 
3 = severe 
4 = life threatening 
5 = lethal 
 
Patients experiencing AEs will be monitored with the relevant clinical evaluations and 
laboratory investigations assessed by the attending physician. All AEs must be monitored 
until satisfactory restitution or stabilization. Results of the monitoring must be recorded in 
the patient chart and eCRF. 
Abnormal laboratory tests are not to be recorded in the eCRF unless they have caused a 
clinical event, resulted in termination of the treatment or otherwise meet the criteria of a 
serious adverse event (see the following). 
Serious Adverse Events 
A serious adverse event (SAE) is to be reported to sponsor within 24 hours and is defined as 
any medical occurrence or effect that occurs at any dose: 
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalization or prolongation of existing patients’ hospitalization; 
- 
results in persistent or significant disability or incapacity; 
- 
leads to a congenital anomaly or birth defect; 
- 
is a significant medical event 
Guidelines for adverse events possible relation to the treatment 
0  
No relation– no temporal relation, other etiologies very likely the cause 
 
1  
Possible relation – less clear temporal relation, other etiologies likely the cause 
 
2  
Probably related – clear temporal relation with recovery at termination of treatment, and not 
reasonably explained by the patient’s known clinical condition 
 
3 
Related – clear temporal relation with laboratory confirmation or a positive retreatment test 
 
If the event is assessed as being caused by the investigational treatment it is classified as an 
adverse reaction (AR) or a serious adverse reaction (SAR). 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
002179-24. Research Ethics Committee file nr. 1704606. Danish Health and Medicines Authorities chart 
nr. 2017061517. 
37 
Adverse Reactions 
An adverse reaction (AR) can be expected if it is described in the IMPD or the relevant product 
summary, or unexpected if the grade or severity does not correlate with the product 
information in the before mentioned documents. 
If the AR is unexpected, meets the criteria of a serious adverse reaction (SAR) and is found 
related to the investigational treatment it is classified as a suspected unexpected serious 
adverse reaction (SUSAR). 
Reporting of Adverse Events and Adverse Reactions 
Investigator reports SAEs, SARs and SUSARs to sponsor within 24 hours. Sponsor reports 
SUSARs to the Danish Medicines Agency within 7 days if considered life threatening or fatal, 
and otherwise within 15 days. Consequences for the study must be reported. Sponsor submits 
a yearly list that summarizes any SAEs and SUSARs as well as a report regarding the study 
patients’ safety to the Danish Medicines Agency and the Research Ethics Committee 
(investigator can report to the Research Ethics Committee as well). 
Sponsor submits a final report to the Danish Medicines Agency at the end of the study, with a 
description of all SAEs, SARs and SUSARs. 
 
The following is not to be reported: 
- 
deaths caused by the malignant disease or progression 
- 
hospitalizations or prolongation of current hospitalization caused by the malignant 
disease: 
o weight loss 
o fatigue 
o electrolyte derangement 
o pain management 
o anxiety 
o palliative hospitalization 
o stay at hospice or terminal care 
o progression of the underlying disease 
- 
hospitalizations or prolongation of current hospitalization if the sole reason for 
hospitalization or prolongation is: 
o fluid treatment or treatment of nausea 


 
 
 
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o blood transfusion 
o platelet transfusion 
o febrile leucopenia/neutropenia 
o administration of investigational procedures 
o placement of a permanent intravenous catheter 
These events are to be registered in the eCRF. 
Known Adverse Reactions 
Chemotherapy 
The adverse reactions to chemotherapy described in the following are all general adverse 
reactions seen when the drugs are given as the primary treatment for oncological and 
hematological diseases. 
In these cases the treatment is often given over several series. In this study, the treatment will 
be given as a single dose, why we expect a milder adverse reactions profile. 
Cyclophosphamide 
The dose limiting toxicities in patients’ receiving treatment with cyclophosphamide are 
myelosuppression (neutropenia, thrombocytopenia and anemia) and urotoxicity (cystitis, 
haematuria and hemorrhagic cystitis). Sufficient treatment with Mesna alongside rehydration 
markedly reduces the frequency and severity of the urotoxicity. 
Other common adverse reactions are alopecia, nausea and vomiting. 
Patients receiving treatment with Cyclophosphamide can experience the following adverse 
reactions described in the product resumé53 found at www.laegemiddelstyrelsen.dk. 
Fludarabine phosphate 
The adverse reactions are myelosuppression (neutropenia, thrombocytopenia and anemia), 
infection including pneumonia, coughing, fatigue, limpness, nausea, vomiting and diarrhea. 
Other common adverse reactions are shivering, edema, malaise, peripheral neuropathy, visual 
disturbances, anorexia, mucositis, stomatitis and rash. Severe opportunistic infections have 
occurred in patients receiving treatment with fludarabine phosphate. Deaths have been 
recorded as a cause of severe adverse reactions. 
Patients receiving treatment with fludarabine phosphate can experience the following 
adverse reactions described in the product resumé54 found at www.laegemiddelstyrelsen.dk. 
Checkpoint inhibition and irAEs 
The antibodies used in this study targets activated T cells in the immune system by blocking 2 
natural brakes in T-cell activation (CTLA-4 and PD-1) which allows a potential immunological 


 
 
 
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reaction against tumor cells. Due to these mechanisms of action, immune related adverse 
events (irAE) can occur, which are often mild, but can aggravate and require treatment. 
Immune related adverse events include skin toxicities (erythema, exema or exanthema with 
generalized itching and urticarial, as well as possible worsening of existing autoimmune skin 
disordes (such as psoriasis and roseaca), gastrointestinal toxicities (diarrhea/colitis), 
endokrinopathias (hypophycitis, hypothyriosis, hyperthyriosis), hepatotoxicity, pulmonary 
toxicity (pneumonitis), ocular toxicity, myocarditis, neuropathia and immune related 
nefrotoxicity.   
Patients can experience irAEs from multiple organ systems and irAEs can also occur late 
during or after the course of treatment.  
See appendix 6 for details regarding handling of irAEs. 
Ipilimumab 
Patients receiving treatment with the CTLA-4 antibody Ipilimumab can experience adverse 
reactions as described above and in the product resumé63 found at www.ema.europa.eu. 
Nivolumab 
Patients receiving treatment with the PD-1 antibody Nivolumab can experience adverse 
reactions as described above and in the product resumé64 found at www.ema.europa.eu. 
TILs 
No SARs are expected due to TIL infusion. The patients might experience transient fever, 
shivering and mild dyspnoea with a few cases of an observed light decrease in saturation.  
There is a theoretical risk of the development of allergic reactions/anaphylactic shock. This 
has not yet been observed according to literature.  
See the IMPD for more details on previous human exposure and anticipated risks. 
Interleukin-2 
Frequency and severity of adverse reactions to IL-2 has generally shown to be related to the 
route of administration, dose and frequency of treatment. Most adverse reactions are self-
limiting and will disappear within 1-2 days after cessation of treatment. Subcutaneous IL-2 
administration can cause a local inflammatory reaction with reddening and induration at the site 
of injection. WHO grade 1 toxicity characterized by flu-like symptoms with muscle soreness, joint 
pains, malaise and a slight increase in temperature with a duration of 12-18 hours has been 
observed when administering IL-2 locally. Experience shows that fatigue is the most common 
adverse event when administering the IL-2 dosage used in this study. 
Patients receiving treatment with IL-2 can experience the following adverse reactions 
described in the product resumé71 found at www.laegemiddelstyrelsen.dk and adverse 
reactions will be treated as described in appendix 4.  


 
 
 
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Risks and disadvantages regarding surgery and test sampling 
Risks associated with removing tumor tissue 
Prior to inclusion it will be assessed whether it is possible to remove some of the patients own 
tumor tissue in a minor operative procedure. Surgery will mainly be performed by physicians 
at the Gynecological Department at Herlev Hospital or by physicians from other specialties if 
necessary. The patient will not be able to participate in the study if no tumor tissue is 
available for removal or if removal will put the patient at a too large risk.  
Risks associated with biopsies 
There is a slight risk of infection and/or bleeding when performing a biopsy. Pain and 
bruising might also occur in the area. 
Risks associated with blood tests 
Pain and bruising can occur in the area. Blood testing will involve frequent hospital visits.  
Monitoring and precautions 
Hematological parameters 
Careful hematological monitoring of blood counts is indicated for all patients during 
treatment. Leukocyte count, platelet count and hemoglobin values will be controlled at fixed 
intervals. Measurements will be made before start of chemotherapy, IL-2 and daily during 
treatment until neutrophile counts is > 500/μl and leukocyte count is > 1000/μl. 
Chemotherapy will not be given to patients with a leukocyte count < 500/μl and/or platelet 
number < 50.000/ μl before the start of chemotherapy. 
Kidney- and urine infections 
Any obstruction of the efferent urinary tracts, cystitis or infection will be resolved before start 
of treatment. Patients will be treated with Mesna and fluid therapy to decrease the frequency 
and severity of bladder toxicity. Treatment will be terminated if cystitis associated with 
micro- or macroscopic haematuria occurs during treatment with Cyclophosphamide. The 
patients’ urine will be controlled for the presence of microscopic haematuria before start of 
treatment with Cyclophosphamide. 
Cardiotoxicity 
Cardiotoxicity is especially seen when administrating high doses of Cyclophosphamide (120-
240 mg/kg body weight). An electrocardiogram will be performed before the start of 
treatment. Patients with known heart disease will not be included in the study. Necessary 
investigational procedures will be performed if the patients experiences symptoms from the 
cardiovascular system (e.g. chest pains, shortness of breath). Myocarditis has been described 


 
 
 
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Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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following high dose Il-2 treatment, if suspected CKMB, TPN, ECG and cardiology consult 
should be performed. 
Immune-related toxicity 
Is monitored and handled as described in appendix 6. 
Infertility 
Patients receiving chemotherapeutic treatment have a risk of affecting their fertility in the 
future. It is not known whether T-cell therapy increases the risk of cross reaction from T cells 
to normal ovarian tissue. 
Live vaccines 
Vaccination with live vaccines is to be avoided prior to- and immediately after treatment with 
chemotherapy because of the immunosuppressive effect. 
Interactions 
Cyclophosphamide inhibits cholinesterase activity which increases the effect of depolarizing 
muscle relaxants such as Suxamethoniumchloride. This can results in prolonged apnea when 
anesthetized. The anesthesiologist is to be informed if the patient has received treatment with 
Cyclophosphamide within 10 days before treatment with Suxamethoniumchloride. The 
combination should be avoided.  
 
The patient is to avoid eating grapefruit or drinking grapefruit juice since grapefruit contains 
a substance that can impair the activation of Cyclophosphamide and thereby its effect. 
 
Transfusion related graft-versus-host reactions have been observed in patients receiving 
treatment with fludarabine phosphate after transfusion with non-radiated/non-filtered blood. 
Patients requiring blood transfusion within ½ a year from treatment with fludarabine 
phosphate is therefore to receive only radiated or filtered blood. An agreement with the blood 
bank at Herlev Hospital has been made so that there will be ordered radiated blood only for 
these patients for ½ a year after treatment. All blood in the Capital Region is filtered. 
 
Effect evaluation, data processing and monitoring 
Effect evaluation 
Primary effect parameter 
Safety and toxicity:  
Registration of all AEs and unexpected events that occur in relation to the treatment will be 
done in accordance with the CTCAE v.4 criteria (appendix 3). 


 
 
 
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Secondary effect parameters 
Immune monitoring:  
Patients will be followed continually with in vitro analysis of the specific T-cell reactivity 
against tumor antigens to evaluate the immunological effect of treatment. The immunological 
response against tumor antigens before and after treatment will be compared. These analyses 
will be done on blood samples and tumor biopsies. 
Clinical evaluation:  
The clinical effect of treatment will be rated using the objective response rate in accordance 
with RECIST 1.1 and overall survival (OS) and progression-free survival (PFS) will be 
described. Furthermore, PERCIST72 and irRC73 will be used exploratively.  
Response Criteria 
RECIST 
Clinical evaluation will be done in accordance with RECIST 1.1 Guidelines74:  
 
Complete response (CR): All lesions disappear. 
Partial response (PR): Defined as a ≥ 30 % reduction in the sum of all measurable parameters 
longest diameter. 
Stabile disease (SD): Defined as a < 30 % reduction in the sum of all measurable parameters 
longest diameter or a < 20 % increase in the sum of all measurable parameters longest 
diameter. 
Progression (PD): Defined as a > 20 % increase in the sum of all measurable parameters 
longest diameter or the appearance of new lesions. 
 
Complete and partial response is to be verified by examination at a minimum of 4 weeks after 
documentation of the response at the earliest. 
Immunological monitoring 
Blood samples for immunological monitoring 
Blood samples with 100 ml heparinized blood for immunological monitoring and 10 ml blood 
in a dry glass for freezing of serum are taken: before treatment with ipilimumab, before start 
of treatment (at ”baseline”), at hospital discharge (approximately 1 week after T-cell infusion) 
and 6 and 12 weeks after T-cell infusion (see the examination plan and follow-up schedule). 
Blood samples will be taken for immunological monitoring every 3rd month at evaluation 
hereafter until the patient is withdrawn from the study. In addition, serum samples (10 ml 
blood sample) will be taken at day 0 before TIL infusion, 2 hours after TIL infusion and every 
2nd day until hospital discharge. 
A total of 500 ml of blood will be collected for research purposes in the period from time of 
surgery until the patient meet for the first evaluation after treatment. These blood samples 


 
 
 
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Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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43 
are taken to assess the effect of treatment on the immune system for research purposes. The 
amount of blood taken during the course of the study does not exceed what the body itself is 
capable of producing between each test. Blood samples for research purposes will not be 
taken if the blood count is not acceptable (> 6 mmol/l). 
 
Mononuclear cells from the peripheral blood (PBMCs) are isolated using 
Lymfoprep/Leucosep density gradient technique. The mononuclear cells are washed and 
resuspended in a freezing media consisting of 90% heat inactivated humane AB serum and 10 
% DMSO. The cells are frozen at -150°C until analysis. A panel of relevant immunological 
assays for testing of antigen specific immune reactivity will be applied, including 
measurements of cytokine production (multimeric fluorescence coloring, ELISpot and ELISA), 
proliferative- and cytotoxic potential. 
Tumor biopsies 
Biopsies are sought taken from accessible tumor lesions or involved lymph nodes depending 
on localization and accessibility. Biopsies are preferred at evaluation 6 weeks after treatment 
and at progression if possible (see the examination plan and follow-up schedule).  
Biopsies will be performed guided by ultrasound and under sterile conditions by the 
Ultrasound Department, Herlev Hospital, if the involved areas are not directly accessible. The 
procedure will be performed in the outpatient clinic and the size will be approximately 5 mm3. 
 
Biopsies will be examined for the concentration of immune cells. Furthermore, TILs will be 
isolated from the lesions and analyzed for clonotype and specificity. 
Specimens for Future Biomedical Research 
Only leftovers from analyses specifically described in the protocol will be transferred to the biobank 
for future biomedical research, as described on page 48 “Research biobank including Future 
Biomedical Research Biobank”. There will be no extra samples to be obtained during this study. 
These specimens may be used to study various causes for how subjects may respond to the 
immunotherapy. These speciemens will be stored to provide a resource for future trials conducted 
by CCIT focused on the study of biomarkers responsible for how immunotherapy works, other 
pathways immunotherapy may interact with, or other aspects of disease. 
Additional research analyses 
Isolation of tumor cells 
Tumor cells are isolated from the tumor fragments or ascites fluid by the use of enzymatic 
processing or seeding of cells from the tumor fragments or ascites fluid and are then frozen 
for later use in determining T-cell anti tumor activity. This exploratory analysis will not 
influence any prior or subsequent surgical decisions or medical treatment of the patients 
enrolled. 


 
 
 
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Tumor-associated lymphocytes (TALs) and chemokines 
Lymphocytes found in ascites will be isolated with gradient centrifugation. A portion will be 
frozen and stored for additional immunological analyses, while a portion will be cultured 
immediately by using the exact methods used with TILs. Remaining ascites fluid will be 
centrifuged, and supernatants will be collected and frozen to study which cytokines present in 
the fluids can influence the activity of TALs. 
Cytokine Release Assay 
TIL cultures is screened for activity against TAA and autologous tumor by determining their 
production of the activating cytokines (INF-γ + TNF-α). The production of the activating 
cytokines is quantified by use of ELISpot technique and intracellular flow cytometric analysis. 
Gene-based arrays 
The aim of the planned gene analyses is to learn more about; 
1. Differences among patients in expression level of a panel of relevant normal genes in 
the tumor microenvironment which could influence the chance of benefit from 
treatment 
2. Expression of tumor/patient specific mutated genes which could influence the chance 
of benefit from treatment 
Analysis for identifying specific tumor gene expression signatures75 and mutations in the 
tumor cells, leading to patient-specific neo-antigens derived from these mutations76, will be 
performed.  
These analyses will contribute to the identification of patients that are most likely to respond 
to treatment as well as contribute to the optimization of T-cell therapy based on the selection 
of neo-antigen specific T cells. These analyses will not benefit the patients included in this 
study, but might benefit future patients. 
Methods: 
Tumor tissue gene expression profiles will be analysed on FFPE tumor tissue. Using Illumina 
targeted RNA sequencing applicable to degraded RNA we will retrieve gene expression data 
on approximately 500 cancer/immunity related genes. 
Next Generation Sequencing (NGS) of tumors and normal cells of the individual patient will be 
used to obtain information on tumor-specific mutations. Gene sequencing will be performed 
on tumor cells (tumor gene profile) and leukocytes (normal gene profile). Tumor specific gene 
expression (neo antigens) will be performed by subtracting the two gene profiles from each 
other. Data regarding potential disease causing genes will be generated as a byproduct of this 
analysis, but this data will not be used or explored further upon, since only data regarding 
tumor specific genes will be processed more closely. Therefore, we do not expect to obtain 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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explicit knowledge regarding disease causing genes. Furthermore, ‘Targeted sequencing’ on a 
limitied number of defined genes will be performed on the tumor tissue to obtain a ‘immune 
profile’ to determine which genes- and consequently which proteins- are expressed in the 
tumor tissue. Data will be handled according to national laws.  
Patients will have the option of being refered to comprehensive genetic counseling prior to 
giving their consent. If by chance these analyses will discover known mutations with potential 
significant impact on patient’s health, the case will be discussed with the Clinical Genetics 
Department, Rigshospitalet, unles the patient has chosen not to be informed as stated in the 
patient information. The following criteria will determine if further actions are indicated. 
• there is a reasonable degree of possibility that a genetic disposition is present, 
• there is solid documentation of a link between the genetic disposition and the 
development of disease, 
• the tests used to determine the genetic disposition are well established, 
• the disease in question can be prevented or treated, and 
• the link between the genetic disposition and the development of disease has 
considerable importance for the patient. 
If indicated, the patient will be contacted and asked for permission to referral to such 
Department for additional information and testing. 
In case that a patient dies/is dead, or do not want information regarding significant health 
issues, a medical assessment, using the 5 above mentioned criteria, will determine whether 
relatives to the patient is to be informed, in accordance with Danish law (sundhedslovens § 
43, stk. 2, nr.2.). 
Statistics  
The study is non-blinded and non-comparative. Descriptive statistics will be used to estimate 
the immunological- and clinical response rate. Descriptive statistics will also be used to sum 
up the duration of response and patient characteristics. The study is designed as a phase I/ 
phase II study and the primary aim is to determine safety and toxicity to the treatment. A 
required sample size that allows determination of primary- as well as secondary- and tertiary 
end points can therefore not formally be calculated.  
Data registration and -analysis 
The patients are given a patient number at inclusion in the study to secure patient anonymity. 
Clinical personnel and selected persons in the laboratory will have access to patient 
information to secure proper treatment. 


 
 
 
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The principal investigator has access to patient charts to obtain information regarding the 
cancer disease to be able to compare this information with the project specific analysis 
performed on cancer tissue and blood tests.  
 
All relevant data is registered in the eCRF (electronic Case Report Form) developed in 
cooperation with the clinical research unit, KFE. The principal investigator has the 
responsibility of manufacturing the eCRF and subsequent recording of data when the 
investigational treatment is finished and eCRFs are reported to sponsor. Sponsor and 
principal investigator are responsible for data analysis on all included patients. Patient data 
and eCRF will be stored for 5 years in accordance with current guidelines for storage of 
personal information. Drafting of a final report will be conducted in collaboration between the 
members of the study group. 
 
Analyses will include: 
• Toxicity (CTC registration) 
• Immunological response  
• Clinical effect parameters 
 
Personal data and remaining tests will be coded at the end of the study. All patients receiving 
T-cell therapy will be included in the statistical analyses. Patients excluded for one of the 
following reasons will not be included in the statistical analyses: 
• Not enough tissue to produce tumor TIL 
• Unable to produce TILs in the laboratory  
• Withdrawal of consent 
• Started other treatment 
End of study report 
Sponsor will inform the Danish Medicines Agency and Research Ethics Committee within 90 
days of study completion. The definition of study completion is 6 months after the last 
patients’ treatment or after exclusion due to progression. In addition, patients will be followed 
for PFS and OS for up to 5 years. If the study is prematurely terminated, the Danish Medicines 
Agency and the Research Ethics Committee will be informed of the reason(s) for the 
termination. Sponsor will submit a final study report to the Danish Medicines Agency and the 
Research Ethics Committee with the study results including publications based on the study 
within a year of study completion. 
Amendments 
An application to the Danish Medicines Agency and the Research Ethics Committee will be 
made if substantial changes to the protocol are to be made. These can be implemented when 
approved. Changes to the protocol are considered substantial in accordance with ‘Vejledning 


 
 
 
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Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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om anmeldelse, indberetningspligt m.v. (sundhedsvidenskabelige forskningsprojekter)’, 
paragraph 6.0. on www.dnvk.dk and the schedule ’Skema om ændringer (ammendmendts) til 
kliniske forsøg’ on sundhedsstyrelsen.dk.  
 
Ethical aspects 
Recruitment of study patients and informed consent 
Eligible patients with advanced ovarian cancer will be referred from the Oncological 
Department at Herlev Hospital or other Oncological centers in Denmark treating patients with 
these cancers. Information about the study will be given at scientific meetings for physicians 
at the relevant departments. 
Referral of patients is to be made to the uro-gynecological (UG team) visitation office, 
Department of Oncology, Herlev Hospital. 
Contact to eligible patients will be done in accordance with the Danish Health Act, § 46, 
paragraph 3. 
 
All patients will be informed about the study according to appendix 1. 
Insurance 
Patients’ participation in the study will be covered by “Patienterstatningen”. 
Ethical aspects 
Despite advances in the chemotherapeutic treatment of patients with relapse of ovarian 
cancer the prognosis is still poor and many of the women are still young and in a good general 
condition, why the need for new effective treatments is great10. The purpose of this study is to 
improve survival for patients with relapse of ovarian cancer. Based on the current knowledge 
and the lack of treatment options, the risks and downsides associated with this study are 
assessed to be acceptable.  
Participation is voluntary and is preceded by oral and written information and the treatment 
will be stopped in case of unacceptable adverse reactions or if the patient wishes so at any 
time. 
The patient will receive treatment after the current guidelines at the department if she does 
not wish treatment according to the protocol. The study is therefore assessed as ethical 
proper.  
 
The study follows the Helsinki agreement and the principal investigator is to obtain 
permission from the Danish Medicines Agency and the Research Ethics Committee. 
 


 
 
 
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For future biomedical research, risks to the subject have been minimized. Risks include those 
associated with venipuncture to obtain the whole blood specimen. This specimen will be obtained at 
the time of routine blood specimens drawn in the main trial. 
Research biobank, including Future Biomedical Research Biobank 
In connection with the current study, blood samples (110 ml/blood sample) and tumor 
biopsies will be stored in coded form at -150 °C in a research biobank at the CCIT in room 
PA102 until all analysis concerning the study is performed. Samples that are not used in the 
study will be transferred to a biobank at CCIT for future biomedical research as described 
below (p. 49). 
 
Analyses will be primarily performed at CCIT. However, some special analyses on tumor 
tissue or blood test samples may be performed at a partner institution after establishing a 
specific written agreement. The Research Ethics Committee will be informed if any such 
agreement is to be established. All patient relevant information will be sent in a codedway. In 
case the patient’s cells will be sent to partner institutions located abroad, these will be 
handled according to national laws and regulations of the specific nation where these have 
been sent. In such a case, all patient information will be communicated in coded form. A 
written data processing agreement will be signed between the data controllers and the data 
processors abroad. If any gene analysis are to be performed abroad, the data processing 
agreement will include the 5 criteria (p. 45) regarding the discovery of known mutations with 
potential significant impact on patient’s health, as well as the requirement, that the partner 
abroad reports back to the primary project managers in Denmark so that relevant actions can 
be taken as described on p. 43. If data processing is to be performed in a third-country, 
permission wil be applied for at the Danish Data Protection Agency, or one of the agencies 
standard contracts will be used. 
 
Samples that are not used in the study will be transferred to a biobank at CCIT for future 
biomedical research for up to 15 years and is accepted by the Danish Data Protection Agency. 
If additional studies in other research areas are to be performed on any samples obtained 
during the conduct of this study/samples that are transferred to a new biobank, a request to 
do so will be submitted to the Research Ethics Committee as per the ‘Act of Processing of 
Personal Data’ §§ 5 and § 10, paragraph 2 and 3. After 15 years, any remaining tissue samples 
will be disposed of according to the local guidelines for destruction of biohazardous waste. 
If a patient withdraws his/her informed consent, all biological material is to be destroyed if 
the patient wishes so.  


 
 
 
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Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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Reporting to the Research Ethics Committee 
The study is reported to the Research Ethics Committee. The law dealing with personal data 
will be respected. Information concerning study patients is protected according to the law 
concerning personal data and the Act on Research Ethics Review of Health Research Projects.  
 
Administrative aspects and publication 
Patient identification 
Patients will be given a number after enrollment in the study. This number will be used to 
identify the patient and will be used in the Case Report Forms (eCRFs). Data and patient 
materials will be treated in code and confidentially. The number is given sequentially after 
enrollment in the study and is not based on the patients’ initials or birthday.   
Publications 
The primary project managers are Inge Marie Svane and Anders Kverneland. The members of 
the project group have joint copyright of the obtained results, given that the Vancouver rules 
are met. Positive results, inconclusive results as well as negative results will be published in 
international journals. Manuscripts will be produced in cooperation with the project 
managers and other members of the study group, with the project managers as primarily 
responsible for the preparation. The project managers are co-authors on publications made 
on the basis of this study. Author succession will be determined based on the individual 
contributions. Use of study data, oral as well as written, at congresses, teaching or the likes, is 
only to take place if accepted by the project managers. The project managers are obliged to 
publish results from the study and are naturally interested in the propagation and 
implementation of the results in clinic. Publications are expected to be completed in 2020. 
Economy 
The study is initiated by Center for Cancer Immune Therapy in cooperation with the 
Department of Oncology, Herlev Hospital, and is partially financed by these two departments. 
In addition, operational- and salary funding is applied for ongoing. At the current stage, 
research grants have been obtained from ‘The Danish Cancer Society’ and ‘OvaCure’.  
 
The Research Ethics Committees will be informed if/when new funds support is obtained. 
None of the physicians involved in the study have any economic interests in the study and 
there is no potential economic gain for the departments or of personnel in connection with 
the study. There are no economic attachments between the financial supporters and the 
project managers. 
The study is part of the principal investigator Anders Kverneland’s PhD project. 
 
 


 
 
 
Protocol GY1721: T cell Therapy in Combination with Checkpoint Inhibitors for Patients with Advanced 
Ovarian-, Fallopian Tube- and Primary Peritoneal Cancer. Version 6. Date: 13-07-2018. EudraCT nr 2017-
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Reference list 
1. 
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