Arginase-1 Peptide Vaccine in Patients With Metastatic Solid Tumors
Phase: PHASE1
Conditions: Non Small Cell Lung Cancer, Urothelial Carcinoma, Malignant Melanoma, Ovarian Cancer, Colorectal Cancer, Breast Cancer, Squamous Cell Carcinoma of the Head and Neck, Metastatic Cancer
Sponsor: Herlev Hospital

 
 
 
Arginase-1 peptide vaccine in patients with 
metastatic solid tumors 
 
NCT: 03689192 
 
01-03-2022 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Project managers and collaborators 
Sponsor 
Inge Marie Svane, MD, Professor, Ph.D., Consultant 
Vice director for Center for Cancer Immune Therapy 
Dept. of Hematology and Oncology 
Herlev & Gentofte University Hospital 
Herlev Ringvej 75, 2730 Herlev 
Phone: +45 38 68 21 31 
E-mail: inge.marie.svane@regionh.dk 
 
Principal Investigator 
Cathrine Lund Lorentzen, MD, Clinicical Assistant 
Center for Cancer Immune Therapy 
Dept. of Hematology and Oncology 
Herlev & Gentofte University Hospital 
Herlev Ringvej 75, 2730 Herlev 
Phone: +45 38 68 93 83  
E-mail: cathrine.lund.lorentzen@regionh.dk 
 
Collaborators 
Mads Hald Andersen, Professor, D.Tech, DMSc 
Vice director for Center for Cancer Immune Therapy 
Dept. of Hematology, Herlev & Gentofte Hospital 
Herlev Ringvej 75, 2730 Herlev 
Phone: +45 38 68 26 02 
E-mail: mads.hald.andersen@regionh.dk 
 
Eva Ehrnrooth, MD, PhD 
CMO for IO-Biotech ApS 
Ole Maaløes Vej 3, 2200 Copenhagen N 
Phone: +45 30 59 60 91 
E-mail: ee@iobiotech.com 
 
Mai-Britt Zocca, Ph.D. 
CEO for IO-Biotech ApS 
Ole Maaløes Vej 3, 2200 Copenhagen N 
Phone: +45 21 94 78 56 
E-mail: mz@iobiotech.com 
 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Clinical Research Center 
Department of Oncology 
Herlev & Gentofte University Hospital 
Herlev Ringvej 75, 2730 Herlev 
Phone: +45 38 68 23 07 
 
 
GCP Unit 
Copenhagen University Hospital GCP Unit 
Bispebjerg Hospital, Bygning 51, 3. sal 
Bispebjerg Bakke 23, 2400 Copenhagen NV 
Phone +45 35 31 38 90 
 
The study will be performed as described in this protocol and in accordance with regulatory 
requirements and Good Clinical Practice. It will be monitored by the GCP unit of the Copenhagen 
University Hospital and can be subject for external audition as quality assessment of the GCP unit. 
The investigator allows direct access to source data and documents including medical records of 
participants, for inspection by the relevant authorities, being the Danish Medicines Agency, GCP 
units and/or other health authorities. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Table of Contents  
Abbreviations..................................................................................................................................................... 6 
Objectives and endpoints .................................................................................................................................. 8 
Protocol synopsis ............................................................................................................................................... 8 
Background .................................................................................................................................................... 8 
Peptide vaccine safety ................................................................................................................................... 9 
Target population .......................................................................................................................................... 9 
Number of participants ............................................................................................................................... 10 
Patient recruitment ..................................................................................................................................... 10 
Treatment plan ............................................................................................................................................ 10 
Assement of safety ...................................................................................................................................... 10 
Immunological evaluation ........................................................................................................................... 10 
Clinical response evaluation ........................................................................................................................ 11 
Background ...................................................................................................................................................... 11 
Metastatic solid cancer ............................................................................................................................... 11 
Immune activation and regulation .............................................................................................................. 11 
Cancer cells induce local tolerance ............................................................................................................. 14 
Mechanism of Action – vaccination therapy ............................................................................................... 15 
Arginase and its potential as a vaccination antigen .................................................................................... 18 
Arginine, the ARG substrate .................................................................................................................... 18 
The ARG enzyme ...................................................................................................................................... 18 
Arginase as an immune suppressor ......................................................................................................... 18 
ARG producing cells ................................................................................................................................. 19 
ARG1 as an immune modulator .............................................................................................................. 20 
The ARG1 peptide vaccine ....................................................................................................................... 21 
Peptide vaccine safety ................................................................................................................................. 26 
Adjuvant ...................................................................................................................................................... 26 
Therapeutic peptide vaccines lack a suitable animal model for safety and dose testing ........................... 27 
Chioce of dosage ............................................................................................................................................. 28 
Number of participants ................................................................................................................................... 29 
Patient recruitment ......................................................................................................................................... 29 
Information and informed content retrieval ................................................................................................... 29 
Study design .................................................................................................................................................... 30 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Inclusion criteria .......................................................................................................................................... 31 
Exclusion criteria.......................................................................................................................................... 32 
Evaluation before the start of treatment .................................................................................................... 32 
Conduct of the study ....................................................................................................................................... 33 
Initial patient enrollment and treatment .................................................................................................... 33 
Detalied treatment and evaluation schedule .............................................................................................. 34 
Follow up ..................................................................................................................................................... 37 
Procedures regarding treatment ................................................................................................................. 38 
Criteria for treatment modifications ........................................................................................................... 38 
Criteria for end of treatment ........................................................................................................................... 39 
Early termination from the study ................................................................................................................ 41 
Concurrent treatment ................................................................................................................................. 41 
Manufacture of the peptide vaccine ............................................................................................................... 41 
Peptide......................................................................................................................................................... 41 
Producing the peptide vaccine .................................................................................................................... 41 
Montanide ISA 51 ........................................................................................................................................ 41 
Preparing the vaccine .................................................................................................................................. 42 
Microbiological control ................................................................................................................................ 42 
Evaluation ........................................................................................................................................................ 42 
Data registration .......................................................................................................................................... 42 
Monitoring ................................................................................................................................................... 43 
Statistical considerations ............................................................................................................................. 43 
Effect parameters ............................................................................................................................................ 43 
Primary effect parameter ............................................................................................................................ 43 
Secondary effect parameter ........................................................................................................................ 44 
Exploratory endpoints and objectives ......................................................................................................... 44 
Immunologic monitoring ................................................................................................................................. 44 
Project blood samples for immunological monitoring ................................................................................ 44 
Tumor biopsies ............................................................................................................................................ 45 
Delayed type hypersensitivity (DTH) & biopsy ............................................................................................ 47 
Biobank and handling of biologic material ...................................................................................................... 47 
Risk evaluation ................................................................................................................................................. 48 
Monitoring of toxicity ...................................................................................................................................... 49 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Adverse events ............................................................................................................................................ 49 
Grading of adverse events ........................................................................................................................... 49 
Serious adverse events ................................................................................................................................ 50 
Adverse reactions ........................................................................................................................................ 50 
Reporting of adverse events and adverse reactions ................................................................................... 51 
Handling adverse events ................................................................................................................................. 52 
Insurance ......................................................................................................................................................... 52 
Ethical aspects ................................................................................................................................................. 52 
Reporting to the Danish Data Protection Agency ........................................................................................... 53 
End of study report .......................................................................................................................................... 53 
Publications ..................................................................................................................................................... 53 
Economy .......................................................................................................................................................... 53 
References ....................................................................................................................................................... 54 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Abbreviations 
AE 
 
Adverse event 
ALAT 
 
P-alanine-aminotransferase 
AML 
 
Acute myeloid leukemia  
ANC 
 
Absolute Neutrophilic Count 
APC 
 
Antigen presenting cell 
AR 
 
Adverse reaction 
ARG 
 
Arginase 
ARG1 
 
Arginase-1 
ASAT 
 
P-aspartattransaminase 
CCIT  
 
Center for Cancer Immune Therapy 
CNS 
 
Central Nerve System 
CR 
 
Complete Response 
CRF 
 
Case Report Form 
CRR 
 
Complete Response Rate 
CRP  
 
C - reactive protein 
CT 
 
Computer tomography 
CTCAE 
 
Common Terminology Criteria for Adverse Events 
CTL 
 
Cytotoxic T lymphocyte 
CTLA-4 
 
Cytotoxic T-Lymphocyte Antigen 4 
DC 
 
Dendritic cell 
DTH 
 
Delayed type hypersensitivity 
ECOG 
 
Eastern Cooperative Oncology Group 
EFEK 
 
Enhed for Eksperimentel Kræftbehandling 
HLA 
 
Human Leukocyte Antigen 
HNSCC 
 
Squamous cell carcinoma of the head and neck 
ICS 
 
Intracellular staining 
IDO 
 
Indolemamine-pyrrole 2, 3-dioxygenase 
IFA 
 
Incomplete Freund’s adjuvant 
IgG 
 
Immunoglobulin G 
 
 
IL-2 
 
Interleukin 2 
INR 
 
International Normalized Ratio 
KFE 
 
Clinical experimental unit 
LDH  
 
Lactate-dehydrogenase 
MDSC 
 
Myeloid-derived Suppressor Cell 
MHC 
 
Major Histocompatibility complex 
MR 
 
Magnetic resonance 
MSV 
 
MilliSievert 
MTD 
 
Maximum tolerated dose 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
NK 
 
Natural killer  
NO 
 
Nitric oxide 
NOS 
 
Nitric oxide synthase 
NSCLC 
 
Non-Small Cell lung cancer 
OR 
 
Objective response 
OS 
 
Overall survival 
PBMCs 
 
Peripheral blood mononuclear cells 
PD-1 
 
Programmed cell death 1 
PD-L1 
 
Programmed cell death ligand 1 
PERCIST 
 
PET Response Criteria in Solid Tumors 
PET 
 
Positron Emissions Tomography 
PFS 
 
Progression free survival 
RECIST 
 
Response Evaluation Criteria in Solid Tumors 
 
PR 
 
Partial Response 
SAE 
 
Serious adverse event 
SAR 
 
serious adverse reaction  
SD 
 
Stabil Disease 
S-HCG 
 
serum-Choriogonadotropin 
SKILs 
 
Skin Infiltrating Lymphocytes 
SOP 
 
Standard operating procedure 
SUSAR 
 
Suspected unexpected serious adverse reaction 
TAMC 
 
Tumor Associated Myeloid Cell  
TCR 
 
T cell receptor 
Treg 
 
Regulatory T cells 
 
 
The peptides mentioned in the protocol:  
 
IO112  
 ARG 171-190;   AKDIVYIGLRDVDPGEHYIL     
 
ARG1-18        
 
     ARG 181-200;   DVDPGEHYILKTLGIKYFSM 
 
ARG1-19 
 
     ARG 191-210;   KTLGIKYFSMTEVDRLGIGK 
 
ARG1-20 
 
 
 
 
 
 
 
 
 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Objectives and endpoints 
The primary objective is to assess the safety and tolerability of a peptide vaccine containing the 
enzyme arginase-1 (ARG1) with Montanide ISA 51 as adjuvant in patients with metastatic and 
treatment-refractory solid tumors. The primary endpoint is adverse events (AE) assessed by 
Common Terminology Criteria for Adverse Events (CTCAE) 4.0 and laboratory monitoring.  
The secondary objective is to evaluate the immune response before, during and after treatment. 
The endpoint is responses of ARG1 specific T cells measured by ELISPOT and cytokine release 
assays.  
The exploratory objective is to evaluate the clinical efficacy of the treatment. The endpoints are 
objective response (OR), progression free survival (PFS) and overall survival (OS). 
Protocol synopsis 
Background 
Metastatic solid tumors are regarded incurable and fatal despite great progresses in medical 
oncology over the past decade. Treatment options are in end-effect palliative and given with the 
purpose of prolonging life. Standard therapies for metastatic solid tumors range from hormone 
suppression to aggressive chemotherapy regimens, but in the last couple of years immunotherapy 
has revolutionized several cancer treatments. This protocol describes a new approach in the 
treatment of patients with metastatic solid cancer by modulating the tumor microenvironment 
using a peptide vaccine. 
 
The unregulated growth of cancer cells is only possible when the immune system does not launch 
an effective antineoplastic response. Cancer cells highjack the system of checks and balances in 
the immune system by exploiting the system of local adaptive immune resistance. One mechanism 
utilized by malignant cells is the overexpression of the immunosuppressive enzyme arginase-1 
(ARG1).  
ARG1 is an enzyme expressed by some cancer cell lines and different myeloid cells in the 
immunosuppressive tumor microenvironment. ARG1 catalyzes the conversion of the amino acid 
arginine to ornithine and urea. A depletion of arginine suppresses T cell function through the 
impairment of the T cell receptor (TCR)-complex resulting in immunosuppression in a cancer 
setting.  
The theoretic background for a peptide vaccine against ARG1 is to boost the activity of ARG1-
specific T cells to eliminate immune cells that express ARG1 as part of the local 
immunosuppressive microenvironment in cancerous tissues. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Center for Cancer Immune Therapy (CCIT) has identified spontaneous T cell reactivity against ARG1 
peptides in peripheral blood mononuclear cells (PBMCs) of cancer patients and healthy donors. 
Thus boosting ARG1-specific T cells may directly modulate immune regulation 1.  
 
Peptide vaccine safety  
The potential peptide vaccine-related safety profile is linked to the presence of the target antigen 
in normal tissue and to the risk of acute anaphylactic reactions.  
However, previous peptide vaccination trials have had very mild side effects, no matter which 
proteins have been targeted (see Risk evaluation). Research groups from CCIT  have documented 
mild side effects testing the indolemamine 2,3-dioxygenase (IDO) peptide vaccine with no CTCAE 
grade 3 or 4 toxicities 2. The ARG1 vaccine and IDO vaccine resemble in many ways.  They are both 
self-proteins with a broad expression and they are lymphocyte suppressive enzymes with an 
overexpression in immunosuppressive cancer associated immune cells.  
Moreover, a phase I trial treating cancer patients with an oral ARG1 inhibitor (CB-1158) presented 
data on drug related adverse effects in April, 2017.  A total of 17 patients with advanced solid 
tumors were treated with CB-1158, and the drug was generally well tolerated with no drug-related 
serious adverse events3.  Even though the ARG1 peptide vaccine and CB-1158 differ in both 
mechanism of action and administration they both target the same enzyme. Therefore, the AEs 
registered for patients treated with CB-1158 could possibly indicate what AEs to expect in the 
ARG1 trial.     
 
Montanide ISA 51 
Montanide ISA 51 can cause local discomfort at the insert site, but does usually not cause any 
systemic side effects with the dose used in this trial. (See “Product Resume Montanide ISA 51”, 
appendix 11)  
 
Target population 
This protocol describes the treatment of patients with solid metastatic cancers. Despite many 
treatment advances in cancer therapy, most metastatic solid cancers are still incurable and most 
patients will at some point find themselves without viable treatment options. The majority of 
deaths are caused by metastatic solid cancers such as lung, colon, breast, and prostate cancer. 
Immunotherapy is an established and effective treatment for an increasing number of cancer 
patients including different metastatic solid cancers.  
The treatment with an ARG1 peptide vaccine has a potential in all metastatic solid cancers with a 
high concentration of ARG1-expressing cells, such as myeloid-derived suppressor cells (MDSCs) 
known for their impairment anti-cancer immunity. These tumors include non-small cell lung 
cancer (NSCLC), colorectal cancer, urothelial cancer, breast cancer, ovarian cancer, malignant 
melanoma, and squamous cell carcinoma of the head and neck (HNSCC) 4,5. We aim to test the 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
ARG1 vaccine in patients who have exhausted conventional treatment options. Hence, patients 
with progressive metastatic solid cancers of the types mentioned above in advances stages are 
eligible to enter this protocol.  
 
Number of participants  
The study is a phase I trial where 10 patients with metastatic solid tumors will be treated. If 3 or 
more patients experience grade 3-4 AE associated with the vaccination, the trial will be stopped.  
 
Patient recruitment 
The trial is expected to begin the inclusion in August 2018. Patients will be recruited from the 
Department of Oncology, Herlev & Gentofte University Hospital, but can also be referred from 
other centers in Denmark. The inclusion period is expected to extend over 12 months.   
 
Treatment plan 
Patients included in the trial will be treated with the ARG1 vaccine every third week for 48 weeks. 
Sixteen vaccines will be given in total.  
At the end of vaccination, patients who are not excluded from the protocol because of progression 
will have follow up appointments after 3 and 6 months in the experimental unit (EFEK), Herlev 
Hospital. 
  
Assement of safety  
A systematic recording of AEs will take place during the course of treatment and follow up in 
accordance with the Common Terminology Criteria for Adverse Events (CTCAE). The AEs will be 
reported by the investigator, who will make a relatedness assessment. Recording will take place 
during patient attendance in the outpatient clinic or at the hospital ward at the Department of 
Oncology, Herlev Hospital. 
 
Immunological evaluation 
Blood samples 
In order to monitor the immune response, ARG1 levels and immune related changes during 
treatment, project blood samples will be taken before, during and after treatment. Project blood 
samples will be taken at the follow up appointments after 3 and 6 months. 
 
Tumor biopsy  
At baseline and after the 4th vaccine, a tumor biopsy will be performed if the tumor is accessible. 
The objective is to evaluate the tumor immune microenvironment of each patient. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Immunohistochemistry and whole exome sequencing to assess initial mutational status will be 
conducted.   
 
Delayed type hypersensitivity  
Delayed type hypersensitivity (DTH) skin-test and punch biopsies from the DTH areas will be done 
after the 4th vaccination for the evaluation of skin infiltrating lymphocytes (SKILs). The SKILs are 
tested for specificity to the ARG1 peptide as a sign of induction of a functional immune response. 
 
Clinical response evaluation 
Clinical response evaluation will be performed with diagnostic imaging according to Response 
Evaluation Criteria In Solid Tumors (RECIST)6, PET Response Criteria in Solid Tumors (PERCIST)7. 
Clinical examination before, during and after treatment (see the Treatment schedule).  
Background  
Metastatic solid cancer  
Despite many and continuous advances in oncology, metastatic solid cancer is still regarded an 
incurable and fatal disease. Treatment options palliative and given with the purpose of prolonging 
life thereby often ameliorating suffering and disability. In Denmark 37.000 patients are diagnosed 
with cancer every year and more than 15.000 die from cancer-related causes 8. The 4 most 
common and most deadly cancers forms are all solid cancers (breast, prostate, lung, and colon 
cancer) that readily metastasize. Combined, these 4 diagnoses alone amount to 55% of all new 
cancer diagnoses and 53% of all cancer-related mortality9.  Standard therapies for these cancers 
range from hormone suppression to aggressive chemotherapy regimens. In the last couple of 
years immune therapy has revolutionized many cancer treatments. In solid tumors, malignant 
melanoma has pioneered the field but it is constantly expanded to other diagnoses. Immune 
therapy was recently approved for lung cancer10 and colon cancer might be next11. The high 
prevalence and morbidity of metastatic solid cancers leaves room for therapeutic improvements. 
 
Immune activation and regulation  
In this section a very brief introduction is given to the adaptive immune system, which is one of 
the two major divisions of the immune system and is very important in the fight against cancer. 
Cells and molecules of the other division of the immune system, called the innate immune system, 
also play their important roles in cancer, but the vaccination therapy described in this protocol has 
its main effects in the adaptive immune system. The adaptive immune system has two branches 
comprised of the humoral and the cellular branch.  
 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
In the humoral branch of the adaptive immune system antibodies are the major effectors. 
Antibodies bind to molecules on their targets, whether the targets are bacteria, virus, fungi or 
malignant cells. Antibodies exert their function by physically blocking surface molecules on the 
target or by one of several ways mediating specific toxicity to the target. When a surface molecule 
is blocked by an antibody, the function of the surface molecule can be inhibited. Antibodies can kill 
targets by cross-linking molecules on the target, by activation of attack-molecules of the 
complement system to make holes in the target’s membrane or by activating other immune cells 
to kill the target.  
 
In the cellular branch of the adaptive immune system the foot soldiers are the T cells. Two 
subgroups of T cells are the CD8+ cytotoxic T cells (CTLs) and the CD4+ helper T cells. While the 
main role of CTLs is to directly kill cells that are virally infected or malignantly transformed, T-
helper cells come in many subtypes and have many different roles in the immune system.  
CTLs are very effective killer cells, and are probably therefore highly regulated. The most 
fundamental level of regulation comes from the principle that CTLs can only kill other cells, if the 
TCR of the CTL recognizes a “peptide-fingerprint” on the target cells. Peptides are small pieces of 
proteins. This fingerprint is a specific peptide epitope bound to a specific major histocompatibility-
class I molecule together making a peptide:MHC-complex. All nucleated cells express these MHC-
molecules that display to the immune system a random sample of what proteins are in the cell. If a 
cell is infected with a virus, viral peptides will be displayed. If a cell has turned malignant, cancer 
peptides will be displayed.  
 
A T cell has many TCRs, but all TCRs on a T cell are identical. Therefore, a CTL is specific to one 
peptide-fingerprint. TCRs are made randomly, but whether a specific TCR is expressed on any T-
cells circulating the body is regulated in the thymus. In the thymus, T cells are positively and 
negatively selected based on what their TCR recognizes and how strongly it is recognized. If the 
TCR binds nothing the T cell dies of neglect. If the T cells TCRs bind self-peptides too strongly, the T 
cell is eliminated.  
 
After the cytotoxic T cell has left the thymus and circulates in the body, another level of regulation 
is added. This second level of regulation is based on the principle that CTLs must be primed by 
antigen presenting cells (APC), often a dendritic cell (DC), to be able to start killing target cells (Fig. 
1). APCs eat antigens, chop up the antigens into peptides, and present them on MHC-molecules. 
Priming happens in lymphoid tissue when a CTL meets an APC that has taken up antigen epitopes 
that the CTL is specific to. The APC delivers a “signal one” to the T cell which is when the APC 
presents the peptide epitope on a MHC-molecule to the T cell. To be activated, the T cell needs a 
“signal 2” which it also gets from the DC because the DC expresses costimulatory proteins that 
activate the T cell. Once a CTL has been primed, it circulates the body is ready to engage target 
cells in the effector phase (Fig. 2). In the effector phase, CTLs kill target cells that on the surface 
have peptide:MHC-complexes that the CTL is specific to. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
A third level of regulation is built into the priming and effector phases. Not only does the killing of 
a target cell depend of a “signal one” and a “signal two” in the priming phase combined with a 
complementary TCR-peptide:MCH match in the effector phase. What is further needed is the 
correct balance of so called co-stimulatory and co-inhibitory signals in both phases. In the effector 
phase one of the very potent inhibitory molecules is programmed death ligand 1 (PD-L1) on the 
target cell. If a CTL recognizes a peptide:MHC-complex but is inhibited by PD-L1 on the target cell 
interacting with its receptor PD-1 on the T cell, the T cell is left in an inactive state (Fig. 2A). The 
negative regulation in the effector phase is an important system to fine-tune the regulation of 
CTLs, shutting down self-reactive T cells that have slipped through the previous levels of 
regulation. The balance between stimulatory and inhibitory molecules acts to ensure that the T 
cells are activated when needed, but prevents T cells from causing damage to normal tissues by 
inhibiting the T cell response. 
 
 
 
Figure 1: Immune checkpoints and checkpoint inhibitors, the priming phase.  In the priming phase, an antigen 
presenting cell activates antigen-specific T cells by stimulating via both the MHC-TCR-pathway (signal one) and the co-
stimulatory B7-CD28-pathway (signal two). In the priming phase, the immune responses can be inhibited if the T cells 
have upregulated the checkpoint molecule CTLA-4. CTLA-4 can be blocked by the checkpoint-inhibiting antibody 
Ipilimumab12.  
 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
 
Figure 2: Immune checkpoints and checkpoint inhibitors, the effector phase. In the effector phase, antigen-specific T 
cells recognize target cells presenting the target peptide-MHC-molecule. In the effector phase, immune responses can 
be inhibited by the checkpoint molecules PD-1/PD-L1. Several antibodies blocking PD-1 or PD-L1 have been marked 12. 
 
Cancer cells induce local tolerance 
The unregulated growth of cancer cells is only possible when the immune system does not launch 
an effective antineoplastic response against the cancer. Impairment of the immune system is thus 
a hallmark of cancer 13. Strikingly, the insufficient immunological activity in cancer patients seems 
not to be a general impairment of the immune system. Most cancer patients do not suffer from 
frequent infections, except when their immune system is very affected by treatment or when the 
cancer has reached a late state with profound impact on the body. Thus, the impairment of the 
immune system must be somewhat specific to the cancer cells and the local microenvironment 
around the tumor. This discrepancy between a near normal overall immune function and 
profound immunosuppression locally can be explained by the increase in immune regulatory 
molecules and immune regulatory cells in and around the tumor.  
 
Cancer cells highjack the system of checks and balances of T cell activation by overexpressing 
inhibitory molecules such as PD-L1 and regulatory enzymes such as IDO. Furthermore, 
immunosuppressive regulatory cells also expressing inhibitory molecules like PD-L1 and ARG1 are 
recruited to the tumor. The expression of ARG1 by immunosuppressive regulatory cells inhibits T 
cells by depleting the microenvironment of the amino acid arginine crucial to T cell signaling and 
functions. 
Combined this causes a very immunosuppressive local environment, skewing the normal balance 
between stimulating and inhibitory signals in the direction of immunosuppression in and around 
the tumor. The co-inhibitory molecules CTLA-4, PD-L1 and PD-1 are checkpoints in the immune 
system. One of the major pillars of the recent impressive advance of the field of immune-oncology 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
is the introduction of the so called immune checkpoint blocking monoclonal antibodies. Antibodies 
blocking the action of CTLA-4 in the priming phase or PD-1/PD-L1 in the effector phase block out 
these negative signals in the interaction of T cells with APC or target cells, respectively (see fig. 1B 
and 2B). Blocking the negative signals in the balance “releases the break” on T cells, allowing them 
to kill malignant cells.  All in all, cancer cells have many mechanisms whereby they escape immune 
attack. 
 
Mechanism of Action – vaccination therapy  
All nucleated cells are characterized by a “peptide-fingerprint” (MHC:peptide complex) on the cell 
surface, which to the outside exposes a mirror of the protein content in the cell. Thus, 
immunosuppressive cells that overexpress ARG will exhibit ARG peptide:MHC complexes on the 
cell surface and can be recognized by T cells 14,15. 
The principle of vaccination is to administer the target (the antigen) recognized by the immune 
system together with an immune enhancer, the adjuvant. The antigen can be composed in many 
different forms. Relevant examples in cancer vaccination are whole tumor cell vaccinations in 
which killed tumor cells are used as the source of antigen. Obviously, such an antigenic source 
comprises a huge number of proteins as potential antigens, many of which will in fact also be 
present in normal cells.  
Administered in combination with a relevant adjuvant, cellular proteins derived from tumor cells 
will be taken up by APCs. The APCs will process the proteins, i.e. cut the proteins into peptides 
that are capable of binding to both class I and class II MHC molecules. MHC:peptide complexes 
expressed on the surface of APCs may elicit T cell responses specific for the individual peptides – 
and these T cells thus have the capacity to recognize cancer cells, which express an identical 
MHC:peptide molecule on the cell surface (Fig. 3). 
At the other extreme, an antigen used for vaccination may simply be a peptide of 9-amino acids 
derived from a protein expressed by cancer cells. This form of antigen negates the need for 
antigen processing step by APC as the peptide is already in a form ready to be presented by the 
APC in the context of MHC. Such peptide antigen may activate T cells capable of recognizing the 
identical peptide sequence expressed on the surface of cancer cells in the context of MHC class I 
molecules. Both approaches have advantages and shortcomings.  
Long peptides of approximately 15 to 20 amino acids have been used in therapeutic vaccination 
studies for more than a decade and can be viewed as an attempt to develop a strictly defined yet 
still broadly applicable target antigen (Fig. 4). A main advantage of the long peptide approach 
compared with the conventional short peptides is that such peptides can encompass epitopes 
binding with both MHC class I and II, i.e. both inducing CD4 T helper responses as well as CD8 
cytotoxic T cell responses. Importantly, peptide binding to MHC class II is rather promiscuous, and 
any given peptide may be capable of binding to several different MHC class II molecules 16,17.  
 
 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
 
 
 
 
 
 
 
 
 
 
 
Figure 3: Principle of therapeutic cancer vaccination. APCs take up antigen at the injection site and 
migrate to a lymph node. During migration to the lymph node they mature to efficient antigen 
presenters, this is improved if a strong adjuvant provides a good “danger”-signal. In the lymph node, 
APCs meet T cells and prime them. When T cells are primed, they leave the lymph node to circulate 
the body patrolling for targets 16. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4: The processing of long peptides in APCs allows stimulation of both CTL and T-helper cells. In 
the right side of the figure, a long peptide is taken up via endocytosis, cut into pieces and presented on 
MHC-class II molecules. This allows it to be presented to CD4-positive T-helper cells. In the left side of 
the figure, a long peptide enters the cytosol. The jury is out as to how it enters the cytosol, but we know 
it happens. From the cytosol, it is processed like a normal protein from the cell. This normal way is by 
degradation in the proteasome, transportation into the ER and in the ER loading onto MHC-class I 
molecules. When a peptide is bound to a MHC-class I molecule, it is presented to CD8-positive CTLs. In 
this manner, a long peptide is not restricted to either class I or class II MHC-molecules. DC: dendritic cell. 
ER: endoplasmatic reticulum. TAP1/2: Transporter associated with Antigen Processing1/2 16. 

18 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Arginase and its potential as a vaccination antigen 
Arginine, the ARG substrate 
Arginine is a conditionally essential amino acid obtained exogenously through diet and 
endogenously through de novo synthesis of cellular protein breakdown. In healthy adults the 
endogenously biosynthesis of arginine from citrulline satisfies metabolic requirements but dietary 
supplements are needed in various cases of metabolic stress and during growth 18,19. Arginine 
serves as a precursor of creatine, citrulline, urea, ornithine, polyamines, proline, and 
glutamate18,20. The amino acid also functions as a substrate for the enzyme nitric oxide synthase 
(NOS) that converts arginine to nitric oxide (NO) and other nitrogen intermediates that are 
important for microbicidal activity (Fig. 5) 21.   
Arginine plays a role in tissue repair and protein synthesis 22. More relevant to this protocol, 
arginine plays an important role in mediating tumor immune regulation23. In a recent mouse 
model, it was demonstrated that activated T cells consume large amounts of arginine, and that an 
arginine supplement enhances T-cell survival and antitumor activity 24. 
 
The ARG enzyme  
Arginine is hydrolyzed to ornithine and urea by the enzyme ARG. In mammals, there are two 
existing ARG isoenzymes: ARG1 and ARG2. The isoenzymes catalyze the same biochemical reaction  
but they differ in cellular expression, location, and regulation 25,26 . The human ARG1 is a 
cytoplasmic enzyme mainly expressed in hepatocytes. It detoxifies ammonia by catalyzing the final 
step in urea cycle. ARG1 is also expressed in various extrahepatic tissues where it regulates 
arginine levels and provides ornithine for biosynthetic reactions 27. The expression of ARG1 by 
some cancer cell lines and regulatory immune cells promote the development of an 
immunosuppressive environment 23,28. The immunotolerogenic impact is partly due to the 
capability of some cells to secrete of ARG1 from intracellular granules into the microenvironment 
29 (Fig. 10).   
 
ARG2 is found in the outer mitochondrial membrane and in the mitochondrial matrix. It is 
predominantly expressed in the prostate, brain, kidney, small intestine, and pancreas. The 
physiological function of ARG2 is still poorly understood compared to that of ARG1 20,23.  In cancer 
research, ARG2 expression has been documented in acute myeloid leukemia (AML) blasts, 
fibroblasts, and various solid cancers such as prostate cancer 30–32.  
 
Arginase as an immune suppressor  
The depletion of arginine by increased ARG expression has emerged as an effective 
immunosuppressive pathway for the mammalian immune system. The reduction of arginine from 
the microenvironment can result in impairment of lymphocyte responses to antigens and arginine-
deprivation has now proved to be a strategy for cancer cells to induce immune tolerance23,25.  This 

19 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
effect of arginine-depletion on T cells was studied in vitro by Rodriguez et al.  and it was 
demonstrated that an arginine deficiency directly affected T cell function through a down 
regulation the main signaling-transduction component of the TCR, the TCR ζ chain (CD3 ζ), in 
human T cells through mRNA instability.  The impairment of T cell function was fully reversed by 
the replenishment of ARG33. At this time, T cell suppression have proved to be the main 
immunoregulatory mechanism of ARG on the tumor environment34.  
 
ARG producing cells 
Cancer cells are capable of  influencing the tumor microenvironment by hijacking local myeloid 
cells and trigger their differentiation into TAMs and MDSCs 35–37.  Both TAMs and MDSCs are highly 
immunosuppressive. They induce a favorable tumor environment, partly, by an ARG1-expression.  
TAMs and MDSCs are also capable of triggering the differentiation of regulatory T cells (Tregs) as 
well as inhibiting the proliferation and activation of natural killer cells (NK cells) and effector T 
cells28,38.   
In addition to ARG1 production induced by anti-inflammatory signals such as IL-4 or IL-13, MDSCs 
and TAMs also express the enzyme nitric oxide synthase (NOS) that catalyzes the production of NO 
and other reactive nitrogen intermediates from arginine when stimulated by pro-inflammatory 
cytokines such as INF-γ (Fig. 5)34. NO has the potential of showing both tumoricidial and tumor 
promoting effects depending on concentration, location, and timing, and a potential ARG1-
induced inhibition of a tumorcidial NO-production by macrophages has been suggested as a 
strategy for cancer cell survival19,21,23,39.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

20 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ARG1 as an immune modulator 
The expression of ARG has been demonstrated in different cancer cell lines 34,40. This protocol is 
focusing on the influence of the ARG1 on the tumor microenvironment, as several studies have 
demonstrated an ARG1 overexpression in the blood and/or tissue of patients with both lung 41 , 
ovarian 42,  renal 43, breast 44,45, esophageal, and gastric cancer46.  
 
The overexpression of ARG1 by macrophages within the tumor environment has proved to favor 
tumor growth in murine lung carcinomas and human renal cell carcinomas43,47. 
It is therefore reasonable to believe that an ARG1 blockage will inhibit tumor growth, and this was 
in fact demonstrated in a murine trial where an injected ARG1-inhibitor reduced the tumor growth 
of lung carcinoma in vivo. In this study, C57BL/6 mice were injected with Lewis lung carcinoma 
(3LL) cells. The mice were devided into six grupous with six mice in each group. During the 
Figure 5: Schematic representation of ARG1- and nitric-oxide-synthase-2-
dependent metabolic pathways. The activities of the enzymes ARG1 and 
nitricoxide synthase 2 (NOS2) are illustrated, together with the 
downstream pathways that are activated by arginine34. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
following 18 days, the mice were injected s.c. with different substances and doses according to 
their group: 6 mice recived PBS, 6 mice recived 20 mg/kg of an ARG1-inhibitor, 6 mice recived 40 
mg/kg of the ARG1-inhibitor, 6 mice recived 80 mg/kg of the ARG1-inhibitor, 6 mice recived the 
ARG1-inhibitor (80 mg/kg) + L-arginine (500mg/kg), and 6 mice recived L-arginine(500mg/kg). The 
mice injected with the ARG1-inhibitor and the mice injected with the ARG1-inhibitor + L-arginine 
had a significantly lower tumor growths than observed in the rest of the groups. The reponses 
were ARG-1 dose-dependent.  
The ARG1-inihitor was also tested in mice lacking function T- and B cells ( C57BL/6-Prkdcscid mice) 
to investigate whether the ARG1-inhibitor-induced anti-tumor effect just described partly was 
mediated by an immune response. Sixteen mice were injected with Lewis lung carcinoma (3LL) 
cells, 8 mice were then injected s.c. with 80 mg/kg of the ARG1-inhibitor and 8 mice were then 
injected s.c. with PBS. The decrease in tumor growth seen in the C57BL/6 mice treated with the 
ARG1-inhibitor was not seen in the treated C57BL/6-Prkdcscid mice indicating that the ARG1-
inhibitor-mediated anti-tumor effect is partly lymphocyte dependend 47.  
The role of ARG1 in the regulation of tumor induced immune tolerance makes it a promising 
therapeutic target. A phase I/II study evaluating an oral ARG inhibitor (CB-1158) as a single agent 
and in combination with Pembrolizumab is currently recruiting, (ClinicalTrials.gov identifier (NCT 
number): NCT02903914)( see Risk Evaluation). 
 
The ARG1 peptide vaccine 
The theoretic background for a vaccine against ARG1 is to boost the activity of ARG1-specific T 
cells (anti-ARG1 T cells) to attack cancer cells and immune cells that express ARG1 as part of the 
local immunosuppressive microenvironment in cancerous tissues.  
 
CCIT therefore recently examined whether sequences of the ARG1 peptide served as a target for 
specific T cells to evaluate whether ARG1 would be a potential new vaccination antigen.  
Naturally present peripheral ARG1-specific T cells were identified in both healthy donors and in 
patients with different cancer diagnoses 1.  
To examine ARG1-specific cells in vitro, a screening of the entire ARG1 sequence was executed. 
Using the ARG1 peptide library and an IFNγ ELISPOT assay it was discovered that T cells recognized 
multiple ARG1 epitopes, including a hot spot region consisting of 50-amino acids that induced the 
most frequent responses (ARG1161-210).  (Fig. 6) 

22 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
 
 
 
 
 
 
Both CD4+ and CD8+ T cell responses against an epitope from the hot-spot region (ARG1161-180) 
were observed in two cancer patients, indicating that HLA class I and II epitopes are present in the 
ARG1 hot spot region (Fig. 7).  
 
Figure 6. A) IFNγ ELISPOT screening of responses against ARG1 peptides in PBMCs from 3 melanoma 
patients. B) IFNγ ELISPOT screening of responses against eight selected ARG1 peptides in PBMCs from 8 
healthy donors. Spot counts are given as a difference between averages of the wells stimulated with the 
peptide and control wells. Peptide and control stimulations were performed in duplicates or triplicates. 

23 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
 
 
 
 
 
To examine the presence of ARG1 specific T cells among tumor infiltrating lymphocytes (TILs), In 
vitro expanded TILs from melanoma patients were stimulated with the following epitopes from 
the hot-spot region: ARG1-17161-180, ARG1-18171-190, and ARG1-19181-200. IFNγ was produced by CD4+ 
T cells in response to all three ARG-peptides in one of the TIL cultures (Fig 8).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 7.  To the right: % of TNFα producing CD4+ and CD8+ T cells in intracellular staining of 
PBMCs from multiple myeloma (MMy9) and breast cancer (BC30) patients stimulated with 
Arg161–180 peptide and non-stimulated control. To the left: dot plots for Arg161–180 peptide 
stimulated. 
Figure 8 - IFNγ production by CD4+ T cells in in vitro 
expanded TILs from a melanoma patient in response to 
Arg161–180, Arg171–190, Arg181–200 peptides 
compared to a non-stimulated control. 

24 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Finally, autologous dendritic cells (DCs) and B cells were transfected with mRNA encoding ARG1 
protein to assess the ability of ARG1 specific CD4+ T cells in recognizing ARG1 expressing immune 
cells. ARG1 specific CD4+ T cells isolated and expanded from two melanoma patients showed 
higher reactivity against DCs and B cells transfected with ARG1 mRNA compared to mock controls 
(Fig. 9).   
 
 
 
 
 
 
 
 
 
 
The patients from which ARG1-specific T cell cultures were generated were selected from a larger 
group of patients that were all screened for responses against ARG1 peptides.  
In total, ARG1 specific T cell responses were screened in 25 cancer patients: 14 melanoma, 1 
breast cancer, 1 renal cell carcinoma, 9 multiple myeloma. 13 of these patients showed a 
Figure 9. ARG1-specific T cells recognize ARG1-expressing immune cells. A),C): IFNγ 
response by ARG1 specific T cell cultures from two melanoma patients (MM01 and 
MM05) to autologous dendritic cells electroporated with irrelevant control mRNA 
(DC Mock) or ARG1 mRNA (DC Arg mRNA), effector to target 10:1. B), D) IFNγ 
response by ARG1-specific T cell cultures from two melanoma patients to 
autologous B cells electroporated with irrelevant control mRNA (B cells Mock 
mRNA) or ARG1 mRNA (B cells Arg mRNA), effector to target 2:1. 

25 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
statistically significant response against one or more of tested ARG1 peptides in IFNγ ELISPOT 
demonstrating that ARG1 specific T cells can be found in patients with different forms of cancer.  
Some of these responses were also further characterized using intracellular staining for IFNγ and 
TNFα in response to ARG1 peptide stimulation, to show that both CD4+ and CD8+ T cell responses 
are present among the ARG1 responding T cells 1. 
 
Overall, ARG1-specific T cells are capable of recognizing ARG1-expressing cells,a vaccine that 
activates ARG1 specific T cells thereby attracting them to the tumor site and reducing ARG1 
expressing cells such as MDCSs could be an effective new approach to reduce tumor burden (Fig. 
10).The Th1 inflammatory response (IFNγ) induced by the ARG1-specific T cells could potentially 
induce IDO and/or PD-L1 expression in the immunoregulatory cells and cancer cells, thereby 
generating targets susceptible to PD1/PD-L1 immunotherapy.  
 
The mechanism of action (MoA) of the ARG1-vaccine differ from the MoA of the ARG1 inhibitors 
presented in this protocol as the ARG1-18,19,20 vaccine will not directly inhibit ARG1. However, 
by sharing the same target-enzyme, both the ARG1-inhibitor and the ARG1-vaccine will inhibit 
ARG1 and increase arginine levels. The anti-tumor results of the ARG1-inhibitor described 
previously therefore indicate that the ARG1-18,19,20 vaccine could induce a potential anti-tumor 
response in the treated patients 47.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 10. We in the CCIT hypothesize that the peptide vaccine consisting of ARG1 will boost the natural 
immunity mediated by ARG1 specific T cells as these T cells will accumulate at the tumor site thereby 
reducing ARG1 expressing regulatory immune cells (such as ARG1 secreting MDSCs). Consequently, we 
hypothesize that the ARG1 induced T cell suppression through arginine depletion will reverse.  
 
 
ARG1-specific T Cell 
Myeloid Dervied Supressor Cell 
  
↑Arginine 
↑ARG1 
↑ARG

26 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Peptide vaccine safety 
Like the PD-L1 and IDO peptide vaccines studied in CCIT, ARG1 specific T cells are not predicted to 
attack normal cells in non-cancerous tissues. It requires a high expression of a certain peptide in a 
cell to produce a high enough amount of peptide:MHC-complexes on the surface to overcome the 
threshold of being killed by peptide specific T cells. The affinity of T cell receptor to MHC:peptide 
complexes are 100-1000 fold lower than that between antibody and antigen. Consequently, many 
peptide:MHC-complexes have to be on the cell surface for the activation signal to be strong 
enough. Furthermore, the activation of peptide specific T cells is a slow process. Only by 
vaccinating several times, the amount of anti-peptide T cells will be sufficient. Finally, it is well 
described that “danger signals” are vital for the effector phase of an immune response. Thus, T 
cells preferentially engage in areas of inflammation with dying cells and other sources of “danger 
signals”. These signals are present in tumors but not in normal, healthy tissue. This further 
explains the general lack of toxicity in anti-cancer vaccinations utilizing self-antigens 48,49. 
 
Since the vaccine is based on a self-antigen, theoretically autoimmunity is a potential risk of the 
treatment. However, the peptide specific T cells are already present in unvaccinated healthy 
individual as well as cancer patients. Vaccinations with the peptides boost the activity of the 
peptide-specific T cells to engage regulatory cells and cancer cells and immune cells which express 
the proteins as part of the local immunosuppressive microenvironment in cancerous tissues.  
As for the ARG1 vaccine, a potential concern is interfering with ARG1 function in the urea cycle, a 
central pathway in the liver for detoxifying ammonia generated from the breakdown of amino 
acids. However, pharmacological ARG-inhibition has been well-tolerated in numerous animal 
studies, including a rat model of hypertension in which the ARG-inhibitor nor-NOHA was injected 
over a period of 10 weeks 50,51.  
Moreover, the oral ARG inhibitor (CB-1158) have been well-tolerated in a phase I trial 
(ClinicalTrials.gov identifier (NCT number): NCT02903914)( see Risk Evaluation). 
All in all, it appears very unlikely that the ARG1-18,19,20 peptide vaccine will initiate a general 
overactivation of the immune system.  
 
Preclinical development:  We conducted an exploratory study with vaccination of mice with the 
humane sequence of ARG1-18,19,20. The mice did not present immune responses to the human 
sequence. Similar results were obtained in murine vaccine studies with human sequence of PD-L1. 
The murine organs have been reviewed by a pathologist without any sign of organ damage (See 
“Preclinical exploratory study of vaccination ARG1 peptides in C57Bl6 mice with Montanide ISA-
51” appendix 09) 
Adjuvant 
A peptide antigen is administered with an immune enhancer known as an adjuvant. The adjuvant 
increases the uptake of the antigen by APCs, and mediates so-called “danger signals” to the APC. 

27 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
When an APC takes up and presents antigen in the context of a danger signal, it will deliver co-
stimulatory signals to T cells, which activates T cells that recognize the antigen.  
The adjuvant to be used is Montanide ISA-51. Montanide is an incomplete Freund’s adjuvant (IFA) 
which forms water in oil emulsion when mixed with an aqueous phase. It is commercially available 
from Seppic S.A, France, which maintains a Drug Master File. Montanide has been used in cancer 
vaccines in other research projects in more than 5000 patients corresponding to more than 50.000 
injections subcutaneously or intramuscularly. IFA is used with success in combination with long 
peptides, where clinically relevant responses can be produced with IFA. For instance in a vaccine 
with a long HPV-16 peptide against vulva cancer 52–54 
 
Montanide has a well-documented safety profile. Frequently reported side effects after injection 
are flu-like symptoms (headache, chills, fever and nausea) while most common local reactions are 
granuloma, local pain, inflammation or erythema. These reactions are generally classified as mild 
to moderate and they are typically transient in nature. 
 
Therapeutic peptide vaccines lack a suitable animal model for safety and dose testing 
Therapeutic peptide cancer vaccines are a group of biopharmaceuticals that lack a useful non-
human model for nonclinical safety evaluation according to the ICH S6(R1): 
 
1.  
To identify initial safe dose and subsequent dose escalation schemes in humans 
2.  
To identify potential target organs for toxicity and for the study of whether such 
toxicity is reversible; and 
3.  
To identify safety parameters for clinical monitoring 
According to the guideline it is recommended that safety evaluation is performed in a relevant 
species where the test material is pharmacologically active alternatively in transgenic animals or 
by use of homologous therapeutics.  
To create an animal model with completely transgenic TCR is not possible.  Furthermore, the 
amino acid sequences of ARG1, the target molecule in question, differ between humans and other 
animals, making the assessment of potential autoimmunity impossible using an animal model.  
Accordingly, it is not feasible to use a transgenic animal model to reproduce the pharmacological 
activity and toxicity that occurs in the human body because of the administration of peptide 
vaccines.  
The numerous limitations on standard preclinical animal studies have been described in a 
consensus paper by a collaboration of international societies for cancer immune therapy 55. This 
consensus paper underscores that “Even if mice were generated that expressed the appropriate 
HLA type and the human antigen sequences, such models might not adequately predict safety or 
autoimmune effects based on the diversity of the other components of antigen processing 
machinery involved.” A clinical development paradigm for cancer vaccines has been proposed by a 
group of more than 50 experts from academia, regulatory bodies and the biotech/pharmaceutical 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
industry from Europe and the US FDA 56. In this development paradigm, it is proposed to “…adopt 
a flexible approach, performing studies likely to be informative, while avoiding tests that 
contribute little valuable information” when performing preclinical safety testing of therapeutic 
cancer vaccines and the combination of cancer vaccines with other agents. Considerations for 
safety studies have been published57. The proposal is to focus on using in silico human expression 
data, and further that it is not feasible to replicate immune responses in animals since immune 
responses are dependent on human MHC-molecules.  
Regarding identification of initial dose, there are likewise no suitable animal models. In the case of 
peptide vaccination, the dosage should be higher than an unknown threshold level, below which 
presentation by antigen-presenting cells will not be sufficient. Doses higher than the threshold 
dose will not increase effectivity, but also do not cause increased toxicity since peptides are 
rapidly degraded by local peptidases to amino acids. The dosages selected for the current study 
are based on experience.  
In conclusion, studies in animals on safety, characterization of the pharmacokinetics, and 
determination of maximum tolerated dose (MTD) are not relevant in the case of therapeutic 
peptide vaccination. 
Chioce of dosage  
Cancer vaccines have emerged as a promising novel modality; however, identifying the maximum 
tolerated dose has been challenging. In contrast to cytotoxic agents, cancer vaccines modulate the 
immune system to target specific antigens on cancer cells and/or other immune cells. This 
mechanism of action is not expected to be directly dose related or to induce severe toxicity. 
Most trials that have been conducted testing the safety and efficacy of therapeutic cancer vaccines 
in dose escalation studies have not provided any correlation between dose escalation and toxicities 
or immune responses (monitored as cellular response) 58.  
In an analysis of clinical trial design suitable for therapeutic cancer vaccines, Rahma et al. found that 
the risk of serious toxicities with therapeutic cancer vaccines are extremely low and that toxicities do 
not correlate with dose levels.  Furthermore, the conventional dose escalation design, with few 
exceptions, is regarded not suitable for cancer vaccines. Therefore, the main goal is to determine a 
dosage that can create an immune response and the method of defining that dose depends on 
whether the vaccine belongs to a class of vaccines that has been previously tested in humans. If the 
vaccine class (e.g, a peptide) has been administered in humans and found to be non-toxic and able to 
induce immune response, then it is proposed using a dosecomparable to the dose used  in the 
previous trials58.  
Based on our previous clinical experience in the CCIT, administration of either 100 µg or 250 µg 
generates an intended immunological response measured by analyzing induced vaccine specific 
cellular response in the treated patients. Especially relevant in these settings is the finding that all 
the patients with stable disease (SD) who responded to treatment in a NSCLC trial exhibited a 
detectable vaccine specific T-cell response prior to vaccination (measured by IFN ELISPOT assay), 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
suggesting that the pre-existence of relatively high frequency anti-IDO T-cells in these patients 
correlate with the clinical efficacy 2,59.  
To conclude, therapeutic cancer vaccines are generally expected to have a relatively safe profile and 
dose-independent efficacy. Based on the above findings, the peptide dosage of a 100 µg of each 
ARG1-18,19,20 peptide has been chosen for the trial, as this dosage is believed to be relevant to 
achieve both immunological and clinical responses.  
Number of participants  
The study is designed as a phase I trial. Ten patients with metastatic solid tumors will be treated.  
If 3 or more patients experience grade 3-4 AE in phase I associated with the vaccination, the trial 
will be stopped.  
Patient recruitment  
Patients will be recruited nationally and included at the Department of Oncology at Herlev 
Hospital. Patients with metastatic, incurable solid tumors (NSCLC, colorectal cancer, urothelial 
cancer, breast cancer, ovarian cancer, malignant melanoma, and HNSCC) can be included 
regardless of what earlier oncological treatment they have received; i.e. surgery, radiation, high-
dose IL-2 or experimental treatment.  
It is estimated that patient inclusion can be completed within 12 months.   
Treating doctors at the departments of oncology can refer patients to the investigator. The 
investigator reads through the referral material and the patients are then invited to a talk about 
the protocol at the Department of Oncology at the experimental unit (EFEK). 
 
Information and informed content retrieval 
At the first appointment at the experimental unit, patients will be informed orally about the 
protocol by a doctor with experience in both oncology and immunotherapy under supervision of 
Professor Inge Marie Svane. The patient is always welcome to bring an assessor (relative, friend 
etc.) Written patient information in the form of ”Participant Information” will be handed out to 
the patient.  
The conversation with the participant takes place in an undisturbed room and 60 minutes is 
allotted.  
The information will be presented in an easily comprehensible language without the use of 
technical or value-laden terms. The consultation and written information will be in Danish, but in 
cases of foreign languages a certified translator will be provided. 
 
Information given during the consultation will be based on the written participant information and 
include the following: 

30 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
- 
Predictable risks, side effects, possible complications, disadvantages, as well as the 
possibility for unpredictable risks and complications 
- 
Other treatment options including advantages and disadvantages, as well as the 
consequences of no treatment and supportive care 
- 
Private and other confidential information contained in the medical record can be 
accessed both during and after the treatment. The medical record information will 
be  reviewed by third party officials during the statutory quality assessment of the 
trial 
- 
Other conditions which might have influence on the patient’s decision on 
participation  
- 
The possibility for the participant to withdraw their informed consent at any time 
after inclusion 
 
If the participant wishes to participate, a new appointment will be booked where the consent is 
signed by the doctor and the participant. The new appointment and time of reflection will be 
within 4-7 days after the first appointment, but if the patient needs more time of reflection this 
can also be accommodated. If the patient wishes to sign the content at the first appointment this 
is also acceptable. If the patient fulfills all in- and exclusion criteria the patient will be included and 
receive treatment immediately after.  
 
After inclusion, the participant will be informed, 
- 
If additional risks, side effects, complications or other disadvantages are discovered 
during the trial 
- 
If major changes are made to the protocol which might affect health or safety 
- 
If other relevant health conditions are discovered during the trial so forth the 
participant has not expressed a wish not to receive such information 
- 
About the results achieved and possible consequences to the patient so forth the 
participant has not expressed a wish not to receive such information 
- 
If the trial is terminated before schedule, the participant will be informed of the 
reason  
 
Information related to the participants will be protected under the legislation of processing of 
personal data and the health legislation, section 3 regarding patient’s rights.   
Patients will be receiving phone numbers to the “contact center” as well as the doctor on duty at 
the Department of Oncology. Patients are assigned a “contact doctor” and a “contact nurse” as 
well as instructions on how to reach these. 
Study design  
The study is a phase I study where 10 patients with metastatic solid tumors will be treated.  

31 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
If 3 or more patients experience drug related grade 3-4 AE in phase I, the trial will be stopped; 
grade ≥ 3 for fatigue and skin related AEs. A minimum of 2 vaccines must be administered before 
inclusion of another patient for each of the 3 first patients included in the study.  All patients will 
receive the same dose vaccine without dose escalation  
 
Inclusion criteria 
1. Age ≥ 18  
2. The patient has metastatic solid tumors (NSCLC, colorectal cancer, urothelial cancer, breast 
cancer, ovarian cancer, malignant melanoma or HNSCC); progressive or recurrent disease 
on or following treatment with standard of care agents 
3. At least one measurable parameter according to RECIST 1.1.  
4. The patient has an ECOG performance status of 0 or 1 
5. Life expectancy of at least 3 months  
6. Prior PD1/PDL-1 allowed 
7. The patient is a female of childbearing potential with negative pregnancy test 
8. For fertile women*: Agreement to use contraceptive methods with a failure rate of < 1 % 
per year during the treatment period and for at least 12 weeks after the treatment. Safe 
contraceptive methods for women are birth control pills, intrauterine device, contraceptive 
injection, contraceptive implant, contraceptive patch or contraceptive vaginal ring.  
9. For men: Agreement to use contraceptive measures and agreement to refrain from 
donating sperm 
10. The patient has met the following hematological and biochemical criteria: 
a. AST and ALT ≤2,5 X ULN or ≤5 X ULN with liver metastases 
b. Serum total bilirubin ≤1,5 X ULN or direct bilirubin ≤ ULN for patient with total bilirubin 
level > 1,5 ULN 
c. Serum creatinine ≤1,5 X ULN 
d. ANC (Absolute Neutrophil Count) ≥1,000/mcL 
e. Platelets ≥ 75,000 /mcL 
f. Hemoglobin ≥ 9 g/dL eller ≥ 5.6 mmol/L 
11. Mandatory provison of archival tissue and blood for biomarker testing at baseline  
12. Mandatory provison of blood for biomarker testing during the study 
13. Signed declaration of consent after oral and written information about the protocol 
 
* A woman is considered of childbearing potential (WOCBP), i.e. fertile, following menarche and until 
becoming post-menopausal unless permanently sterile. Permanent sterilisation methods include 
hysterectomy, bilateral salpingectomy and bilateral oophorectomy. A postmenopausal state is defined as no 
menses for 12 months without an alternative medical cause60 
 
 

32 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Exclusion criteria 
1. The patient has not recovered from surgery or is less than 4 weeks from major surgery 
2. The patient has a history of life-threatening or severe immune related adverse events on 
treatment with another immunotherapy and is considered to be at risk of not recovering 
3. The patient is expected to require any other form of systemic antineoplastic therapy or 
radiation therapy while receiving the treatment. However, radiation therapy treatment of 
non target lesion is allowed. 
4. The patient has a history of severe clinical autoimmune disease 
 
5. The patient has a history of pneumonitis, organ transplant, human immunodeficiency virus 
positive, active hepatitis B or hepatitis C 
6. The patient requires systemic steroids for management of immune-related adverse events 
experienced on another immunotherapy  
7. The patient has any condition that will interfere with patient compliance or safety 
(including but not limited to psychiatric or substance abuse disorders) 
8. The patient is pregnant or breastfeeding 
9. The patient is unable to voluntarily agree to participate by signed informed consent or 
assent 
10. The patient has an active infection requiring systemic therapy 
11. The patient has received a live virus vaccine within 30 days of planned start of therapy 
12. Significant medical disorder according to investigator; e.g. severe asthma or chronic 
obstructive lung disease, dysregulated heart disease or dysregulated diabetes mellitus 
13. Concurrent treatment with other experimental drugs 
14. Concurrent treatment with Valproate or Xanthin Oxidase inhibitors  
15. Known side effects to Montanide ISA-51 
16. Any active autoimmune diseases e.g. autoimmune neutropenia, thrombocytopenia or 
hemolytic anemia, systemic lupus erythematosus, scleroderma, myasthenia gravis, 
autoimmune glomerulonephritis, autoimmune adrenal deficiency, autoimmune thyroiditis 
etc. 
17. Severe allergy or anaphylactic reactions earlier in life  
 
Evaluation before the start of treatment 
The following parameters have to be performed within a month of the start of vaccinations (blood 
tests within two weeks):  
• 
Medical history and clinical examination 
• 
Performance status according to the ECOG scale 
• 
Toxicity evaluation 
• 
Concurrent medical intake 
• 
Electrocardiogram 

33 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
• 
Blood tests:  
O Hemoglobin, leukocyte differentiation count and platelets 
O Potassium, Sodium, Creatinine, albumin, uric acid, lactate dehydrogenase, Alkaline 
Phosphatase, Alanine transaminase (ALAT), amylase, bilirubin, ionized calcium, C 
Reactive Protein (CRP) 
O Hepatitis B, hepatitis C (IgG), HIV, HTLV-1(IgG), Epstein-Barr Virus EBV and 
Treponema.  
• 
Pregnancy test: Women of childbearing potential must have a pregnancy test 
performed at the time of screening. This involves women who are not surgically 
sterilized or postmenopausal  
• 
Diagnostic imaging: PET-CT, CT, or MRI scan of neck, thorax, abdomen, pelvis and 
optionally CNS and extremities depending on the cancer diagnosis.  
 
Conduct of the study 
Initial patient enrollment and treatment 
A minimum of 2 treatments with the peptide vaccine have to be administered before inclusion of 
another patient for each of the first 3 patients included in the study.  
 
 
 
1st patient 1st dose
2nddose
3rd dose
4th dose
5th dose
6th dose
7th dose
9th dose 
10th dose
Safety 
evaluation
Go/no go 
for next 
patient
2nd 
patient
1st dose
2nddose
3rd dose
4th dose
5th dose
6th dose
Safety 
evaluation
Go/no go 
for next 
patient
3th patient 
1st dose
2nddose
3rd dose
Safety 
evaluation 
before 
remaining 
patients’ 
inclusion 
for 
remaining 
patients
Go/no go 
for 
remaining 
patients
Dosing of 
remaining 
patients 
can start 
in parallel
x

34 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Treatment plan and treatment schedule 
Patients included in the protocol are treated with the ARG1-vaccine every third week for 48 
weeks, whereupon no additional vaccinations will be given. In total, 16 vaccines will be 
administered. Each vaccine consists of 100 µg ARG1-18, 100 µg ARG1-19, and 100 µg ARG1-20 in 
500 µl sterile water and 500 µl Montanide. In case of disease progression, patients are referred 
back to the oncology team, from which they were referred initially. Further treatment after 
disease progression will not be affected by participation in the study.Patients who receive all 
vaccines without progressive disease will have follow up appointments after 3 and 6 months in the 
experimental unit (EFEK) Herlev Hospital.  
Detalied treatment and evaluation schedule 
 
Treatment every third week 
Evaluation after every fourth vaccine 
Series no. 
Screening  
1.  
2.  
3.  
4. 
 
  
Day: 
1 
1 
1 
1 
21 days  
Year: 20___ 
  
   
  
  
Treatment 
    
  
    
    
    
ARG1-peptide vaccine  
0 
0 
0 
0 
  
Signed consent form 
0 
 
 
 
 
Anamnesis  
0 
 
 
 
 
Physical examination 
0 
0 
0 
0 
0 
Hight and weight 
0 
  
CTCAE 4.0 
0 
0 
0 
0 
0 
  
ECOG Performance status  
0 
0 
0 
0 
0 
  
BP, Puls, Tp. 
0 
0 
0 
0 
0 
  
Mediaction intake 
0 
0 
0 
0 
0 
  
Blood test 
  
 
  
Ipilimumab male/female* 
0 
0 
0 
0 
0 
  
12 HER EKS 
 0 
0 
p-HCG (if relevant) 
0 
  
  
  
  
MM1413/MM1414 screening* 
0 
   
  
  
  
Tumor evaluation 
  
   
   
  
CT scan of the cerebrum 
0 
  
  
  
  
CT/PET-CT/MRI scan 
0 
  
  
  
   
0 
Other 
 
 
 
 
 
 
EKG 
0 
  
  
  
  
Tumor biopsy 
0 
  
  
   
0 
DTH 
  
  
  
0 
 

35 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
 
 
Series no. 
5.  
6.  
7.  
8. 
 
Day: 
1 
1 
1 
1 
21 days  
Year: 20___ 
  
   
  
  
Treatment 
    
  
    
    
    
ARG1-peptide vaccine  
0 
0 
0 
0 
  
Signed consent form 
 
 
 
 
Anamnesis  
 
 
 
 
Physical examination 
0 
0 
0 
0 
Hight and weight 
0 
  
CTCAE 4.0 
0 
0 
0 
0 
  
ECOG Performance status  
0 
0 
0 
0 
  
BP, Puls, Tp. 
0 
0 
0 
0 
  
Mediaction intake 
0 
0 
0 
0 
  
Blood test 
  
 
  
Ipilimumab male/female* 
0 
0 
0 
0 
  
12 HER EKS 
  
0 
p-HCG (if relevant) 
  
  
  
  
MM1413/MM1414 screening* 
   
  
  
  
Tumor evaluation 
  
   
   
  
CT scan of the cerebrum 
  
  
  
  
CT/PET-CT/MRI scan 
  
  
  
   
0 
Other 
 
 
 
 
 
EKG 
  
  
  
  
Tumor biopsy 
  
  
   
DTH 
  
  
  
 
Series no. 
9.  
10. 
11.   
12. 
 
Day: 
1 
1 
1 
1 
21 days  
Year: 20___ 
  
   
  
  
Treatment 
    
  
    
    
    
ARG1-peptide vaccine  
0 
0 
0 
0 
  
Signed consent form 
 
 
 
 
Anamnesis  
 
 
 
 
Physical examination 
0 
0 
0 
0 
Hight and weight 
0 
  
CTCAE 4.0 
0 
0 
0 
0 
  
ECOG Performance status  
0 
0 
0 
0 
  
BP, Puls, Tp. 
0 
0 
0 
0 
  
Mediaction intake 
0 
0 
0 
0 
  

36 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Blood test 
  
 
  
Ipilimumab male/female* 
0 
0 
0 
0 
  
12 HER EKS 
  
0 
p-HCG (if relevant) 
  
  
  
  
MM1413/MM1414 screening* 
   
  
  
  
Tumor evaluation 
  
   
   
  
CT scan of the cerebrum 
  
  
  
  
CT/PET-CT/MRI scan 
  
  
  
   
0 
Other 
 
 
 
 
 
EKG 
  
  
  
  
Tumor biopsy 
  
  
   
DTH 
  
  
  
 
Series no. 
13.  
14.  
15.   
16. 
 
Evaluation  
Day: 
1 
1 
1 
1 
21 days 
1 
Year: 20___ 
  
   
  
  
 
Treatment 
    
  
    
  
    
 
ARG1-peptide vaccine  
0 
0 
0 
0 
  
 
Signed consent form 
 
 
 
 
 
Anamnesis  
 
 
 
 
 
Physical examination 
0 
0 
0 
0 
0 
Hight and weight 
0 
  
 
CTCAE 4.0 
0 
0 
0 
0 
  
0 
ECOG Performance status  
0 
0 
0 
0 
  
 
BP, Puls, Tp. 
0 
0 
0 
0 
  
 
Mediaction intake 
0 
0 
0 
0 
  
 
Blood test 
  
 
  
 
Ipilimumab male/female* 
0 
0 
0 
0 
  
 
12 HER EKS 
  
0 
 
p-HCG (if relevant) 
  
  
  
  
 
MM1413/MM1414 screening* 
   
  
  
  
 
Tumor evaluation 
  
   
   
  
 
CT scan of the cerebrum 
  
  
  
  
 
CT/PET-CT/MRI scan 
  
  
  
   
0 
 
Other 
 
 
 
 
 
 
EKG 
  
  
  
  
 
Tumor biopsy 
  
  
   
 
DTH 
  
  
  
 
 

37 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
*Detalied blood test description  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Follow up  
Patients who complete the treatment of vaccines for all 48 weeks will be followed in the 
specialized unit at EFEK 3 and 6 months after treatment.   
 
Mont  
3 
6 
Day: 
1 
1 
Year: 20_____ 
 
 
Toxicity evaluation and performance status 
0 
0 
Clinical examination  
0 
0 
Ipilimumab male/female  
0 
0 
Project blood samples 
0 
0 
Radiologic imaging with CT, MR or PET-CT scan according to 
cancer diagnosis. 
0 
0 
 
Duration of treatment:  
  
MM1413/1414 Screening 
Ipilimumab female 
Ipilimumab male 
Analysis  
B-Hæmoglobin  
B-Hæmoglobin  
B-Hæmoglobin  
B-Trombocytter 
B-Trombocytter 
B-Trombocytter 
B-Leukocytter 
B-Leukocytter 
B-Leukocytter 
B-Leukocyttype 
B-Leukocyttype 
B-Leukocyttype 
P-Creatinin 
P-Creatinin 
P-Creatinin 
P-Natrium 
P-Natrium 
P-Natrium 
P-Kalium 
P-Kalium 
P-Kalium 
P-Calcium-ion 
P-Calcium-ion 
P-Calcium-ion 
P-Fosfat 
 
 
P-Magnesium 
 
 
P-ALAT  
P-ALAT  
P-ALAT  
P-ASAT 
P-ASAT 
P-ASAT 
P-Basisk Phosphatase  P-Basisk Phosphatase  P-Basisk Phosphatase  
P-Bilirubin 
P-Bilirubin 
P-Bilirubin 
P-Albumin 
P-Albumin 
P-Albumin 
P-LDH 
P-LDH 
P-LDH 
P-CRP 
P-LH 
P-testosteron 
P-APTT 
P-FSH 
 
P-Koag II-VII-X 
P-Estradiol 
 
P-Koag II-VII-X (INR) 
P-Prolactin 
 
P-TSH 
P-TSH 
P-TSH 
P-Frit T4 
P-Frit T4 
P-Frit T4 
P-Frit T3 
 
 
HIVCOMBO 
P-Carbamid 
P-Carbamid 
P-HBVSAG 
P-Hydrogencarbonat 
P-Hydrogencarbonat 
P-HBVSAB 
P-Albumin 
P-Albumin 
P-HBVCAB 
P-Urat 
P-Urat 
P-HCVAB 
P-Amylase 
P-Amylase 
P-HTLVIGG 
P-Glucose 
P-Glucose 
P-EBV 
P-Cortisol 
P-Cortisol 
P-TREPONE 
 
 

38 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Treatment with the ARG1 peptide vaccine is maximum 48 weeks.  
Treatment will be stopped if there is a confirmed disease progression according to the immune-
related response criteria or by unacceptable toxicity  
 
Concurrent treatment:  
Vaccination with live virus vaccines are contraindicated during treatment and initiation treatment 
must be at least one month after any such vaccinations. Local palliative radiotherapy or surgery of 
solitary metastases can be performed during treatment.  
 
Procedures regarding treatment  
The vaccinations will be given at the Unit for Experimental Cancer Therapy Unit (EFEK) at the 
department of Oncology, Herlev and Gentofte University Hospital. EFEK is a unit specialized in 
conducting phase I first-in-human clinical trials. A 500 µl aqueous solution of 100µg ARG1-18, 
100µg ARG1-19, and 100µg ARG1-20 peptide dissolved in sterile water is mixed to an emulsion 
with 500µl Montanide ISA-51 and given as a subcutaneous injection on the lateral side of the 
upper arm preceded by disinfection. The site of injection will alternate between the left and right 
arm for each vaccination. 
 
During administration of the first 3 vaccines, the patient is observed for 30 minutes with 
measurements of pulse and blood pressure before and after the vaccine is given. The remainder of 
vaccines can be administered with observation of the patient for 15 minutes. Patients are 
observed for acute toxicities in the form of allergic reactions, including anaphylactic shock. Allergic 
reactions and/or anaphylactic shock will be treated per hospital guidelines.   
The vaccines are ordered after contact to the project nurse at the clinical experimental unit (KFE) 
and to the manufacturing unit. (SOP: ”Bestilling og håndtering af ARG1-18,19,20 peptid vaccine til 
patienter i protokol AA1809”, appendix 14). When the vaccine is received, the “receipt of 
transport” is signed. (SOP: “Transport og udlevering af ARG1-18,19,20 peptid og Montanide, 
AA1809” appendix 15). 
 
If the patient is prevented in attending the planned treatment day, the treatment can be moved to 
the following Tuesday or Thursday. If the treatment is postponed more than 6 weeks, patients will 
be excluded.   
   
 
 
 
Criteria for treatment modifications 
 

39 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Delay of treatment 
The treatment will be postponed if the patient experiences non-hematological AE grade ≥ 2, 
except fatigue and skin related AEs such as local reaction at the injection site. Otherwise the 
following criteria are applicable:   
 
Blood samples 
 
 
Criteria 
Leukocytes  
< 1,5 x 109 (grade 3) 
Neutrophilic granulocytes 
< 1,0 x 109 (grade 3) 
Thrombocytes 
< 50 x 109 (grade 3) 
Hemoglobin 
< 5 mmol/l (grade 3) 
Creatinine 
>3 x ULM (grade 3) 
ASAT 
>5 x ULN (grade 3) 
ALAT 
>5 x ULN (grade 3) 
Bilirubin 
>3 x ULN (grade 3) 
Other AEs 
 
Skin-related AE 
≥ grade 3 
Fatigue AE 
≥ grade 3 
All other AE  
≥ grade 2 
 
The treatment will be postponed until CTCAE grade < 2.  
Criteria for end of treatment 
1. Unacceptable toxicity (see adverse events) 
2. Disease progression 
3. On patient request 
4. Clinical judgement 
5. Other anti cancer treatment. However, radiotherapy treatment of to non target lesions is 
allowed. 
6. Pregnancy 
7. Delayed treatment 
 
Unacceptable toxicity:  
If treatment related AE grade 3-4 is not reduced appropriately within 12 weeks, the treatment will 
be stopped and the patient is excluded. Shorter delays can occur multiple times with no 
consequences for the patient or the trial.  
The patient will be excluded subject to the following toxicities:   
 
Blood sample 
Criteria 
Leukocytes 
< 1,0 x 109 (≥grade 4) 

40 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Neutrophilic granulocytes 
< 0,5 x 109 (≥grade 4) 
Thrombocytes 
< 25 x 109 (≥grade 4) 
Hemoglobin 
< 4 mmol/l (≥grade 4) 
Creatinine 
>4,5 x ULM (≥grade 4) 
ASAT* 
>8 x ULN (≥grade 4)* 
ALAT* 
>8 x ULN (≥grade 4)* 
 
Other Adverse Events 
Bronchospasms or hypersensitivity reactions 
≥ grad 3 
Skin related AE 
≥ grad 4 
All other AE 
≥ grad 3 
Any eye pain or reduced visual of grade 2 or above with no restitution within two weeks will 
lead to exclusion 
 
 
*ASAT/ALAT grade 3 with bilirubin > 1,5 x ULN 
** Bilirubin > 3 x ULN with ASAT/ALAT > 2,5 x ULN 
 
Disease progression: Treatment is terminated if disease progression occurs accorting to RECIST 
1.1  at predefined evaluation time points.  
Patient request: The treatment can be stopped at any time if the patient wishes so. 
Clinical judgement: The treatment can be stopped because of medical conditions at any time if 
the investigator finds it in the patients’ best interest. 
Other treatment: Patients will be excluded at any time a new treatment with an experimental 
drug or other systemic anticancer treatment is initiated after inclusion in this protocol. The patient 
will be excluded if systemic treatment with corticosteroids is initiated unless it is on vital indication 
and in agreement with the physician responsible for the protocol. Supportive treatment is given 
on usual medical indication and is to be specified in the patient chart and flow sheets. 
Pregnancy: A pregnancy test is to be performed before women of childbearing potential can be 
included in the study. Women of childbearing potential are to contact investigator immediately if 
pregnancy is suspected (absent or delayed menstruation etc.) at any point during the course of 
treatment and for 3 months after the end of treatment. A pregnancy test is performed if 
pregnancy is suspected and the participants can receive no more vaccination treatments and is 
terminated from the study if it turns out positive. A pregnant participant and the course of the 
pregnancy will be followed closely in the outpatient clinic. 
 
Delayed treatment: The patient is terminated from the study if the treatment is delayed more 
than 6 weeks. It is the responsibility of the principle investigator to assess the cause of delay and 
define the time point for the start of the delay. A patient can be delayed several times during the 
cause of treatment without being excluded. 
 

41 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Early termination from the study 
Participants, which are excluded from the study, will be referred back to the oncology team from 
which they came, for standard treatment. Patients that are excluded before the second vaccine 
will be replaced with new participants.  
 
Concurrent treatment 
• 
Supportive treatment is given based on clinical judgement and should be noted in 
the patient charge in accordance with existing practice 
• 
Local palliative radiotherapy is allowed 
Manufacture of the peptide vaccine 
Peptide 
For detailed description of peptide mixture production (see SOP “Fremstilling af ARG1-18,19,20 
peptid til protokol AA1809” appendix 12. 
 
The peptide sequence:  
IO112  
 
ARG 171-190;   AKDIVYIGLRDVDPGEHYIL 
ARG1-18        
ARG 181-200;   DVDPGEHYILKTLGIKYFSM 
ARG1-19 
ARG 191-210;   KTLGIKYFSMTEVDRLGIGK 
ARG1-20 
 
 
Peptides will be provided with above 90 % purity from external producer. Before administration 
they will be dissolved in sterile water, mixed and sterile filtrated through a 0,22 µm (30 mm Ø) 
filter. 
Producing the peptide vaccine 
The final production of the peptide vaccine will take place under GMP approved laboratory 
conditions (approved by the Danish Medicines Agency) which quality assures a sterile therapeutic 
product. It will take place in JM702, Herlev and Gentofte Hospital, authority number: 24223.  
 
Montanide ISA 51 
Montanide ISA 51 is produced and sold by Seppic INC: www.seppic.com. France. Montanide is 
delivered sterile and ready for use in ampules of 3 ml. Montanide ISA 51/Vaccine adjuvant is based 
on vegetable oil. See product resume or link: http://www.seppic.com/human-health/vaccine-
adjuvant/montanide-isa-51-@/1018/view-1042-seproduit.html  
 

42 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Preparing the vaccine 
The peptide mixture and Montanide is delivered to the dept. of hematology shortly before use by 
a project nurse or laboratory staff from Hematologic Laboratory 54P4. The peptide mixture 
constitutes 500 µl consisting of 100 µg ARG1-18, 100 µg ARG1-19, 100 µg ARG1-20 dissolved in a 
watery solution. Montanide ISA-51 also constitutes 500 µl and just before injection, the two 
solutions are mixed through an I-connector Luer lock into a 1 ml syringe. The vaccine preparation 
is marked with patient name, personal security number, date and time of preparation. 
For detailed description of the peptide preparation see SOP “Klargøring af ARG1-18,19,20 
peptidvaccine til protokol AA1809.”appendix 13. See also example of labels for the vaccine in 
“Eksempel på forsøgslægemiddeletiketter” appendix 06.  
 
Microbiological control 
Endotoxin and cultivation tests of the peptide preparations are performed at the Department of 
Clinical Microbiology, Herlev and Gentofte Hospital before use of the vaccines. 
Evaluation  
Data registration  
The patients are given a patient number at inclusion in the study to secure patient anonymity. 
Clinical personal and selected persons in the laboratory will have access to patient information, 
but only in form of personal ID and name, to secure proper treatment.  
The principal investigator has access to patient charts to obtain information regarding the cancer 
disease to be able to compare this information with the project specific analysis performed on 
cancer tissue and blood tests.  
All relevant data is registered in case rapport form in collaboration with the clinical research unit 
(KFE). The principal investigator is responsible for manufacturing of the case report form (CRF) and 
data registration in the CRF after the course of treatment. CRFs will be reported to sponsor. 
Sponsor and principal investigator are responsible for data analysis on all included participants. 
Participant data and CRF will be kept for 5 years in accordance with current guidelines for storage 
of sensitive data. Completion of a final report will be done in collaboration between the members 
of the project group.  
 
The analysis will include: 
1. 
Adverse events registration 
2. 
Immunological response 
3. 
Clinical endpoints 
 
Sensitive data and any other remaining samples will be anonymized at the end of the trial. 

43 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Following an approval from the Danish Data Protection Agency the Danish biotech company IO 
Biotech will gain access to pseudonymized trial data.   
 
Monitoring  
Monitoring of the trial will be done by the GCP unit at the University of Copenhagen. It is expected 
that the responsible GCP unit will contact and visit the principal investigator on a regular basis and 
request for access to monitoring of the different source documents in relation to the trial (Case 
Report Forms and other relevant documents). 
Audit from the Danish Medicines Agency and other health authorities are likewise allowed on 
request. Monitor has access to laboratory test results and other participant information needed to 
verify the entries made in the Case Report Forms. The principal investigator (or his/her substitute) 
agrees to cooperate with the monitor so that potential problems discovered during the monitoring 
process will be solved. 
 
Statistical considerations  
The study is a phase I trial with the primary objective to evaluate safety of the combination 
therapy. Since the trial is a first in human study, a formal sample size calculation cannot be 
performed. From experience with similar studies, it is deemed appropriate to include 10 
participants. 
Ten participants are deemed sufficient to evaluate safety and considered safe if less than 20 % 
experience grade 3-4 AE.  
 
All participants that are included and treated according to the protocol will be included in the 
statistical analyses. Participants who are excluded before receiving treatment will not be included 
in the statistical analyses:  
• 
Where the inclusion criteria at baseline are not fulfilled 
• 
Patients withdraw their consent 
• 
Have started other treatment 
 
For immunological responses, the 95 % confidential interval will be calculated for patient data and 
for response rates descriptive statistics will be used.  
 
Effect parameters  
Primary effect parameter 
Primarily, safety and tolerability of the combination therapy is assessed. This is done by 
registration of all adverse events and possible unwanted events that occur in relation to the 

44 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
treatment and in accordance with the CTCAE criteria. Data collection will take place in accordance 
with the data source list.  
 
Secondary effect parameter 
For the assessment of the immunological response, ARG1-specific T cells responses will be 
identified by the use of ELISPOT technology and antigen specific populations will be characterized 
for their cytokine secretion pattern. 
We will further examine the effect of the vaccine in different subtypes of immune cells during 
therapy. Flow cytometry analysis will be performed prior to, during and after treatment. The 
numbers of different subtypes of immune cells will be registered and compared e.g. memory T 
cells, Treg, Natural Killer (NK) and MDSC.  
 
Exploratory endpoints and objectives  
The clinical effect of the treatment will be assessed by measure of tumor responses by standard 
radiologic imaging with CT, MR, PET-CT, or a MR scan according to cancer diagnosis. Before 
inclusion a recent scan must to be available for review. Before hospital admission baseline imaging 
will be performed. The evaluation will be evaluated according to RECIST 1.1 and PERCIST with the 
endpoints being objective response (OR), progression free survival (PFS) and overall survival (OS).  
 
RECIST 
Clinical evaluation will be done in accordance with RECIST 1.1 Guidelines6 .  
Complete response (CR): All lesions disappear. 
Partial response (PR): Defined as ≥ 30% reduction in the sum of all measurable parameters’ 
longest diameter. 
Stabile disease: defined as < 30% reduction in the sum of all measurable parameters’ longest 
diameter or a < 20% increase in the sum of all measurable parameters’ longest diameter. 
Progression: Defined as a > 20% increase in the sum of all measurable parameters’ longest 
diameter or the appearance of new lesions. 
CR and PR is to be verified by examination at a minimum of 4 weeks after documentation of the 
response at the earliest. 
Immunologic monitoring  
Project blood samples for immunological monitoring 
110 ml blood will be sampled in Sodium-heparin prepared vials where mononuclear cells are 
isolated. 8 ml blood will be sampled in a dry medium for serum to be frozen. Plasma samples for 
gene analysis are made from 8 mL EDTA blood. Project blood samples will be taken 5 times the 
first year and thereafter at followup after three and six months.  

45 
 
Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Immune cells are isolated from the collected blood samples by use of leucosep/lymphopreb-
technique and are frozen in a biobank for later analyses; immune assays on viable immune cells. 
Antigen specific immune reactivity will be tested by use of a panel of relevant immunological 
assays including ELISPOT, ELISA, proliferation assays, cytotoxic assays, intracellular staining (ICS), 
and multimeric staining of ARG1 specific CD8 T cells. Blood and serum samples are processed and 
stored at Center for Cancer Immune Therapy (CCIT). 
 
Tumor biopsies  
Biopsies from the available tumor lesions or involved lymph nodes will be performed before the 
1st vaccine and after the 4th vaccine. Depending on the localization and accessibility, different 
types of biopsies will be performed. If the involved areas are not directly accessible, the biopsy will 
be conducted under sterile conditions via ultrasound.  
 
Tumor biopsies will be used for explorative assays including:  
• Immunoscore CR assay will be performed for quantification of CD3 and CD8 T cells in the core of 
the tumor for biopsy samples or in both the margin and the core of the tumor for resected tumors. 
This is done to asses “Immunoscores” predictive and prognostic value, which in turn will help 
select the patients who will benefit from the treatment and vice versa in the future.  
• Immunoseek CR assay will be performed to detect CD8+ lymphocytes and PD-L1+ immune and 
tumor cells by a multiplexed IHC assay on the same FFPE slid if possible. Standardized Image 
analysis will allow quantifying cells by digital pathology with the support of a pathologist.  
• IHC staining PD-1, will consist of positive PD-1 T cells quantification by digital pathology of a 
single FFPE biopsy slide.  
• IHC staining ARG1-18,19,20 will consist of ARG1-18,19,20 protein quantification by digital 
pathology on a single FFPE biopsy slide. 
Gene-based assays:  
• Immunosign CR and Pan Cancer Immune profiling on NanoString platform will be used for testing 
patient sample biopsies for gene expression quantification of different immune genes. 
• A whole exome sequencing performed on tumor samples and corresponding healthy tissues on 
pre-treated patient samples to assess initial mutational status for each patient.  
The aim of the planned gene analyses is to learn more about;  
1.  Differences among patients in expression level of a panel of relevant normal genes in the tumor 
microenvironment which could influence the chance of benefit from treatment.  
2.  Expression of tumor/patient specific mutated genes which could influence the chance of 
benefit from treatment.  
 
Analysis for identifying specific tumor gene expression signatures and mutations in the tumor 
cells, leading to patient-specific neo-antigens derived from these mutations will be performed. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
These analyses will contribute to identification of patients that are most likely to respond to 
treatment.  
 
 
 
Methods in gen-based assays 
A whole exome sequencing will be performed on tumor samples and corresponding healthy 
tissues on pre-treated patient samples to assess initial mutational status for each patient. 
 
This testing will be performed on Illumina NGS platform per batch of 14 samples.  
Total genomic DNA will be extracted from fresh frozen biopsy or from 1 FFPE biopsy block to 
prepare 1 FFPE slide biopsies of 20µm. The best would be to have fresh frozen biopsies. 
If a biopsy of healthy tissue cannot be provided then genomic DNA will be extracted from 5ml 
whole blood. 
 
Data regarding potential disease causing genes will be generated as a byproduct of this analysis, 
but this data will not be used or explored further upon, since only data regarding tumor specific 
genes will be processed more closely. Therefore, we do not expect to obtain explicit knowledge 
regarding disease causing genes. Furthermore, ‘Targeted sequencing’ on a limited number of 
defined genes will be performed on the tumor tissue to obtain a ‘immune profile’ to determine 
which genes- and consequently which proteins- are expressed in the tumor tissue. Data will be 
handled according to national laws.  
 
If by chance these analyses will discover known mutations with potential significant impact on 
patient’s health, the case will be discussed with the Clinical Genetics Department, Rigshospitalet, 
unless the patient has chosen not to be informed as stated in the patient information. If the 
patient wishes genetic counseling it will be offered by specialized doctors, if mutations with 
potential significant impact on the patient’s health are discovered. The following criteria will 
determine if further actions are indicated. 
• There is a reasonable degree of possibility that a genetic disposition is present, 
• There is solid documentation of a link between the genetic disposition and the development of 
disease, 
• The tests used to determine the genetic disposition are well established, 
• The disease in question can be prevented or treated, and 
• The link between the genetic disposition and the development of disease has considerable 
importance for the patient. 
If indicated, the patient will be contacted and asked for permission to referral to such Department 
for additional information and testing. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
In case that a patient dies/is dead, or do not want information regarding significant health issues, 
a medical assessment, using the 5 above mentioned criteria, will determine whether relatives to 
the patient is to be informed, in accordance with Danish law (sundhedslovens § 43, stk. 2, nr.2.). 
Delayed type hypersensitivity (DTH) & biopsy 
Delayed type hypersensitivity will be tested after the 4th vaccination. The test consists of up to a 
total of 3 intradermal injections of either an aqueous solution of ARG1 or the negative control 
saline. After 48 hours, the diameter of the reaction is measured, and 6mm punch biopsies are 
taken from the injection sites. The biopsies are divided in two, half is frozen for later investigation, 
half is used to grow skin-infiltrating lymphocytes (SKILs). SKILs are tested for specificity to the 
ARG1 peptides as a sign of induction of a functional immune response. The patient can refuse to 
undergo the DTH-testing without exclusion of the trial. 
Biobank and handling of biologic material 
In connection with the current study, blood samples (110 ml/blood sample) and tumor biopsies 
will be stored in coded form at -150 °C in a research biobank at the CCIT in room PA102 until all 
analysis concerning the study is performed. Blood samples will be taken during the 48 week 
treatment period and thereafter at the followups after three- and six months. The material is 
hereafter transferred to a biobank for future biomedical research for up to 15 years if accepted by 
the Danish Data Protection Agency, after which the remaining material will be destroyed.   
 
Analyses will be primarily performed at CCIT. However, some special analyses on tumor tissue or 
blood test samples will be performed at a partner institution after establishing a specific written 
agreement. All patient relevant information will be sent in an anonymous way. In case the 
patient’s cells will be sent to partner institutions located abroad, these will be handled according 
to national laws and regulations of the specific nation where these have been sent. In such a case, 
all patient information will be communicated in coded form. A written Data Processing Agreement 
will be signed between the data controllers and the data processors abroad. If any gene analysis 
are to be performed abroad, the data processing agreement will include the 5 criteria (See 
immunologic monitoring) regarding the discovery of known mutations with potential significant 
impact on patient’s health, as well as the requirement, that the partner abroad reports back to the 
primary project managers in Denmark so that relevant actions can be taken. If data processing is 
to be performed in a third-country, permission will be applied for at the Danish Data Protection 
Agency, or one of the agencies standard contracts will be used. 
 
Samples that are not used in the study will be transferred to a biobank at CCIT for future 
biomedical research for up to 15 years and if accepted by the Danish Data Protection Agency. If 
additional studies in other research areas are to be performed on any samples obtained during the 
conduct of this study/samples are transferred to a new biobank, a request to do so will be 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
submitted to the Scientific Ethics Committee, Capital Region of Denmark, as per the ‘Act of 
Processing of Personal Data §§ 5 and § 10, paragraph 2 and 3. After 15 years, any remaining tissue 
samples will be disposed of according to the local guidelines for destruction of biohazardous 
waste. 
If a patient withdraws his/her informed consent, all biological material is to be destroyed if the 
patient wishes so.  
Risk evaluation  
Evaluation of potential risks associated with the peptide vaccines are based on clinical experiences 
from other cancer vaccination trials.  
 
Acute reactions 
Subcutaneous injections can in theory cause anaphylactic reactions as any other vaccine, but so far 
it has not occurred for cancer vaccines.  
The kinetics of immune induction by cancer vaccines is slow, - that is weeks to months as 
compared to check point inhibitors.  
 
Autoimmunity 
There is a small theoretical risk of inducing autoimmunity when/if the vaccine eliminates the 
immunosuppressive arginase positive cells because arginase is a self-antigenpresent in various cell 
types in both unvaccinated healthy individuals as well as cancer patients.  
However, all previous trials with peptide vaccinations have had very mild side effects, no matter 
which proteins have been targeted, as reviewed by Rahma and colleagues 58 .  
The ARG1 vaccine is in many ways similar to the IDO vaccine tested at CCIT, and there are no 
apparent reasons the toxicity should be higher when vaccinating against ARG1. Both ARG1 and 
IDO are self-proteins with a broad expression. Both molecules are lymphocyte-suppressive they 
are all are overexpressed in cancer cells and immunosuppressive cancer-associated immune cells. 
The adverse events in our trials with IDO vaccination have been at as low a level as with other 
peptide vaccines; in the study by Iversen et al, no grade 3-5 toxicities were seen 2. 
A phase I trial treating cancer patients with an oral ARG1 inhibitor (CB-1158) presented data on 
adverse effects in April, 2017. A total of 17 patients with advanced solid tumors have been treated 
with CB-1158, and the drug was generally well tolerated with no drug-related serious adverse 
events.  Treatment related adverse events were limited to a single case of Grade 1 anemia, 
fatigue, increased ALT and myalgia.  No Grade 3 treatment related adverse events were reported. 
Plasma levels of arginase were inhibited >90% in all patients, and in 10 of 11 patients plasma 
arginine increased 1.5 fold or more3. As mentioned earlier (see Assement of safety)  the ARG1 
peptide vaccine and CB-1158 differ in both mechinsm of action and administration way, making 
them very hard to compare. However, they both target the same enzyme and the AEs registered 
for patients trateted with CB-1158 could possibly indicate what AEs to expect in the ARG1 trial.  

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
 
Tolerance 
In contrast to autoimmunity there is also a theoretical risk of tolerance development leading to 
increased immune suppression and tumor tolerance and thus increased disease aggressiveness. 
However, this phenomenon is purely theoretical and the eventual risk is unknown.  
 
 
Procedures and examinations 
Patients included in the trial will have additional blood tests performed compared to patients 
receiving standard treatment. The amount of blood and the specific procedure when taking a 
blood sample are not associated with any significant risk. There is a minimal risk of bleeding and 
infection and pain and bruising can occur in the area.  
 
Tumor biopsies will be taken by experienced doctors, and is not associated with any significant 
risk; again there is a small risk of bleeding and infection and pain and bruising might also accur in 
the area.  
 
CT scans and PET-CT scans are not considered a risk based on the low dose radiation. Both full 
body CT and PET-CT scans give a radiation doses of around 15 milliSievert (mSv), which is about 5 
times the annual background radiation61,62. The contrast used can very rarely cause allergic 
reactions and brief reduction in renal function; therefore patients are screened by standard 
procedures prior to the examination.  
 
Monitoring of toxicity 
Adverse events 
Adverse events (AE) are defined as any undesirable experience occurring to a subject during a 
clinical trial, whether or not considered related to the investigational treatment. All AEs reported 
spontaneously by the subject or observed by the investigator or his staff will be recorded and 
described in the patient chart and the electronic Case Report Form (eCRF). The severity and 
consequences will be recorded for each AE. The severity and relation to the study medication will 
be assessed in accordance with the guidelines described in the following. 
The investigator must attempt to identify all clinical and objective events from patients receiving 
treatment and determine their relation to the study medication. The investigator determines the 
relationship between AEs and treatment using the following guidelines: 
 
Grading of adverse events  
The severity of an AE refers to the intensity of the reaction. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Events are graded using CTCAE version 4.0. The following scale can be used if this grading is not 
applicable:  
 
1 = light 
2 = moderate 
3 = severe 
4 = life threatening 
5 = lethal 
 
Patients experiencing AEs will be monitored with the relevant clinical evaluations and laboratory 
investigations assessed by the attending physician. All AEs must be monitored until satisfactory 
restitution or stabilization. Results of the monitoring must be recorded in the patient chart and 
eCRF. 
Abnormal laboratory tests are not to be recorded in the eCRF unless they have caused a clinical 
event, resulted in termination of the treatment or otherwise meet the criteria of a serious AE (see 
the following). 
 
Serious adverse events 
A serious adverse event (SAE) is to be reported to sponsor within 24 hours and is defined as any 
medical occurrence or effect that occurs at any dose: 
 
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalization or prolongation of existing patients’ hospitalization; 
- 
results in persistent or significant disability or incapacity; 
- 
leads to a congenital anomaly or birth defect; 
- 
is a significant medical event 
 
Adverse reactions 
An adverse reaction (AR) can be expected if described in the IMPD or the relevant product 
summary, or unexpected if the grade or severity does not correlate with the product information 
in the before mentioned documents.  
If the AR is unexpected, meets the criteria of a serious adverse reaction (SAR) and is found related 
to the investigational treatment, it is classified as a suspected unexpected serious adverse reaction 
(SUSAR). As this is a First in human trial, the SARs are difficult to classifiy as “expected” or 
“unexpected.” Therefore, SARs will be reported as SUSARs.    
 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
Reporting of adverse events and adverse reactions 
Investigator reports SAEs, SARs and SUSARs to sponsor within 24 hours. Sponsor reports SARs and 
SUSARs to the  Danish Medicine Agencywithin 7 days if considered life threatening or fatal, and 
otherwise within 15 days. Consequences for the study must be reported. Sponsor submits a list 
annually that summarizes any SARs and SUSARs as well as a report regarding the study patients’ 
safety to the Danish Medicine Agency and the Research Ethics Committee (investigator can report 
to the Research Ethics Committee as well). 
Sponsor submits a final report to the Danish Medicine Agency at the end of the study, with a 
description of all SAEs, SARs and SUSARs. 
 
The following is not to be reported: 
 
- 
Deaths caused by the malignant disease or progression 
- 
Hospitalizations or prolongation of current hospitalization caused by the malignant 
disease: 
o 
weight loss 
o 
fatigue 
o 
electrolyte derangement 
o 
pain management 
o 
anxiety 
o 
palliative hospitalization 
o 
stay at hospice or terminal care 
o 
progression of the underlying disease 
 
- 
Hospitalizations or prolongation of current hospitalization if the sole reason for 
hospitalization or prolongation is: 
o 
fluid treatment or treatment of nausea 
o 
blood transfusion 
o 
platelet transfusion 
o 
febrile leucopenia/neutropenia 
o 
administration of investigational procedures 
o 
placement of a permanent intravenous catheter 
 
These events are to be registered in the eCRF. 
 
Known adverse reactions  
Montanide ISA-51: 
Montanide ISA-51 has been used in more than 150 clinical trials. Montanide has a well-
documented safety-profile. Frequently reported side effects after injection are flu-like symptoms 
(headache, chills, fever and nausea) while most common local reactions are granuloma, local pain, 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
inflammation or erythema. These reactions are generally classified as mild to moderate and they 
are typically transient in nature.  
Reference is made in attached drug information leaflet (appendix 11).   
 
Arginase peptide:  
The peptide has not been tested in humans, so no adverse reactions have been documented.  
Handling adverse events 
Autoimmune side effects will be handled according to a local guideline for handling side effects for 
checkpoint inhibitors based on recommendations by Weber et al63. This guideline is in Danish and 
named “Vurdering og håndtering af bivirkninger relateret til behandling med ipilimumab, 
nivolumab og pembrolizumab” and is available online and in print for the physician at duty. In the 
highly unlikely case that the vaccine induces a cytokine release syndrome, also known as a 
cytokine storm, the patient will be treated in accordance with the guideline on treatment of 
cytokine release syndrome recently published by Brudno et al64.  
Patients will be admitted at the Department of Oncology, Herlev Hospital, in case of occurrence of 
adverse events related to the investigational treatment that requires hospitalization.  
The intensive care unit at Herlev Hospital will be informed of the study, since intensive care 
support could potentially be required if a treated patient develops anaphylactic shock. The 
information will be given prior to the start of patient inclusion in the protocol and treatment will 
not begin before the department management at the intensive care unit has confirmed that the 
information is received. 
Insurance 
Patients’ participation in the study will be covered by the hospital’s liability insurance. 
Ethical aspects 
This project will potentially add to existing knowledge in the field of immune therapy and help 
improve treatments and thus prognosis of prognosis of metastatic solid tumors.  
There does not seem to be unacceptable risks or disadvantages regarding the planned treatment 
based on the current knowledge on peptide vaccines (see peptide vaccine safety). 
 
Participation is voluntary and is preceded by oral and written information and the treatment will 
be stopped in case of unacceptable adverse reactions or if the patient wishes so at any time. The 
patient will receive treatment after the current guidelines at the department or referring 
department if he/she does not wish treatment according to the protocol. The study is therefore 
assessed as ethically proper. 

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
The study follows the Helsinki agreement and the principal investigator is to obtain permission 
from the Danish Medicine Agencyand the Research Ethics Committee. 
Reporting to the Danish Data Protection Agency 
The study is reported to the Danish Data Protection Agency when approvals from the Danish 
Medicine Agency and the Research Ethics Committee are obtained. The law dealing with personal 
data will be respected. Information related to study patients is protected according to the law on 
personal data and the Act on Research Ethics Review of Health Research Projects. 
End of study report 
Sponsor will inform the Danish Medicine Agency and Research Ethics Committee within 90 days of 
study completion. The definition of study completion is 6 months after the last patients’ treatment 
or after exclusion due to progression. If the study is prematurely terminated, the Danish Medicine 
Agency will be informed of the reason(s) for the termination. Sponsor will submit a final study 
report to the Danish Medicine Agency and the Research Ethics Committee with the study results 
including publications based on the study within a year of study completion. 
Publications 
Cathrine Lund Lorentzen, Mads Hald Andersen and Inge Marie Svane constitute the group 
responsible for the project.  
Under the circumstance that the Vancouver rules are met, the group responsible for the project 
holds equal rights to the achieved results. The use and presentation of the data in any form, orally 
or written, can only take place with accept from everybody in the group. Positive, negative and 
inconclusive results will be reported in scientific journals. The order of authorship is determined by 
effort and the investigators will be co-authors on further publications derived from this study. The 
main analysis will be done when all patients have been treated which is expected to be during the 
year 2020/21.  
Economy  
The trial is fully financed. Funding has been acquired partly through the Department of Oncology 
and through IO Biotech ApS. IO Biotech is a biotech company established in the light of peptide 
vaccine patents from CCIT and “Region Hovedstaden”. Lundbeck, Novo and Sunstone have 
invested in IO Biotech. The project is co-funded through a research agreement between the capital 
region and IO Biotech with 2.500.000 Danish crowns over 3 years. The money will be transferred 
to a research account with a doctor salary of 3 years and 700.000 Danish crowns for immune 
analyses for the vaccinated patients. IO Biotech also finances the vaccine products, while the 
remaining expenses are covered by CCIT and the department of oncology.  

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Protocol: AA1809, Arginase-1 peptide vaccine in patients with metastatic solid tumors. Copenhagen, Date: 
20-08-2018 , version: 2.3,   Eudra CT no: 2018-000719-26 
The project has been initiated by Mads Hald Andersen, Inge Marie Svane and Cathrine Lund 
Lorentzen 
The ARG1 vaccine is developed by Mads Hald Andersen who is a part of the management at the 
CCIT. By Danish law on public inventions at public institutions, the capital region holds the patent, 
which is licensed for commercialization through the industrial partner IO Biotech. In the case of a 
sale of the patent, by law the income will be distributed with 1/3 for the inventor, 1/3 for CCIT, 
1/6th to Herlev and Gentofte Hospital and 1/6th to Tectra, the technology transfer unit of the 
capital region.     
 
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