Clinical trial NCT04904185
Conditions: Malignant Melanoma
Keywords: Adoptive Cell Therapy Immune Therapy Multiple Antigen Specific Endogenously derived T cells
Sponsor: Inge Marie Svane
Start date: 2021-09-17; Completion date: 2024-06-07
Enrollment: 8 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
With the introduction of checkpoint inhibitors substantial improvements have been made in the treatment of malignant melanoma (MM). Despite this still a a subset of patients, approximately 50 %, experience no response to therapy.

One of the strategies to overcome these obstacles have been ACT with tumour infiltrating lymphocytes (TILs). Most TIL based ACT products are non-specifically expanded providing growth preference to co-infiltrated virus specific T cells, and it is currently challenging to expand T cells in an antigen-specific manner, while at the same time obtaining the ideal functional characteristics for specific and strong tumour-killing capacity with sufficient persistence.

In this phase I trial artificial antigen-presenting scaffolds for antigen-driven T cell expansion are used. These scaffolds will generate a MASE-T cell product enriched for selected specificities towards antigens known to be expressed by melanoma cells The aim of the study is to demonstrate that treatment with af MASE-T cell product i safe and feasible. Further the study will elucidate whether treament with the MASE-T cell product leads to objective responses and improves progression free survival (PFS).

Detailed description:
There are around 350-400 new cases of patients with metastatic melanoma (MM) per year in Denmark. MM is a very aggressive cancer with a poor prognosis. Traditional oncological treatments such as surgery, chemotherapy and radiation therapy have a poor effect, and the 5-year overall survival has hitherto been less than 10 %.Substantial improvements have been made in the treatment of MM; especially immunotherapy is showing promising results with checkpoint inhibitors (CPI) such as programmed cell death protein 1 (PD-1) and Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) blocking antibodies administered as standard treatment in the frontline. The 5-year overall survival has now reached 52 %, 44 % and 26 % in nivolumab/ipilimumab, nivolumab, and ipilimumab respectively. However, a subset of patients - approximately 50 % experience no response to therapy, with clear primary resistance. One of the strategies to overcome these obstacles have been ACT with tumour infiltrating lymphocytes (TILs). A crucial condition for optimal ACT based on TILs is the generation of sufficient numbers of tumourreactive T cells. However, the expansion of TILs requires extensive ex vivo culturing often at the cost of T cell differentiation and functional activity. Most TIL based ACT products are non-specifically expanded providing growth preference to co-infiltrated virus specific T cells, and it is currently challenging to expand T cells in an antigen-specific manner, while at the same time obtaining the ideal functional characteristics for specific and strong tumour-killing capacity with sufficient persistence. Recent data suggest that the majority of tumour specific T cells responsible for tumour rejection under CPI are recruited from peripheral blood and lymph system, while not present in the tumour prior to treatment. This is supported by the finding that most tumour resident T cells are dysfunctional.

To overcome the current limitations in the treatment of malignant melanoma artificial antigen-presenting scaffolds for antigen-driven T cell expansion, generating a MASE-T cell product enriched for selected specificities towards antigens known to be expressed by melanoma cells has been designed. The antigen-scaffolds will ensuring optimal T cell stimulation by mimicking the in vivo stimulation of T cells by dendritic cells in the lymph nodes. The scaffolds contain both the antigen specific element - in the form of a peptide-MHC molecule and cytokine (IL2 and IL21), to provide growth and functional signals to the antigen specific T cell. As a result of this T cell expansion strategy, we can obtain a T cell product enriched for tumourantigen specific T cells. Superior functional activity towards tumor cells and antigen recognition compared to conventional T cell expansion strategies has been demonstrated in-vitro. Importantly, antigen-specific T cells in the MASE-T cell product possess a 'younger' phenotype, which has previously been described to correlate with improved in vivo persistence.

The study is a phase 1, non-randomized study. The trial will be conducted in two parts (A and B). Patients will be treated as followed:

* Part A (6 patients): Lymphodepleting chemotherapy (cyclophosphamide 500 mg/m2/day i.v. on day -4, -3, -2 and fludarabine 30 mg/m2/day i.v. on day -4, -3) followed by i.v. infusion of the MASE-T product on day 0. If the production of the MASE-T cell product was feasible for the majority (≥50%) of patients intended to treat in Arm A and the toxicity was acceptable, six patients will further be included in part B.
* Part B (6 patients): Lymphodepleting chemotherapy (cyclophosphamide 500 mg/m2/day i.v. on day -4, -3, -2 and fludarabine 30 mg/m2/day i.v on day -4, -3) followed by i.v infusion of the MASE-T product on day 0. Pembrolizumab 2 mg/kg will be administered on day -1 and day +21.

The primary objective is to evaluate the safety and feasibility of the MASE-T treatment alone or in combination with Pembrolizumab in patients with stage IV metastatic melanoma according to Common Terminology Criteria for Adverse Events (CTCAE version 5.0).

The secondary objectives are to evaluate T cell profile and persistence in vivo from tumor biopsies and blood samples as well as evaluation of the clinical efficacy of the treatment according to RECIST 1.1 and iRECIST. In addition, best overall response (BOR), duration of response (DOR), overall survival (OS), progression-free survival (PFS) will be monitored.

Arms: arm 1: Six patients will be included in Part A. After inclusion 300 mL blood will be drawn from the patients for the production of the MASE-T cell product. Four days prior to MASE-T infusion the patient will receive lymphodepleting chemotherapy (cyclophosphamide 500 mg/m2/day i.v. on day -4, -3, -2 and fludarabine 30 mg/m2/day i.v. on day -4, -3) followed by i.v. infusion of the MASE-T product on day 0. arm 2: Six patients will be includede in Part B. After inclusion 300 mL blood will be drawn from the patients for the production of the MASE-T cell product. Four days prior to MASE-T infusion the patient will receive lymphodepleting chemotherapy (cyclophosphamide 500 mg/m2/day i.v. on day -4, -3, -2 and fludarabine 30 mg/m2/day i.v on day -4, -3) followed by i.v infusion of the MASE-T product on day 0. Pembrolizumab 2 mg/kg will be administered on day -1 and day +21.
Interventions: intervention 1: Cyclophosphamide intervention 2: Fludarabine Phosphate intervention 3: Multiple Antigen Specific Endogenously derived T cells intervention 4: Pembrolizumab
Intervention descriptions: intervention 1: Cyclophosphamide 500 mg/m2 is administered i.v. on day -4, -3 and -2 intervention 2: Fludarabine Phosphate 30 mg/m2 is administered on day -4 and -3 intervention 3: Antigen specific, ex vivo expanded T cells derived from peripheral blood T cells intervention 4: Pembrolizumab 2 mg/kg is administered on day -1 and on day 21. The medicine is administered over 30 minutes

Protocol documents:
Prot_SAP_000.pdf: 


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Project managers and collaborators 
Sponsor 
Inge Marie Svane, professor, M.D., Professor 
Head of National Center for Cancer Immune Therapy (CCIT-DK) 
Phone: 38 68 21 31 
 
 
 
Principal Investigator 
Tine Juul Monberg, M.D., Ph.D. student 
National Center for Cancer Immune Therapy (CCIT-DK) 
Phone: 38 68 29 83 
 
 
Additional collaborators 
Özcan Met, MSc, PhD 
National Center for Cancer Immune Therapy (CCIT-DK) 
Department of Hematology and Oncology 
Herlev Hospital, University of Copenhagen 
Phone: 38 68 92 29 
 
Sine Reker Haderup, Sektionsleder, Professor 
Institut for Sundhedsteknologi 
DTU Kemitorvet, Bygning 203, rum 154 
2800 Kgs Lyngby  
Phone: 35 88 62 90 
 
Monitoring 
GCP-enheden 
GCP-unit at University of Copenhagen 
Bispebjerg Hospital, Bygning 51, 3.sal 
Bispebjerg Bakke 23 
2400 København NV 
Phone: 38 63 56 20 
 
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. 
 


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1. Table of Contents 
1. 
Table of Contents ............................................................................................................................. 3 
1 Synopsis ................................................................................................................................................. 6 
1.1 Rationale ......................................................................................................................................... 6 
1.2 Objectives and endpoints ............................................................................................................. 7 
1.3 Study design ................................................................................................................................... 7 
1.4 Population ....................................................................................................................................... 8 
1.5 Toxicity ............................................................................................................................................ 8 
1.6 Evaluation of toxicity and clinical response ............................................................................... 9 
1.7 Translational research and immunological response evaluation............................................ 9 
2 Introduction and rationale ................................................................................................................... 9 
2.1 Current treatment landscape for MM patients in Denmark .................................................... 9 
2.2 Tumour immunology and cancer immunotherapy ................................................................... 10 
2.3 Adoptive cell therapy (ACT) in patients with Metastatic Melanoma ................................... 11 
2.3.1 ACT with TILs ........................................................................................................................ 11 
2.3 Tumour antigens .......................................................................................................................... 11 
2.4 ImmPACT scaffolds for expansion of MASE-T product ......................................................... 12 
2.5 ACT with Multiple Ag specific T cells (MASE-T) .................................................................... 14 
2.6 The supportive regimen .............................................................................................................. 14 
2.7 CCIT-DK is a leading center for ACT ........................................................................................ 15 
3. Hypothesis, objectives and endpoints ............................................................................................ 15 
3.1 Hypothesis .................................................................................................................................... 15 
3.2 Primary objective and endpoint ................................................................................................. 15 
3.3 Secondary objectives and endpoints ........................................................................................ 16 
4. Trial design ......................................................................................................................................... 16 
4.1 Trial period .................................................................................................................................... 16 
4.2 Statistical consideration ............................................................................................................. 16 
4.3 Study population .......................................................................................................................... 17 
4.4 Inclusion criteria .......................................................................................................................... 17 
4. 5 Exclusion criteria ........................................................................................................................ 17 
5. Conduct of study ................................................................................................................................ 18 
5.1 Overview ........................................................................................................................................ 18 
5.2 Screening ...................................................................................................................................... 18 


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5.3 Treatment plan ............................................................................................................................. 19 
5.4 Overview of blood tests and evaluation during the treatment course ................................. 21 
5.5 Medicinal products used in the protocol .................................................................................. 22 
5.5.1 Cyclophosphamide (sendoxan®) ........................................................................................ 22 
5.5.2 Fludarabine phosphate (“Actavis” or “Ebewe”) ............................................................... 22 
5.5.3 Pembrolizumab ..................................................................................................................... 22 
5.5.4 The MASE-T product ............................................................................................................ 22 
5.5.5 Pegfilgratim (Neulasta) ........................................................................................................ 22 
5.6 Guidelines for supportive and concomitant therapy ............................................................... 23 
5.7 Follow-up ...................................................................................................................................... 24 
5.8 End of study .................................................................................................................................. 24 
5.9 Early termination of the treatment ............................................................................................ 25 
5.10 Criteria for treatment modifications ....................................................................................... 25 
5.11 Concurrent treatment................................................................................................................ 25 
6. 
Manufacturing of the MASE-T product with ImmPACT technology ....................................... 25 
6.1 Expansion of PBMC cultures to MASE-T product .................................................................. 25 
6.2 Handling and transportation of the MASE-T product ............................................................ 26 
7 
Adverse events, toxicity and precautions ................................................................................... 26 
7.1 Adverse events and reactions .................................................................................................... 26 
7.2 Reporting of Adverse Events and Adverse Reactions ............................................................ 27 
7.3 Toxicity related to lymphodepleting chemotherapy ................................................................ 28 
7.3.1 Cyclophosphamide ............................................................................................................... 28 
7.3.2 Fludarabine phosphates ...................................................................................................... 28 
7.4 Toxicity related to MASE-T infusion ......................................................................................... 28 
7.5 Toxicity related to Pegfilgastrim ................................................................................................ 28 
7.6 Toxicity related to Pembrolizumab ............................................................................................ 28 
7.8 Risks and disadvantages regarding invasive procedures ...................................................... 29 
7.9 Monitoring of organ function...................................................................................................... 29 
7.10 Risks and precautions ............................................................................................................... 29 
8 Translational research ....................................................................................................................... 30 
8.1 Research blood tests ................................................................................................................... 30 
8.2 Tumour biopsies .......................................................................................................................... 31 


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8.2.1 Gene-based arrays ............................................................................................................... 31 
8.2.2 Cell lines and cell cultures ..................................................................................................... 31 
9 Data handling and reporting ............................................................................................................. 32 
9.1 Electronic case report form ........................................................................................................ 32 
9.2 End of study .................................................................................................................................. 32 
10 
Research partner institutions ................................................................................................... 32 
11 Ethical considerations ...................................................................................................................... 33 
11.1 Recruitment of patients and informed consent..................................................................... 33 
11.2 Patient identification ................................................................................................................. 33 
11.3 Insurance .................................................................................................................................... 33 
11.4 General ethical aspects ............................................................................................................ 33 
11.5 Ethical considerations regarding gene-based research ....................................................... 34 
11.6 Approvals .................................................................................................................................... 34 
11.7 Amendments .............................................................................................................................. 34 
12 Biobank and handling of biological material................................................................................. 35 
12.1 Research biobank ...................................................................................................................... 35 
12.2 Biobank for future research ..................................................................................................... 35 
12.3 Disposal of biological material ................................................................................................ 35 
13 Administrative aspects and publications ...................................................................................... 35 
13.1 Publications ................................................................................................................................ 35 
13.2 Economy ...................................................................................................................................... 35 
14. References ........................................................................................................................................ 37 
14 
Appendix 1: CTCAE v 5.0 ........................................................................................................... 40 
Common Terminology Criteria for Adverse Events v5.0 ............................................................... 40 
15 
Appendix 2: Guidelines for informed consent ........................................................................ 41 
Retningslinjer for informeret samtykke (Guidelines for informed consent) .............................. 41 
16 
Appendix 3: ECOG ...................................................................................................................... 43 
17 
Appendix 4: Guideline for treating febrile neutropenia ......................................................... 44 
 


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1 Synopsis 
1.1 Rationale 
This protocol describes the application of Adoptive Cell Transfer (ACT) of Multiple Antigen (Ag) 
Specific Endogenously derived T cells (MASE-T) alone or in combination with Pembrolizumab in 
patients with metastatic melanoma.  
 
There are around 350-400 new cases of patients with metastatic melanoma (MM) per year in 
Denmark. MM is a very aggressive cancer with a poor prognosis. Traditional oncological treatments 
such as surgery, chemotherapy and radiation therapy have a poor effect, and the 5-year overall 
survival has hitherto been less than 10 %.1  
Substantial improvements have been made in the treatment of MM; especially immunotherapy is 
showing promising results with checkpoint inhibitors (CPI) such as programmed cell death protein 1 
(PD-1) and Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) blocking antibodies 
administered as standard treatment in the frontline.2,3,4 The 5-year overall survival has now reached 
52 %, 44 % and 26 % in nivolumab/ipilimumab, nivolumab, and ipilimumab respectively.5  
 
However, a subset of patients – approximately 50 % experience no response to therapy, with clear 
primary resistance. The reasons for primary resistance include inadequate T-cell infiltration into the 
tumour, as well as immunosuppressive factors within the tumour microenvironment.6,7 Emerging data 
also demonstrates that patients who fail responding to CPI, do not mount a sufficient T cell response 
towards the tumour. 
One of the strategies to overcome these obstacles have been ACT with tumour infiltrating 
lymphocytes (TILs).8  
A crucial condition for optimal ACT based on TILs is the generation of sufficient numbers of tumour-
reactive T cells.9,10  
However, the expansion of TILs requires extensive ex vivo culturing often at the cost of T cell 
differentiation and functional activity. Most TIL based ACT products are non-specifically expanded 
providing growth preference to co-infiltrated virus specific T cells, and it is currently challenging to 
expand T cells in an antigen-specific manner, while at the same time obtaining the ideal functional 
characteristics for specific and strong tumour-killing capacity with sufficient persistence.11,12,13  
 
Recent data suggest that the majority of tumour specific T cells responsible for tumour rejection 
under CPI are recruited from peripheral blood and lymph system, while not present in the tumour 
prior to treatment.14 This is supported by the finding that most tumour resident T cells are 
dysfunctional.15,16 
 
1.1.1 Expansion of Multiple Ag specific T cells (MASE-T) with ImmPACT technology 
To overcome the mentioned limitations, we have designed artificial antigen-presenting scaffolds for 
antigen-driven T cell expansion, generating a MASE-T cell product enriched for selected specificities 
towards antigens known to be expressed by melanoma cells. The antigen-scaffolds will ensuring 
optimal T cell stimulation by mimicking the in vivo stimulation of T cells by dendritic cells in the  lymph 
nodes. The scaffolds contain both the antigen specific element – in the form of a peptide-MHC 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
molecule and cytokine (IL2 and IL21), to provide growth and functional signals to the antigen specific 
T cell. As a result of this T cell expansion strategy, we can obtain a T cell product enriched for tumour-
antigen specific T cells. Superior functional activity towards tumor cells and antigen recognition 
compared to conventional T cell expansion strategies has been demonstrated in-vitro. Importantly, 
antigen-specific T cells in the MASE-T cell product possess a ‘younger’ phenotype, which has 
previously been described to correlate with improved in vivo persistence.17 Similar antigen 
presenting scaffolds have been successful in the expansion of both antigen-specific T cells and 
chimeric antigen receptor T cells (CAR-Ts).18 
 
1.1.2 Infusion of Multiple Ag Specific T cells (MASE-T) 
Hence, we aim to infuse a T cell product for ACT derived from peripheral blood T cells. The 
melanoma-antigen specific T cells in the peripheral blood sample will engage with the Ag-scaffold to 
receive preferential expansion signals. The Ag-scaffold will ensure the presence of all required T cell 
stimulation factors to form an immunological synapse for efficient T cell stimulation.  
 
The MASE-T cell product will be administered alone in the first six patients. If tolerated, it will be 
combined with the standard treatment Pembrolizumab, a PD-1 blocking antibody, with the rationale 
to enhance the MASE-T cell product anti-tumour activity by blocking inhibitory signals of T-cell 
activation in the tumour microenvironment.  .  
 
1.2 Objectives and endpoints 
The primary objective is to evaluate the safety and feasibility of the MASE-T treatment alone or in 
combination with Pembrolizumab in patients with stage IV metastatic melanoma according to 
Common Terminology Criteria for Adverse Events (CTCAE version 5.0).  
 
The secondary objectives are to evaluate T cell profile and persistence in vivo from tumor biopsies 
and blood samples as well as evaluation of the clinical efficacy of the treatment according to RECIST 
1.1 and iRECIST. In addition, best overall response (BOR), duration of response (DOR), overall 
survival (OS), progression-free survival (PFS) will be monitored.  
 
1.3 Study design 
The study is a phase 1, non-randomized study. The trial will be conducted in two parts, six patients 
are enrolled in part A. If the production of the MASE-T cell product was feasible for the majority 
(≥50%) of patients intended to treat in Arm A and the toxicity was acceptable, six patients will further 
be included in part B.   
Patients will be included and treated at the Department of Oncology at Herlev Hospital. Patients can 
also be referred from other oncology centres in Denmark. The inclusion period is expected to run 
over approximately 2 years, starting in Spring 2021. We expect all patients to finish treatment within 
3 years.   
 


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At the time of inclusion peripheral mononuclear cells (PBMCs) will be collected from the patient in 
order to produce the MASE-T product. Production is expected to take 2 weeks. The cell product will 
be cultivated with 30 pre-selected melanoma tumour antigens for HLA-A2 positive patients.  
 
Patients will be treated as followed:  
Part A (6 patients): Lymphodepleting chemotherapy (cyclophosphamide 500 mg/m2/day i.v. on day 
-4, -3, -2 and fludarabine Phosphate 30 mg/m2/day i.v. on day -4, -3) followed by i.v. infusion of the 
MASE-T product on day 0.  
Part B (6 patients): Lymphodepleting chemotherapy (cyclophosphamide 500 mg/m2/day  i.v. on day 
-4, -3, -2 and fludarabine Phosphate 30 mg/m2/day i.v on day -4, -3) followed by i.v infusion of the 
MASE-T product on day 0. Pembrolizumab 2 mg/kg will be administered on day -1 and day +21.    
 
Blood samples for research purposes will be collected at baseline, during treatment, and at time of 
progression. Tumour tissue will be collected if possible, at baseline and after the first evaluation scan 
and at progression.  
 
Patients will be closely monitored and upon clinical or radiological signs of progression, patients will 
be transferred to standard of care and followed for overall survival by medical journal entry.  
Patients who respond to the treatment will be followed with clinical evaluation and blood samples for 
5 years. With the treatment period (expected 3 years) and follow-up period (5 years) we expect end 
of study 8 years after initiation.   
 
The study is monitored by the Good Clinical Practice (GCP) unit and will be reported to the Danish 
Medicines Agency, the Research Ethics Committee and the Danish Data Protection Agency. 
 
1.4 Population 
 HLA-A2 positive patients with stage IV metastatic melanoma are eligible for inclusion. Other 
inclusion criteria are performance status (PS) 0-1, at least one measurable lesion according to 
RECIST 1.1, acceptable kidney- and liver functions and absence of major co-morbidities. Patients 
with brain metastasis may be excluded.  
Importantly, the treatment will only be completed on patients, where we can manufacture an 
acceptable MASE-T product (see IMPD). A total of 12 patients will be treated with the MASE-T cell 
product this study. It is only the patients who receive the MASE-T infusion that counts in the trial.  
 
1.5 Toxicity 
The MASE-T ACT product has never been tested in humans but multiple clinical trials with adoptive 
cell transfer of autologous ex vivo expanded TILs have been carried out and is reasonably well 
tolerated.19  
The MASE-T ACT product is derived from the autologous PBMCs, and though it is enriched with 
tumour reactive T cells, the MASE-T product share a similar phenotype with conventional TIL-derived 
cell products, and we expect a comparable safety and toxicity profile. Additionally, in comparison to 
conventional ACT, the patients in this trial are treated with a milder regime with less lymphodepleting 


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chemotherapy and without Interleukin 2 (IL-2) stimulation. The National Center for Cancer Immune 
Therapy (CCIT-DK) at the oncology department at Herlev Hospital have treated over 100 patients 
with TIL-derived ACT and is experienced in managing side effects.  
 
Pembrolizumab is a standard treatment in patients with MM. For patients treated with checkpoint 
inhibitor monotherapy (PD-1 or PD-L1 antibodies) CTC grade 3-4 adverse events (AE) occur at 
frequencies of about 10% and include rashes, gastrointestinal problems, hepatitis and hypophysitis. 
The oncology department at Herlev Hospital treat patients with Pembrolizumab daily and are 
experienced in managing side effects.  Patients included in the study have already been treated with 
standard pembrolizumab or nivolumab. If the patients experienced grade 3-4 adverse events on the 
PD-1 treatment they are excluded from this trial (see exclusion criteria).  
 
Combining ACT with lymphodepletion and checkpoint inhibitors  
Several clinical studies have already combined ACT with lymphodepletion and checkpoint inhibitors 
and has been reported as reasonably well tolerated. (NCT01993719, NCT01174121, NCT02500576, 
NCT02621021) 
 
1.6 Evaluation of toxicity and clinical response 
Toxicity and safety are assessed by CTCAE v. 5.0 at fixed time points. Baseline imaging is performed 
at baseline, 6 and 12 weeks after MASE-T infusion, and thereafter every third or sixth month 
according to the follow-up schedule and will be evaluated according to RECIST 1.1. The follow-up 
period will continue for up to 5 years with regular blood tests and diagnostic imaging (RECIST 1.1).  
 
1.7 Translational research and immunological response evaluation 
To evaluate the MASE-T treatments and the immunological response, blood samples for 
experimental analyses are drawn at fixed time points. Blood samples will be collected at: 1) baseline 
2) 1 week after MASE-T infusion 3) 3 weeks after MASE-T infusion and at the clinical evaluations. 
Core needle biopsy will be collected if possible, at baseline and at the first evaluation scan and will 
be analysed for T cell infiltration and signs of tumour cell killing. At signs of progression a new tumour 
biopsy will be performed for further analyses. 
 
2 Introduction and rationale  
2.1 Current treatment landscape for MM patients in Denmark  
In Denmark there are approximately 2300 new cases of melanoma each year and approximately 350 
patients are diagnosed with metastatic melanoma (MM).20,21  
In the metastatic setting, the most widely used first line therapy is immunotherapy comprising the 
checkpoint inhibitors (CPIs) pembrolizumab or nivolumab for patients with tumours expressing PD-
L1 >1 %, while ipilimumab (also a CPI) in combination with nivolumab are mostly used in patients 
with tumours expressing PD-L1 < 1%.22 This is based on data demonstrating that PD-1 inhibitor 
monotherapy is superior to ipilimumab and is equivalent in objective response rates and survival 


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compared to the combination therapy for patients with tumours expressing PD-L1 >1 %, but is 
significantly less toxic.23  
Second line therapy consists of ipilimumab, if PD-1 inhibitor monotherapy was administered in first 
line. If the combination was administered in first line, second line treatment for patients with BRAF 
wildtype tumours is IL-2 infusion or oral temozolomide, or entry into a clinical trial.24,25  
Another treatment option exists for the roughly 40% of MM patients with BRAF mutated tumours; 
combination treatment with BRAF inhibitor dabrafenib in combination with the MEK inhibitor 
trametinib.26 
Due to huge advances in the treatment of MM, OS has increased from median 9-10 months in 2009 
to multiple trials reporting 2-years survival rates in the range of 50 %. 22,26,27However, the patients 
enrolled in these trials are highly selected, and the benefit of these advances in the real-world 
population remains to be prospectively validated.21  
Immunotherapy is therefore showing promising results, but new and improved treatment strategies 
are still needed.  
 
2.2 Tumour immunology and cancer immunotherapy  
In recent years there has been immense progress in the understanding of the interplay between the 
immune system and cancer cells. It has become clear that the immune system recognizes and reacts 
against cancers and that an effective immune response against cancer cells is associated with a 
better prognosis.28 The presence of an immune response can be clearly illustrated by TILs that often 
exist in an inactivated state or at an immunological equilibrium with the cancer cells in the tumour 
microenvironment. The inactive state of the TILs is characterized by abnormal intracellular signaling, 
apoptosis and reduced proliferative capabilities.29 It is widely accepted that cancer cells at some 
point escape the immunological equilibrium in a process called immuno-editing that is an evolution-
like process where cancer cells mutate and progressively find ways to avoid immune recognition or 
activation.30 The escape results in a progressive cancer disease that requires external intervention. 
Cancer immunotherapy is defined as the approach to combat cancer by generating or augmenting 
an immune response against cancer cells. Over the past decade, two types of immunotherapy have 
emerged as particularly effective in cancer treatment; the use of CPIs to enhance natural anti-tumour 
activity and the administration of specific anti-tumour immune cells via ACT.  
The treatment with CPIs relies on boosting a pre-existing population of potentially tumour-reactive T 
cells in the patient. Thus, in poorly immunogenic patients, CPIs alone are likely to fail.31 In this regard 
the administration of tumour-recognizing T cells via ACT would enable immune-based therapies for 
poorly immunogenic tumours and potentially augment responses in tumours that are unresponsive 
to CPI.  
 
 
 


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2.3 Adoptive cell therapy (ACT) in patients with Metastatic Melanoma 
The goal of ACT is to generate a robust immune mediated anti-tumour response via infusion of ex 
vivo manipulated T cells. ACT-based strategies utilizing T cells to destroy tumours can be divided 
into a) isolation of naturally occurring tumour specific T cells from existing tumour masses (TILs) and 
b) the genetic modification of blood-derived T cells to allow specific recognition of tumour cells. In 
both settings, T cells are manipulated ex vivo followed by expansion and later reinfusion into the 
lymphodepleted patient. ACT with TIL is visualized in figure 1.  
 
Figure 1. Adoptive T cell transfer approach to harness the immune system to treat cancer: Adoptive cell therapy (ACT) with 
TILs (tumor infiltrating lymphocytes) 
2.3.1 ACT with TILs 
ACT with TILs has been tested in multiple trials world-wide and also at CCIT, Herlev Hospital, 
reporting objective response rates of 40-50% in MM patients, including complete tumour regression 
in 10 to 25 % of the patients.8 It has also been shown that patients refractory on anti-PD-1 still respond 
to an infusion of TILs.32 Treatment with TILs is only applicable when a tumour biopsy of at least 2 cm 
can be removed. Unfortunately, a large group of patients does not have surgically respectable tumors 
and cannot be offered treatment.   
 
2.3 Tumour antigens   
T cells recognise cancer cells by binding to cancer-related peptides presented on a MHC molecule. 
Some tumour antigens are highly tumour specific while others are with lower tumour specificity:   
Antigens of high tumour specificity:  


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Three types of tumour antigens have the potential to elicit immune responses that are strictly tumour 
specific:  
• 
Neo-antigens are derived from spontaneous mutations acquired during carcinogenesis and 
are present in most tumour types. The contribution of these antigens to tumour 
immunogenicity is expected to vary according to the mutation rate (tumour mutational burden 
(TMB)).33 The contribution is expected to be higher in melanomas due to a high MTB induced 
by UV radiation and in lung carcinomas that arise from tobacco smokers.34  
• 
Cancer-germline genes are not expressed in normal tissue but are expressed in male and 
female germline cells, in trophoblastic cells and in a substantial fraction of tumours. The 
mechanism of activation involves demethylation of the promotor of these genes.35,36 Cancer-
germline genes (MAGE, BAGE, GAGE, LAGE-1, NY-ESO-1) are frequently expressed in 
melanoma, bladder, head and neck and lung carcinomas.  
• 
Viral antigens have been shown for a subset of human tumours including cervical carcinoma, 
nasopharyngeal carcinoma, adult T cell leukaemia and hepatocarcinoma and has been useful 
for cancer prevention.  
Antigens of low tumour specificity  
Two types of tumour antigens a known to have low tumour specificity:  
• 
Differentiation antigens (tissue-specific expression) is found in melanoma patients where T 
cells recognize both tumour cells and normal melanocytes. The antigens are melanocyte-
specific proteins such as tyrosinase, Melan-A (MART1) and GP100.37  
• 
Overexpression of proteins in tumours provide an opportunity for a specific T cell response, 
that is if tumour cells present an amount of peptide-HLA complexes that is above the 
threshold of normal cells, a specific antitumoral T cell response could occur. Examples of 
overexpressed proteins are HER2 (in breast and ovarian cancer) and WT1 (in leukaemia).38,39  
 
2.4 ImmPACT scaffolds for expansion of MASE-T product 
To expand MASE-T we have established ImmPACT scaffolds. ImmPACT scaffolds are antigen-
presenting scaffolds based on dextran polymers with bound biotinylated peptide-MHC monomer, IL-
2 and IL-21 in an optimized stoichiometric ration (figure 2B). Expansion of antigen-specific cells are 
based on the ability of the T cells to recognize and bind peptide-MHC complexes on the dextran 
polymer, which is the main driver of the interaction. Upon interaction of the ImmPACT scaffold to the 
TCR, the T cell is further stimulated with IL-2 and IL-21, both of which are important for T cell 
expansion, survival and memory. The interaction between ImmPACT scaffolds and T cells leads to 
specific expansion of T cells, with a favorable phenotype and ability to recognize and eradicate 
melanoma tumours (figire 2A). Using a library of 30 antigens (both cancer germline genes and 
melanocyt specific antigens) for different ImmPACT scaffolds we can create a multitargeted and 
polyclonal T cell product. (Table 1)   
 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
 
Figure 2. A) T cell activation and proliferation (expansion) in humans requires presentation of an antigen through the MHC 
molecule on an antigen presenting cell (APC), co-stimulation and cytokine release is also mandatory. B) In vitro T cell 
activation and expansion of Ag specific T cells for ACT through ImmPACT scaffolds consisting of a dextran backbone with 
bound peptide MHC monomer, IL-2 and IL21 (cytokines). 
Table 1: Top 30 shared melanoma antigens and HLA-A2 sequences 
No. 
Protein 
Sequence 
No. 
Protein 
Sequence 
1 
MAGE-A2 
LVHFLLLKY 
16 
MC1R 
TILLGIFFL 
2 
gp100 / Pmel17 
IMDQVPFSV 
17 
Melan-A / MART-1 
ILTVILGVL 
3 
CDKN1A 
GLGLPKLYL 
18 
TRP-2 
FVWLHYYSV 
4 
gp100 / Pmel17 
YLEPGPVTA 
19 
KIF20A 
AQPDTAPLPV 
5 
Melan-A / MART-
1 (WT) 
EAAGIGILTV 
20 
MAGE-A10 
GLYDGMEHL 
6 
MAGE-C2 
KVLEFLAKL 
21 
p53 
RMPEAAPPV 
7 
MAGE-A10 
SLLKFLAKV 
22 
SSX-2 
KASEKIFYV 
8 
gp100 / Pmel17 
KTWGQYWQV 
23 
STEAP1 
FLYTLLREV 
9 
STEAP1 
MIAVFLPIV 
24 
TRP-2 
SVYDFFVWL 
10 
Telomerase 
RLFFYRKSV 
25 
GnTV 
VLPDVFIRCV 
11 
LAGE-1 
MLMAQEALAFL 
26 
Livin (ML-IAP) 
SLGSPVLGL 
12 
MAGE-A2 
YLQLVFGIEV 
27 
MAGE-A1 
YLEYRQVPV 
13 
MAGE-C2 
LLFGLALIEV 
28 
Meloe-1 
TLNDECWPA 
14 
STAT1-alpha/ß 
KLQELNYNL 
29 
NY-ESO-1 / LAGE-
2 
SLLMWITQC 
15 
TAG-1 
SLGWLFLLL 
30 
TRAG-3 
ILLRDAGLV 
 
 
A 
B 
APC 
T cell 
T cell 
MHC 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
2.5 ACT with Multiple Ag specific T cells (MASE-T) 
Adoptive cell therapy with the MASE-T product has never been tested in patients but is anticipated 
to have a great potential. The technique is simpler than with TIL; with expansion of T cells from blood 
(no need of tumour biopsy for production of T cells) and only requires one step expansion. The 
MASE-T product targets multiple tumour specific antigens and is therefore anticipated to be more 
tumour directed than ACT with TILs.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.6 The supportive regimen  
Traditionally, the ACT is assisted by a supporting regime. Lymphodepleting regimens are applied in 
most trials with ACT. The lymphodepletion provides an immunological window where sufficient 
stimulatory cytokines and growth factors are readily available for the infused TILs while also depleting 
regulatory and suppressive cells. 40 
 
Following MASE-T infusion, patients also receive a single dose pegfilgrastim to help reconstitute 
bone marrow function after the lymphodepleting chemotherapy.  
 
Besides the well-established supportive regime of lymphodepletion, the efficacy of ACT may be 
further supported by immune checkpoint inhibition. Immune checkpoints are surface receptors on 
immune cells that serve as regulatory brakes in the immune system to avoid autoimmunity. The 
checkpoints are exploited by cancer cells to avoid immune recognition and activation. In recent years 
checkpoint inhibitors (especially anti-PD-1) have yielded impressive results in many cancer forms 
Patient 
ImmPACT 
MASE-T Cell 
product 
Blood 
Figure 3 ACT with MASE-T.  


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
and have now become standard therapy in malignant melanoma.41 Emerging studies show that 
immunotherapy in contrast to traditional cancer medicines might be more dependent on the 
mutational load of the individual patient rather than the cancer diagnosis. 42,43 This could mean that a 
more personalized approach to cancer therapy like ACT is needed.  
Both ACT and checkpoint inhibition work by tipping the immunological balance in favor of activation 
and away from suppression or avoidance by the cancer cells. Due to the increasing success of 
checkpoint inhibitors, it is speculated that combination with ACT would provide a synergistic 
immunological benefit to cancer treatments - much like combining different checkpoint inhibitors has 
been  - and scientific evidence is aligning with this theory.44 
 
It has been shown repeatedly that tumour-specific TILs frequently express PD-1  and that PD-1 
inhibition increase their tumour killing capacities.45,46,47 This fits the general perception that PD-1 
predominantly potentiates the effector functions of T cells48. The results are so promising that several 
clinical studies combining PD-1 blockade with ACT are already ongoing (NCT01993719, 
NCT01174121, NCT02500576, NCT02621021). 
 
Based on the available evidence and in order to optimize the anti-tumour activity of the infused 
MASE-T product, ACT is combined with pembrolizumab in patient in group B. To maximize the in 
vivo efficacy, we administer pembrolizumab just before and three weeks following the MASE-T 
infusion.  
 
Two trials at CCIT-DK, Herlev Hospital has been conducted where combination of ACT with TILs has 
been combined with both lymphodepleting chemotherapy, IL-2 injections and CPIs, which was 
tolerable and feasible for all 30 patients. (NCT03296137, NCT03287674)  
 
2.7 CCIT-DK is a leading center for ACT  
Center of Cancer Immune therapy (CCIT-DK) at Herlev Hospital is a leading European center of TIL-
based ACT. Despite the promising results of this therapy, widespread application and testing of ACT 
is limited by the considerable expertise, facilities and resources required for the TIL expansion, 
infusion and supportive regime. Due to this complexity, only a handful of centers in the world are 
currently able to perform ACT. At CCIT we have many years of experience, and we have over the 
years built up the required expertise and effectiveness in all steps of the process.  
3. Hypothesis, objectives and endpoints  
3.1 Hypothesis  
Our hypothesis is that the treatment with lymphodepleting therapy and MASE-T alone or in 
combination with Pembrolizumab is well tolerated and a feasible treatment option in patients with 
MM. 
 
3.2 Primary objective and endpoint 
The primary objective is to asses tolerability and feasibility of the treatment. The primary endpoint 
is adverse events assessed by CTCAE 5.0 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
 
3.3 Secondary objectives and endpoints  
To characterize T cell profile and persistence in vivo from tumor biopsies and blood samples and 
to characterize biomarkers for immunological response to the treatment. 
To evaluate the clinical efficacy. Endpoints are Best Overall Response (BOR) according to RECIST 
1.1 and iRECIST, Duration of response (DOF), Progression Free Survival (PFS) and Overall Survival 
(OS).  
 
RECIST 1.1 Guidelines:  
• 
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  
 diameter or a < 20 % increase in the sum of all measurable parameters 
longest diameter. 
• 
Progressive disease (PD): Defined as a > 20 % increase in the sum of all measurable 
parameters longest diameter or the appearance of new lesions. 
• 
CR and PR are to be verified by a subsequent evaluation after no less than 4 weeks. 
 
4. Trial design  
The trial is designed as a phase I, single-centre, non-randomized clinical trial. The trial will be 
conducted in two parts, six patients are enrolled in group A. If the treatment is possible to produce 
in 3 out of 6 patients and is feasible for all patients, six patients will be treated in group B.  
Part A: Lymphodepleting chemotherapy and MASE-T infusion  
Part B: Lymphodepleting chemotherapy and MASE-T infusion combined with Pembrolizumab  
 
4.1 Trial period 
The trial is expected to start Spring 2021 and patients are expected to be enrolled during the course 
of 2 years. All 12 patients are expected to have ended treatment during the course of 3 years. 
Patients will be followed for up to five years. End of trial is defined as the date of last patients’ last 
visit which is expected 8 years after initiation.  
 
4.2 Statistical consideration  
The study is non-blinded and non-comparative. Descriptive statistics will be used to estimate the 
immunological response and clinical response rate. Descriptive statistics will also be used to sum up 
the duration of response and the patient characteristics. A power calculation for the required sample 
size to determine of primary and secondary endpoints cannot be formally calculated. A samples size 
of 12 participants is chosen due to practical considerations that include a realistic time frame as well 
as the limited available facilities and trained personal.  
 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
4.3 Study population 
HLA-A2 positive patients with histologically confirmed metastatic or inoperable melanoma who have 
progressed on standard therapy. Importantly, the treatment can only be completed in patients, where 
we are able to produce an acceptable cell product from the drawn blood sample.  
 
4.4 Inclusion criteria 
1. Age ≥ 18 ≤ 75  
2. Progressive disease on or after anti-PD-1/anti-PD-L1 monotherapy or progressive disease on or 
after anti PD-1 plus anti-CTLA-4 therapy  
3. The patient has histologically confirmed metastatic cutaneous melanoma. Patients with 
metastatic ocular/mucosal or other non-cutaneous melanoma cannot be included 
4. The patient is HLA-A2 positive  
5.  ≥ 10 % of lymphocytes in peripheral blood are CD8+  
6. At least one measurable parameter according to RECIST version 1.1 guidelines 
7. ECOG performance status of 0 or 1 
8. No significant toxicity from previous cancer treatments (CTC ≤ 1)  
9. Women of childbearing potential: Negative serum pregnancy test and must use effective 
contraception. This applies from screening and until 6 months after treatment. Birth control pills, 
spiral, depot injection with gestagen, subdermal implantation, hormonal vaginal ring and 
transdermal depot patch are all considered effective contraceptives 
10. Men with female partner of childbearing potential must use effective contraception from 
screening and until 6 months after treatment. Effective contraceptives are as described above 
for the female partner. In addition, documented vasectomy and sterility or double barrier 
contraception are considered effective contraceptives 
11. Signed statement of consent after receiving oral and written study information 
12. Willingness to participate in the planned treatment and follow-up and capable of handling 
13. The patient has met the following haematological and biochemical criteria: 
a) AST and ALT ≤2,5 X ULN or ≤5 X ULN with liver metastases 
b) Serum total bilirubin ≤1,5 X ULN or direct bilirubin ≤ ULN for patient with total bilirubin 
level > 1,5 ULN 
c) Serum creatinine ≤1,5 X ULN 
d) ANC (Absolute Neutrophil Count) ≥1,000/mcL 
e) Platelets ≥ 75,000 /mcL 
f) Hemoglobin ≥ 9 g/dL or ≥ 5.6 mmol/L 
4. 5 Exclusion criteria  
1. Another malignancy or concurrent malignancy unless disease-free for 3 years 
2. Requirement for immunosuppressive doses of systemic corticosteroids (>10 mg/day 
prednisone or equivalent) or other immunosuppressive drugs within the last 3 weeks prior to 
screening 
3. Prior treatment with adoptive transfer of Tumor Infiltrating T cells (TIL) 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
4. Grade 3-4 adverse events upon treatment with PD-1 checkpoint inhibitors (only phase B) 
5.  Patients who have any CNS lesion that is symptomatic, greater than 1 cm in diameter or 
show significant surrounding edema on MRI scan will not be eligible until they have been 
treated and demonstrated no clinical or radiologic CNS progression for at least 2 months. 
However, patients with subclinical brain metastasis with a maximum of 4 metastasis < 1 cm 
can be included. 
6. The patient has any condition that will interfere with patient compliance or safety (including 
but not limited to psychiatric or substance abuse disorders)  
7. The patient is pregnant or breastfeeding  
8. The patient has an active infection requiring systemic therapy  
9. The patient has received a live virus vaccine within 30 days of planned start of therapy 
10. Significant medical disorder according to investigator; e.g severe asthma or chronic 
obstructive lung disease, dysregulated heart disease or dysregulated diabetes mellitus.  
11. Concurrent treatment with other experimental drugs  
12. Any significant active autoimmune disease  
13. Severe allergy or anaphylactic reactions earlier in life 
14. Known hypersensitivity to one of the active drugs or one or more of the excipients. 
15. Unrelieved lower urinary tract obstruction 
 
5. Conduct of study 
5.1 Overview 
The study is a phase I clinical trial enrolling patients with metastatic melanoma who are refractory to 
PD-1 antibody treatment. 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. The study is 
monitored by the Good Clinical Practice unit (GCP-unit) at the Capital Region (RegionH) and will be 
reported to the Danish Medicines Agency, the Research Ethics Committee and the Danish Data 
Protection Agency. 
Referral of patients is to be made to the visitation office of clinic 5at the department of oncology, 
Herlev Hospital. 
We aim to include 12 patients. 6 in part A and 6 in part B. The inclusion is estimated to start in Q2 of 
2021 and be completed within three years. Primary feasibility and toxicity are thus scheduled within 
3 years of trial initiation.  
Patients are screened, treated and entering follow-up as described in the following sections.  
 
5.2 Screening 
After signing informed consent according patients are screened for eligibility. 
The screening is divided in two phases:  
Phase 1: Patients will be screened for HLA-A2 specificity by flowcytometry. HLA-A2 specificity test 
by flowcytometry is completed within two days. Further the frequency of CD8+ lymphocytes in 
peripheral blood will be tested by flowcytometry. If the patient is HLA-A2 positive and have ≥ 10 % 
CD8+ lymphocytes in peripheral blood they will enter the second phase of screening. If the patient 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
is HLA-A2 negative or have < 10 % CD8+ lymphocytes in peripheral blood they will be excluded. 
Furthermore, the first six patients will additionally be screened for the presence of tumor-antigen 
specific CD8 T cells, corresponding to the 30 tumor-antigen-derived peptides included in the 
ImmPACT scaffold. Only patients who are HLA-A2 positive, have ≥ 10 % CD8+ lymphocytes and 
have >0.002% (with a limit of 10 detectable events) tumor-antigen tetramer positive CD8 T cells will 
continue for phase 2 screening.   
Phase 2: Patients will further be screened for the following parameters and HLA-A2 specificity will 
be validated.  
• 
Complete physical examination including height, weight, performance status, vital signs 
including blood pressure, heart rate, saturation and temperature.  
• 
Pathologic confirmation of metastatic melanoma (archival tissue may be used) 
• 
HLA-A2 specificity 
• 
PET-CT of chest, abdomen and pelvis  
• 
MR cerebrum  
• 
ECG 
• 
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 
contraceptions regularly within the last 6 months 
• 
Comprehensive blood test panel including:  
o Hematology: Hemoglobin, leukocytes with diff-count and platelets 
o Liver: ASAT, ALAT, albumin, alkaline phosphatase, bilirubin, LDH, INR, APPT. 
o Kidney: Sodium, potassium, creatinine, carbamide, phosphate, Ca-ion 
o Other: CRP 
o Chronic infections: HIV, Hepatitis B surface antigen, Hepatitis B surface antibody, 
Hepatitis B core antibody, Hepatitis C IgG, HTLV IgG, EBV IgG, Trepone,  
o Endocrine: TSH, T4, T3, Cortisol, Oestradiol (female), prolactin (female), Testosterone 
(male) 
• 
Kidney function (clearence)  
• 
Reviewing the checklist for inclusion/exclusion for treatment. 
5.3 Treatment plan  
12 patients will be included in total. The first six patients will be included in group A and the 
following 6 in group B. Enrollment of the first three patients in group A will be staggered; the 
second and third patient will not be included until the previous patient completes day 7 of the 
treatment without intolerable toxicity  
If the following criteria are met six additional patients can be included in part B: 1. production of the 
MASE-T cell product was feasible for at least 50% of patients intended to treat in part A. 2. 
Acceptable toxicity observed in part A patients defined as MASE-T related grade III/IV toxicity in no 
more than 1 of 6 patients.   
 
For both groups a peripheral blood sample of 300 mL will be drawn right after inclusion to 
manufacture the MASE-T product. Around ten days after, patients will be hospitalized and treated 
with 3 days of lymphodepleting chemotherapy (day -4, -3 and -2). On day 0 the MASE-T product will 


20 
MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
be administered (figure 4). Two hours after MASE-T infusion, pegfilgrastim is given to help 
reconstitute bone marrow function.   
If the treatment is feasible for all six patients in group A, pembrolizumab will be administered at day 
-1 and day 21 in group B. (table 2)  
 
 
 
 
Treatment schedule (Table 2) 
Hospital 
Outpatient clinic 
Day 
-4 
-3 
-2 
-1 
0 
21 
Cyclophosphamide 500 mg/m2  IV 
x 
x 
x 
  
  
  
Fludarabine Phosphate 30 mg/m2  IV 
x 
x 
  
  
  
  
MASE-T IV infusion 
  
  
  
  
x 
  
Pegfilgrastim 6 mg s.c 
  
  
  
  
x 
  
Pembrolizumab 2 mg/kg ONLY PART B 
  
  
  
x 
  
x 
 
 
Figure 4 Flow chart treatment plan  
 
Light Blue arrows indicate conditioning with cyclophosphamide 500 mg/m
2
/dose on days -4, -3, and -2 and Fludarabine 
Phosphate 30 mg/m2/dose on day -4 and -3.  
Dark blue arrows indicate PD-1 inhibitor, pembrolizumab, given at 2 mg/kg/dose on days -1 and 21.  
Green arrows indicate infusion of MASE-T product  
Respons to therapy evaluation is completed with PET/CT scans.  
 
.  
 
 
 
 
 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
 
5.4 Overview of blood tests and evaluation during the treatment course  
The patients are continuously monitored on several parameters before, during and after MASE-T 
infusion. Group B are also monitored before every treatment with Pembrolizumab.  
 
Time point 
Screening 
Peripheral blood drawn for MASE-T production 
At admission  
Before Lymphodepleting chemotherapy  
Group A: No treatment 
Group B: 1st of 2 Pembrolizumab 
MASE-T infusion 
After MASE-T infusion 
Group A: No treatment  
Group B: Pembrolizumab 2nd of 2 
First clinical evaluation  
Second clinical evaluation 
Third clinical evaluation  
Day 
-21 -15 -5 
-4-2 -1 
0 
+6 
+21 
+42 
+84 +168 
BP, P, Tp 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Toxicity assessment 
x 
 
x 
 
x 
x 
x 
x 
x 
x 
x 
PS, clinical assessment 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Weight 
x 
 
x 
x 
x 
 
 
 
 
 
 
Screening blood tests 
x 
 
 
 
 
 
 
 
 
 
 
Research blood samples* 
 
 
x 
 
 
x 
x 
x 
x 
x 
x 
Routine blood tests 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
ECG 
x 
 
 
 
 
 
x 
 
 
 
 
Kidney function 
x 
 
 
 
 
 
 
 
 
 
 
Urine sample 
 
 
x 
 
 
 
 
 
 
 
 
Tumour biopsy 
 
x 
 
 
 
 
 
 
x 
 
 
PET-CT  
x 
 
 
 
 
 
 
 
x 
x 
x 
MR cerebrum  
x 
 
 
 
 
 
 
 
 
 
 
* See section 8.1, “Research blood tests” 
 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
5.5 Medicinal products used in the protocol  
5.5.1 Cyclophosphamide (sendoxan®)  
Cyclophosphamide (sendoxan®) is an alkylating drug that works by creating covalent bindings 
with biologically important macromolecules. In cancer therapy, it binds and links DNA strings 
preventing cell division. The effect can be opposed by cell DNA repair systems that are usually 
impaired in cancer cells. The binding also damages important cellular functions and leads to cell 
death. Cyclophosphamide is given as an intraveneous infusion for three consecutive days in a 
dosage of 500 mg/m2. Patients are treated according to local guidelines.  
5.5.2 Fludarabine phosphate (“Actavis” or “Ebewe”)  
Fludarabine phosphate (Fludarabinphosphat “Actavis”® or Fludarabinphosphat “Ebewe”®) is 
a pro-drug that is concerted to the active triphosphate 2-fluoroara-ATP. It is an anti-metabolite that 
inhibits DNA synthesis while simultaneously reducing RNA and protein synthesis. Fludarabine 
phosphate is used in the treatment of hematological diseases as chronic lymphatic leukemia. 
Fludarabine phosphate is given as an intravenous infusion for 2 consecutive days in parallel with 
cyclophosphamide infusion in a dosage of 30 mg/m2 body surface. Patients are treated according to 
local guidelines.  
 
The lymphodepleting regimen is milder than normally administered when we treat patients with TIL 
therapy. Heczey et al have treated patients with neuroblastoma with this regimen and found it safe 
and manegable, also in combination with CAR T therapy and anti-PD-1 therapy.49  
5.5.3 Pembrolizumab  
Pembrolizumab (Keytruda®) is an anti-PD-1 antibody that inhibits the PD-1/PD-L1 pathway on T 
cells. PD-1 is a surface receptor on activated T cells and when it binds PD-L1 on target cells, it 
induces the cell death of the activated T cell. Cancer cells use PD-L1 expression to avoid immune 
recognition. Pembrolizumab is approved by the EMA for treatment of metastatic melanoma. Patients 
in group B will be treated with 2 mg/kg Pembrolizumab the day before infusion of MASE-T (day -1) 
and on day +21. Patients are treated according to local guidelines.  
5.5.4 The MASE-T product  
The MASE-T product contains melanoma tumour antigen specific T cells. They are infused IV into 
the patient on day 0. The number of cells in the product varies between patients. It usually consist of 
20-200 million cells. See the Investigational Product Medicinal Dossier (IMPD) for more information.  
5.5.5 Pegfilgratim (Neulasta)  
Pegfilgratim (Neulasta) is an analog human granulocyte colony stimulatory factor (GM-CSF). It 
stimulates 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 chemotherapy. 
Pegfilgrastim helps the patients recover from the lymphodepleting chemotherapy by a single dose 
of 6 mg s.c. at 2 hours after MASE-T infusion.  


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
Medicinal products are only administered by trained medical staff and are labelled according to 
Annex 13 in the GMP regulations.  
 
5.6 Guidelines for supportive and concomitant therapy 
Supportive treatment is given on ordinary medical indications estimated by the clinician responsible 
for the treatment. Any measures should be specified in the patient chart and flow sheet. The following 
is meant as a guideline and other medications can be administered if appropriate.  
During the lymphodepleting chemotherapy the following are given to protect mucosa of the bladder 
and relieve dehydration, nausea or vomiting: 
Supplementary fluid therapy during cyclophosphamide treatment.    
Fixed:  
• 
1 L isotonic saline on day -5, -4, -3, -2. The patient should additionally drink 2 liters 
fluid a day during chemotherapy.  
• 
Inj. Mesna, (25% of cyclophosphamide dosage) IV x 4 on day -4 and -3 and -2. 
• 
Inj. Aloxi 250 μg IV on day -4 and -2. 
• 
Tabl. Emend, 125 mg x 1 on day -4 and 80 mg on day  -3 and-2.  
If needed:  
Tabl. Motilium, 20 mg if needed max x 3. 
Tabl. Temesta, 1-2 mg if needed, max x 4. 
Tabl. Pantoloc, 40 mg if needed max x 2. 
 
To prevent opportunistic infections:  
Tabl. Sulfamethizole with Trimethoprime, 400/80 mg x 1 daily for 6 months from day -4. 
Tabl. Aciclovir, 400 mg x 2 for 6 months from day 0. 
Tabl. Diflucan, 100 mg x 1 on day 0 and until the neutrophile count is > 1000/µl. 
 
After MASE-T infusion: 
Inj. Pegfilgrastim, 6 mg x 1 s.c. on day 0, two hours after TIL infusion. 
Tabl. Pethidin, 25 mg if needed max x 4. 
 
Oxygen on nasal catheter if needed. 
In case of fever during neutropenia 
o Patients are treated with antibiotics according to local guidelines for febrile neutropenia 
described in appendix 4. 
In case of anemia or trombocytopenia 
o Transfusions with filtered and radiated blood should be administered in line with institutional 
guidelines  
o Transfusion with platelets is indicated if platelets < 20/μl or if it is otherwise clinically indicated. 
In case of diarrhea: 
o Patients are treated according to local guidelines that include loperamide and fecal samples.  
o Loss of fluid and electrolytes are corrected orally or intravenously and evaluated daily. 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
In case of immune related adverse events (irAEs) to checkpoint inhibitors local guidelines are 
followed and e.g. corticosteroid can be administered: 
o IrAEs are managed according to national guidelines that are available at https://immuntox.dk.  
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.  
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. 
 
5.7 Follow-up 
After completion of treatment patients enter the follow-up phase. At follow-up visits patients are 
evaluated clinically, with blood samples, project blood samples and PET/CT scans. Follow-up visits 
will be scheduled after 6 weeks, 12 weeks and thereafter every third month for the first two years 
and every six months thereafter, or as clinically indicated, for a total of 5 years.  
 
1 Tumour biopsy only at progression 
  
 
5.8 End of study  
Criteria for end of treatment are:  
• 
Completion of treatment  
• 
Unacceptable toxicity  
• 
Patients request  
• 
Progressive disease  
• 
Stopping treatment for other reasons at the discretion of the treating physician 
In case patients stop treatment but are not in progression they will enter the follow-up phase unless 
they request otherwise. In case of progression patients are excluded from the trial and offered 
standard of care treatment according to local guidelines.  
 
 
 
Follow-up (5 years) 
Months from MASE-T infusion 1.5 
3 
6 
9 
12 
15 
18 
21 
24 
30 
36 
42 
48 
52 
60 
PS, clinical assessment 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
CTCAE 5.0 
0 
0 
0 
 
 
 
 
 
 
 
 
 
 
 
 
Routine blood tests 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Research blood sample  
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
PET/CT  
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Tumour biopsy 
0 
01 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
5.9 Early termination of the treatment 
Several reasons can lead to early termination of the planned treatment:  
• 
Failure to manufacture the MASE-T product from peripheral blood sample 
• 
Patient’s request  
• 
Unexpected medical conditions if the principal investigator finds it to be in the patient’s best 
interest 
• 
Patients will end the study treatment if they start treatment with another experimental drug or 
other systemic anticancer treatment 
• 
Treatment is terminated in case of serious adverse events that make completion of the 
treatment impossible 
• 
Patients who are for any reason excluded after initiating study treatment will still be offered 
treatment of any adverse events that occurs in relation to their participation in the protocol.  
 
5.10 Criteria for treatment modifications  
At the discretion of the treating physician, treatment with Pembrolizumab (group B) might be delayed 
or stopped if the patient experiences toxicity. Treatment modifications are performed following 
standard of care guidelines, which are accessible at https://free.mymedcards.dk/?q=immuntox.  
 
5.11 Concurrent treatment  
Supportive treatment can be administered based on clinical judgement and should be noted in the 
patient chart according to existing practice.  
Local radiotherapy or surgery is allowed if the lesion was present at baseline and if not involving a 
target lesion. 
 
6. Manufacturing of the MASE-T product with ImmPACT 
technology 
 
Acquisition of blood sample for PBMCs two weeks prior to treatment. 300 mL blood is drawn from 
the patient to manufacture the MASE-T product. Blood will be drawn at the outpatient clinic (Clinic 5 
at Herlev Hospital) by experienced nurses and collected in a 300 mL blood bag. The blood bag is 
immediately transported to the GMP lab.   
 
6.1 Expansion of PBMC cultures to MASE-T product  
The ImmPACT Ag-scaffold strategy has been designed to enable ex vivo expansion of ag-specific T 
cells directly from patient blood and without the need for tumor biopsy. Utilizing the ImmPACT Ag-
scaffolds presenting the top 30 shared antigens, we can successfully expand ag-specific CD8+ T 
cells from patient PBMCs and generate higher numbers of ag-specific CD8+ T cells after day 14 of 
culture compared to culture initiation.  


26 
MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
The MASE-T product consists of expanded antigen specific T cells from the patients own blood. 
Importantly, no residual Ag-scaffold is detected in the final T cell product . See IMPD for more details.  
 
6.2 Handling and transportation of the MASE-T product  
The infusion bag with the MASE-T product is labelled with patient ID and a patient specific transport 
form is filled out. The infusion bag and transport form are placed in a secure hatch for immediate 
pickup by a trained clinician, who validates the information on the infusion bag and the transport 
form. The transport form is signed, and the infusion bag is transported directly to the patient. Before 
administration, the information on the infusion bag is revalidated by the treatment staff and matched 
to the ID of the patient through patient identification.  
 
7 Adverse events, toxicity and precautions  
7.1 Adverse events and reactions  
Adverse events (AE) are defined as an undesirable event in a participant during a clinical trial – 
regardless of whether or not the event is considered related to the investigational treatment. All AEs 
reported spontaneously by the subject or observed by the investigator or 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 and the severity and relation to the study medication will 
be assessed in accordance with the guidelines described here. 
Relation to the treatment will be determined by investigator in accordance with the following 
guideline: 
0: Unrelated– no temporal relation, other etiologies very likely the cause 
1: Related - clear temporal relation with laboratory confirmation or a positive retreatment test 
Grading of Adverse Events refers to the intensity and severity of the reaction. Events are graded 
using CTCAE version 5.0 - see appendix 1. The following scale can be used if this CTCAE is not 
applicable:  
1: light 
2: moderate 
3: severe 
4: life threatening 
5: lethal 
Patients who experience AEs will be monitored by relevant clinical and paraclinical evaluation 
assessed by the attending clinician. 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 recorded in the eCRF unless they have caused an AE, resulted in 
termination of the treatment or meet the criteria of a serious adverse event (see the following). 
Serious Adverse Events (SAEs) are defined as any medical events or affects that: 
o results in death 
o is life threatening (at the time of the event) 
o requires hospitalization or prolongation of existing patients’ hospitalization 
o results in persistent or significant disability or incapacity 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
o leads to a congenital anomaly or birth defect 
o is an otherwise significant medical event 
Adverse Reactions (ARs) can be either expected if they are described in the IMPD or in the relevant 
product summary, or unexpected if the grade or severity does not fit the available product 
information. If an event is assessed to be caused by the investigational treatment, it is classified as 
an adverse reaction (AR). An AR can be classified as a Serious Adverse Reaction (SAR) by the same 
criteria as a SAE. 
Suspected unexpected serious adverse reaction (SUSAR) is an unexpected adverse reaction that is 
deemed related to the investigational treatment. The section describing known side effects and their 
frequencies in the prescribing information of the used medicinal products will be used as reference 
for classifying an AE as unexpected. Side effects from the MASE-T product will be classified as 
unexpected using “Reference Safety information” section in the IMPD. 
 
7.2 Reporting of Adverse Events and Adverse Reactions  
The investigator reports SAEs andSARs to the sponsor within 24 hours. Sponsor judges whether a 
SAR fulfills the criteria of being a SUSAR and reports SUSARs to the Danish Medicines Agency within 
7 days if fatal or considered life threatening, and otherwise within 15 days. Consequences for the 
study must be reported. Sponsor submits a yearly list that summarizes any SARs and SUSARs as 
well as a report regarding the safety of study participants 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 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 
including: 
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 
o hospitalizations or prolongation of current hospitalization if the sole reason for 
hospitalization or prolongation is one or more of the following: 
o fluid treatment or treatment of nausea 
o blood transfusion 
o platelet transfusion 
o febrile leucopenia/neutropenia 
o administration of investigational procedures 
o placement of a permanent intravenous catheter 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
These events are registered in the eCRF (RedCAP). 
 
7.3 Toxicity related to lymphodepleting chemotherapy  
7.3.1 Cyclophosphamide  
Cyclophosphamide is a classical chemotherapeutic drug and its usual limiting toxicities are 
myelosuppression (neutropenia, thrombocytopenia and anemia) and urotoxicity (cystitis, haematuria 
and hemorrhagic cystitis). Concomitant Mesna therapy along with sufficient rehydration markedly 
reduces the frequency and severity of the urotoxicity. The most common adverse reactions are 
alopecia, nausea and vomiting. Further information is available from the Danish Medicines Agency.  
7.3.2 Fludarabine phosphates  
Fludarabine phosphates most common side effects are myelosuppression (neutropenia, 
thrombocytopenia and anemia), infections including pneumonia, cough, fatigue, limpness, nausea, 
vomiting and diarrhea. Other adverse reactions are shivering, edema, malaise, peripheral 
neuropathy, visual disturbances, anorexia, mucositis, stomatitis and rash. Severe opportunistic 
infections and death have also been reported. Further information is available from the Danish 
Medicines Agency.  
 
7.4 Toxicity related to MASE-T infusion 
Toxicity related to ACT with MASE-T product are theoretically comparable to treatment with ACT 
with tumour infiltrating lymphocytes (TILs). The most common toxicities during TIL therapy are due 
to the effects of the lymphodepleting preparative regimens and the subsequent IL-2 after TIL infusion.  
TIL-related toxicity is less common, but patients may develop, mostly transient, hypoxia, dyspnoea, 
hypotension, fatigue, nausea and chills and fever shortly after infusion of TILs.8  
Other signs of toxicity develop later after infusion may consist of melanoma associated autoimmune 
disease such as vitiligo, diffuse erythematous full-body rash, hearing loss or uveitis, of which latter 
promptly responds to topical corticosteroids. See reference safety information in IMPD.   
 
7.5 Toxicity related to Pegfilgastrim  
The most common side effects are bone and muscle pain, which are generally mild to moderate in 
severity and can be treated with standard painkiller.  
 
7.6 Toxicity related to Pembrolizumab 
Pembrolizumab is a checkpoint inhibitor and can cause toxicities by an overactivation of the immune 
system called immune related adverse events (irAEs). They include skin toxicities (erythema, eczema 
or exanthema with generalized itching and urticarial, worsening of existing autoimmune skin 
disorders 
(such 
as 
psoriasis 
and 
roseaca), 
gastrointestinal 
toxicities 
(diarrhea/colitis), 
endocrinopathies (hypophysitis, thyroiditis etc.) hepatotoxicity, pneumonitis, ocular toxicity, 
myocarditis, neuropathy and immune related nephrotoxicity. The most common side effects are 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
fatigue, diarrhea, skin rash and itching affecting about 10 % of patients. Further information is 
available from the EMA.   
 
Heczey et al have treated patients with neuroblastoma with the same lymphodepleting regimen in 
combination with CAR T cells and anti-PD-1 therapy and have found it manageable and feasible.49    
 
7.8 Risks and disadvantages regarding invasive procedures 
Tumour biopsies have a slight risk of infection and bleeding. Pain and bruising might also occur in 
the area. Other risks are highly dependent on anatomical location. Biopsies will only be performed if 
deemed safe by relevant specialist. 
 
Blood tests can cause local pain and bruising. Blood testing will involve frequent hospital visits.  
 
7.9 Monitoring of organ function 
Hematological monitoring of blood counts is indicated for all patients throughout treatment. 
Leukocyte count, platelet count and hemoglobin values will be checked daily at fixed intervals. Blood 
cell counts will be performed on day -5 before start of chemotherapy and every day during 
hospitalization  until neutrophil counts is >500/μl and leukocyte count is >1.000/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 functions are closely monitored. Any obstruction of the 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 from chemotherapy. The patients’ 
urine will be tested for microscopic hematuria before start of cyclophosphamide and this drug will 
be discontinued if cystitis associated with micro- or macroscopic hematuria occurs during treatment. 
 
7.10 Risks and precautions  
Infertility can occur during chemotherapeutic treatment and there is also a risk of a permanent 
affection of the fertility.  
 
Vaccination with live vaccines should be avoided prior to- and 6 months after treatment with 
Pembrolizumab (Group B). 
 
Depolarizing muscle relaxants that inhibits the cholinesterase activity interact with 
cyclophosphamide and in concomitant treatment could modulate the effect of drugs such as 
suxamethonium. This can result in prolonged apnea when anesthetized and the combination should 
be avoided. The anesthesiologist should be informed if the patient has received treatment with 
cyclophosphamide within 10 days before putting into general anesthesia.  
 
Grapefruit or grapefruit juice should be avoided since grapefruit contains a substance that can 
impair the activation of cyclophosphamide and thereby hinder its effect. 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
 
Transfusion related graft versus-host reactions have been observed in patients receiving 
treatment with fludarabine phosphate after transfusion with non-radiated and non-filtered blood. 
Participants in need of blood transfusions within 6 months after receiving with fludarabine phosphate 
should only receive radiated and filtered blood. To this end, the blood bank will be informed of the 
patients’ enrolment in the protocol and the patient will then only receive radiated blood for 6 months 
after treatment. All blood in the Capital Region is filtered. 
 
8 Translational research  
Blood samples and tumour tissue are collected for research purposes.  
 
8.1 Research blood tests  
In total 350 ml of blood will be collected for research purposes in the period from initiation of 
treatment until the patient meets for the first evaluation after treatment. At each evaluation 78 mL will 
be collected and potentially another 78 mL at progression. These blood samples are collected to 
assess the effect of treatment on the immune system and persistence of infused T cells. 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.  
 
Immune monitoring is performed on heparinized blood by flow cytometry.  
These will be collected at:  
o 100 mL at baseline (day -5)  
o 60 ml on day +5  
o 60 ml on day +21  
o 60 ml on day +42  
o 60 ml on day +84  
o and thereafter 60 ml every third month at time of clinical evaluation and at 
progression.  
Mononuclear cells from the heparinized 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 type AB serum and 10% DMSO. The cells are frozen at 
-150°C until analysis. Analyses will primarily focus on evaluation of treatment induced quantitative 
changes in T-cells and will include quantitative and qualitative analyses of blood T cells measuring 
concentration and phenotypic stage of T-cells by flow cytometry using surface marker CD3, CD4, 
CD8, CD28, CD95, CCR7, and CD45RA amongst others. Furthermore, regulatory T cells, myeloid 
derived suppressor cells (MDSC), NK cells, NKT cells, dendritic cells, and monocytes will be 
quantitated by flow cytometry. Specific attention is given to the number and phenotype of the 
antigens specific T cells coming from the infusion product. These will be traced based on their 
antigenicity to determine the kinetics and persistence over time. Furthermore, selected subgroups 
of cell may be subjected to single-cell analyses.  


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
ELISpot IFN-γ analyses and new advanced technologies including combinatorial coding flow 
cytometry will be employed for quantification of tumour antigen specific T-cells where feasible and 
otherwise to asses general immune status.  
Serum samples (9 mL) and plasma samples (9 mL) will be collected at the same time points as the 
heparinizied blood and also on day 0, 1 and 2. Serum and plasma samples are centrifuged and frozen 
for later use. 
 
8.2 Tumour samples 
Biopsies are performed from accessible tumour lesions at baseline, at the first clinical evaluation 
(after 6 weeks) and in case of progression (only performed for patients treated at Herlev University 
Hospital). Biopsies will only be performed if deemed safe and without unreasonable risk for the 
patient. Biopsies will be paraffin embedded, tumour cell lines and TILs will be grown and used for 
later immune analyses.  
Avaliable tumor samples in the clinical biobank “Patobank” can be used. These samples will only be 
used if enough material is available that this usage will have no consequence for the future cancer 
therapy of the patient 
8.2.1 Gene-based arrays  
The aim of the gene analyses is to learn about: 
Differences among patients in expression level of a panel of relevant normal genes in the tumour 
microenvironment which could influence the chance of benefit from treatment, and expression of 
tumour/patient specific mutated genes which could influence the chance of benefit from treatment, 
including specific tumour gene expression signatures50 and mutations in the tumour cells and patient-
specific neo-antigens derived from these mutations51. 
These analyses will contribute to the identification of patients who are most likely to respond to 
treatment. 
Tumour tissue gene expression profiles will be analyzed on either cryopreserved or FFPE-preserved 
tumour tissue. We will retrieve gene expression data on approximately 500 cancer/immunity related 
genes using Illumina targeted RNA sequencing applicable to degraded RNA. Targeted sequencing 
on a limited number of defined genes will be performed on the tumour tissue to obtain an immune 
profile to determine which genes are expressed in the tumour tissue. 
Next Generation Sequencing (NGS) with extensive mapping and whole genome sequencing of 
tumours and normal cells of the individual patient will be used to obtain information on tumour-
specific mutations. Gene sequencing will be performed on tumour cells (tumour gene profile) and 
leukocytes (normal gene profile) and by subtracting the two the tumour specific gene expression 
(neo-antigens) is found.  
8.2.2 Cell lines and cell cultures  
Establishment of autologous short term in vitro cultured tumor cell lines (as in ref. 52and53, or 
equivalent methods) and lymphocyte cultures (as in ref. 54or equivalent methods) will be attempted 
using these samples. These cultures will be primarily used to perform correlates of tumor-
recognition in vitro (as in ref.54) with immune parameters and clinical outcomes following therapy 
on an exploratory basis. The informed consent sheet will contain information about the possibility to 


32 
MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
generate cell lines and cell cultures, that under pseudonymized or anonymized form can be used 
for health research purposes in multiple countries, according to the Danish National Ethics 
Committee guidelines 
 
9 Data handling and reporting  
The participants are allocated a study number at inclusion to ensure that they cannot be directly 
identified. The PI will create an allocation key with study numbers and social security numbers that 
will be securely stored in an access-restricted folder created by the Capital Regions IT department 
“CIMT”. Selected collaborators at CCIT will be able to gain access to patient personal information to 
secure proper treatment and evaluation. All handling of personal data will be reported and approved 
by the Danish Data Protection Agency “Videnscenter for dataanmeldelser” through Herlev Hospital 
and according to Danish law. The PI has access to the patient chart including medical records and 
medication list before inclusion in the study in order to obtain relevant information and ensure patient 
safety. 
 
9.1 Electronic case report form  
All relevant data is registered in the eCRF (electronic Case Report Form) developed in cooperation 
with the Clinical Research Unit (KFE). The PI is responsible for creating the eCRFs and the 
subsequent recording and reporting of data. The sponsor and PI are both responsible for data 
analysis on all included patients. 
Patient data and eCRF will be stored for 25 years after study completion in accordance with current 
guidelines for storage of personal information in clinical trials. A final report will be drafted in 
collaboration between the members of the study group. 
The eCRF will include: 
o Toxicity (CTC registration) 
o Immunological response 
o Clinical effect parameters. 
 
9.2 End of study  
The study sponsor will inform the Danish Medicines Agency and Research Ethics Committee within 
90 days of the study completion. If the study is prematurely terminated, the Danish Medicines Agency 
and the Research Ethics Committee will be informed of the reason for the termination. 
 
10 Research partner institutions  
Laboratory analyses are performed at DTU or at CCIT or other locations within RegionH. Other 
analyses on either tumour tissue or blood samples can be performed at partner institutions but only 
after establishing a specific written agreement for transfer and handling of materials and data.  
In cases where patient samples are to be sent abroad, they will be handled according to national 
laws and regulations in the receiving country. All personal information will be pseudononymized, and 


33 
MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
a data processing agreement will be signed between the sponsor or PI and the data processors 
abroad. If any gene analysis is performed abroad, the data processing agreement will include the 
five criteria described later in “Ethical considerations regarding gene-based research” in the section 
“Ethical aspects”. If the data processing is performed in another country or location within Denmark, 
permission will be applied for at the Danish Data Protection Agency. 
 
11 Ethical considerations  
 
11.1 Recruitment of patients and informed consent  
In Denmark, metastatic melanoma patients are treated exclusively at the three participating 
Departments of Oncology. Referred patients will be screened for eligibility and invited to an 
informative consultation with the project manager or a collaborator. 
 
The patients will receive oral and written information and signed consent of participation will be 
obtained before inclusion. The oral information includes information and counseling about the 
planned genetic analyses to enable patients to decide on whether or not to receive information about 
unexpected findings. The study will adhere strictly to the Helsinki declaration. The patient information 
follows the guidelines listed in appendix 2. Any contact to eligible patients will be done in accordance 
with the Danish Health Act, § 46, paragraph 3. 
 
11.2 Patient identification  
Patients are allocated a study number at inclusion. The number is given sequentially and is not based 
on the patients name or birthday. This number will be used to identify the patient and will be used in 
the eCRFs. Data and patient material will be treated pseudo anonymized and confidential.  
 
11.3 Insurance 
Participants of the study are covered by insurance from the Danish Patient Compensation 
Association “Patienterstatningen”. 
 
11.4 General ethical aspects  
Despite many treatment advances the majority of patients with MM are still incurable. Most patients 
will at some point find themselves without viable treatment options. The purpose of this study is to 
investigate whether treatment with MASE-T infusion in combination with lymphodepleting 
chemotherapy with or without pembrolizumab could benefit these patients.  
Based on the current knowledge and when the described precautions are taken, we find that the 
potential benefit of participating in this study outweighs the potential risks and downsides. At the 
individual level, eligible patients have advanced disease and suffered progression on traditional 
treatments. MASE-T may represent an additional treatment option and that may for many patients 
outweigh a potential risk of toxicity. Participation is always voluntary and is preceded by oral and 


34 
MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
written information of the study. The treatment will be stopped at any time in case of unacceptable 
adverse reactions or at the patients request.  
 
11.5 Ethical considerations regarding gene-based research  
Data regarding potential disease-causing genes may be generated as a byproduct of certain modern 
laboratory techniques. Byproduct data will not be used or explored further since only data regarding 
tumour specific genes are processed more closely. Therefore, we do not expect to obtain explicit 
knowledge regarding disease-causing genes. If by chance these analyses will discover known 
mutations with potential significant impact on patient’s health, the case will be discussed in an 
independent expert workgroup in collaboration with the Clinical Genetics Department at 
Rigshospitalet, unless the patient has chosen not to be informed as stated in the patient information. 
The following criteria will determine if further actions are indicated: 
o A reasonable degree of possibility that a genetic disposition is present. 
o Solid documentation linking the genetic disposition and the development of disease. 
o The tests used to determine the genetic disposition are well established. 
o The disease in question can be prevented or treated. 
o The link between the genetic disposition and the development of disease has considerable 
importance for the patient. 
If indicated, the patient will be contacted for referral to the relevant Genetics Department for 
additional information and testing. In cases where a patient is deceased, 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 
(Sundhedsloven § 43, stk. 2, nr.2.). 
 
11.6 Approvals  
The PI is to obtain permission from the Danish Medicines Agency “Lægemiddelstyrelsen”, the 
National Research Ethics Committee “National Videnskabsetisk komité” and the Danish Data 
Protection Agency “Videnscenter for dataanmeldelser” before starting the study. 
The law dealing with personal data will be respected. Information concerning study participants is 
protected according to the law concerning personal data and the Act on Research Ethics Review of 
Health Research Projects.  
 
11.7 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 changes can then be implemented after 
approval. Changes to the protocol are considered substantial in accordance with guidelines 
described 
in 
“Vejledning 
om 
ændringer 
af 
et 
godkendt 
forskningsprojekt” 
on 
www.nvk.dk/forsker/forskervejledning and ”Skema om ændringer til kliniske forsøg” on 
www.sundhedsstyrelsen.dk.  
 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
12 Biobank and handling of biological material 
12.1 Research biobank  
In connection with the current study, blood samples and tumour tissue will be stored at -80 or -140 
°C in a research biobank at the CCIT in room PA102. They will be stored until all analyses concerning 
the study are finished. In case of surplus samples after all planned analyses are finished, these 
samples will be transferred to a biobank for future research. 
 
12.2 Biobank for future research  
Surplus samples are stored in a biobank for future research at CCIT. For research in other areas with 
material in the biobank, new approvals from the Research Ethics Committee and Danish Data 
Protection Agency will be needed.  
 
12.3 Disposal of biological material  
When approval for storage of biological samples expires, remaining material will be fully anonymized 
or disposed of according to the local guidelines for disposal of biohazardous waste. 
If a patient withdraws his/her informed consent, all biological material will be destroyed at the 
patients’ request. 
13 Administrative aspects and publications  
13.1 Publications  
Inge Marie Svane, Sine Reker Hadrup and Tine Juul Monberg constitute the group responsible for 
the project. Under the circumstance that the Vancouver rules are met, the group responsible for the 
project holds equal rights to the achieved results. The use and presentation of the data in any form, 
orally or written, can only take place with accept from everybody in the group. Positive, negative and 
inconclusive results will be reported in scientific journals. Author succession will be determined 
based on individual contributions and the investigators will be co-authors on further publications 
derived from this study. The main analysis will be done when all patients have been treated. 
Additional interim analyses can be done after treatment of respectively the first 3 and the first 6 
subjects. These results might be published.   
No later than a year after the trial has ended, the trial results must be entered in EudraCT and 
subsequently, data will be published on www.clinicaltrialsregister.eu. Further, the results will be 
reported to the Danish Medicines Agency.  
 
13.2 Economy 
This project has been initiated by Inge Marie Svane from the Center for Cancer Immune Therapy, 
Denmark, in collaboration with Sine Reker Hadrup from the Technical University of Denmark, dept 
of Health technology. The project is supported via a grant from ‘immunterapi-puljen på finansloven’  
with the project title: Empowering cancer immunotherapy in Denmark, applied by Professor, MD 
Inge Marie Svane, j.nr. 4-1612-236/8. From this grant the budget for DTU is 960.681 dkr (incl. 44% 
overhead to DTU) and the budget for CCIT-DK is 1.141.208 dKr (incl 3.1% overhead to hospital) 


36 
MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
 
The Research Ethics Committee will be informed in case further funding is obtained. The technology 
behind ImmPACT is developed in Sine Reker Hadrups research group and patent protected by DTU. 
DTU has made a licence agreement with the spin-out PokeAcell to make use of this technology for 
development of a cell therapy product. This licence agreement has been signed by president of DTU 
Anders Bjarklev and CEO at PokeAcell Anne Cordt. Sine Reker Hadrup is co-founder of PokeAcell. 
PokeAcell will also financially support the production of the Ag scaffold as part of their development 
process towards commercially viable cell product. The financial contribution from PokeAcell is 1.5-2 
mio dkr. IMS is associated with PokeAcell as an independent advisor and provide consultancy 
services in connection with clinical development of the ImmPACT technology. The collaboration 
between the three parties in the study is regulated by a contract.  
 
 


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MASE-T Protocol version 2.3, date:  27 March 2023. EudraCT: 2020-000939-46 
14. References  
1. 
Bhatia, S., Tykodi, S. S. & Thompson, J. a. Treatment of metastatic melanoma: an overview. 
Oncology (Williston Park). 23, 488–96 (2009). 
2. 
Hodi, F. S. et al. Improved survival with ipilimumab in patients with metastatic melanoma. N. 
Engl. J. Med. 363, 711–23 (2010). 
3. 
Robert, C. et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N. Engl. J. Med. 
372, 2521–32 (2015). 
4. 
Larkin, J. et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated 
Melanoma. N. Engl. J. Med. 373, 23–34 (2015). 
5. 
LBA68_PR ‘5-year survival outcomes of the CheckMate 067 phase 3 trial of nivolumab plus 
ipilimumab (NIVO+IPI) combination therapy in advanced melanoma‘ will be presented by 
James M. Larkin during the Proffered Paper session on Saturday, 28 September, 08:3. 
6. 
Hugo, W. et al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in 
Metastatic Melanoma. Cell 165, 35–44 (2016). 
7. 
Spranger, S. et al. Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor 
microenvironment is driven by CD8(+) T cells. Sci. Transl. Med. 5, 200ra116 (2013). 
8. 
Andersen, R. et al. Long-Lasting complete responses in patients with metastatic melanoma 
after adoptive cell therAndersen, R., Donia, M., Ellebaek, E., Borch, T. H., Kongsted, P., 
Iversen, T. Z., … Svane, I. M. (2016). Long-Lasting complete responses in patients with 
metasta. Clin. Cancer Res. 22, 3734–3745 (2016). 
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