Adoptive Therapy With TCR Gene-engineered T Cells to Treat Patients With MAGE-C2-positive Melanoma and Head and Neck Cancer
Phase: PHASE1
Conditions: Melanoma, Melanoma, Uveal, Head and Neck Cancer
Sponsor: Erasmus Medical Center

NL69911.000.19 / MC2TCR 
 
 
Short title: MC2 TCR T cell therapy 
 
 
 
PROTOCOL TITLE  
Adoptive therapy with TCR gene-engineered T cells to treat patients with MAGE-C2-
positive melanoma and head and neck cancer 
 
Protocol ID 
NL69911.000.19 
Short title 
MC2TCR 
EudraCT number  
2019-000657-31 
Version 
4.0 
Date 
9 September 2019 
Principal investigator 
 
 
 
 
 
 
 
Project leaders 
 
 
 
 
 
Dr. A.A.M van der Veldt  
Department of Medical Oncology 
Erasmus MC Cancer Institute 
Dr. Molewaterplein 40 
The Netherlands 
3015 GD Rotterdam 
Tel: +31 10 7034447 
Email: a.vanderveldt@erasmusmc.nl 
 
Dr. C.H.J. Lamers 
Dr. R. Debets 
Laboratory of Tumor Immunology 
Department of Medical Oncology 
 
Co-investigators 
Prof. dr. S. Sleijfer 
Department of Medical Oncology 
 
Sponsor  
Erasmus MC  
Dr. Molewaterplein 40 
3015 GD Rotterdam 
 
Subsidising party 
Dutch cancer society (grant EMCR 2015-7741)  
Postbus 75508 
1070 AM Amsterdam 
 
Independent expert (s) 
P.G.N.J. Mutsaers, MD  
Department of Hematology 
Erasmus MC 
 

NL69911.000.19 / MC2TCR 
 
 
Short title: MC2 TCR T cell therapy 
 
 
 
Laboratory sites 
Laboratory of Tumor Immunology 
 (PI: dr. R. Debets) 
Department of Medical Oncology 
Erasmus MC 
 
 
Statistician 
Dr. E. Oomen 
Department of Medical Oncology 
Erasmus MC 
 
Qualified persons for the manufacturer’s license of Erasmus MC, register nr. 108517F 
 
Pharmacy  
Dr. L. Ruijgrok 
Department of Pharmacy 
Erasmus  MC 
Erasmus MC ATMP facility 
Dr. E. Braakman 
Department of Hematology 
Erasmus  MC 

NL69911.000.19 / MC2TCR 
 
 
Short title: MC2 TCR T cell therapy 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Sponsor or legal representative 
Head of Department 
Prof. dr. S. Sleijfer 
 
 
 
Principal Investigator 
Dr. A.A.M van der Veldt  
 
 
 
 
 
Project leaders 
Dr. C.H.J. Lamers 
 
 
Dr. R. Debets 
 
 
 
 
 
 
 

NL69911.000.19 / MC2TCR 
 
 
Short title: MC2 TCR T cell therapy 
 
 
 
TABLE OF CONTENTS 
PROTOCOL TITLE ............................................................................................................... 1 
TABLE OF CONTENTS ........................................................................................................ 4 
LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS ................................................ 7 
SUMMARY 9 
1. 
INTRODUCTION AND RATIONALE ..............................................................................14 
1.1 Clinical background .......................................................................................................14 
1.2 Adoptive T cell transfer (AT): previous clinical successes ..............................................14 
1.2.1 
AT using tumor-infiltrating Lymphocytes (TILs) ................................................14 
1.2.2 
AT using chimeric antigen receptor (CAR) T cells ............................................15 
1.2.3  
AT with CAR-T cells: a pioneering study at Erasmus MC .................................15 
1.3 AT with TCR T cells .......................................................................................................15 
1.3.1 
Clinical experience and challenges ..................................................................15 
1.3.2 
Unique elements of current Erasmus MC trial with TCR T cells .......................16 
1.3.4 
Safety considerations of Erasmus MC AT trial with TCR T cells .......................20 
2. 
OBJECTIVES ................................................................................................................21 
2.1 Phase I 21 
2.1.1 
Primary objectives ............................................................................................21 
2.1.2  
Secondary and exploratory objectives ..............................................................22 
2.2 Phase II 22 
2.2.1 
Primary objective .............................................................................................22 
2.2.2 
Secondary objectives .......................................................................................22 
2.2.3 
Explorative objectives ......................................................................................22 
3. 
STUDY DESIGN............................................................................................................23 
3.1 Description of the study design ......................................................................................23 
3.1.1 
Phase 1: dose escalating phase ......................................................................23 
3.1.3 
Phase II: expansion phase ...............................................................................24 
4. 
STUDY POPULATION ..................................................................................................24 
4.1 Population .....................................................................................................................24 
4.2 Inclusion criteria.............................................................................................................24 
4.3 Exclusion criteria ...........................................................................................................25 
4.4 Sample size calculation .................................................................................................26 
5. 
TREATMENT OF SUBJECTS .......................................................................................27 
5.1 Investigational product/treatment ...................................................................................27 
5.2 Use of co-intervention (if applicable) ..............................................................................27 
5.3 Escape medication (if applicable) ..................................................................................27 
6. 
INVESTIGATIONAL PRODUCT ....................................................................................28 
7. 
METHODS ....................................................................................................................30 
7.1 Study endpoints .............................................................................................................30 
7.1.1 
Phase I - primary study endpoints ....................................................................30 
7.1.2 
Phase II - primary study endpoint .....................................................................30 
7.1.3 
Phase II - secondary endpoints ........................................................................31 

NL69911.000.19 / MC2TCR 
 
 
Short title: MC2 TCR T cell therapy 
 
 
 
7.1.4 
Phase I and phase II - Secondary and exploratory endpoints ..........................31 
7.2 Randomisation, blinding and treatment allocation ..........................................................31 
7.3 Study procedures ..........................................................................................................31 
7.3.1 
Pre-study screening .........................................................................................31 
7.3.2 
Procedures of TCR T cell treatment .................................................................32 
7.3.2 
On study evaluation .........................................................................................33 
7.3.3 
Additional immune-related research evaluations ..............................................33 
7.3.4 
Post-treatment and off-study evaluation ...........................................................34 
7.3.5 
Treatment of treatment-related toxicity .............................................................35 
7.3.6 
Re-treatment ....................................................................................................35 
7.3.7  
Off-study criteria...............................................................................................35 
7.4 Withdrawal of individual subjects ...................................................................................36 
7.4.2 
Specific criteria for withdrawal ..........................................................................36 
7.4.3 
Replacement of individual subjects after withdrawal ........................................36 
7.4.3 
Follow-up of subjects withdrawn from treatment ..............................................36 
7.5 Premature termination of the study ................................................................................36 
7.6 Long-term follow-up .......................................................................................................36 
8. 
SAFETY REPORTING ..................................................................................................39 
8.1 Temporary halt for reasons of subject safety .................................................................39 
8.2 Adverse and serious adverse events .............................................................................39 
8.2.1 
Adverse events (AEs) ......................................................................................39 
8.2.2 
Serious adverse events (SAEs) .......................................................................39 
8.2.3 
Reporting of SAEs ...........................................................................................40 
8.3 Suspected unexpected serious adverse reactions (SUSARs) ........................................40 
8.3.1 
Reporting of SUSARs ......................................................................................40 
8.4 Annual safety report ......................................................................................................40 
8.5 Follow-up of AEs ...........................................................................................................41 
8.6 Data Monitoring Committee (DMC) ................................................................................41 
9. 
STATISTICAL ANALYSIS .............................................................................................41 
9.1 Primary study parameter(s) ...........................................................................................41 
9.2 Secondary study parameter(s) .......................................................................................42 
9.3 Toxicity analysis ............................................................................................................42 
9.4 Interim analysis (if applicable)........................................................................................42 
10. ETHICAL CONSIDERATIONS ......................................................................................42 
10.1 
Regulation statement ................................................................................................42 
10.2 
Recruitment and consent ..........................................................................................43 
10.3 
Objection by minors or incapacitated subjects...........................................................43 
10.4 
Benefits and risks assessment, group relatedness ....................................................43 
10.5 
Compensation for injury ............................................................................................44 
10.6 
Incentives ..................................................................................................................45 
11. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .............................45 
11.1 
Handling and storage of data and documents ...........................................................45 
11.2 
Monitoring and Quality Assurance .............................................................................46 

NL69911.000.19 / MC2TCR 
 
 
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11.3 
Amendments .............................................................................................................46 
11.4 
Annual progress report ..............................................................................................46 
11.5 
Temporary halt and (prematurely) end of study report ...............................................46 
11.6 
Public disclosure and publication policy ....................................................................47 
12. STRUCTURED RISK ANALYSIS ..................................................................................47 
12.1 
Potential issues of concern .......................................................................................47 
12.2 
Synthesis ..................................................................................................................49 
13. REFERENCES ..............................................................................................................50 
14. APPENDICES ...............................................................................................................54 
Appendix 1: Eastern Cooperative Oncology Group (ECOG) Performance Status ................54 
Appendix 2: Investigational product label .............................................................................55 

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Short title: MC2 TCR T cell therapy 
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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
ABR 
ABR form, General Assessment and Registration form, is the application form that is 
required for submission to the accredited Ethics Committee; in Dutch: Algemene 
Beoordeling en Registratie 
AE 
Adverse Event 
AR 
AT 
AZA 
Adverse Reaction 
Adoptive T cell therapy  
5-azacitidine  
BORIS 
Brother Of the Regulator of Imprinted Sites (= ligand for CTCF) 
CA 
CGA 
Competent Authority 
Cancer Germline Antigen  
CCMO 
 
CRS 
CT 
Central Committee on Research Involving Human Subjects; in Dutch: Centrale 
Commissie Mensgebonden Onderzoek 
Cytokine Release Syndrome 
Computed Tomography 
CTCAE 
Common Terminology Criteria for Adverse Events (CTCAE) 
CTCF 
11-zinc finger protein or CCCTC-binding factor (= transcriptional repressor)  
CV 
Curriculum Vitae 
DLT 
Dose Limiting Toxicity 
DMC 
Data Monitoring Committee (a.k.a. Data Safety Monitoring Board (DSMB)) 
ECOG 
Eastern Cooperative Oncology Group 
EMC 
Erasmus university Medical Center 
EU 
European Union 
EudraCT 
HNSCC 
European drug regulatory affairs Clinical Trials  
Head and Neck Squamous Cell Carcinoma 
GCP 
Good Clinical Practice 
IB 
Investigator’s Brochure 
IC 
IL-2 
Informed Consent 
Interleukin-2 
IMP 
Investigational Medicinal Product  
IMPD 
MC2 
Investigational Medicinal Product Dossier  
MAGE-C2  
METC 
  
MNC 
MRI 
MTD 
PBMC 
Project team 
Medical research ethics committee (MREC); in Dutch: medisch ethische toetsing 
commissie (METC) 
Mononuclear Cell 
Magnetic Resonance Imaging 
Maximum Tolerated Dose 
Peripheral Blood Mononuclear Cell 
PI, co-investigator and project leaders 

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PFS 
QP 
RECISTv1.1 
OOS 
OS 
Progression Free Survival 
Qualified Person 
Response Evaluation Criteria in Solid Tumors, version 1.1 
Out Of Specification 
Overall survival 
(S)AE 
(Serious) Adverse Event  
SPC 
Summary of Product Characteristics; in Dutch: officiële productinfomatie IB1-tekst 
Sponsor 
The sponsor is the party that commissions the organisation or performance of the 
research, for example a pharmaceutical company, academic hospital, scientific 
organisation or investigator. A party that provides funding for a study but does not 
commission it is not regarded as the sponsor, but referred to as a subsidising party.  
SUSAR 
TCR 
Suspected Unexpected Serious Adverse Reaction 
T Cell Receptor 
VP 
Valproic Acid 
WBP 
Personal Data Protection Act; in Dutch: Wet Bescherming Persoonsgegevens 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 

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SUMMARY 
Rationale 
 
In patients with advanced melanoma and other tumor types, prior clinical studies have shown 
efficacy of T lymphocytes directed towards tumor antigens. To this end, tumor reactivity can be 
imposed by the transfer of T cell receptor (TCR) genes into previously non-reactive T cells. 
 
MAGE-C2 (MC2) is a Cancer Germline Antigen (CGA) that is highly expressed in melanoma and 
head and neck squamous cell carcinoma (HNSCC), but not in normal mature tissues. MC2 can 
evoke clinically effective T cell responses, as demonstrated in previous vaccination studies.  
 
In this phase I/II study, we will investigate the safety of adoptive T cell therapy (AT) with T cells 
gene-engineered to express MC2-specific TCRs in patients with advanced melanoma or HNSCC. 
 
Objectives 
Phase I 
 
Primary objectives: 
o 
to study the safety and feasibility of AT with autologous MC2 TCR T cells, combined with 
epigenetic drug treatment, in patients with advanced melanoma or HNSCC. Adverse events 
(AEs) will be documented according to CTCAE version 5.0; 
o 
to define the maximum tolerated dose (MTD) of MC2 TCR T cells in the combination 
treatment. 
 
Secondary and exploratory objectives: 
o 
to study presence and function of MC2 TCR T cells in peripheral blood samples after 
treatment; 
o 
to study the systemic release of inflammatory cytokines after administration of autologous 
MC2 TCR T cells; 
o 
to document MC2 expression in tumor tissues (when available) prior to and after epigenetic 
drug treatment; 
o 
to document the induction of global DNA hypomethylation and histone acetylation in peripheral 
blood mononuclear cells (PBMC) by epigenetic drug treatment; 
to study immune parameters, in particular T cell parameters, in blood and tumor tissues (when 
available) prior to and during treatment. 
 
Phase II 
 
Primary objectives: 
o 
to assess the efficacy of AT with autologous MC2 TCR T cells at MTD, combined with 
epigenetic drug treatment, in patients with advanced melanoma or HNSCC; 
o 
tumor response will be evaluated using RECIST v1.1.[1]  Response is defined as complete 
remission (CR) and partial response (PR) according to RECIST v1.1. 
 
Secondary objectives: 
o 
to determine the following outcomes in patients treated with MC2 TCR T cells at MTD: 
- 
progression free survival; 

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- 
duration of response; 
- 
overall survival. 
 
Exploratory objectives: 
o 
to study presence and function of MC2 TCR T cells in peripheral blood samples after 
treatment; 
o 
to study the systemic release of inflammatory cytokines after administration of autologous 
MC2 TCR T cells; 
o 
to document MC2 expression in tumor tissues (when available) prior to and after epigenetic 
drug treatment; 
o 
to document the induction of global DNA hypomethylation and histone acetylation in peripheral 
blood mononuclear cells (PBMC) by epigenetic drug treatment; 
o 
to study immune parameters, in particular T cell parameters, in blood and tumor tissues (when 
available) prior to and during treatment. 
 
Study design 
 
This is a single institution phase I/II trial consisting of an accelerated titration phase I design and a 
subsequent single arm (2-stage) phase II study. In the phase I part, the recommended T cell 
number for phase II will be determined. 
 
Eighteen patients with advanced melanoma or HNSCC, a HLA-A2 genotype and MC2-positive 
tumors will be included in the study.  
 
Screening will consist of genotyping for HLA-A2 and MC2 immunohistochemistry on tumor tissue.  
 
Prior to T cell transfer (day 0), patients will be treated with the epigenetic drugs valproic acid (VP, 
dose 50 mg/kg/d, 7d; days -9 to day -3) and 5-azacitidine (AZA, dose 75mg/m2/d, 7d; days -9 to 
day -3). 
 
Phase I: patients will be treated with one single intravenous administration of TCR T cells at 5 
different escalated doses of 5x107, 5x108, 5x109,1.0x1010, and the total number of cultured TCR T 
cells (i.e. usually 1.0 – 5.0 x1010 TCR T cells). MC2 TCR Tcell infusions will be supported by low 
dose of IL-2 administrations (s.c. 5x105 IU/m2 2qd for 5 days). 
 
In case of dose limiting toxicities (DLT) in the accelerated titration phase, the phase I part will 
continue in a classical 3+3 design and dose-escalation will be semi-logaritmic. 
 
In case 1 (or 3) patient(s) has(ve) been treated in a particular dose level, but not before 3 weeks 
after treatment of the (3rd) patient, AEs will be evaluated and the project team (PI, co-investigator 
and project leaders) will decide whether the trial will continue to the next dose level in phase I or 
will continue using the current dose level in a classical 3+3 design. 
 
If DLT is observed in 2 out of 6 patients (3+3 extended cohort), the previous (semi-logaritmic) 
dose level will be the maximum tolerated dose (MTD) (see § 4.4). 
 
Phase II: patients will be treated with TCR T cells at the MTD. In case no DLTs were observed, 
patients will be treated with the total number of cultured TCR T cells, which is usually 1.0 – 5.0 
x1010 TCR T cells. 

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 
In both phase I and II, peripheral persistence, differentiation state and tumor recognition of 
peripheral and intra-tumoral TCR T cells will be evaluated, as well as additional immune and T cell 
parameters in blood and tumor tissue. 
 
Study population 
Patients with unresectable stage IIIc and IV melanoma (including unknown primary, mucosal and 
uveal melanoma), and unresectable recurrent/metastatic (R/M) HNSCC who are ≥18 years of age are 
eligible. Melanoma patients must have progressive disease after standard treatment including for 
instance immune checkpoint inhibition, BRAF/MEK-inhibition and chemotherapy. R/M HNSCC patients 
who are ineligible for or unwilling to get platinum-based chemotherapy or for whom no standard 
treatment is available are eligible. Only patients with a HLA-A2 genotype and MC2-positive tumors will 
be included.  
 
Intervention 
Autologous T cells, obtained by leukapheresis, will be transduced with a retroviral vector encoding the 
MC2 TCR. Subsequently, these TCR T cells will be expanded ex vivo in the presence of defined 
cytokines. Following pre-treatment with epigenetic drugs, TCR T cells will administered intravenously 
with support of low-dose IL-2. For safety measures, AEs will be documented according to CTCAE 5.0. 
Tumor response will be evaluated using RECIST v1.1. Tumor biopsies will be taken before treatment 
(to assess MC2 expression) and where possible at 3 weeks after MC2 TCR T cell infusion and at 
progressive disease to evaluate therapy induced changes of the tumor microenvironment. An optional 
tumor biopsy may be taken after epigenetic pre-treatment and prior to MC2 TCR T cell infusion to 
document the in vivo upregulation of the MC2 expression. 
 
Main study parameters/endpoints: 
Primary endpoints 
Phase I 
 AEs according to CTCAE 5.0; 
 recommended Phase II dose; 
 feasibility to deliver this sequence of treatment. 
Phase II 
 objective response rate according to RECIST v1.1; 
 PFS; 
 OS. 
 
Secondary and exploratory endpoints 
Phase I & II 
 persistence and function of MC2-specific T cells in peripheral blood; 
 systemic release of inflammatory cytokines after administration of autologous MC2 TCR T cells; 
 MC2 expression in tumor tissues prior to and after epigenetic drug treatment; 

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 global DNA hypomethylation and histone acetylation in PBMCs after epigenetic treatment and 
administration of autologous MC2 TCR T cells; 
 immune parameters, in particular T cell parameters, in blood and tumor tissues (when available) 
prior to and during treatment. 
 
Nature and extent of burden risks associated with participation, benefit group relatedness 
For patients included in this phase I/II trial, no standard therapies are available in the Netherlands. 
Previous trials have shown efficacy of AT in several patient populations.  
In patients with metastatic melanoma or synovial carcinoma, AT with a CGA NY-ESO TCR T cells has 
shown very limited AEs and significant objective clinical responses of 55 and 61%, respectively.[2, 3] 
In patients with multiple myeloma, infusion of NY-ESO TCR T cells 2 days after an autologous stem 
cell transplant (preceded by Melphalan) resulted in a clinical response of 80%, thereby inducing 
various serious AEs (SAEs), which were reversible.[4]  
In patients with melanoma, AT using autologous TCR T cells with affinity enhanced TCRs for MAGE-
A3 has shown clinical responses, but also fatal on- and off target toxicities as a result of either 
targeting a common epitope of the MAGE-A antigen superfamily or a TCR with a non-restricted 
binding motif.[5, 6] In the current study, these (S)AEs are not expected as the MC2-specific TCR has a 
restricted binding motif without enhanced affinity. Nevertheless, in case patients may experience a 
cytokine release syndrome (CRS) with severe symptoms in the current study, patients will be treated 
with high dose corticosteroids, the anti-IL6R antibody tocilizumab [7], and the anti-CD52 antibody 
alemtuzumab, depending on the severity of the symptoms. In addition, the doses of the administered 
epigenetic drugs are well tolerated and are only associated with mild transient leucopenia and 
neutropenic fever.[8] Toxicities of epigenetic drugs and IL-2 are expected to be mild and managible by 
standard supportive care.  In this patient population, the potential burden and AEs are justified by the 
substantial chance of (durable) tumor responses as a result of the proposed treatment. 
 
 
The study proposal contains the following unique elements (see Figure 1): 
1. targeting MC2 by TCR T cells; 
2. a new T cell processing method to generate young T cells; 
3. pretreatment with epigenetic drugs to increase MC2 expression in the tumor; 
4. no chemotherapy prior to T cell infusion, which are usually administered in other trials to support 
modified T-cells, but which also substantially increase toxicity. 
 
 

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Figure 1: 
Adoptive T cell therapy with MC2 TCR T cells - overview of procedures and treatment. 
 
 
Figure 2: 
Adoptive T cell therapy with MC2 TCR T cells - schedule of laboratory and clinical 
procedures. For monitoring schedule, see Table 1b 
 
 

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1. INTRODUCTION AND RATIONALE 
1.1 
Clinical background 
In 2018, 7000 novel cases of melanoma were diagnosed in The Netherlands and incidence rates are 
still increasing.[9]  Until 2012, the OS of patients with unresectable stage IIIc and metastatic 
melanoma was only 6-10 months.[10, 11] The introduction of targeted therapy (BRAF and MEK 
inhibitors) and immune checkpoint inhibitors (anti-CTLA-4 and anti-PD-1 antibodies) has significantly 
improved the perspectives and survival of these patients.[12-20]. Nevertheless, approximately 50% of 
patients with cutaneous and mucosal melanoma does not have (long-term) benefit and eventually 
experience progressive disease.[11] Although novel therapies have proven efficacy in cutaneous and 
mucosal melanoma, there is still a large fraction of patients that show non-durable responses.[11] On 
top of that, systemic therapy is still disappointing for metastatic uveal melanoma; in fact, standard 
systemic therapy is lacking for patients with metastatic uveal melanoma and their OS is poor with 8-13 
months.[21, 22] Each year over 800 patients die of melanoma in The Netherlands.[11] 
 
In 2018, 3100 novel cases of HNSCC were diagnosed in The Netherlands (NKR) and incidence rates 
are gradually increasing.[23]  After initial diagnosis and multidisciplinary treatment, a substantial 
proportion of patients are diagnosed with recurrent/metastatic (R/M) HNSCC. For patients with R/M 
HNSCC cetuximab added to platinum-5-fluorouracil is the only approved treatment with significant 
overall survival benefit.[24]However, the life expectancy still is less than 1 year and the toxicity of this 
regimen is considerable. In patients with R/M HNSCC, PD-1 blockade is a promising new treatment 
option. In a randomized phase III trial, treatment with nivolumab resulted in a longer survival than 
treatment with chemotherapy.[25] Pembrolizumab as first-line treatment significantly improved overall 
survival over platinum/5-fluorouracil/cetuximab in patients with R/M SCCHN with a PD-L1 combine 
positive score (CPS) ≥20.[26] Despite the therapeutic benefit of these new treatments, the OS of 
patients with R/M HNSCC remains poor. 
 
In conclusion, there is a need to develop new treatment strategies for a substantial number of patients 
with advanced and metastatic melanoma and HNSCC. In the current phase I/II trial, adoptive T cell 
transfer targeting MAGE-C2 (MC2), a tumor specific target in melanoma and HNSCC, will be 
evaluated. 
 
1.2 
Adoptive T cell transfer (AT): previous clinical successes 
1.2.1 
AT using tumor-infiltrating Lymphocytes (TILs) 
There is substantial clinical evidence that AT leads to objective responses in melanoma patients. In 
the past 15 years, several clinical trials have demonstrated that AT with TILs is a safe and effective 
strategy for patients with metastatic melanoma.[27-32] These treatments yielded impressive objective 
response rates of 50% with a five year OS rate of ~20%.[32, 33] Recently, tumor-reactive TILs also 
have been successfully grown from other tumor types, including renal cell, ovarian, breast, bile duct 
and cervical cancer.[34-37] In patients with bile duct and cervical cancer, TIL therapy was also 

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effective and demonstrated control of tumor growth.[38] [34] To date, generation of TILs from solid 
tumors is successful in only about half of the patients.[32] 
 
1.2.2 
AT using chimeric antigen receptor (CAR) T cells 
A more universal strategy to generate tumor-specific T cells for clinical application is based on the 
transfer of genes encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CARs are 
antibody-based receptors and recognize extracellular antigens in a major histocompatibility (MHC) 
unrestricted fashion (representing about 25% of all antigens), generally with high binding strength. 
 
After breakthrough studies using CD19 CAR T cells for refractory B cell malignancies [39, 40], the 
number of clinical applications for CAR-T cells are rapidly expanding.[41-44] Over the last 2 years, 2 
products of CD19-CAR T cells have been granted FDA approval for various B cell malignancies, i.e., 
tisagenlecleucel (KYMRIAH®, Novartis) and axicabtagene ciloleucel (YESCARTA®, Kite 
Pharmaceuticals).[44] Until now, tumor responses with CAR T cells in patients with B cell 
malignancies have not yet been consistently reproduced for patients with solid tumors (see § 1.2.3). 
However, CAR-T cell development for patients with solid tumors is still in its early stages with an 
extending number of ongoing and expected clinical trials (www.clinicaltrials.gov). 
 
1.2.3  AT with CAR-T cells: a pioneering study at Erasmus MC 
Between 2003-2010, we have treated 12 patients with autologous T cells redirected against renal cell 
carcinoma (RCC) via a CAR specific for carboxy-anhydrase IX (CAIX) that is over-expressed by RCC. 
Per original protocol 3 patients, and per amendment another 5 patients were treated with 1x109 CAIX 
CAR-T cells and low dose IL-2. Four out of these 8 patients presented with severe, but transient liver 
toxicities, which were most likely due to CAIX expression on the surface of epithelial cells lining the 
bile ducts in the liver (albeit at a low level) [45, 46]. A second amendment enabled pre-treatment with 5 
mg of the humanized anti-CAIX mAb. Based on the favorable kinetics of this antibody, pre-infusion 
was expected to cover CAIX expressed by normal tissue, thereby leaving CAIX expression in RCC 
targetable for T cell treatment [46]. With this adapted regimen consisting of pre-infusion with 
humanized anti-CAIX mAb, treatment with CAR-T cells (1x109 - 2x109 ) and low dose IL-2, patients 
(n=4) did not experience any adverse events.[45, 46] However, CAR-T cells did not persist in the 
circulation for more than 30 days following infusion. T cell persistence and anti-tumor responses may 
have been limited because of the low T cell doses as well as the mature differentiated status of the 
infused T cells.[47] As several new treatment options for RCC were introduced in the clinic from 2005 
onwards, patient accrual was hindered and this T cell study was prematurely discontinued in 2010. 
 
1.3 
AT with TCR T cells 
1.3.1 
Clinical experience and challenges 
Whereas CARs recognize extracellular antigens in a MHC unrestricted fashion (representing about 
25% of all antigens), with high binding strength, TCRs recognize both extra- and intracellular antigens 

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(representing all antigens) with low-to-medium binding strength. As a result, TCRs have a wider 
breadth of targetable antigens. To date, a limited number of clinical trials has applied TCR T cells for 
the treatment of solid tumors (see [48, 49], and about a few dozen of these trials are ongoing 
(www.clinicaltrials.gov). 
In previous clinical trials, TCRs were usually restricted to the HLA-A1 or A2 genotype and directed 
against differentiation antigens (i.e. MART-1, gp100, CEA, and p53), Cancer Germline Antigens (i.e. 
MAGE-A3 and NY-ESO-1), and viral antigens. Collectively, these trials demonstrated responses in 12 
to 80% of patients with metastatic melanoma, colorectal carcinoma, synovial sarcoma, and multiple 
myeloma.[2-4] However, treatment-related AEs became evident from studies with TCRs, in particular 
for TCRs with high-affinity, directed against antigens with over-expression in both tumors and healthy 
tissue.[5, 50-52]. Two previous clinical studies applied TCRs directed against MAGE-A3 (2 different 
epitopes) and reported fatal neurological and cardiovascular AEs [5, 52]. These AEs were most likely 
caused by the use of affinity-enhanced TCRs that recognize a shared or highly similar epitope 
compared to the cognate epitope. As previously mentioned in § 1.2.3, the above-mentioned AEs 
require careful selection and preclinical assessment of T cell target antigens.[53, 54] Besides limiting 
toxicity, durable tumor responses need to be achieved upon treatment with TCR T cells.[48, 55] In 
previous studies, short duration of tumor responses was often associated with a short persistence of 
TCR T cells in blood.[56, 57] 
 
Collectively, findings in previous clinical trials demonstrate that TCR T cell therapy is promising and 
indicate that further development of TCR T cell therapy should focus on (see § 1.3.2 for more detail): 
 
Selection and validation of effective and safe target antigens for T cells 
 
Strategies to enhance TCR T cell persistence and anti-tumor responses 
 
1.3.2 
Unique elements of current Erasmus MC trial with TCR T cells 
1.3.2.1 
MAGE-C2 is a ‘safe’ target antigen 
MAGE-C2 (MC2) is a tumor specific target in melanoma and HNSCC. We have developed T cells 
which are gene-engineered to express MC2-specific TCRs. For treatment with these MC2 TCR T 
cells, risk of severe AEs is considered negligible since we do not use an affinity-enhanced TCR, as the 
TCR is derived from a patient without signs of toxicity. In addition, on- or off-target recognition was not 
observed in our preclinical studies (see below for details). 
 
We have selected the HLA-A2-restricted MC2336-344 epitope ALKVDVEERV as a target for AT for the 
following reasons: 
 
MC2 protein is expressed in 40% of metastatic melanomas and 20% of HNSCCs and its 
expression is associated with poor clinical outcome in these tumor types 
(http://www.cta.lncc.br)[58-60]; 
 
MC2 is not expressed in healthy tissues, with the exception of gonads. Nevertheless, the gonads 
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expression. This is supported by the observation that the presence of high numbers of MC2 T 
cells (in a vaccinated patient) was not associated with AEs [61, 62]; 
 
MC2 protein actively contributes to the development of malignancies by facilitating angiogenesis 
and migration of tumor cells, and suppressing p53-dependent apoptosis and epithelial-to-
mesenchymal transition (EMT) [63]; 
 
MC2 protein is immunogenic and is associated with clinical responses. In at least 2 melanoma 
patients several MAGE-C2 specific T cell clones were identified that showed increased 
frequencies in blood and regressing tumor lesions following vaccination.[61, 64] 
 
MC2 ALK peptide is significantly bound by HLA-A2, a property that may facilitate cross-
presentation and contribute to effective T cell responses [65]; 
 
MC2 ALK peptide is, in contrast to other antigenic peptides, favored by immune proteasomes 
[66]; 
 
MC2 ALK peptide is an immunogenic epitope as demonstrated by its ability to induce a T cell 
response with durable tumor response and without AEs [61, 62]. 
 
In melanoma patients with durable tumor responses following MAGE-A3 vaccination, T cell clones 
targeting MC2 epitopes have been identified. From melanoma patient EB81 with a > 3 yrs complete 
response, amongst others, the T cell clone EB81-CTL16 was isolated, targeting the MC2336-344 epitope 
ALKVDVEERV.[61, 62] CTL EB81-CTL16 demonstrated a 1000-fold increase in numbers in a 
regressing cutaneous metastasis and represented about 10% of T cells present in an invaded lymph 
node. These observations strongly suggested a role of this T cell clone in the observed tumor 
response. In the same patient, other T cell clones, specific for the same and other MC2 epitopes, also 
showed increased frequencies in blood and a regressing metastasis.[61, 62] In another melanoma 
patient (LB2586) with tumor response after MAGE-A vaccination, the most frequent anti-tumor T cell 
clone was again directed against a MC2 epitope.[64] 
 
The MC2 TCRαβ genes derived from EB81-CTL16 (provided by our collaborator: prof. Pierre Coulie, 
Univ Louvain, Brussels, Belgium) were sequence characterized (Vα3.01/Jα35.01/Cα; Vβ28.01/Jβ1-
6.01/Cβ1), codon optimized (GeneArt, Regensburg, Germany) and cloned into the pMP71 vector 
separated by an optimized T2A ribosome skipping sequence. When introduced into primary human T 
cells, the MC2 TCRαβ genes were functionally expressed. Our in vitro studies revealed that the MC2 
TCR16 displays a low-to-intermediate ligand-binding affinity (ED50=0.75 nM [54, 67]) and a 
dependency for the CD8α co-receptor. Furthermore, MC2 TCR16 T cells, when incubated with native 
MC2/A2+ tumor cells, displayed antigen-specific cytolysis and INF production.[54, 67] TCR 
expression and function could be maintained in cells kept in culture for longer than 3 weeks.  
 
Besides efficacy, we have also evaluated the toxicity risks for the MC2 target antigen as well as its 
corresponding TCR16. First, expression of MC2 antigen in healthy human tissues was assessed. 
Quantitative PCR analysis on a cDNA library of 48 healthy human tissues demonstrated absence of 
MC2 mRNA in healthy tissues. In addition, using an MC2-specific antibody, we confirmed the 
presence of MC2 protein in testis and melanoma as well as its absence in multiple healthy tissues, 

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such as brain, heart, intestine, and lung.[54] For TCR16, the recognition motif toward its cognate 
peptide and the cross-reactivity toward non-cognate peptides were evaluated. These analyses 
showed a highly stringent TCR recognition motif towards the cognate antigen (7 of 9 amino acids: 
xLKDVEERx) with cross-reactivity limited to MAGE-C1, i.e. a related CGA without expression in 
healthy non-gonadal tissues. Collectively, these analyses showed absent MC2 expression in somatic 
healthy tissues, pointing towards a beneficial safety profile for MC2 TCR16 (further MC2 TCR).[54] 
 
1.3.2.2 
Therapeutic T cells have a ‘young’ phenotype and warrant improved peripheral 
persistence 
Furthermore, we have defined a T cell activation and processing method that includes T cell activation 
with soluble anti-CD3/CD28 mAbs in the presence of both IL-15 and IL-21 prior to TCR gene transfer 
and which results in enhanced proportions of T cells with a ‘young’ phenotype.[68-70] In more detail, T 
cells generated according to this processing method demonstrated enhanced binding of pMHC [HLA-
A2/MC2ALK],  enhanced frequency of CD27+, CD62L+, CD45RA+ T cells, and enhanced in vitro MC2-
specific T cell function. This T cell processing method has been included in the existing GMP protocol 
[71, 72] to generate MC2 TCR T cells  for clinical application. 
 
1.3.2.3 
Combination treatment with epigenetic drugs 
To enhance the efficacy of MC2 TCR T cells, epigenetic treatment is promising, as epigenetic 
treatment can induce increased de novo and sustained expression of CGAs, such as MAGE-A1, -A3, -
A4, NY-ESO-1 in tumor cell lines ex vivo and in human tumors in vivo.[73, 74] Effective and safe 
clinical regimes with the epigenetic drugs valproic acid (VP) and 5-azacitidine (AZA) have been 
developed for the treatment of patients with advanced solid tumors, acute myeloid leukemia (AML) 
and high risk myelodysplasia syndromes (MDS).[8, 75, 76]  Yet the AZA+VP treatment regimen come 
with AEs in more than 10% of patients, in particular (febrile) neutropenia, leukopenia and 
nausea/vomiting, for which effective anti-emetic prophylaxis can be applied.[8, 76, 77]   In AML 
patients, AZA+VP treatment was able to induce an anti-MAGE T cell response.[75] Furthermore, DNA 
hypo-methylation is a beneficial prognostic marker in patients with stage IIIC melanoma.[78] 
 
We have extended these observations and demonstrated that pre-treatment with AZA+VP significantly 
increased MC2 gene expression in melanoma and HNSCC tumor cell lines and enhanced their ability 
to elicit an IFN response by MC2 TCR T cells. After treatment with AZA+VP, we showed selective 
induction of gene and protein expression of MC2 in tumor cells, but not in normal cells.[79] Literature 
studies show that de-repressed expression in CGAs, including NY-ESO1, MAGE-A1 and MAGE-A3, 
the is related to epigenetic changes, one of them being a shift from the transcription factor CTCF to 
BORIS. Normal cells do not express BORIS and potentially have additional multiple silencing 
mechanisms still intact, making them less sensitive to epigenetic drugs than tumor cells.[80-83] 
 
The proposed doses and schedule of the two epigenetic drugs in this protocol have been assessed 
and applied in several clinical studies [8, 76, 84-86] and consist of subcutaneous administration of 

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AZA at an dose of 75mg/m2/d;q7d and oral administration of VP at a dose of 50 mg/kg/d;q7d. With 
respect to pharmacokinetics, AZA reaches maximal concentration in blood (Cmax) at 30 min after 
subcutaneous administration with an elimination half-life of 41 min and sub-therapeutic levels at 
approximately 3h (= 4-5 x half-life). After oral administration, VP reaches Cmax in blood after 1-6 
hours with an elimination half-life of 10-15 hours and sub-therapeutic levels at approximately 2 days. 
In PBMCs, this combination of AZA and VP has shown to induce global DNA hypomethylation and 
histone acetylation.[8] 
 
1.3.2.4 
No pre-treatment with chemotherapy 
For TIL-based therapy, pre-treatment with non-myeloablative chemotherapy and (high dose) IL-2 after 
T cell infusions were required to achieve efficacy.[28, 33, 87] This strategy has been incorporated in 
most recent trials with gene-engineered T cells, despite the fact that its benefit was formally not 
investigated. Since chemotherapy increased the risk of AEs we consider it worthwhile to revisit the 
need for this pre-treatment, which is supported by two clinical reports. First, Butler and co-workers [88] 
showed that MART1-specific T cells, generated using co-stimulatory artificial antigen presenting cells 
(aAPC), survived for prolonged periods in advanced stage melanoma patients without previous 
chemotherapy or high-dose IL-2 treatment. We argue that a mild myelosuppression/leukopenia 
induced by AZA and VP [8, 77, 89] as well as the generation of young T cells  [68] are expected to 
eliminate the need for prior chemotherapeutic lymphodepletion. 
 
1.3.2.5 
Low-dose Interleukin-2 
IL-2 is a T cell growth factor that supports the proliferation, function and survival of T cells both in vitro 
and in vivo. Initially, the anti-tumor activity of IL-2 was investigated in trials applying high-dose IL-2 
(15-24 x 106 IU/m2/day), delivered systemically, either as a bolus injection or as a 24 hr continuous 
infusion. However, the high-dose systemic IL-2 was associated with severe toxicities.[90-94] 
Subsequent studies showed that treatment with low-dose IL-2 administered subcutaneously (s.c.) was 
well tolerated with only mild toxicity (grade I-II) ([95] and systematic review [96]) and that a low-dose of 
IL-2 was sufficient to achieve immuno-modulatory effects such as pre-activation of T-cells.[97, 98] 
 
In this study we apply a low-dose of 1x106 IU IL-2/m2 daily for a period of 5 days following the MC2 
TCR T cell administration. In our former CAIX CAR T cell trial (P00.0040C) we have applied the same 
IL-2 dose for up to 20 days, without noticeable IL-2 related toxicities ([46]). In addition, similar IL-2 
dose and schedules are presently applied in several  clinical TCR T cell studies (see clinicaltrial.gov: 
NCT03462316 and NCFT03029273 (NY-ESO-1- TCR T cells); NCT02550535 (WT1 TCR T cells) 
NCT03686124 and NCT03247309 (ACTengine T cells). 
 

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1.3.4 
Safety considerations of Erasmus MC AT trial with TCR T cells 
1.3.4.1 
No expected on- and off-target toxicity 
On-target toxicity is not expected as MC2 is not expressed by somatic human cells in healthy 
individuals and cancer patients.[54] MC2 is only expressed by germline cells in the gonads, which are 
immune privileged organs. Treatment with AZA+VP has been demonstrated to increase CGA 
expression in tumors [79], yet no signs have been reported of auto-immunity towards gonads following 
patient treatment with these drugs. Therefore, AZA+VP treatment is highly unlikely to induce gonadal 
recognition by MC2 TCR T cells. In addition, the MC2 TCR was isolated from a melanoma patient who 
showed a complete remission following a MAGE-A1 and MAGE-A3 vaccination regimen, without any 
clinical toxicity. 
 
Also, off-target toxicity is not expected as TCR16, recognizing the ALK epitope in the context of HLA-
A2, has a very restricted recognition motif towards the ALK epitope, and TCR cross-reactivity was only 
found towards MAGE-C1, a related CGA that is not expressed in healthy non-gonadal tissues.[54] In 
AT with TCR T cells directed against the melanocyte differentiation antigen MART-1, melanocyte 
destruction in skin, eye and ear (leading to vitiligo, uveitis and hearing loss) have been reported([50, 
51] NKI: NL.37327.000.11). No such toxicities are expected for TCR16 T cells since MC2 is not 
expressed in normal melanocytes or other cells derived from normal tissues.[54] 
 
1.3.4.2 
No expected toxicity associated with retroviral integrations 
Retroviral transductions may potentially lead to malignant transformation when the therapeutic gene 
integrates in/nearby oncogenes. This genotoxicity was observed in a gene therapy trial of XSCID with 
the intent to gene correct hematopoietic stem cells, where 5 children developed leukemia upon 
retroviral integration in the LMO2 oncogene.[99, 100] However, the risk of transforming events upon 
infusion of TCR T cells is likely to be negligible when compared to the X-SCID trial. To date, dozens of 
clinical AT studies with retro- or lentiviral gene-modified T lymphocytes have been performed with no 
reports regarding toxic viral integrations. The recently reported leukemia in a patient treated with CD19 
CAR T cells appeared to be derived from a transduced leukemic B cell that was present in the 
leukapheresis product at start of treatment but not from treatment with CAR T cells.[101] 
 
1.3.4.3 
No expected toxicity related to activated and expanded T-cells in vivo 
In several studies using TILs or CARs, the AT of T cells has been complicated by the cytokine release 
syndrome (CRS) with high plasma levels of cytokines such as IL-6 and 
IFN-γ. In some patients, CRS can induce high fevers, rigors, hypotension, and heart failure.[39, 42, 
102] In case of severe deterioration of the patient’s condition, escape medication consisting of high 
dose corticosteroids in conjunction with anti-CD52 antibody (alemtuzumab) may be given.[103] Severe 
and potentially life-threatening CRS can effectively be treated with the anti-IL-6 receptor antibody 
tocilizumab.[104, 105] Although we formally cannot exclude the chance that CRS may occur, CRS has 

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mainly been described for CAR-T cells in patients with high tumor load in the blood compartment. 
Therefore, CRS is not or to a lesser extent expected in patients with solid tumors. 
 
1.3.4.4 
No severe toxicity expected for pre-treatment with AZA+VP  
In clinical practice, AZA is administered for the treatment of several hematological malignancies and is 
associated with AEs in more than 10% of patients, in particular (febrile) neutropenia and leukopenia.[8, 
76, 77]. In clinical practice, VP is primarily prescribed for the treatment of epilepsy, bipolar disorder, and 
prevention of migraine headaches. During VP treatment, toxicities are limited, and hematological 
toxicities are rare. 
Hematological toxicity is the most frequently reported severe toxicity for the combination AZA and VP. 
In a phase I study investigating the combination of VP (maximum dose 60 mg/kg/d for 28 days) and 
AZA, the maximum tolerated dose for AZA was 75mg/m2/d for 10 days.[8] At that dose, which was 
expanded, 3 of 16 patients (18%) showed dose-limiting grade 3-4 toxicity (neutropenic fever). Other 
toxicities were grades 1 and 2 somnolence (six patients), tremor (six patients), hypomagnesemia (one 
patient), anemia (two patients), and vomiting (one patient). In the current study, VP will be administered 
at a lower dose (50 mg/kg/d) and a shorter treatment duration (7d), whereas the treatment duration of 
AZA is also shorter (7d). Therefore, the toxicity of the combination of AZA and VPis expected to be 
limited in the current study.  
In the current study, MC2 TCR T cells will be infused 2 days after the last AZA+VP dose. Regarding the 
short half-life of AZA, no direct effect of peripheral AZA can be expected on infused T cells. Of note, 
Cruz and colleagues [73] showed that in vitro treatment of T cells with AZA did neither compromise T 
cell phenotype nor function. The waning blood levels of VP in absence of AZA are also not expected to 
affect the T cells. 
 
1.3.4.5 
No severe toxicity expected for low-dose IL-2 
The applied dose of IL-2 is chosen such that only minor toxicities can be expected. From the 
systematic review [96] is concluded that administration of low-dose rIL-2 can be considered safe for 
clinical and experimental use in humans since an overall low rate of severe adverse events was 
observed, limited to thrombocytopenia. Most frequently local reactions at the injection sites were 
reported and other (grade I-II AE)  reactions included, influenza-like syndrome, headache and 
dyspnea. 
 
 
2. OBJECTIVES 
2.1 
Phase I 
2.1.1 
Primary objectives 
 
to study the safety and feasibility of AT with autologous MC2 TCR T cells, combined with 
epigenetic drug treatment, in patients with advanced melanoma or HNSCC. AEs will be 
documented according to CTCAE version 5.0; 

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 
to determine the recommended phase II dose, based on the MTD of MC2 TCR T cells in the 
combination treatment.  
 
2.1.2  Secondary and exploratory objectives 
 
to study presence and function of MC2 TCR T cells in peripheral blood samples after treatment.  
 
to study systemic release of inflammatory cytokines after administration of autologous MC2 TCR 
T cells; 
 
to document MC2 expression in tumor tissues (when available) prior to and after epigenetic drug 
treatment; 
 
to document the induction of global DNA hypomethylation and histone acetylation in PBMCs by 
the epigenetic drug treatment; 
 
to study immune parameters, in particular T cell parameters, in blood and tumor tissues (when 
available) prior to and during treatment. 
 
2.2 
Phase II 
2.2.1 
Primary objective 
 
to study the efficacy of AT with autologous MC2 TCR T cells at MTD, combined with epigenetic 
drug treatment, in patients with advanced melanoma or HNSCC. 
 
2.2.2 
Secondary objectives 
To determine the following outcomes in patients treated with MAGE-C2 TCR T cells at MTD: 
 
progression free survival; 
 
duration of response; 
 
overall survival. 
 
2.2.3 
Explorative objectives 
 
to study persistence of MC2 TCR T cells, their differentiation state and ability to recognize tumor 
cells at regular intervals (see § 7.3.3) following start of T cell treatment. 
 
to document MC2 expression in tumor tissues (when available) prior to and after epigenetic drug 
treatment.  
 
to document the induction of global DNA hypomethylation and histone acetylation in PBMCs. 
 
to assess presence of immune and T cell parameters in blood, and micro-environment of tumor 
tissues using advanced flow cytometry and in situ stainings. 
 
to assess response rate according to immune related Response Criteria (irRC).[106] 
 
 

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3. STUDY DESIGN 
3.1 
Description of the study design 
In this open label non-randomized single institution phase I/II trial, the safety and efficacy of epigenetic 
drugs followed by transfer of TCR T cells will be evaluated in patients with advanced melanoma or 
HNSCC.   
 
This study consists of two parts: 
 
phase I: dose escalating; 
 
phase II: expansion. 
 
Patients will be screened for HLA-A2 by genotyping and tumors will be screened for MC2 by 
immunohistochemistry. Eligible patients will undergo leukapheresis to retrieve sufficient T cells which 
will be MC2 TCR gene-engineered and expanded in the laboratory as described (see IMPD). Patients 
will be treated with an epigenetic drug regimen consisting of AZA (75 mg/m2/day s.c. x 7 days), and 
VP (50 mg/kg/day p.o. x7 days). Two days following this regimen, one single intravenous adoptive 
transfer of autologous MC2 TCR T cells will be administered,  starting with the first dose level. The 
treatment schedule is depicted in Figure 2. 
 
3.1.1 
Phase 1: dose escalating phase 
Patients will be treated according to an accelerated titration phase I trial design.[107] This design 
incorporates a rapid dose escalation, reduces the number of patients who are undertreated, facilitates 
completion of the phase I trial, and provides significant information on tolerability and the 
establishment of a recommended T-cell number for further testing in the phase II study. 
 
Patients will be treated in subsequent cohorts of 1 patient according to the following dose-escalation 
scheme: 
dose level 1: 
5.0x107 MC2 TCR T cells; 
dose level 2: 
5.0x108 MC2 TCR T cells; 
dose level 3: 
5.0x109 MC2 TCR T cells; 
dose level 4: 
1.0x1010 MC2 TCR T cells; 
dose level 5: 
the total number of cultured cells, being typically 1.0 – 5.0 x1010 MC2 TCR T cells. 
 
After 1 (3) patient(s) has/have been treated in each dose level, but not before 3 weeks after the patient 
has been infused with TCR T cells, the project team (PI and co-investigators) will decide, based the 
observed AEs, whether the trial will be continued at the next dose level or will continue in the current 
dose level (see § 4.4). From the moment onwards that AEs are observed, next dose escalations will 
be  semi-logarithmic.  
DLT is defined as any CTCAE grade 4 and non-manageable grade 3 toxicity within a 21 day DLT 
period.  
 

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Clinical evaluation for toxicity and efficacy will consist of comprehensive clinical follow-up, laboratory 
tests and conventional imaging (e.g. computed tomography and magnetic resonance imaging). At 6 
and 12 weeks and every 3 months thereafter response evaluation will be performed. See § 7.3 for 
scheduled visits and specific procedures. 
 
For translational analyses, peripheral blood samples and tumor biopsies will be collected as well (see 
Figure 3). 
 
3.1.3 
Phase II: expansion phase 
A Simon-2 stage single arm phase II design will be applied.[108] The study will continue the first stage 
at the recommended TCR T cell dose number defined in phase I until a total of 6 patients have been 
enrolled and treated. Clinical responses and safety data of these first 6 patients will be evaluated by 
the DMC. If no clinical responses will be observed, the trial will be discontinued and the conclusion will 
be that in the applied setting the TCR lacks efficiency; otherwise, the trial will continue to its second 
stage.  
 
In the second stage, overall 12 patients will be enrolled (including the first stage): if the total number of 
responses for the two stages combined is less than 3, the trial will be discontinued as soon this is 
evident and the conclusion will be that in the applied setting the TCR lacks efficiency. Clinical 
responses and safety data of the patients will be evaluated by the DMC. 
 
 
4. STUDY POPULATION 
4.1 
Population 
In this trial two groups of patients will be enrolled: 
1) 
patients with inoperable stage IIIc/IV melanoma with progressive disease after standard 
therapy; 
2) 
patients with R/M HNSCC who are ineligible for or unwilling to get platinum-based 
chemotherapy or for whom no standard treatment is available. 
 
4.2 
Inclusion criteria 
Subjects eligible for inclusion in this study have to meet all of the following criteria: 
 
written informed consent must be obtained prior to any screening procedures. 
 
male or female must be ≥ 18 years of age at time of providing informed consent.  
 
one of the following three malignancies:  
1. 
previously treated for unresectable or metastatic cutaneous or mucosal melanoma for whom 
no standard treatment is available (anymore); 
2. 
metastatic uveal melanoma, progressing after standard of care therapy, if available; 
3. 
R/M HNSCC for whom no standard treatment is available anymore; 

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 
patients must be HLA-A2*0201 positive; 
 
primary tumor and/or metastasis (archival or fresh biopsy) is positive for MC2 (> 5% of tumor 
cells) according to immunohistochemistry; 
 
measurable disease according to RECIST v1.1; 
 
at least one lesion, suitable for sequential mandatory tumor biopsies;  
 
ECOG performance status of 0 or 1. Life expectancy ≥ 12 weeks (see Appendix 1);  
 
patients with melanoma must have had objective evidence of disease progression while on or after 
standard systemic therapy. The last dose of prior therapy (e.g. anti-PD-1, chemotherapy) must 
have been received more than 4 weeks prior to the start of study treatment. For melanoma patients 
who are treated with BRAF- and MEK inhibitors, an interval of 2 weeks between discontinuation of 
BRAF- and MEK inhibition and start of study treatment is sufficient; 
 
patients with R/M HNSCC must have had objective evidence of disease progression and are 
ineligible for or unwilling to get platinum-based chemotherapy or for whom no standard treatment 
is available; 
 
patients of both genders must be willing to practice a highly effective method of birth control 
during treatment and for four months after receiving the preparative regimen; 
Patients must meet the following laboratory values at the screening visit in the absence of growth 
factors and/or transfusion support: 
 
hematology: 
o absolute neutrophil count greater than 1.5x109/L; 
o platelet count greater than 75x109/L; 
o hemoglobin greater than 5 mmol/L or 8.0 in g/dl; 
 
chemistry: 
o serum ALAT/ASAT less than 3 times the upper limit of normal (ULN), unless patients have 
liver metastases (< 5 times ULN); 
o serum creatinine < 1.5 ULN; 
o total bilirubin less than or equal to 20 micromol/L, except in patients with Gilbert’s Syndrome 
who must have a total bilirubin less than 50 micromol/L; 
 
serology: 
o seronegative for HIV antibody; 
o seronegative for hepatitis B antigen, and hepatitis C antibody; 
o seronegative for lues. 
 
4.3 
Exclusion criteria 
Subjects who meet any of the following criteria will be excluded from participation in this study: 
 
presence of symptomatic brain metastases. Note: Subjects with symptomatic brain 
lesions who have been definitively treated with stereotactic radiation therapy, surgery or gamma 
knife therapy are eligible; 
 
presence of active brain metastases defined as new or progressive brain metastases at the time 
of study entry. Note: Subjects with treated or stable brain metastases are eligible; 
 
presence of leptomeningeal metastases; 

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 
presence of malignant pleural effusion or ascites; 
 
systemic chronic steroid therapy (˃ 10mg/day prednisone or equivalent) or any other 
immunosuppressive therapy a within 7 days prior to leukapheresis or 72 hours prior to infusion of 
the MC2 TCR T cells. Note: Local steroids such as topical, inhaled, nasal and ophthalmic steroids 
are allowed; 
 
active, known or suspected autoimmune disease or a documented history of autoimmune 
disease. Note: Subjects with vitiligo, controlled type I diabetes mellitus on stable insulin dose, 
residual autoimmune-related hypothyroidism only requiring hormone replacement or psoriasis not 
requiring systemic treatment are permitted; 
 
any active systemic infections, coagulation disorders or other active major medical illnesses, such 
as active autoimmune diseases requiring anti-TNF treatment; 
 
history of myocardial infarction, cardiac angioplasty or stenting, unstable angina, or other clinically 
significant cardiac disease within 6 months of enrollment; 
 
AEs of previous treatment. Toxicities associated with prior systemic and non-systemic treatment 
must have recovered to a grade 1 or less. Patients may have undergone minor surgical 
procedures or palliative radiotherapy (for non-target lesions) within the past 4 weeks, as long as 
all toxicities have recovered to grade 1 or less; 
 
women who are pregnant or breastfeeding. A negative pregnancy test before inclusion in the trial 
is required for all women of child bearing age; 
 
use of any live vaccines against infectious diseases within the last 3 months; 
 
active infection requiring systemic antibiotic therapy at start of study treatment; 
 
prior allogenic bone marrow or solid organ transplant; 
 
history of known hypersensitivity to any of the investigational drugs used in this study; 
 
malignant disease, other than being treated in this study. Exceptions to this exclusion include the 
following: malignancies that were treated curatively and have not recurred within 2 years prior to 
start of study treatment; completely resected basal cell and squamous cell skin cancers and any 
completely resected carcinoma in situ. 
 
4.4 
Sample size calculation 
In phase I, patients will be treated according to an accelerated titration phase-I trial [107] in 
subsequent cohorts of 1 patient. In case a DLT is encountered at a given dose level, the study is 
converted to a conventional 3+3 phase-I trial design. From that moment on, dose escalation will be 
semi-logarithmically to MTD (up-escalation, with DLT in 1 of the 6 patients, and 1 or 2 down-
escalations with DLT in 2 of the 6 patients). 
If in the 3+3 phase I trial design, a DLT is encountered in one of the first 3 patients at a given dose level, 
additional patients to a maximum of 6 are enrolled at that dose level. Escalation to the next higher dose 
level only occurs when the DLT rate is below 33%. The MTD is defined as the highest dose that can be 
safely administered yielding no DLT or a DLT rate below 33%. 
 
 
 

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In phase II, patients will be treated at the MTD or highest T cell dose evaluated according an optimal 
Simon-2 stage approach. Sample size calculation is based on the percentage of patients with an 
objective clinical response according the RECIST v1.1. The following statistical values will be adhered 
to: p0 = 0.05 (probability of spontaneous clinical response); p1 = 0.35 (expected probability of therapy-
induced clinical response); alpha = 0.05 and power 0.8. The p0 value is set at 0.05 since we expect 
that without epigenetic pretreatment no spontaneous clinical responses will be observed. 
 
In case no clinical responses are observed in the first 6 evaluable patients, the present study will be 
closed and the conclusion will be that in the applied setting T cell treatment lacks efficiency; at 1 or 
more clinical responders, patient numbers are extended to 12 (including the first stage). If the total 
number of responses for the two stages combined is less 3, the trial will be stopped as soon as this is 
evident and the conclusion will be that in the applied setting the T cell treatment lacks efficiency; at 3 
or more responses in 12 evaluable patients the treatment is considered to show activity and warrants 
further investigations. Again safety data will be evaluated by the DMC at the end of the trial. 
Patients who cannot be evaluated for response will be replaced by new patients. 
  
 
5. TREATMENT OF SUBJECTS 
5.1 
Investigational product/treatment 
Patients will first undergo leukapheresis for retrieval of T cells. Once transduction with the retroviral 
vector encoding the MC2 TCR has been successfully employed (meeting the in-process quality 
criterion, i.e., ≥10% surface expression of TCR transgenes on T cells), patients will be treated 
according to the protocol. This protocol consists of treatment with the epigenetic drugs AZA and VP to 
upregulate the expression of MC2 in tumor cells and to induce a mild state of lymphopenia, followed 
by infusion of autologous TCR T cells, as shown in Figure 2. 
 
5.2 
Use of co-intervention (if applicable) 
Patients will be treated exactly according to protocol (see § 7). 
 
5.3 
Escape medication (if applicable) 
Nevertheless, In case patients may experience a cytokine release syndrome with severe symptoms in 
the current study, patients will be treated with high dose corticosteroids, the anti-IL6R antibody 
tocilizumab[7], and the anti-CD52 antibody alemtuzumab, depending on the severity of the symptoms. 
 

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6. INVESTIGATIONAL PRODUCT  
Patients will receive autologous MC2 TCR T cells, which will be produced according to the IMPD (see 
IMPD: MC2 TCR transduced T cells, version 2.0). Transduction and expansion of T cells will be 
performed at EMC, again according to IMPD. After the expansion phase, T cells are washed, 
concentrated and released by the QP gene therapy (pharmacist). Within 6 hours, MC2 TCR T cells will 
be intravenously administered to the patient, without further controls and/or processing, which has 
been previously validated (see IMPD). 
 
For this IMP resides under environmental licences IM-MV 19-004 (LG 19-003) – application under 
consideration since February 21, 2019 
 
Name and description of investigational product(s) 
The Investigational product is : MC2 TCR T cells. 
The MC2 TCR T cells are autologous T cells that have been transduced with the pMP71 retroviral 
vector expressing the codon optimized MC2336-344 specific T cell receptor TCR16. The α and β chains 
of the TCR16 are expressed in equimolar amounts from the same vector using the picornavirus 2A 
cleavage element. 
TCR16-positive CD4+ and CD8+ T cells can be visualized in flow cytometry (FCM) analysis, using the 
TCR16Vb-specific mAb (anti-TCRVb3). As TCR16 is CD8-dependent, FCM analysis using the TCR16-
specific pMHC-multimer (HLA-A2/MC2ALK), accurately enumerates TCR16+CD8+ T cells, but not 
TCR16+ CD4+ T cells.[54, 67, 68] The assessment of the T cell dose for patient treatment will be 
based on the FCM analysis of the TCRVb3+ (CD4+ and CD8+) T cells (corrected for the naturally 
occurring number of TCRVb3+ T cells in that particular patient) 
 
Summary of findings from non-clinical studies 
When introduced into primary human T cells, the MC2 TCR16αβ genes were functionally expressed 
on the cell membrane. Our in vitro studies revealed that the MC2 TCR16 displays a low-to-
intermediate ligand-binding affinity (ED50=0.75 nM) and a dependency for the CD8α co-receptor. 
Furthermore, MC2 TCR16 T cells, when incubated with native MC2/A2+ tumor cells, displayed 
antigen-specific cytolysis and INF production. TCR expression and function could be maintained in 
cells kept in culture for longer than 3 weeks.  
Pre-treatment with AZA+VP significantly increased MC2 gene expression in melanoma and HNSCC 
tumor cell lines, but not normal cells, and enhanced their ability to elicit an INF response by MC2 
TCR T cells. 
Evaluation of toxicity risks for the MC2 target antigen as well as its corresponding TCR16 revealed 
that: 
- MC2 is not expressed on/in non-gonadal somatic healthy tissues; 
- TCR16 has a highly stringent TCR recognition motif towards the cognate antigen 

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Collectively, these analyses point towards a beneficial safety profile for MC2 TCR16. 
 
Summary of findings from clinical studies 
This is the first in man application of MC2 TCR T cells, so no clinical data are available. 
In previous clinical trials, TCRs were usually restricted to the HLA-A1 or A2 genotype and directed 
against differentiation antigens (i.e. MART-1, gp100, CEA, and p53), Cancer Germline Antigens (i.e. 
MAGE-A3 and NY-ESO-1), and viral antigens. Collectively, these trials demonstrated responses in 12 
to 80% of patients with metastatic melanoma, colorectal carcinoma, synovial sarcoma, and multiple 
myeloma.[2-4] However, treatment-related AEs became evident from studies with TCRs, in particular 
for TCRs with high-affinity, directed against antigens with over-expression in both tumors and healthy 
tissue.[5, 50-52]. Two previous clinical studies applied TCRs directed against MAGE-A3 (2 different 
epitopes) and reported fatal neurological and cardiovascular AEs [5, 52]. These AEs were most likely 
caused by the use of affinity-enhanced TCRs that recognize a shared or highly similar epitope 
compared to the cognate epitope.  
Summary of known and potential risks and benefits 
For patients with unresectable stage IIIc or stage IV melanoma and patients with unresectable stage III 
Head and Neck Squamous Cell Cancer (HNSCC), who have failed standard therapies (including 
immune check point blockade), prognosis is generally poor. TCR gene therapy could provide an 
attractive alternative since it is a strategy that enables the fast and efficient generation of large numbers 
of tumor reactive T cells. In patients with melanoma, AT using autologous TCR T cells with affinity 
enhanced TCRs for MAGE-A3 has shown clinical responses, but also fatal on- and off target toxicities 
as a result of either targeting a common epitope of the MAGE-A antigen superfamily or a TCR with a 
non-restricted binding motif. In the current study, these (S)AEs are not expected as the MC2-specific 
TCR has a restricted binding motif without enhanced affinity. In this patient population, the potential 
burden and AEs are justified by the substantial chance of (durable) tumor responses as a result of the 
proposed treatment. See also §1.3.4. 
 
Description and justification of route of administration and dosage 
The in vitro generated and expanded MC2 TCR cell product is resuspended in a volume of 200 ml 
Ringers lactate w/ 1% HSA for direct intravenous administration. Administration of blood products, 
including (gene modified) T cell products, is by default intravenously. Administration of the fresh 
product optimally preserves (gene modified) T cell properties. The staring dose of in the phase I part is 
5x107 MC2 TCR T cells. This low dose is chosen as this is the first in man application of MC2 TCR T 
cells and SAEs have been reported at 100-fold higher doses of TCR T cells, albeit with other 
specificity.  As MC2 has been selected after careful evaluation of safety aspects of the receptor 
TCR16, no AEs are expected at this dose.  
 
Dosages, dosage modifications and method of administration 
The phase I starts at an MC2 TCR T cell dose of  5x107 and will be further escalated, based on 
toxicities and may be escalated up to 5x1010 MC2 TCR T cells. The MC2 TCR Tcells will be 

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administered in a single intravenous infusion at treatment day 0, with support of IL-2 administrations 
(s.c. 5x105 IU/m2 2qd) at treatment days 0 to 4. 
  
Preparation and labelling of Investigational Medicinal Product 
The MC2 TCR T cell product is produced at the Erasmus MC Facility for Cell- and Gene-Therapy. At 
the end of production the MC2 TCR T cells are harvest from culture and resuspended in 200 ml of 
infusion medium (Ringer Lactate containing 1% human serum albumin) in an infusion bag, for direct 
administration. Patient product specific labels will be adhered to the infusion bag and the ‘outer bag’, 
in which the infusion bag is transported (for product label, see appendix 2; for QP release form, see 
IPMD Appendix 6). 
 
 
Drug accountability 
The production of the  MC2 TCR T-cell product is performed by qualified ATMP production personnel.  
At the end of production, the head of production will summarize the results of the production and QC 
tests on the release form. The head of QA/QC will verify whether the analyses have been performed 
correctly. The release of the MC2 TCR T cell product is performed by the QP for cell and gene therapy 
products. The release of the MC2 TCR T cell end-product is based on results of control tests from both 
intermediate cell products and the end-product. The MC2 TCR T cell end-product will only be released 
when pre-set release criteria are met (see IMPD Table 11). 
 
Out of Specification (OOS) 
In case the Investigational product (autologous MC2 TCR T cell) characteristics are out of specification 
(regarding cell number, viability or proportion MC2 TCR+ T cells), yet with a positive benefit/risk-
analysis, the QP and treating physician may still decide to release the product for patient 
administration. Patients receiving an OOS investigational product will be included in the safety 
analysis, but excluded from efficacy analysis. An OOS investigational product administration will be 
notified to the competent authorities (see also SOP CK07.1150). 
 
 
7. METHODS  
7.1 
Study endpoints 
7.1.1 
Phase I - primary study endpoints 
 
AEs according to CTCAE v5.0; 
 
recommended phase II dose; 
 
feasibility to deliver this sequence of treatment. 
 
7.1.2 
Phase II - primary study endpoint 
 
objective response rate according to RECIST v1.1 Response is defined as complete remission 
(CR) and partial response (PR) according to RECIST v1.1. 
 

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7.1.3 
Phase II - secondary endpoints 
 
PFS, defined from the date of administration of TCR T cells to the date of first documented 
progressive disease according to RECIST v1.1 or death. Patients who are alive and progression-
free at the time of the analysis will be censored at their last available assessment date; 
 
OS, defined from the date of administration of TCR T cells to the date of death. Patients who are 
alive at the time of the analysis will be censored at the date they were last known to be alive. 
 
7.1.4 
Phase I and phase II - Secondary and exploratory endpoints 
 
persistence of MC2 TCR T cells, their differentiation state and ability to recognize tumor cells at 
regular intervals (see § 7.3.3) following start of T cell treatment; 
 
presence of immune and T cell parameters in blood, and micro-environment of tumor tissues 
using advanced flow cytometry and in situ stainings; 
 
MC2 expression in tumor tissues (when available) prior to and after epigenetic drug treatment; 
 
global DNA hypomethylation and histone acetylation in PBMC after epigenetic drug treatment; 
 
response rate according to immune related Response Criteria (irRC).[106] 
7.2 
Randomisation, blinding and treatment allocation 
Patients fulfilling eligibility criteria will be enrolled in the study. Patients have to be registered before 
leukapheresis is performed. 
 
7.3 
Study procedures 
A brief overview of the study procedures is presented in Figures 2 and 3.  
 
Figure 3. Treatment and monitoring scheme, for more detail see Tables 1a/b 
 
 
7.3.1 
Pre-study screening 
Screening will comprise two steps, first subjects will be screened for HLA-A2 status; and if positive, 
subsequently for MC2 expression in the tumor. The screening period begins once written informed 
consent is provided. An attempt will be made to complete all procedures within 30 days of enrollment.  
However, given the multiple screening tests and the pre-study procedures to assess patient eligibility, 
tests older than 30 days may be acceptable to limit unnecessary test repetition. If older than 30 days, 

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at least laboratory tests and CT scans need to be repeated. Other key tests can be repeated at the 
discretion of the treating physicians. 
 
The following screening tests will be performed. 
Step 1: 
 
HLA typing; 
 
in case no archival tumor material is available a fresh biopsy of a melanoma or HNSCC lesion 
accessible to sampling will be collected; 
 
tumor evaluation by immunohistochemistry for MC2 protein expression. 
 
Step 2: in case patient is HLA-A2 positive and has >1% MC2 expression in tumor tissue, the 
remaining baseline parameters as defined in § 4.2 and 4.3 are evaluated and include:  
 
complete physical examination including height, weight, performance status, and vital signs; 
 
laboratory evaluations: 
o 
hematology: hemoglobin, Ht, erythrocytes, reticulocytes, platelets, WBC and differential; 
o 
blood chemistry: Na, K, creatinine, Ca, P, Mg, bilirubin, AF, GT, ASAT, ALAT, LDH, glucose, 
APTT, INR, fibrinogen; 
o 
urine analysis (pH, protein, erythrocytes, leucocytes, glucose, ketones, nitrites) 
o 
TSH and FT4; 
o 
b-HCG pregnancy test for all women of child-bearing age; 
 
baseline CT of the chest, abdomen pelvis and brain MRI. Additional imaging may be performed if 
clinically indicated based on patient’s signs and symptoms; 
 
ECG; 
 
HIV, HCV, CMV, HAS antibody titers, HbsAG, EBV panel and lues panel; 
 
in case no fresh biopsy has been collected for screening, a baseline excision or biopsy of a 
melanoma or HNSCC lesion accessible to sampling will be collected. 
 
7.3.2 
Procedures of TCR T cell treatment 
Pre-treatment phase - leukapheresis and T cell transduction 
Prior to study treatment, at day -13, patients will undergo a 10 liter leukapheresis to collect a minimum 
of 1x109 mononuclear cells (MNCs) and 300 ml plasma. In addition, 70 ml blood in serum tubes will be 
collected. MNCs are isolated from the leukapheresis product by Ficoll gradient separation; at least 
6x108 of MNCs are freshly processed to prepare the TCR T cells, the remainder is cryopreserved and 
serves as back-up. The plasma and the serum are both used as autologous cell culture medium 
additive during the preparation of the TCR T cells. Patient MNCs will be transduced and processed in 
accordance with the cell production facility’s SOPs as described in the IMPD (days -14 to day 0), and 
have to be released for clinical application by a Qualified Person gene therapy. In process and final 
product samples are cryopreserved for retrospective testing and archive. 
 
Treatment schedule 

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Treatment schedule will be according the following scheme, which will comprise a course of therapy 
from day -13 through day 0 (also see Tables 1a/b). 
Day -9 to day -3 
5-azacitidin (AZA): 75 mg/m2/day subcutaneously for 7 days. 
Valproic acid (VP): 50mg/kg/day per os for 7 days. 
Granisetron: 1 mg/day per os (supportive care medication during AZA and VP treatment). 
Note: patients with R/M HNSCC that are unable to swallow, will receive Valproic acid in feeding by 
stomach tube and granisteron 10-40 μg/kg. i.v. 
Day 0 
TCR T cells: depending on dose level (phase I) and MTD (phase II), a single dose of between 5x107 
and 5x1010 T cells will be administered intravenously over 20 to 30 minutes. Cell preparation is 
detailed in the IMPD. 
Day 0 to day 4 
Interleuki-2 (IL-2): 5x105 IU/m2/evry 12 hours subcutaneously for 5 days. 
7.3.2 
On study evaluation 
During the preparative epigenetic regimen: every 2 days 
 
physical examination, laboratory evaluations (Hematology and Chemistry) and assessment of 
AEs, see § 7.3.1. 
 
Prior to T cell infusion: day 0 
 
physical examination, laboratory evaluations (Hematology and Chemistry) and assessment of 
AEs, see § 7.3.1. 
 
After T cell Infusion 
 
vital signs will be monitored hourly until stable and then routinely every 4 hours up to 24 hours. 
Thereafter, every day until hospital discharge, unless otherwise clinically indicated; 
 
laboratory evaluations (Hematology and Chemistry), see § 7.3.1; daily until hospital discharge; 
 
blood samples (4ml) are obtained to study systemic release of inflammatory cytokines and 
presence of MC2 TCR T cells, i.e., daily until hospital discharge. Thereafter, these assays will be 
performed on blood samples obtained as described in § 7.3.3. 
 
7.3.3 
Additional immune-related research evaluations 
At regular intervals prior to and after T cell infusion 40ml blood samples will be obtained for immune-
related exploratory testing and archiving, i.e., at day -13, day 0 (prior to infusion of TCR T cells), and 
post T cell infusion at days  7, 14, 21, 28, 60 and 90 subsequently every 3 months until 1 year, and 
thereafter yearly until 5 year after T cell infusion. From blood PBMC and plasma are isolated and 
cryopreserved. Blood samples will be analyzed as follows:  
 
Blood and PBMC 

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 
freshly collected blood samples will be subjected to multiplex flowcytometry to assess: 
o  numbers of 18 leukocyte subsets, and 
o Presence of MC2 TCR T cells by pMHCHLA-A2/ALK  binding and qPCR; 
 
cryopreserved PBMC will be subjected to multiplex flowcytometry for in-depth T cell immune 
profiling, including, amongst others, assessment of differentiation, expression of inhibitory as well 
as stimulatory co-signaling molecules; 
 
immune profile, as above, of MC2 TCR T cells; 
 
cryopreserved PBMC will be tested for MC2 TCR mediated immune-functions, e.g., interferon- 
production upon encounter of MC2-expressing tumor cells, and  
 
TCR repertoire (by RNAseq). 
 Plasma 
 
plasma samples will be tested with ELISA or multiplex assays for cytokines (such as IFNγ, IL-2, 
TNFα, IL-6, IL-10, CXCL10 or CXCL9). IL-6 will be measured every day during hospitalization 
and will continue up to 1 month after T cell infusion. At that time, the IL-6 levels are normalized 
again in all previous patients. 
Tumor biopsy 
An 18G needle biopsy or a thru-cut biopsy will be taken from a metastasis before and after treatment 
(at 3 weeks after T cell infusion and at progression; optional following epigenetic treatment), when 
feasible from the same lesion. Tissue will be processed for future NGS analysis using in silico tools to 
assess mutational burden, TCR repertoire and expression of immune-related genes. In addition, tissue 
will be processed for multiplex in situ stainings using the Vectra technology to assess the immune 
micro-environment of tumor tissue (such as presence of immune effector cells, among which TCR T 
cells; immune suppressor cells; immune checkpoints; and MC2 antigen). 
Tumor biopsies are per protocol, but may be derogated from in consultation with the PI. 
 
7.3.4 
Post-treatment and off-study evaluation 
After re-infusion of T cells, response evaluation will be performed at 6 weeks (± 1 week), 12 weeks (± 
1 week), and then every 3 months (± 1 weeks). Patients with progressive disease will be monitored at 
the same time points until death or next systemic treatment. 
 
At each follow-up moment, the following evaluations will be performed: 
 
physical examination including vital signs and ECOG status; 
 
toxicity assessment; 
 
CT of the chest, abdomen and pelvis; this evaluation will be used to determine tumor response. If 
clinically indicated, other scans may be performed, e.g. MRI of the head and neck region and/or 
brain; 
 
laboratory evaluations: hematology and chemistry, see § 7.3.1; 
 
other tests will be performed as clinically indicated. 
 

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7.3.5 
Treatment of treatment-related toxicity 
7.3.5.1 
Treatment of TCR T cell-related toxicity 
Although this study is designed to limit chances of on- or off-target toxicities, we cannot formally 
exclude the development  of T cell-related AEs, which usually resemble autoimmune disorders.   
In addition, and again against expectations, patients may develop signs of CRS with high fevers, 
chills/rigors and hypotension. In case these patients are not responding to supportive measures, 
patients will be treated with high doses of corticosteroids (prednisone 1-2 mg/kg). In case of high 
serum levels of IL-6 (> 200 pg/ml), patients will be treated with tocilizumab (8 mg/kg with maximum of 
800 mg). In case of an acute deterioration of the patient’s condition, not due to septic shock syndrome, 
patients will additionally be treated with alemtuzumab (3 mg day 1, 10 mg day 2, 30 mg day 3 and 
later), until T cell count drops to <50 CD3 cells/microliter) and the patient’s situation is stabilized. 
 
7.3.5.2 
Treatment of epigenetic durg-related toxicity 
Patients will receive anti-emetic medication. Depending on the nature of the observed toxicity 
appropriate additional supportive measures will be taken. 
7.3.5.2 
Treatment of IL-2-related toxicity 
Depending on the nature of the observed toxicity appropriate additional supportive measures will be 
taken. 
 
7.3.6 
Re-treatment 
A single re-treatment (including pretreatment with epigenetic drugs) is permitted for patients with 
relapse after an initial complete or partial response after treatment with TCR T cells . Period between 
first treatment cycle and re-treatment should be at least 6 months. Patients must meet all original 
eligibility criteria (including hematological recovery) and have additional T cells available. The eligibility 
criteria can be found in § 4.2 and 4.3. 
 
7.3.7  Off-study criteria 
Patients will be taken off-study when one of the following criteria is met: 
 
patient voluntarily withdraws; 
 
deterioration of performance status not being able to undergo (pre-)treatment; 
 
the final product of TCR T cells does not meet all release criteria; 
 
significant patient non-compliance; 
 
progressive disease without eligibility for re-treatment; 
 
if the treating physician is convinced that it is in the best interest of the patient to discontinue 
study participation.   
 
up2 

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7.4 
Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The investigator or treating physician can decide to withdraw a subject from the study 
for urgent medical reasons. 
 
7.4.2 
Specific criteria for withdrawal 
If, after having provided informed consent but before initiation of the treatment, patients do not fulfil the 
eligibility criteria anymore, patients will be withdrawn from the study. 
 
7.4.3 
Replacement of individual subjects after withdrawal 
Patients who are withdrawn prior to initiation of the treatment may be replaced. 
 
7.4.3 
Follow-up of subjects withdrawn from treatment 
Follow-up of patients withdrawn from the study will continue at least until 6 weeks after completion of 
treatment or until treatment-related toxicity has resolved to grade 2 or less. Thereafter, patients will 
receive standard follow-up. 
 
7.5 
Premature termination of the study 
 
If 2 patients develop a non-manageable grade 4 toxicity due to the treatment, which does not 
show improvement within 48 hours with supportive measures, or are clearly not known as side 
effects of the preparative regimen or not related to events external to participation in this study, 
accrual to the study will be halted pending discussions with the DMC. 
 
In case of a grade 5 toxicity not attributable to disease progression or more than one 
prolonged grade 4 major organ toxicity (lasting more than a month after treatment) prior to 
completion of the study will halt patient treatments pending a review of those toxicities by CCMO 
and DMC. 
 
7.6 
Long-term follow-up 
Per protocol follow-up is 5 years after T cell infusions.  

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Table 1a. Schedule of assessments 
 
Parameters 
Day 
Baseline 
Screening 
-28 
Leuka-
feresis 
-13 
Epigenetic  
therapy 
-9 to -2 
T cell 
infusion 
0 
Day 
  
1 to 71 
Follow-
up2 
Informed consent 
X 
 
 
 
 
 
HLA typing 
X 
 
 
 
 
 
Pathological confirmation of melanoma or HNSCC and 
MAGE-C2 expression in primary tumor and/or metastasis 
(archival or fresh biopsy) 
X 
 
 
 
 
 
Physical examination3 
X 
X4 
X5 
X6 
X6 
X 
Hematology: ANC, Hb, platelet count 
X 
X 
X 
X 
X 
X 
Chemistry7 
X 
X 
X 
X 
X 
X 
Thyroid: TSH and FT4 
 
X 
 
 
 
 
Coagulation: PT/INR and aPTT 
 
X 
 
 
 
 
b-HCG pregnancy test, if applicable8 
X 
X 
 
 
 
 
Urinalysis and culture9 
 
X 
 
 
 
 
Serology10 
X 
 
 
 
 
 
CT chest, abdomen, pelvis and evaluation of skin lesions 
X 
 
 
 
 
X  
Brain MRI11 
X 
 
 
 
 
X 
ECG  
X 
 
 
 
 
 
Adverse events 
 
 
X 
X 
X 
X 
Biopsy of metastasis12 
 
X 
 
(X) 
 
X 
Blood for immunological testing13 
 
X 
 
X 
X 
X 
 
1  Every day until release from hospital, expectedly up to day 7 
2  At days 7, 14, 21, 28, 60 and 90 subsequently every 3 months until 1 year, and thereafter yearly until 5 year after T cell infusion. 
3  WHO, PS, weight, temperature, pulse, blood pressure 
4  May be repeated at the discretion of investigator if screening exams are older than 30 days 
5  At time of epigenetic treatment: ECOG should be 0 to 1 
6  After cell infusion: vital signs to be monitored hourly until stable, then routinely (every 4 hours) unless otherwise clinically indicated 
7  Na, K, Cl, Ca, Mg, HCO3-, Phosphate, Alkaline Phosphatase, Direct bilirubin, LD, Total Protein, CK, Uric acid. At screening only ALAT, ASAT, serum creatinine, total bilirubin, CRP. 
8  Within 1 week of epigenetic treatment 
9 If clinically indicated 
10 HIV, HbsAG, HCV, CMV, HSV, EBV and lues   
11 Needs to be repeated for melanoma patients if older than 30 days at start of study treatment. In that case, CT is allowed instead of MRI. Of note: at other time points, CT is not sufficient for
 
brain imaging and MRI brain is required. 
12 Biopsy of accessible tumor site (18G or thru-cut) to be taken for translational research before and after treatment (at 3 weeks after AT and at progression), when possible of same site; 
 
Optional: an additional biopsy may be taken at day 0, i.e, after epigenetic treatment and before MC2TCR T cell infusion. Tumor biopsies are per protocol, but may be derogated from in 
 
consultation with the PI.  
13 Samples with a maximum of 40ml EDTA blood 
 

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Table 1b Treatment phase schedule and event monitoring calendar Days -14 to +9 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood collections: 
13 Samples with a maximum of 40ml EDTA blood 
14 PBMC harvest including 300 ml plasma from 10 liter leukapheresis 
15 70ml blood in serum tubes, for MC2 TCR T cell generation 
16 Samples with a maximum of 3 ml EDTA blood 
 
 
 
Parameters 
                           Day 
Day of Treatment 
-14 
-9 
-8 
-7 
-6 
-5 
-4 
-3 
-1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Inpatient care 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
According to clinical need 
Outpatient care  
X 
X 
X 
X 
X 
X 
X 
X 
 
Epigenetic drugs 
A/V 
A/V 
A/V 
A/V 
A/V 
A/V 
A/V 
MC2 TCR T cells 
X 
Interleukin-2 
 
 
 
 
 
X 
X 
X 
X 
X 
 
Physical examination 
Adverse events 
X 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
According to clinical need 
Hematology 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
According to clinical need 
Chemistry 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
According to clinical need 
Coagulation screen 
 
X 
 
X 
  According to clinical need 
Leukapheresis14 
X 
 
 
 
 
 
 
 
 
According to clinical need 
Blood (serum)15 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for cytokine assays 
T cell persistence16 
 
X 
X 
X 
X 
X 
Untill discharge 
Blood for immunological 
testing and archive13 
X 
X 
X 
 
 
 
 
 
 
X 
 
 
 

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8. SAFETY REPORTING 
8.1 
Temporary halt for reasons of subject safety 
In accordance to § 10, subsection 4, of the Medical Research Involving Human Subjects Act (WMO), 
the sponsor will suspend the study if there is sufficient ground that continuation of the study will 
jeopardise subject’s health or safety. The PI will notify the CCMO without undue delay of a temporary 
halt including the reason for such an action. The study will be suspended pending a further positive 
decision by the CCMO. The investigator will take care that all subjects are kept informed.  
 
8.2 
Adverse and serious adverse events 
An investigator who is a qualified physician will evaluate all AEs according to the NCI Common 
Terminology for Adverse Events (CTCAE), version 5.0. The CTCAE grade at onset and the maximum 
CTCAE grade of any AE will be recorded on AE case report forms/worksheets. 
 
8.2.1 
Adverse events (AEs) 
AEs are defined as any undesirable experience occurring to a subject during the study, whether or not 
considered related to the investigational product/trial procedure/the experimental intervention. All AEs 
reported spontaneously by the subject or observed by the investigator or his staff will be recorded. 
 
8.2.2 
Serious adverse events (SAEs) 
A SAE is any untoward medical occurrence or effect that: 
 
results in death; 
 
is life threatening (at the time of the event); 
 
requires hospitalization or prolongation of existing hospitalization; 
 
results in persistent or significant disability or incapacity; 
 
is a congenital anomaly or birth defect; or 
 
any other important medical event that did not result in any of the outcomes listed above due to 
medical or surgical intervention but could have been based upon appropriate judgement by the 
investigator. 
 
Hospitalization is part of the treatment procedure, starting with the administration of the T cell product, 
and will therefore not be reported as SAE. If the patient experiences an event that falls under one of 
the above-mentioned criteria and/or the event requires IV or other treatment that cannot be given 
outside the hospital and/or the patient is admitted to an Intensive Care Unit, the event will be reported 
as an SAE. An elective hospital admission will not be considered as SAE. 
 
Note: Suspected unexpected serious adverse reactions (SUSARs) and SAEs are not only those linked 
with the IMP itself (MC2 TCR T cells), but also those in relation to the following aspects: quality 
defects of the IMP leading or potentially leading to serious events, serious events occurring during the 

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donation (leukapheresis), treatments carried out in view of the treatment with the IMP (epigenetic drug 
treatment), and any other procedures which do not take place outside of the clinical trial. 
 
8.2.3 
Reporting of SAEs 
The investigator will report the SAEs through the web portal ToetsingOnline to the CCMO that 
approved the protocol (CCMO) within 7 days of first knowledge of the SAEs that result in death or are 
life threatening, which is followed by a period of maximally 8 days to complete the initial preliminary 
report. All other SAEs will be reported within a period of maximum 15 days after the sponsor has first 
knowledge of the SAE. 
 
8.3 
Suspected unexpected serious adverse reactions (SUSARs) 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. the event must be serious (see chapter 8.2.2); 
2. there must be a certain degree of probability that the event is a harmful and an undesirable 
reaction to the IMP, regardless of the administered dose; 
- 
the adverse reaction must be unexpected, that is to say the nature and severity of the adverse 
reaction are not in agreement with the product information (see IMPD). 
 
8.3.1 
Reporting of SUSARs 
The sponsor will provide expedited reporting of SUSARs and any follow-up information electronically 
through the above-mentioned web portal ToetsingOnline.  
SUSARs that are fatal or life-threatening will be reported to the authorities within 7 days for a 
preliminary report, with another 8 days for completion of the report. All other SUSARs will be reported 
not later than 15 days after the investigator has first knowledge of the SUSAR. 
Additional information can be sent as soon as possible. 
 
8.4 
Annual safety report 
The PI will submit, once a year throughout the clinical trial, a safety report to the CCMO and 
competent authority. 
This safety report consists of: 
 
a list of all suspected (unexpected or expected) serious adverse reactions, along with an 
aggregated summary table of all reported serious adverse reactions, ordered by organ system; 
 
a report concerning the safety of the subjects, consisting of a complete safety analysis and an 
evaluation of the balance between the efficacy and the harmfulness of the IMP under 
investigation. 
 

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8.5 
Follow-up of AEs 
All AEs will be followed until they have abated, or until a stable situation has been reached. Depending 
on the event, follow-up may require additional tests or medical procedures as indicated, and/or referral 
to the general physician or a medical specialist. 
SAEs need to be reported till end of study. 
 
8.6 
Data Monitoring Committee (DMC) 
A DMC will be installed and will be comprised of at least 3 (inter)national experts, including at least 2 
medical oncologists, in the field of adoptive T cell therapy, and will be installed once the protocol has 
been approved by CCMO. Members of the DMC are Prof. Carl June, Parker Institute for Cancer 
Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA; Fiona Thistlethwaite, The Christie 
NHS Foundation Trust, Manchester, UK and Prof John Haanen, NKI, Amsterdam. 
 
The DMC will evaluate the safety data after completion of phase I part, after the first stage in the 
phase II part (6 patients) and at the end of the second stage of the phase II part (12 patients) 
according § 9.1 and DMC charter version 1.0 dated 3 june 2019. DMC may also be consulted at the 
discretion of the treating physicians.  
 
The DMC will meet in TCs and will decide on the recommended dose for the phase II part and whether 
further patient inclusion for phase II, second stage is warranted. Rules for stage evaluation and pre-
termination of the study due to AEs can be found in § 8.3 of the protocol.  
 
The advice(s) of the DMC will only be sent to study project team. If the project team decides not to 
fully implement the advice of the DMC, the project team will send the advice to the CCMO, including a 
note to substantiate why (part of) the advice of the DMC will not be followed. 
 
9. 
STATISTICAL ANALYSIS 
All analyses will be done in accordance with the per protocol principle, restricted to eligible and 
evaluable patients. 
 
9.1 
Primary study parameter(s) 
With respect to the main endpoint: A binomial probability test will be used to evaluate 
whether this regimen is worth further study. A 90% confidence interval for the percentage of response 
will be presented (α = 0.10). 
 
In phase I, patients will be treated according to an accelerated titration phase-I trial [107] in 
subsequent cohorts of 1 patient. In case a DLT is encountered at a given dose level, the study is 
converted to a conventional 3+3 phase-I trial design. If in the 3+3 phase I trial design, a DLT is 

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encountered in one of the first 3 patients at a given dose level, additional patients to a maximum of 6 are 
enrolled at that dose level. Escalation to the next higher dose level only occurs when the DLT rate is 
below 33%. The MTD is defined as the highest dose that can be safely administered yielding no DLT or a 
DLT rate below 33%. 
 
In phase II, patients will be treated at the MTD or highest T cell dose and evaluated according an 
optimal Simon-2 stage approach. Sample size calculation is based on the percentage of patients with 
an objective clinical response according to RECIST v1.1. The following statistical values will be 
adhered to: p0=0.05 (probability of spontaneous clinical response); p1=0.35 (expected probability of 
therapy-induced clinical response); alpha =0.05 and power 0.8. The p0 value is set at 0.05 since we 
expect that without epigenetic pretreatment no spontaneous non-T cell related clinical responses will 
be observed. 
 
In case no clinical responses are observed in the first 6 evaluable patients, the present study will be 
closed and the conclusion will be that in the applied setting T cell treatment lacks efficiency; at 1 or 
more clinical responders, patient numbers are extended to 12 (including the first stage). If the total 
number of responses for the two stages combined is less than 3, the trial will be stopped as soon as 
this is evident and the conclusion will be that in the applied setting T cell treatment lacks efficiency; at 
3 or more responses in 12 evaluable patients the treatment is considered to show activity and 
warrants further investigations. 
 
9.2 
Secondary study parameter(s)  
PFS, OS and duration of response will be explored by means of the Kaplan-Meier method. For the 
exploratory study parameters, descriptive statistics will be applied. 
 
9.3 
Toxicity analysis 
The analysis of treatment toxicity will be done primarily by tabulation of the incidence of 
AEs, see § 7.5. 
 
9.4 
Interim analysis (if applicable) 
Not applicable. 
 
10. 
ETHICAL CONSIDERATIONS 
10.1 Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki, (Declaration of 
Helsinki, 59th WMA General Assembly, Seoul, October 2008) and in accordance with the Medical 
Research Involving Human Subjects Act (WMO).The protocol has been written, and the study will be 
conducted according to the ICH Harmonized Tripartite Guideline for Good Clinical Practice 

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(ref:http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6_R1/ 
Step4/E6_R1__Guideline.pdf). 
The protocol must be approved by the CCMO and competent authority (CA) prior to starting this study. 
 
10.2 Recruitment and consent 
Recruitment 
It is the responsibility of the investigator to give each patient, prior to entering the trial, full and 
adequate verbal and written information (see below). Confidentiality-related information will be 
provided. The written information must be given to each patient. Patients will be given sufficient time to 
consider the provided text. An independent physician will be available for consultation. It is the 
responsibility of the investigator to obtain signed informed consent from every patient prior to the start 
of any study-related procedure(s). 
 
Informed consent (IC) 
All patients will be informed about the aims of the study, the possible adverse events, the procedures 
and possible hazards to which he/she will be exposed, and the mechanism of treatment allocation 
(patient information letter). They will be informed as to the strict confidentiality of their patient data, but 
that their medical records may be reviewed for trial purposes by authorized individuals other than their 
treating physician. It will be emphasized that the participation is voluntary and that the patient is 
allowed to refuse further participation in the protocol whenever he/she decides to do so. This will not 
prejudice the patient’s subsequent care. Documented informed consent will be obtained for all patients 
included in the study before they are registered as having entered the study.  
The IC procedure will conform to the ICH guidelines on Good Clinical Practice. This implies that “the 
written IC form will be signed and personally dated by the patient or by the patient’s legally acceptable 
representative”. 
 
The written patient information is part of the documentation reviewed by the CCMO. The patient 
information letter and informed consent form are attached as a separate document  
 
10.3 Objection by minors or incapacitated subjects 
Not Applicable. 
 
10.4 Benefits and risks assessment, group relatedness 
The prognosis of patients with unresectable stage IIIc or metastatic stage IV cutaneous and mucosal 
melanoma has improved in the past 5 years as result of of targeted therapy and immune checkpoint 
inhibitors. With these developments the poor prognosis has improved considerably. Still part of the 
patients, do not benefit from these novel therapies. In addition, treatment options for patients with 
metastatic uveal melanoma are still very limited.  
 

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Although treatment options for patients with R/M HNSCC have increased,  the median overall survival 
still is less than 1 year. Besides, treatment of R/M HNSCC often is accompanied by considerable 
toxicity. 
 
In patients with metastatic melanoma, synovial carcinoma and multiple myeloma, adoptive transfer of 
TCR T cells has shown limited AEs and significant objective clinical responses of 55, 61 and 80%, 
respectively[1-3].  In the present protocol, we target MC2 using TCR16 T cells in patients with 
advanced melanoma or HNSCC that express MC2. To sensitize tumor cells to T cell treatment, 
patients will be pretreated with the epigenetic drugs AZA and VP. T cell treatment will be supported 
with low-dose IL-2 administrations. 
 
Epigenetic treatment-related toxicity has been demonstrated to be well manageable. In fact, the doses 
of the administered epigenetic drugs are precedented and well tolerated and have been reported to 
induce only a mild transient leucopenia and neutropenic fever.[8] 
In case vigorous in vivo activation of infused T cells occurs, patients may (although given the design of 
the study not expected) experience some auto-immunity and/or CRS. Auto-immune reactions are 
treated by standard of care. Depending on the severity of CRS (fever, chills, hypotension) patients 
may be treated with high dose corticosteroids whether or not in conjunction with anti-CD52 antibody 
alemtuzumab. In case of severe deterioration of the patient’s condition,escape medication consisting 
of anti-IL6R antibody tocilizumab.[7] may be given. 
Low-dose IL-2 –related toxicities are limited and well managible. [96] In fact, the doses of the 
administered IL-2 are precedented and well tolerated and have been reported to induce only a 
transient thrombocytopenia and mild malaise (flu-like symptoms). 
The fact that these patients may have a substantial chance of durable objective responses, which 
otherwise would not occur, justifies for the burden and possible toxicities. 
10.5 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of the WMO. 
This insurance provides cover for damage to research subjects through injury or death caused by the 
study. The insurance applies to the damage that becomes apparent during the study or within 4 years 
after the end of the study. 
1. 
€ 450.000,-- (i.e. four hundred and fifty thousand Euro) for death or injury for each subject who 
participates in the Research; 
2. 
€ 3.500.000,-- (i.e. three million five hundred thousand Euro) for death or injury for all subjects 
who participate in the Research; 
3. 
€ 5.000.000,-- (i.e. five million Euro) for the total damage incurred by the organisation for all 
damage disclosed by scientific research for the Sponsor as ‘verrichter’ in the meaning of said Act 
in each year of insurance coverage. 
 

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10.6 Incentives 
Not applicable. 
 
11. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
11.1 Handling and storage of data and documents 
Subject identification and registration 
Prior to enrollment in the study and prior to any study related procedure, the patient must personally 
sign and date the informed consent form. Each patient will be given a unique sequential study subject 
number not based on the patient’s initials or birthdate. The key to the patient study number is 
safeguarded by the local investigator.  
 
Patient registration will done at the Erasmus MC Clinical Trial Center and will only be accepted from 
authorised investigators or through their authorised data managers or authorised staff members. A 
patient can be registered only after verification of eligibility. Patients have to be registered before 
leukapheresis is performed. During the registration procedure eligibility criteria and items of patients’ 
informed consent are checked. 
 
The handling of personal data complies with the EU General Data Protection Regulation and the 
Dutch Act on Implementation of the General Data Protection Regulation (in Dutch: Uitvoeringswet 
Algemene verordening gegevensbescherming (AVG). Collaboration with a third party outside the EU 
requires a privacy assurance agreement complying with these regulations. 
 
The Informed Consent Form will include a statement by which the patient allows the Sponsor’s duly 
authorized personnel, the ethics review committee (IRB/ERC) or similar or expert committee, and the 
regulatory authorities to have direct access to original medical records which support the data on the 
CRFs (e.g. patient’s medical file). This personnel, bound by professional secrecy, must maintain the 
confidentiality of all personal identity or personal medical information, according to confidentiality and 
personal data protection rules and in compliance with all applicable privacy laws, rules, and 
regulations. 
 
Storage of patient material 
Patient biomaterials will be coded (anonymized) and will be stored for a up to 30 years to be used 
current translational research or for future cancer research by the Erasmus MC (EMC) or the EMC in 
collaboration with third parties within The Netherlands, within the EU or outside the EU. After that 
period, materials may be destroyed as per responsibility of the PI. 
 
Data management  
Data will be collected using an eCRF (electronic case report form) designed for this study. According 

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 to ICH guidelines for Good Clinical Practice (GCP), the trial monitor will check the specific CRF 
entries against the source documents, as specified in the study specific monitoring plan.  
 
11.2 Monitoring and Quality Assurance  
The study will be conducted and data are generated, documented, and reported in compliance with 
the accepted standards of GCP. Steps to be taken to ensure the accuracy and reliability of data 
include the selection of qualified personnel and review of protocol procedures before the study. Data 
collected on the eCRF will be verified for accuracy by the local trial monitor according to the Monitor 
Plan. All data management procedures will be documented in detail in a study-specific Data 
Management Plan. 
 
11.3 Amendments  
A ‘substantial amendment’ is defined as an amendment to the terms of the CCMO application, or to 
the protocol or any other supporting documentation, that is likely to affect to a significant degree: 
- the safety or physical or mental integrity of the subjects of the trial; 
- the scientific value of the trial; 
- the conduct or management of the trial; or 
- the quality or safety of any intervention used in the trial. 
All substantial amendments will be notified to the CCMO and to the competent authority. 
Non-substantial amendments will not be notified to the CCMO and the competent authority, but will be 
recorded and filed by the investigator. 
 
11.4 Annual progress report 
The investigator will submit a summary of the progress of the trial to the CCMO once a year. 
Information will be provided on the date of inclusion of the first subject, numbers of subjects included 
and numbers of subjects that have completed the trial, serious adverse events/serious adverse 
reactions, other problems, and amendments.  
 
11.5 Temporary halt and (prematurely) end of study report 
The investigator will notify the CCMO of the end of the study within a period of 90 days. The end of the 
study is defined as the last patient’s last visit.  
The sponsor will notify the CCMO immediately of a temporary halt of the study, including the reason of 
such an action. In case the study is ended prematurely, the sponsor will notify the CCMO within 15 
days, including the reasons for the premature termination. 
Within one year after the end of the study, the investigator/sponsor will submit a final study report with 
the results of the study, including any publications/abstracts of the study, to the CCMO.  
 

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11.6 Public disclosure and publication policy 
Prior to initiation, the study will be submitted to the Dutch National Trial register, which is a recognized 
register that is acknowledged by the WHO and International Committee of Medical Journal Editors 
(ICMJE) and to the NCI’s PDQ® Cancer Clinical Trials Registry. 
All the results will officially be published. Study investigators will be informed in writing prior to any 
written communication or oral presentation about the study and invited to give 
comments. 
 
12. 
STRUCTURED RISK ANALYSIS 
12.1 Potential issues of concern 
a. Level of knowledge about mechanism of action 
The mechanism of action is immunologic. The treatment applies autologous T cells that are 
genetically modified ex vivo to express a patient derived MC2-specific TCR. Upon reinfusion into the 
patient the MC2 TCR T cells will recognize and kill MC2-expressing tumor cells.  
The MC2 TCR recognizes the immunogenic HLA-A2 restricted MC2ALK epitope. MC2 is not expressed 
in healthy tissues (with the exception of gonads, but these sites are immune-privileged and have no 
MHC expression). The expression of MC2 on tumor cells in vivo is upregulated by a preparative 
regimen of epigenetic drugs prior to the MC2 TCR T cell infusion. MC2 TCR T cell infusions will be 
followed by low-dose IL-2 administrations to support proliferation, function and survial of T cells in vivo. 
 
b. Previous exposure of human beings with the test product(s) and/or products with a similar 
biological mechanism 
This is the first in man application of MC2 TCR T cells. MC2 has not be targeted before with 
adoptive T cell therapy (AT). Clinical TCRs tested so far in AT were mostly HLA-A1 or A2-restricted 
and directed against differentiation antigens (i.e. MART-1, gp100, CEA and p53), CGAs (i.e. MAGE-
A3 or NY-ESO-1) and viral antigens. Collectively, these trials demonstrated tumor responses in 
patients with metastatic melanoma, colorectal carcinoma, synovial sarcoma and multiple myeloma that 
ranged from 12 to 80%.[3, 4] However, treatment-related toxicity became evident from studies with 
TCRs, in particular for TCRs with high-affinity, directed against antigens that were over-expressed on 
tumors but were also expressed (in some cases a highly similar antigen) on healthy cells.[5, 50-52]  
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human 
cell material? 
The primary mechanism of MC2 TCR T cells has been illustrated by in vitro recognition and the killing 
of MC2 positive, but not MC2-negative human tumor cell lines.[54, 67, 68] In patients, direct tumor cell 
killing by MC2 TCR T cells will be assessed directly in tumor biopsies and indirectly by assessing pro-
inflammatory cytokines in blood. 
 
 
 

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d. Selectivity of the mechanism to target tissue in animals and/or human beings 
The CGA MC2 has a restricted expression profile and is only expressed by various tumors cells, but 
not in healthy tissues, with the exception of gonads, which are immune-privileged having no MHC 
expression. 
The MC2-specific TCR16, recognizing the ALK epitope in the context of HLA-A2, has a very restricted 
recognition motif towards the ALK epitope, and TCR cross-reactivity was only found towards MAGE-
C1, a related CGA that also shows no expression in healthy non-gonadal tissues.[54] 
 
e. Analysis of potential effect 
In the phase I-part of the study, DLT and the recommended phase II dose (at MTD) will be assessed. 
The optimal dose of a particular T cell product may vary per type of T cell preparation (TIL, CAR T 
cells, and TCR T cells), patient preparative treatment and expression of the target antigen. As MC2 is 
not expressed in normal somatic tissues, no ‘on-target’ toxicities are foreseen (see also “c”). 
Therefore, we anticipate that the MTD / recommended phase II dose will be in the higher dose range.   
 
f. Pharmacokinetic considerations 
The duration of  anti-tumor responses is related to the level of persistence of TCR T cells in the blood 
.[56, 57] Therefore, we have incorporated in the protocol strategies to enhance TCR T cell 
persistence.[68] There are no special issues regarding elimination of MC2 TCR T cells. 
 
g. Study population 
Adult patients with unresectable stage IIIc and IV melanoma (including unknown primary, mucosal and 
uveal melanoma), and unresectable recurrent/metastatic (R/M) HNSCC for whom no standard 
treatment is available (anymore).  
 
h. Interaction with other products 
Patients will receive an epigenetic drug preparative regimen consisting of AZA (75 mg/m2/day s.c. x 7 
days), and VP (50 mg/kg/day p.o. x 7 days). Two days following this regimen, patients will receive one 
single intravenous adoptive transfer of autologous MC2 TCR T cells. The epigenetic drug regimen will 
enhance MC2-protein expression in tumor cells, but will not affect MC2 TCR T cell activity. Following 
TCR T cell transfer, no systemic immune suppressive agents  (˃ 10mg/day prednisone or equivalent) 
are allowed. Low-dose IL-2 administrations following MC2 TCR T cell infusions will support 
proliferation, function and survival of T cells in vivo. 
 
i. Predictability of effect 
In patients, tumor cell killing by MC2 TCR T cells will be assessed directly in tumor tissue and 
indirectly by assessing pro-inflammatory cytokines in blood. In addition, persistence of MC2 TCR T 
cells in blood will be assessed (mirrors clinical response in similar studies); as well as immune and T 
cell parameters in blood, and micro-environment of tumor tissues. To document the effect of the 
epigenetic drug preparative regimen (i) the MC2 expression in tumor biopsies taken prior to and after 

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epigenetic drug treatment and (ii) the global DNA hypomethylation and histone acetylation in PBMCs 
will be assessed. 
 
j. Can effects be managed? 
In case patients may experience CRS with severe symptoms, patients will be treated with high dose 
corticosteroids, the anti-IL6R antibody tocilizumab [7], and the anti-CD52 antibody alemtuzumab, 
depending on the severity of the symptoms. Toxicities of epigenetic drugs and IL-2 are expected to be 
mild and managible by standard supportive care. 
 
12.2 Synthesis 
In patients with advanced melanoma and other tumor types, prior clinical studies have shown efficacy 
of T lymphocytes directed towards tumor antigens. To this end, tumor reactivity can be imposed by the 
transfer of TCR genes into previously non-reactive T cells. The development of TCR T cell therapy 
critically depends on selection and validation of effective and safe T cell target antigens. 
We selected the CGA MC2, that is highly expressed in melanoma and HNSCC, but not in normal 
mature tissues. MC2 can evoke clinically effective T cell responses, as demonstrated in previous 
vaccination studies. We confirmed MC2 expression in a series of tumor cells and its absence in 
somatic healthy tissues. In addition, we have shown that MC2 expression in tumor cells, but not 
normal somatic cells, can be epigenetically upregulated.   
 
Next, we isolated the MC2-specific TCR16 from a melanoma patient who showed a complete 
remission following a MAGE-A1 and MAGE-A3 vaccination regimen, without any clinical toxicity. Upon 
transfer of TCR16 to primary human T cells, these T cells express TCR16 and are able to recognize 
and kill MC2-expressing tumor cells. CR16 has a highly restricted recognition motif toward its cognate 
peptide and cross-reactivity is only limited to MAGE-C1, i.e., a related CGA without expression in 
healthy non-gonadal tissues. Collectively, these analyses showed absent MC2 expression in somatic 
healthy tissues and a beneficial safety profile for MC2 TCR16 [54], therefore the development of on- or 
off-target toxicities in patients treated with autologous MC2 TCR16 T cells is highly unlikely.  
 
In this phase I/II study, we will investigate the safety and feasibility of AT with autologous MC2 TCR T 
cells, combined with epigenetic drug treatment, in patients with MC2-positive advanced melanoma or 
HNSCC. The epigenetic preparative regimen is well established and induces mild side effects. Based 
on the tumor-restricted expression of MC2 and the beneficial safety profile for MC2 TCR16, the 
development of on- or off-target toxicities in patients treated with autologous MC2 TCR16 T cells is 
highly unlikely. Nevertheless, in case patients may experience CRS with severe symptoms, patients 
will be treated with high dose corticosteroids, the anti-IL6R antibody tocilizumab, and the anti-CD52 
antibody alemtuzumab, depending on the severity of the symptoms. Toxicities of epigenetic drugs and 
IL-2 are expected to be mild and managible by standard supportive care. 
In this patient population, the potential burden and AEs are well justified by the substantial chance of 
(durable) tumor responses as a result of the proposed treatment. 

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13. 
REFERENCES 
1. 
Eisenhauer, E.A., et al., New response evaluation criteria in solid tumours: revised RECIST guideline 
(version 1.1). Eur J Cancer, 2009. 45(2): p. 228-47. 
2. 
Robbins, P.F., et al., Tumor regression in patients with metastatic synovial cell sarcoma and melanoma 
using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol, 2011. 29(7): p. 917-24. 
3. 
Robbins, P.F., et al., A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell 
receptor: long-term follow-up and correlates with response. Clin Cancer Res, 2015. 21(5): p. 1019-27. 
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14. 
APPENDICES 
Appendix 1: Eastern Cooperative Oncology Group (ECOG) Performance Status 
Grade Description 
0  
Fully active, able to carry on all pre-disease performance without restriction. 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or 
sedentary nature (e.g., light housework, office work). 
2  
Ambulatory and capable of all self-care but unable to carry out any work activities. Up and about 
more than 50% of waking hours. 
3  
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours. 
4  
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair. 
5  
Death 
 
To be eligible for inclusion, patients have to have an ECOG performance status of 0 to 1.The 
ECOG performance status will be recorded throughout the study. 
 
 
 

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Appendix 2: Investigational product label 
 
 
    INVESTIGATIONAL PRODUCT 
    MAGE-C2 TCR16 getransduceerde autologe T cellen 
    Studie: NL69911.000.19     Studienummer: XXX 
 
    Patiëntnaam  
 
: 
…………………….. 
    PID nummer patiënt 
: 
…………………….. 
    Product 
 
 
:   
MC2 TCR16 T cellen 
    Aantal getransduceerde T cellen:  ……. x 108 
    Volume product 
 
: 
……. ml* 
    Datum uitgifte  
 
: 
….. / …. / …….. 
    Houdbaar tot  
 
:  
… uur  
    
    Bewaartemperatuur product tussen 2-8ºC 
    Houdbaarheid product: maximaal 6 uur tussen 2 – 8ºC, waarvan 
     maximaal 1 uur op kamertemperatuur 
         
    *Infusie vloeistof: Ringers lactaat met 1% humaan serum albumine 
   
     Product alleen geschikt voor toediening bij deze patiënt 
     Alleen voor intraveneuze toediening 
     Identificeer nauwkeurig de bedoelde ontvanger van dit product  
     Product niet bestralen en geen leukocytenreductiefilter gebruiken 
     Uitsluitend voor gebruik binnen klinische trial 
     Investigators:  
Dr. A. van der Veldt / Dr. C. Lamers 
       
Dr. Molewaterplein 40, 3015 GD Rotterdam 
       
Interne Oncologie, Tumor Immunologie lab. (Be430C) 
       
Tel: 010-7034447 / 010-7041771