Conditions: Metastatic Melanoma
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 48
Healthy volunteers: no

Brief summary:
Combination therapy is becoming more and more general in the treatment of oncological diseases. In this clinical trial combination the standard immunotherapeutic treatment; the programmed death 1 (PD-1) regulatory antibody Nivolumab and a peptide vaccine consisting of programmed death ligand 1 (PD-L1) and Indoleamine 2,3-dioxygenase (IDO) peptides will be tested in patients with metastatic melanoma. Patients will be treated with Nivolumab every second week as long as there is clinical benefit. The PD-L1/IDO peptide vaccine is given from start of Nivolumab and every second week for the first 6 vaccines and thereafter every fourth week up to 1 year.

Detailed description:
Background:

Huge advances have been made in the treatment of metastatic melanoma (MM) the past 5 years. Especially immunotherapy has shown promising results.

Cancer cells are naturally attacked by cells of the immune system, but can induce a state of tolerance whereby they escape from immune attack. This escape is brought about by many mechanisms. An important one is the programmed death pathway (PD-1/PD-L1). PD-L1 is commonly overexpressed on cancer cells. Interaction of PD-1 on activated T cells and PD-L1 on cancer cells lead to inhibition of the cytotoxic T cells. Another important mechanism is through overexpression of the metabolic enzyme IDO on cancer cells. Activation of IDO also inhibits cytotoxic T cells.

Investigators have recently identified spontaneous T cell reactivity against PD-L1 and IDO in the tumor microenvironment and in the peripheral blood of patients with MM and healthy donors. Both IDO and PD-L1 reactive CD8 T cells are cytotoxic and can kill cancer cells and immune regulatory cells in vitro.. Thus boosting specific T cells that recognize immune regulatory proteins such as IDO and PD-L1 may directly modulate immune regulation.

Due to distinct mechanisms of action, the combination of treatment with a monoclonal antibody targeting PD-1 (Nivolumab) and a vaccine with peptides against PD-L1 and IDO may have a synergistic effect.

Investigators have previously reported a phase I trial where, the IDO peptide was tested in 15 patients with MM in combination with Ipilimumab, and no grade 3-4 toxicity was seen. The PD-L1 peptide is currently being tested in a first-in-man study in patients with multiple myeloma.

Methods:

A two-step clinical phase I/II trial design will be used, starting out with a pilot study including 6 patients with MM to test feasibility and tolerability. If the treatment is found feasible the study will be extended to a phase II study with 24 patients. The objective is to describe anti-tumor immune responses and objective responses using RECIST 1.1.

Patients will be treated with Nivolumab in accordance with standard regimen, which involves outpatient IV infusions every second week as long as there is clinical benefit. The PD-L1/IDO peptide vaccine is given from start of Nivolumab and every second week for the first 6 vaccines and thereafter every fourth week up to 1 year. 15 vaccines will be administered in total.

Patients will be followed with clinical controls and diagnostic imaging every 12 weeks. Patients who receive all vaccines will have follow up after 3 and 6 months in parallel with standard of care treatment for Nivolumab.

Arms:
arm 1: All patients receive the same treatment. Patients included in the protocol are treated with Nivolumab according to usual guidelines, implying outpatient IV infusions of 3 mg/kg biweekly until progression.

The vaccine is administered on the same day as the administration start of Nivolumab. The vaccination is given biweekly for a total of 6 times, then every fourth week up to week 47, whereupon no additional vaccines will be given. In total, 15 vaccines will be administered. A vaccine consist of 100 μg IDO long peptide, 100 μg PD-L1 long1 peptide and 500 microliters Montanide as adjuvant.

Patients who complete all vaccines will continue Nivolumab treatment after standard guidelines.

Interventions: intervention 1: Nivolumab intervention 2: PD-L1/IDO peptide vaccine
Intervention descriptions: intervention 1: Nivolumab 3 mg/kg is administered biweekly as long as there is clinical benefit. intervention 2: The vaccine is administered biweekly for a total of 6 times, then every fourth week up to 47 weeks, whereupon no additional vaccinations will be given. A total of 15 vaccines will be administered. A vaccine consists of 100 μg PD-L1 long1 peptide, 100 μg IDO long peptide and 500 μl Montande as adjuvant.

Protocol document:
Prot_SAP_000.pdf: 
4-10-2022
Combination therapy with Nivolumab and
PD-L1/IDO peptide vaccine to patients with
metastatic malignant melanoma
A phase I/II trial
EudraCT number: 2016-004527-23
Sponsor
Inge Marie Svane, MD, Professor, Ph.D.
Vice director for Center for Cancer Immune Therapy
Dept. of Oncology
Herlev & Gentofte University Hospital
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev
Phone: +45 38 68 21 31
E-mail: inge.marie.svane@regionh.dk
Principal Investigator
Cathrine Lund Lorentzen, MD, Pd.D. student
Center for Cancer Immune Therapy
Dept. of Oncology
Herlev & Gentofte University Hospital
Borgmester Ib Juuls Vej 25C, 5th floor, 2730 Herlev
Phone: +45 38 68 93 83
E-mail: Cathrine.lund.lorentzen@regionh.dk
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Sponsor
Inge Marie Svane, MD, Professor, Ph.D., Consultant
Vice director for National Center for Cancer Immune Therapy, CCIT-DK
Dept. of Oncology, Herlev Hospital
Borgmester Ib Juuls Vej 25C, 5th floor
DK-2730 Herlev
Phone: +45 38 68 21 31
E-mail: inge.marie.svane@regionh.dk
Principal Investigator
Cathrine Lund Lorentzen, MD
National Center for Cancer Immune Therapy, CCIT-DK
Dept. of Oncology, Herlev Hospital
Borgmester Ib Juuls Vej 25C, 5th floor
DK-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 National Center for Cancer Immune Therapy, CCIT-DK
Dept. of Oncology, Herlev Hospital
Borgmester Ib Juuls Vej 25C, 5th floor
DK-2730 Herlev
Phone: +45 38 68 26 02
E-mail: mads.hald.andersen@regionh.dk
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
Clinical Research Center
Department of Oncology, Herlev Hospital
Borgmester Ib Juuls Vej 25C, 5th floor
DK-2730 Herlev
Phone: +45 38 68 23 07
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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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Table of Contents
1.1                Objectives and endpoints ..................................................................................................................................... 9
1.2                Background................................................................................................................................................................. 9
1.3                Toxicity........................................................................................................................................................................10
1.4                Target population...................................................................................................................................................10
1.5                Number of participants........................................................................................................................................11
1.6                Patient recruitment................................................................................................................................................11
1.7                Treatment plan ........................................................................................................................................................11
1.8                Toxicity evaluation .................................................................................................................................................11
1.9                Immunological evaluation ..................................................................................................................................11
1.10              Clinical response evaluation...............................................................................................................................12
2.1                Malignant melanoma............................................................................................................................................13
2.2                Standard treatment of malignant melanoma .............................................................................................13
2.1                Immune activation and regulation ..................................................................................................................14
2.2                Cancer cells induce local tolerance .................................................................................................................16
2.3                IDO and its potential as a vaccination antigen...........................................................................................17
2.3.1             IDO and its function.........................................................................................................17
2.3.2             Preclinical data on IDO ...................................................................................................17
2.4                PD-L1 and its potential as a vaccination antigen ......................................................................................19
2.4.1             PD-1/PD-L1 axis.................................................................................................................19
2.4.2             Preclinical data on PD-L1...............................................................................................20
2.5                Mechanism of Action – vaccination therapy ...............................................................................................22
2.6                PD-1 and PD-L1 blocking antibodies.............................................................................................................25
2.7                Adjuvant.....................................................................................................................................................................25
2.8                Rationale behind the peptide vaccine with IDO & PD-L1......................................................................27
2.9                Rationale behind the combined treatment strategies.............................................................................28
2.10              CCIT’s experience with IDO and PD-L1 peptide vaccines.......................................................................28
2.10.1           NSCLC and IDO peptide vaccine................................................................................29
2.10.2           MM and IDO peptide vaccine in combination with Ipilimumab..................30
2.10.3           Multiple myeloma and PD-L1 peptide vaccine after high dose
chemotherapy...............................................................................................................................................30
2.11              Therapeutic peptide vaccines lack a suitable animal model for safety and dose testing .........30
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5.1                Inclusion criteria......................................................................................................................................................33
5.2                Exclusion criteria .....................................................................................................................................................34
5.3                Evaluation before the start of treatment.......................................................................................................35
7.1                Initial patient enrollment and treatment.......................................................................................................36
7.2                Treatment plan and treatment schedule.......................................................................................................36
7.4                Follow up ...................................................................................................................................................................39
7.5                Procedures regarding treatment......................................................................................................................39
7.7                Criteria for treatment modifications ...............................................................................................................41
8.1                Early termination from the study .....................................................................................................................43
8.2                Concurrent treatment...........................................................................................................................................43
10.1              Peptides......................................................................................................................................................................44
10.2              Producing the peptide vaccine .........................................................................................................................44
10.3              Montanide ISA 51...................................................................................................................................................44
10.4              Preparing the vaccine ...........................................................................................................................................44
10.5              Microbiological control........................................................................................................................................45
12.1              Data registration.....................................................................................................................................................45
12.2              Monitoring ................................................................................................................................................................46
12.3              Statistical considerations.....................................................................................................................................46
12.4              Effect parameters....................................................................................................................................................47
12.4.1           Primary effect parameter...............................................................................................47
12.4.2           Secondary effect parameter .........................................................................................47
12.4.3           Tertiary effect parameter ...............................................................................................47
12.5              Immunologic monitoring ....................................................................................................................................48
12.5.1           Blood samples for immunological monitoring....................................................48
12.5.2           Tumor biopsy for tumor immune microenvironment evaluation ...............48
12.5.3           Delayed type hypersensitivity (DTH) & biopsy ....................................................50
12.6              Biobank and handling of biologic material..................................................................................................51
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12.7              Risk evaluation.........................................................................................................................................................52
13.1              Adverse events ........................................................................................................................................................55
13.2              Grading of adverse events..................................................................................................................................55
13.3              Serious adverse events.........................................................................................................................................55
13.4              Guidelines for adverse events’ possible relation to the treatment.....................................................56
13.5              Adverse reactions ...................................................................................................................................................56
13.6              Reporting of adverse events and adverse reactions ................................................................................56
13.7              Known adverse reactions.....................................................................................................................................57
13.7.1           Montanide ISA-51.............................................................................................................57
13.7.2           IDO long peptide...............................................................................................................57
13.7.3           PD-L1 long1 peptide........................................................................................................57
13.7.4           Nivolumab............................................................................................................................58
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Abbreviations
ALAT
ANC
ASAT
CCIT
CNS
CR
CRR
CRF
CRP
CT
CTCAE
CTL
CTLA-4
DMSO
EMA
ECOG
MHC
IDO
IDO long
IgG
IL-2
INR
irRC
LDH
MART
MDSC
MHC
MM
MR
NSCLC
ORR
OS
PBS
PD-1
PD-L1
PD-L1 long1
PD-L2
PERCIST
P-alanine-aminotransferase
Absolute Neutrophilic Count
P-aspartattransaminase
Center for Cancer Immune Therapy
Central Nerve System
Complete Response
Complete Response Rate
Case Report Form
C - reactive protein
Computer tomography
Common Terminology Criteria for Adverse Events
Cytotoxic T lymphocyte
Cytotoxic T-Lymphocyte Antigen 4
Dimethylsulfoxid
European Medicines Agency
Eastern Cooperative Oncology Group
Human Leukocyte Antigen
Indolemamine-pyrrole 2, 3-dioxygenase
Indolemamine-pyrrole 2, 3-dioxygenase long
Immunoglobulin G
Interleukin 2
International Normalized Ratio
Immune Related Response Criteria
Lactate-dehydrogenase
Melanoma Antigen Recognized by T cells
Myeloid-derived Suppressor Cell
Major Histocompatibility complex
Malignant melanoma
Magnetic resonance
Non-Small Cell lung cancer
Objective Response Rate
Overall survival
Phosphate buffered saline
Programmed cell death 1
programmed cell death ligand 1
programmed cell death ligand 1 long1
programmed cell death ligand 2
PET Response Criteria in Solid Tumors
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PET
RECIST
PR
SD
S-HCG
SKILs
SOP
TAA
TADC
TNM
Treg
UG-team
Positron Emissions Tomography
Response Evaluation Criteria in Solid Tumors
Partial Response
Stabil Disease
serum-Choriogonadotropin
Skin Infiltrating Lymphocytes
Standard operating procedure
Tumor Associated Antigens
Tumor Associated Dendritic Cells
Tumor-Node-Metastases
Regulatory T cells
Urologisk-Gynækologisk team
The peptides mentioned in the protocol: (same peptide has different names)
IO101
→IDO short
IO102
→IDO long
IO103
→PD-L1 long1
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Protocol synopsis
1.1
Objectives and endpoints
The primary objective is to assess tolerability and safety of the peptide vaccine containing the peptides
Indolemamine 2,3-dioxygenase long (IDO long) and programmed death ligand-1 long1 (PD-L1 long1) with
Montanide ISA 51 as adjuvant to patients with metastatic malignant melanoma (MM) in combination with
the immune checkpoint blocking antibody Nivolumab. The endpoint is adverse events (AE) assessed by
CTCAE 4.0.
The secondary objective is to evaluate the immune response before, during and after treatment. The
endpoint is responses of PD-L1 and IDO specific T cells measured by ELISPOT and cytokine release assays.
The tertiary objective is to evaluate the clinical efficacy of the treatment. The endpoints will be objective
response (OR), progression free survival (PFS) and overall survival (OS).
1.2
Background
MM is the 5th most common cancer in Western Europe. Contrary to other solid tumors which primarily
affect elderly people, MM is known to affect younger and middle-aged people. The incidence has increased
over the last 15 years with about 3-5 % per year. In Europa alone, 20.000 deaths in 2008 were caused by
MM.1
While traditional oncological treatments such as chemotherapy and radiation therapy have a poor effect on
MM,2 immunotherapy has shown very promising results. One of the novel immunotherapeutic
treatments, Nivolumab, was in 2015 approved as first-line standard therapy to patients with MM.
Cancer cells are naturally attacked by cells of the immune system, among others the cytotoxic T cells. Cancer
cells can induce a state of tolerance whereby they can escape from the immune attack.
This escape is brought about by many different mechanisms; some of the most important are through
overexpression of the programmed cell death 1 (PD-1)/PD-L1 molecules and the metabolic enzyme IDO.
The PD-1 receptor is expressed on T cells amongst other cells. The blocking of PD-1 on T cells with PD-1
blocking antibodies protects the T cells from the inactivation signal from PD-L1 expressed by cancer cells
or immune regulatory cells. Nivolumab is a PD-1 blocking antibody, which has shown an objective clinical
response rate in 30 % of the patients with MM.3
The expression of the metabolic enzyme IDO is induced on tumor cells upon exposure to IFN-γ. Activation of
IDO inhibits cytotoxic T cells by depleting the microenvironment of amino acids crucial to the function of
lymphocytes.4
Center for Cancer Immune Therapy (CCIT) has identified spontaneous T cell reactivity against PD-L1 and
IDO in the tumor microenvironment and in the peripheral blood of various cancer patients and healthy
donors. The IDO reactive CD8+ T cells were cytotoxic and could kill both cancer cells and immune regulatory
dendritic cells in vitro. The PD-L1 reactive CD8+ T cells were also cytotoxic and were able to kill cancer cells
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and myeloid derived suppressor cells (MDSCs). Thus boosting specific T cells that recognize immune
regulatory proteins such as IDO and PD-L1 may directly modulate immune regulation. 5,6,7,8,9,10,11
Due to the distinct mechanisms of action, the combination of treatment with a monoclonal antibody
targeting PD-1 and peptide vaccination with peptides against PD-L1 and IDO will have synergistic effects.
Nothing suggests that combining Nivolumab with the experimental peptide vaccine consisting of IDO long
and PD-L1 long1 peptides should be more toxic than treatment with Nivolumab alone (see section 12.7).
1.3
Toxicity
IDO
The IDO peptide has been tested in patients with non-small-cell-lung-cancer (NSCLC) (short peptide
sequence of 9 amino acids) and MM (long sequence of 21 amino acids). There were minimal toxicity and no
grade 3-4 toxicities which could be associated with the IDO vaccine. 12,13 (See IMPD, appendix 5)
PD-L1
PD-L1 peptide is currently being tested at CCIT in a first-in-human phase I clinical trial in patients with
multiple myeloma. Generally peptide vaccines are considered to possess very low risk of toxicity.14 (See
IMPD, appendix 5)
Since PD-L1 has a similar expression profile and mode of induction (IFN-γ) nothing suggests that the side
effects of a PD-L1 peptide vaccine will differ from IDO vaccines.
Montanide ISA 51
Montanide ISA 51 can cause local discomfort at the insert site but does not usually cause any systemic side
effects with the dose used in this trial. (See product resume, appendix 12)
Nivolumab
Grade 3-4 adverse events are seen in around 10-15 % of the patients treated with monotherapy Nivolumab.
Half of the side effects involve the gastrointestinal tract, lungs, the endocrine glands, the liver or the skin.
The side effects are generally manageable, and the patients are observed closely by an experienced staff at
the Department of Oncology. (See product resume, appendix 13)
1.4
Target population
Patients with metastatic malignant melanoma, who according to usual guidelines and clinical evaluation
are suited for treatment with Nivolumab.
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1.5
Number of participants
The study is designed as an open phase I/II trial. In phase I, 6 patients with MM will be treated. Before phase II
can start, all 6 patients must receive the first 4 treatments without any grade 3-4 adverse events, other than
those expected with Nivolumab.
If 3 or more patients experience grade 3-4 AE in phase I associated with the vaccination, the trial will be
stopped. In phase II, additionally 44 patients will be included.
Three patient cohorts will be included; Cohort A: Anti PD-1/PD-L1 naïve patients (30 patients). Cohort B:
Patients with progressive disease ON anti‐PD‐1 antibody monotherapy (10 patients). Cohort C: Patients
with progressive disease during follow up OFF anti-PD-1 after clinical benefit (SD/PR/CR) on anti‐PD‐1
antibody therapy (10 patients).
1.6
Patient recruitment
The trial is expected to begin inclusion in March 2017. Patients with malignant melanoma will be recruited
from the Department of Oncology, Herlev & Gentofte University Hospital, but can also be referred from
other centers in Denmark. Patients will receive vaccines for up to 47 weeks and will be followed until
progression. The inclusion period is expected to extend over 24 months.
1.7
Treatment plan
Patients included in the trial will be treated with Nivolumab in accordance with the standard regimen, which
at the moment involves outpatient IV infusions every second week as long as there is a clinical effect.
The PD-L1/IDO vaccine is given from the start of Nivolumab and every second week for the first 6 vaccines
and thereafter every fourth week up 47 weeks. 15 vaccines will be given in total.
At the end of vaccination, patients who are not excluded from the protocol because of progression will
continue treatment with Nivolumab in accordance with the usual guidelines.
Patients who receive all vaccines will have follow up appointments after 3 and 6 months in the experimental
unit (EFEK) Herlev Hospital in parallel with the standard of care treatment for Nivolumab.
1.8
Toxicity evaluation
Systematic recording of possible adverse events will take place during the course of treatment and follow up
in accordance with the Common Terminology Criteria (CTCAE). Recording will take place during patient
attendance in the outpatient clinic or the hospital ward at the Department of Oncology, Herlev Hospital.
1.9
Immunological evaluation
Blood samples
In order to monitor the immune response and immune related changes during treatment, project blood
samples will be taken before, during and after treatment. Project blood samples will be taken every third
month up to 5 years. (See 12.5.1)
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Tumor biopsy
At baseline and after the sixth vaccine, tumor biopsy will be done if the tumor is accessible. The objective is
to evaluate the tumor immune microenvironment of each patient. Immunohistochemistry, gene
expression quantification on different immune genes and whole exome sequencing to assess initial
mutational status will be conducted. 15,16 (See 12.5.2)
Delayed type hypersensitivity
Delayed type hypersensitivity (DTH) skin-test and punch biopsy from the DTH area will be done after the
sixth vaccination for the evaluation of skin infiltrating lymphocytes (SKILs) reactive to PD-L1 and IDO. (See
12.5.3)
1.10 Clinical response evaluation
Clinical response evaluation will be performed with diagnostic imaging according to RECIST 1.117, PERCIST18
and immune related response criteria (irRC)19 as well as clinical examination before, during and after
treatment. For further details, see the treatment schedule.
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Background
2.1
Malignant melanoma
Malignant melanoma (MM) is the 5th most commonly diagnosed cancer in Western Europe. Unlike
other solid tumors, which mainly affect elderly adults, melanoma also significantly affects young and
middle-aged people. Incidence rates have been increasing for the last 15 years of about 3-5 %. In
Denmark, 2200 people are diagnosed with malignant melanoma (2014).20 5-10 % of these patients
have metastases by the time of diagnoses and around 10-15 % develop metastases later in the course
of disease.21 There are around 350-400 new cases of patients with metastatic malignant melanoma
per year in Denmark. MM is a very aggressive cancer with a bad prognosis where traditional
oncological treatments such as surgery, chemotherapy and radiation therapy have a poor effect, and
the 5 year overall survival has hitherto been less than 10 %.22
2.2
Standard treatment of malignant melanoma
Huge advances have been made in the treatment of MM in the past 5 years with multiple drugs
being approved. In Denmark, 5 treatments are currently approved; Pembrolizumab/Nivolumab,
Ipilimumab, Dabrafenib/Trametinib, Temodal, Interleukin-2 (IL-2) + Peginterferon.
Currently, a PD-1 blocking antibody (Pembrolizumab or Nivolumab) is first line treatment and yields a
response rate of about 30 % with about 5 % complete response rate and a 3-year overall survival rate
of 40 %. 23,24
Ipilimumab, another immune checkpoint blocking antibody against cytotoxic T lymphocyte
associated protein 4 (CTLA-4) is used in the second line and yields a response rate of about 10-15 %,
but importantly a fraction of treated patients experience long term stabilization of disease with
about 20 % being alive more than 8 years. 25,26
Combining Nivolumab (PD-1 antibody) and Ipilimumab for the treatment of MM is already EMA
approved and is has recently been approved by the Danish authorities as first line treatment to a
group of MM patients.27
Patients with BRAF mutated melanomas have an additional treatment option after the approval of
multiple BRAF and MEK inhibitors. Currently, the combination of Dabrafenib and Trametinib is the
treatment of choice, with a response rate of 70 % and significant prolongation of overall survival.
Unfortunately, the anti-tumor effect of this treatment is only temporary and eventually all patients
progress. 28,29
Temozolomide is a chemotherapeutical treatment which has an objective response rate (ORR) of 15
% but no general effect on overall survival.30
IL-2 and peginterferon are immune stimulating cytokines. Combination of IL-2 and peginterferon is
applied for patients under 70 years in a good health condition and has shown an ORR of 15 % and
complete response in less than 5 %.31
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2.1
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 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.
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 cytotoxic T cells (CTLs) and the 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 T cell receptor
(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 which 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, to be able to start killing target cells (see Figure 1). APCs
eat antigens they meet, 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 dendritic cell because the dendritic cell expresses costimulatory proteins
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that activate the T cell. Once a CTL has been primed, it circulates the body and is ready to engage
target cells in the effector phase (see Figure 2). In the effector phase, CTLs kill target cells that on the
surface have peptide:MHC-complexes that the CTL is specific to.
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 1 and a signal 2 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 (see Figure 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 Ipilimumab. (Ribas, NEJM 201532)
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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. (Ribas, NEJM 201532)
2.2
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.33 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 that also express inhibitory molecules like PD-L1 and IDO are recruited to the tumor.
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 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 Figures 1B and 2B). Blocking the negative signals in the
balance “releases the break” on T cells, allowing them to kill malignant cells.
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All in all, cancer cells have many mechanisms whereby they escape immune attack. Two of the mechanisms,
i.e. the immune inhibitory pathway PD-1/PD-L1 and the metabolic enzymes will be described further in
section 2.3 and 2.4.
2.3
IDO and its potential as a vaccination antigen
2.3.1 IDO and its function
IDO is an immunoregulatory enzyme that has crucial functions in immune suppression and homeostasis,
and is implicated in suppressing T cell immunity in cancer.34 IDO is a major inhibitor of the effector phase of
the immune response, and suppresses effector T cells directly by degradation of the essential amino acid
tryptophan. The biological effect of IDO is mediated in part through local depletion of tryptophan, but also
via immune-modulatory tryptophan metabolites.4 Regulation of tryptophan metabolism by IDO in
dendritic cells (DC) is a highly adaptable modulator of immunity: IDO+DC are capable of inducing
immune suppression and anergy in antigen-specific T cells in the lymph node draining the injection site.
Effector T cells starved of tryptophan are unable to proliferate and go into G1 cell cycle arrest via activation
general control nonderepressible 2 kinase (GCN2) pathway. GCN2 is a stress-response kinase that is
activated by elevations in uncharged transfer RNA (tRNA), as would occur if the T cells were deprived of
tryptophan. Finally, IDO enhances local regulatory T cell mediated immune suppression: constitutive IDO
expression in DC provides T cells with regulatory properties that block T cell responses to antigenic
stimulation.35,36
IDO expression is widely dysregulated in cancer patients. IDO contributes in critical manner to inhibit or
terminate inflammation and is highly over-expressed in cancer. In cancer patients, elevation of IDO
expression occurs in a subset of plasmacytoid DC in tumor-draining lymph nodes.37 In addition, IDO may be
expressed within the tumor microenvironment, including tumor stromal cells, where it inhibits the
effector phase of immune responses.38 Activation of IDO in either tumor cells or nodal-regulatory DC each
appears to be sufficient to facilitate immune escape of tumors. In this regard, it has been described that
expression of IDO in tumor cells is associated with an impaired prognosis. Tumor cells transfected with
IDO become resistant to immune eradication in a murine model where a fully protective immune response
had been established by immunization.39 IDO expressing CD19+ plasmacytoid DC isolated from tumor-
draining LN mediates profound immune suppression and T cell anergy in vivo, whereas plasmacytoid DC
from normal LNs and spleen do not express IDO.
Conclusively, IDO is a critical cellular factor contributing to the suppression of anti-cancer immunity and as
such, IDO is most likely a crucial mechanism in cancer progression. Hence, IDO has become a very
attractive target for the design of new anticancer drugs.
2.3.2 Preclinical data on IDO
Demonstration that T cells recognize IDO-derived peptides
A shorter peptide, IO101, which is an HLA-A2-restricted, IDO derived CD8+ T cell epitope (IDO199-207;
ALLEIASCL), was initially identified. IO101 contains only MHC class I-restricted CD8+ T cell epitope that is
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contained within the longer IO102 (also named IDO long) sequence (which contains both CD8+ T cell
epitopes as well as CD4+ T cell epitopes; IDO194-214 DTLLKALLEIASCLEKALQVF).
Spontaneous T cell reactivity towards both of these IDO-derived peptides have been detected in cancer
patients bearing different tumor types (i.e. melanoma), which was measured by flow cytometry using
HLA:peptide tetramers, as well as in ELISPOT assays after in vitro stimulation but also in direct ex vivo
assays. In addition, it was demonstrated that T cell reactivity against both IO101 and IO102 to a lesser
extent exists in healthy individuals. Interestingly, in all patients, we detected both CD4 and CD8 T cell
responses against IDO, suggesting that class I- and II restricted IDO responses co-develop. 40,41,42
Demonstration that IDO-specific T cells can kill tumor cells
IDO-reactive CD8+ T cells are peptide specific, cytotoxic effector cells: IDO specific T cells can effectively
lyse IDO+ cancer cell lines of different origins, such as melanoma. Interestingly, circulating IDO specific T
cells can be isolated from not only cancer patients but also healthy individuals, and these T cells exhibit
similar cytotoxic functions against IDO-expressing malignant cells. Furthermore, the addition of known
IDO-inducers like IFN-gamma cause the expansion of IDO specific T cells among PBMCs, thus the level of
IDO expression in PBMCs positively correlates with the frequency of IDO-specific T cells. Finally, the
relevance of IDO-specific CD4+ T cells in cancer was further emphasized by the findings that IDO specific T
cells reacted towards dendritic cells (DC) pulsed with IDO+ tumor lysates. 40,41,42
Demonstration that IDO-specific T cells enhance activities of other effector T cells
IDO specific cytotoxic T cells can boost activation of other T cells towards viral and tumor-associated
antigens. This “supportive” effect on T cell immunity by IDO specific T cells was mediated in several direct
and indirect manners. First of all, IDO specific T cells were capable of killing IDO-expressing regulatory cells
thereby directly targeting the IDO-dependent counter-regulatory pathway. Thus, when IDO-specific T cells
were activated, the IDO activity decreased, the level of tryptophan was elevated, and T cell activities were
enhanced. Furthermore, IDO specific T cells enhanced overall production of pro-inflammatory cytokines,
like TNF-α and IL-6, but a decrease in IL-10. Moreover, the metabolites of tryptophan are known to be
toxic to CD8+ T cells and CD4 Th1 cells, but not Th2 cells. Hence, increased IDO activity appears to favor the
polarization of helper T cells toward a Th2 phenotype. Conversely, activation of IDO-specific cytotoxic T cells
may drive T-helper polarization in the Th1-direction, which is considered beneficial for protective immunity
in cancer. Lastly, IDO produces kynurenine, which may effectively hamper the immune response by binding
the aryl hydrocarbon receptor favoring the local formation of Tregs. Hence, targeting IDO-positive T cells
should decrease the number of Tregs. Indeed the frequency of Tregs decreased when IDO-specific T cells
were activated. 40,41,42
Conclusion on preclinical data on IDO peptide
In conclusion, IDO-specific T cells are able to directly recognize and kill IDO+ cancer cells and IDO
expressing regulatory T cells. IDO specific T cells are naturally present in cancer patients and to a lesser
extent in healthy donors. IDO will likely serve as a new and widely applicable target for immunotherapeutic
strategies.
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Vaccination with an IDO peptide has been tested in humans, see section 2.10.1 and 2.10.2
2.4
PD-L1 and its potential as a vaccination antigen
2.4.1 PD-1/PD-L1 axis
The PD-1 receptor was discovered in 1992, as a protein that was upregulated during the apoptosis of
lymphocytes, hence the name.43 The main action of PD-1 is negative regulation of T cell activity. PD-1 is
expressed on monocytes, dendritic cells, T cells, B cells and natural killer (NK) cells. PD-1 is upregulated on T
cells when they are activated, but persistent expression of PD-1 is a marker of T cell exhaustion, an
unresponsive state of inactivity.44
The PD-1 ligand PD-L1 (B7-H1) was discovered in 1999 and is a 290-amino acid transmembrane protein
encoded by the CD274 gene.45,46 The extracellular portion of PD-L1 comprises IgV- and IgC-like domains,
while the intracellular part comprises a 30-amino acid tail.45 The expression of PD-L1 in normal non-
inflamed tissues has reported either not expressed47–49 or only expressed in low amounts50–53 apart from on
macrophages which are clearly PD-L1 positive.47 Whether PD-L1 expression can be found when
investigating normal tissues, can be boiled down to the sensitivity of the technique. PD-L1 expression is
induced in normal cells if they are in an inflammatory environment by stimulation with the inflammatory
cytokines such as interferon-γ (IFN-γ).47–53
When PD-L1 interacts with PD-1 on a T cell, this inhibits the activation of the T cell via interfering with the
signaling from the TCR.54,55 Furthermore, via several other mechanisms, PD-1 stimulation inhibits
proliferation, differentiation and cytotoxic functioning of the T cell as well as diminishes its release of
inflammatory cytokines and leads to poor contact with dendritic cells.55 PD-L1 plays a critical role in the
conversion of naïve T cells to regulatory T cells (Tregs).56 Signaling into the cancer cell, PD-L1 acts in a
manner to reduce apoptosis.57
When an activated CTL commences the killing process of a target cell, it excretes the cytokine IFN-γ and
upregulates its own expression of PD-1 (Figure 3). This has major implications in the local
microenvironment. IFN-γ upregulates both MCH-class I molecules and PD-L1 molecules on the cells near
the CTL. More MCH-class I molecules mean better presentation of peptides from the cells in the
neighborhood, which improves the chance that CTLs will find suited target cells. But the upregulation of
PD-L1 combined with the activation induced PD-1 on the T cell itself pulls the balance in the direction of
shutting down the immune response. This is a normal limitation of T cell activity.
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Figure 3: Interferon upregulates PD-L1. When CTLs recognize their specific tumor antigen, both PD-1 on the T cell
and PD-L1 on the tumor cell are expressed due to IFN-γ secreted by the CTLs. MHC-expression is also increased, not
shown in this figure. (Ribas, Cancer Discovery 201558)
2.4.2 Preclinical data on PD-L1
Demonstration that T cells recognize PD-L1-derived peptides
The PD-L1 long1 (PD-L19-27) peptide contains both CD8+ T cell epitopes as well as CD4+ T cell epitopes.
Spontaneous T cell reactivity towards PD-L1 long1 peptide have been detected in both healthy individuals
and cancer patients including MM, which was measured by flow cytometry using HLA:peptide tetramers,
as well as in ELISPOT assays after in vitro stimulation but also in direct ex vivo assays. The T cell reactivity
was higher in cancer patients, and both CD4 and CD8 T cell responses against PD-L1 long 1 was detected.
These PD-L1 specific CD8+ T cells release IFN- γ and TNF-α. Notably, a few individuals in whom we were
able to measure specific T cell responses directly ex vivo had a relatively high number of PD-L1 specific T
cells.9
This is especially noteworthy as detection of conventional tumor-associated antigen specific T cells, with a
few exceptions, are not possible to detect in PBMC from cancer patients directly ex vivo without prior in
vitro peptide stimulation.59
Demonstration that PD-L1 long1 T cells can kill tumor cells
Further analyses demonstrated that the PD-L1 specific T cells in PBMCs were cytolytic effector cells using
Granzyme B ELISPOT assay. In addition, PD-L1 specific T cell cultures were generated by re-stimulating
PBMCs with the PD-L1 peptide in vitro and showed that the subsequent T cell lines were PD-L1 specific.
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Furthermore, we found that the PD-L1 specific CD8+ T cells were cytolytic effector cells that can recognize
and kill PD-L1-expressing melanoma cells.10
Demonstration that PD-L1 specific T cells directly and indirectly augment other T cell responses
The PD-L1/PD-1 pathway is important for the regulation of both viral and anticancer CTL responses. PD-L1
specific CTLs were therefore used to influence antiviral immunity. The addition of PD-L1 specific CTLs in
culture 1 week after virus epitope stimulation resulted in an increase in the number of virus-specific CD8+ T
cells. An increase was also found in cultures co-stimulated with the PD-L1 peptide epitope compared to
cultures that were co-stimulated with an irrelevant epitope from HIV-1. Hence, PD-L1 specific CTLs may
efficiently augment the effector phase of the immune response by suppressing PD-L1 expressing
regulatory cells that restrain PD-1-expressing effector T cells.60,61
PD-L1 long1 enhances the immune response towards a DC vaccine
A DC vaccine was recently tested in patients with stage IV melanoma. In the study, patients were vaccinated
with allogeneic DCs transfected with mRNA encoding the tumor-associated antigen p53, survivin and
telomerase (called DCvacc in the following). However, clinical benefit was limited with low reactivity
towards DCvacc in patient PBMCs.
To investigate if PD-L1 long1 could enhance the reactivity towards DCvacc, in vitro co-stimulation
experiments where PBMCs from patients with malignant melanoma were stimulated with DCvacc and PD-L1
long1, or DCvacc and an irrelevant control peptide.
The results show a significant increase in the T cell reactivity against DCvacc when patient PBMCs were co-
stimulated with PD-L1-derived peptides. Reactivity of CD4+ T cells increased the most, but CD8+ T cell
reactivity was also significantly boosted by co-stimulation.
This suggests that the addition of PD-L1 epitopes to a cancer vaccine could strengthen immune responses
against the vaccine in vivo.
Conclusion on preclinical data on the PD-L1 peptide
PD-L1 specific T cells are naturally occurring in both cancer patients and to a lesser extent in healthy donors.
PD-L1 long1 can induce broad CD4 and CD8 T cell drive immune responses towards PD-L1 expressing cells
including cancer cells and cells of the immune system. Adding PD-L1 specific T cells to virus specific T cells, or
as co-stimulation with DC-vaccine results in enhanced expansion of the virus or DC-vaccine specific T cells.
Vaccination with the PD-L1 peptide is currently being tested in a first-in-man study in patients with multiple
myeloma, see section 2.10.3.
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2.5
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, cancer cells as well as
immunosuppressive immune cells that overexpress PD-L1 and IDO will exhibit PD-L1 peptide:MHC and IDO
peptide:MHC complexes on the cell surface and can be recognized by T cells. 62,63
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 antigen presenting cells (APC). APC 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 APC 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.
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. 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.
Indeed, both the IDO long peptide and the PD-L1 long1 peptide can be recognized by both CD4 and CD8
T cells.
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Figure 5: 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. APC: antigen-presenting cell. DNA:
Deoxyribonucleic Acid. IDO: Indoleamine 2,3-deoxydase. MDSC: myeloid derived suppressor cell. SLP: synthetic long
peptide. RNA: Ribonucleic Acid. Treg: regulatory T cell. (Melief, JCI 2015) 64
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Figure 6: 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. (Melief, JCI 201565)
PD-L1 and IDO are normal proteins which are overexpressed by cancer cells including melanoma cells. In
the case of this vaccine against PD-L1 and IDO, we have found PD-L1 and IDO specific T cells in both
healthy donors and cancer patients. Interestingly, we have previously described that patients harboring a
spontaneous T cell response towards IDO prior to vaccination performed clinically better when vaccinated
with an IDO peptide13.
Once PD-L1 or IDO expressing cancer cells are attacked by T cells recognizing PD-L1 or IDO, remnants of
the killed cancer cells will be taken up by APCs. The APCs will process antigens from the cancer cells and
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present their cancerous neoantigens to other T cells. This process is called epitope spreading and is
thought to be one of the underlying causes to the success of cancer immune therapy.
Vaccine activated PD-L1 or IDO specific T cells will engage at the tumor micro-environment and attack
cancer cells. However, PD-L1 or IDO specific T cells do not attack normal cells in non-cancerous tissues,
since the expression level of PD-L1 and IDO is much lower. It requires a high expression of PD-L1 and IDO in
a cell to produce a high enough amount of PD-L1-peptide:MHC-complexes on the surface to overcome the
threshold of being killed by PD-L1 or IDO 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 PD-L1-
peptide:MHC-complexes and IDO-peptide:MHC-complexes have to be on the cell surface for the activation
signal to be strong enough. Furthermore, the activation of PD-L1 or IDO -specific T cells is a slow process.
Therefore, only by vaccinating several times, the amount of anti-PD-L1 or anti-IDO 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”66. 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.
2.6
PD-1 and PD-L1 blocking antibodies
Multiple studies of anti-PD-1 and anti-PD-L1 blockade report the subsequent restoration of T cell effector
function and proliferation as well as increased infiltration of tumors by cytotoxic T lymphocytes (CTLs). This
alters the balance between cytotoxic cells and immunosuppressive cells and ultimately results in the death of
tumor cells. The blockade of either PD-1 or PD-L1 by monoclonal antibodies has led to outstanding
clinical responses, and the Food and Drug Administration (FDA) recently approved the anti-PD-1 antibodies
Pembrolizumab (Keytruda) and Nivolumab (Opdivo) in September and December of 2014, respectively.
Blocking the PD-1 pathway shows great clinical promise, and there is high commercial interest and intense
competition among drug companies to develop agents that target PD-1 or PD-L1.
2.7
Adjuvant
As described in section 2.6, a peptide antigen is administered with an immune enhancer, an adjuvant. The
adjuvant increases the uptake of the antigen by APCs, and mediates so-called “danger signals” to the APC.
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.67,68,69
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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.
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2.8
Rationale behind the peptide vaccine with IDO & PD-L1
Cancer cells and other immune regulatory cells, such as tumor-associated dendritic cells (TADC) and
MDSC's express inhibitory surface molecules (e.g., PD-L1) and metabolic enzymes (e.g. IDO), which limits
the anti-tumor activity of specific anti-tumor T cells in the tumor microenvironment. PD-L1 specific or IDO
specific T cells recognize MHC-bound PD-L1 or IDO peptides, and are able to eliminate PD-L1 expressing or
IDO expressing immunoregulatory cells and cancer cells. Thus, activation of PD-L1 or IDO specific T cells
through vaccination with the PD-L1 and IDO peptides will boost a natural killing of cancer cells and
counteract immunoregulatory mechanisms in the tumor microenvironment. In addition, the vaccine should
attract specific pro-inflammatory helper T cells to the tumor microenvironment. Thus, IDO or PD-L1 specific T
cells may both directly support anti-cancer immunity by killing target cells as well as indirectly by
releasing pro-inflammatory cytokines in the microenvironment to boost additional anti-cancer immunity
(Figure 7).
Fig. 7: The peptide vaccine consisting of PD-L1 long1 and IDO long peptide boosts the natural immunity
mediated by PD-L1 and IDO specific T. These can attack and kill regulatory immune cells and cancer cells as
well as support additional anti-cancer immunity by the release of helper cytokines.
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2.9
Rationale behind the combined treatment strategies
As described earlier, antibodies blocking PD-1 (Nivolumab) have shown an OR in 30 % of MM patients, but
by combining this treatment with a peptide vaccine containing IDO and PD-L1, we aim to antagonize
additional immunosuppressive pathways and thereby boost the effect of the PD-1 blocking antibody,
Nivolumab. The hypothesis is that the PD-L1/IDO peptide vaccine and Nivolumab will complement each
other, since their mechanisms of action differ.
Effective peptide vaccination results in a slowly increasing number of cytotoxic T cells specific to the antigen
over the course of multiple repeated vaccinations. In the case of a PD-L1/IDO peptide vaccine, PD-L1
specific and IDO specific cytotoxic T cells will target PD-L1 and IDO expressing cells in the tumor. When
cytotoxic T cells engage target cells, the T cells secrete INF-γ to the surroundings and upregulate PD-1 on
their surface. When the surrounding cells in the tumor microenvironment are exposed to INF-γ, they further
upregulate PD-L1 and IDO. In this manner, the action of PD-L1- and IDO-specific T cells will be self-limiting.
Combining peptide vaccination against PD-L1 and IDO with antibodies against PD-L1 will prolong the effect
of PD-L1 specific and IDO specific T cells, and thus increase the effect of the vaccination (Figure 8).
Trials with vaccination with IDO peptides have been conducted as monotherapy and in combination with
the anti-CTLA-4 checkpoint blocking antibody Ipilimumab and likewise a trial with vaccination with PD-L1
long1 as monotherapy is undergoing. Like all previous peptide vaccination studies, toxicity has been very
limited. When combining IDO long peptide vaccination with Ipilimumab, no increase in toxicity was seen
compared with what would be expected with Ipilimumab alone.
Fig. 8: The self-limiting effects of peptide vaccination-stimulated cytotoxic T cells can be mitigated by
concomitant treatment with checkpoint blocking antibodies. A: Cytotoxic T cells recognize target cells via peptide-
MHC-complex and secrete effector cytokines. B: Activated T cells upregulate PD-1, and via INF-γ upregulate PD-L1 on
cells around them. PD-L1 specific T cells thus have a self-limiting activity. C: Concomitant treatment with immune
checkpoint blocking antibodies will act in synergy to prolong the effect of vaccination therapy.
2.10 CCIT’s experience with IDO and PD-L1 peptide vaccines
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2.10.1 NSCLC and IDO peptide vaccine
A peptide vaccine containing IDO has already been tested in humans. To evaluate the efficiency and safety of
IDO-based vaccinations, CCIT conducted a phase I first-in-man clinical vaccination trial. The study
comprised 15 patients with advanced non-small-cell-lung-cancer that were vaccinated with an IDO-derived
short 9-amino acid peptide in Montanide adjuvant. The vaccine was very tolerated with no toxicities above
grade 2.
In the study, the median overall survival (OS) was > 2 years, which was higher than expected for this patient
group. In 1 patient with liver metastasis an objective response (PR) was observed. The patient had
continuous tumor regression on vaccine treatment for 1 year before qualifying as a partial response. He
and one other patient are still enrolled in the trial on the 5th year without signs of progression.
The vaccine comprised an MHC-A2-restricted epitope from IDO. Hence, prior to inclusion in the study, MHC
tissue typing was performed. Therefore, it was possible to compare the OS of the MHC-A2+ patients that
were vaccinated with the patients that were otherwise eligible for the study but were excluded, since they
did not express MHC-A2. The MHC-A2+ patients that were vaccinated had a median survival of 25.9 months
demonstrating significantly longer OS compared to the MHC-A2- patients who had a median OS of 7.7
months. Importantly, is was recently described in a large study that expression of MHC-A2 is an unfavorable
prognostic factor in stage I NSCLC patients, which underlines the potential importance of the significantly
longer OS observed in vaccinated MHC-A2 patients compared to unvaccinated MHC-A2 negative NSCLC
patients, though these data need confirmation in large clinical trials.70
Immune monitoring revealed that IDO specific T cells were detectable in all patients, thus not only in
patients that seem to benefit clinically. In 2 SD patients an IDO-specific T cell response was detected during 1
year of treatment, suggesting sustained long-term IDO reactivity.13
Figure 9: Toxicity and duration of treatment in 15 patients with metastatic lung cancer vaccinated with a
short IDO-peptide. Of note is the absence of grade ≥3 toxicities. Duration of treatment was at the time of
publication in 2014. Two patients (numbers 17 and 18, Figure D) still receive vaccinations at present time - 4.5 years
after initiation. (Iversen, Clin Cancer Res 201413)
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2.10.2 MM and IDO peptide vaccine in combination with Ipilimumab
A first-in-man study with IDO long (21-amino acid peptide) was performed in 10 melanoma patients in
combination with the standard treatment at that time which was the anti-CTLA4 checkpoint blocking
antibody Ipilimumab. The primary objective of the trial was to assess safety and tolerability of the
combination treatment. Each patient was vaccinated a total of 7 times. The first 4 vaccines were given
weekly and the subsequent 3 vaccines were administered biweekly. Ipilimumab was administered IV at a
dose of 3 mg/kg every 3 weeks for a total of 4 treatments. Vaccine related toxicities were below grade 3 as
in the previous IDO vaccine study, but as expected, patients experienced toxicity to Ipilimumab. One patient
developed presumed Ipilimumab-induced colitis, which initially responded to corticosteroids, but later
relapsed while the patient due to unfortunate circumstances was admitted to a local hospital where the
patient died after receiving suboptimal therapy. Colitis is a known side effect to Ipilimumab which requires
intensive management. Vaccine-specific responses were demonstrated by ELISPOT in 3 of 10 patients.12
2.10.3 Multiple myeloma and PD-L1 peptide vaccine after high dose chemotherapy
The PD-L1 peptide is currently being tested in patients with multiple myeloma in a first in man phase I
study (EudraCT number 2016-000990-19). There is not yet data on toxicity.
In exploratory MOA murine studies, the human sequence as expected does not induce activation of PD-L1
specific T cells in mice, whereas, vaccination with the murine sequence induces strong responses. No
toxicity was found in mice vaccinated with either human PD-L1 long1 & murine PD-L1 long1. Therapeutic
peptide vaccines lack a suitable model for safety, see section 2.11.
Vaccination with PD-L1 peptide will resemble IDO vaccination in several ways:
·
It is also given with the adjuvant Montanide
·
The target protein is also an immunosuppressive self-molecule, which is widely expressed on
cancer cells, antigen-presenting cells, and immunosuppressive cells
·
IDO is also broadly induced on non-immune cells when their tissue is inflamed e.g. by IFN-γ
Nothing suggests that vaccination against PD-L1 should be more toxic than vaccination against IDO.
2.11 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
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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 PD-L1, 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.71 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 industry from Europe and the US FDA72. 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 published73. 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.
Patient recruitment
Patients will be recruited nationally and included at the Department of Oncology at Herlev Hospital. Patients
with metastatic melanoma who are candidates to receive Nivolumab monotherapy can be included
regardless of what earlier oncological treatment they have received; i.e. surgery, radiation, high-dose IL-2 or
experimental treatment.
The majority of patients with metastatic melanoma in Denmark are treated at Herlev, Odense and Aarhus
University Hospitals. We expect around 30-40 patients per year to be candidates for monotherapy with
Nivolumab. It is therefore estimated that patient inclusion can be completed within 24 months.
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Treating doctors at the departments of oncology and plastic surgery 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:
-
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 might 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
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-
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 designed as an open phase I/II study. In phase I, 6 patients with metastatic MM will be treated.
Around 10-15 % of the patients treated with Nivolumab have grade 3-4 AE.
Before phase II can start, all 6 patients must receive the first 4 treatments without any grade 3-4 adverse
events, other than those expected with Nivolumab.
If 3 or more patients experience grade 3-4 AE in phase I associated with vaccination, the trial will be stopped. A
minimum of 2 vaccines must be administered before inclusion of another patient for each of the 3 first
patients included in the study.
In phase II, additionally 44 patients will be included. All patients will receive the same dose vaccine without
dose escalation in combination with Nivolumab. A total of 50 patients will be included.
5.1
Inclusion criteria
1. Age ≥ 18
2. The patient has locally advanced or metastatic melanoma with progressive, persistent or recurrent
disease on or following treatment with standard of care agents.
Patients belonging to one of the following patient groups will be enrolled:
Cohort A: Anti PD-1/PD-L1 naïve patients (30 patients).
The patient is a candidate for Nivolumab monotherapy. Prior anti‐PD‐1/anti‐PD-L1 antibody
treatment is not allowed. OR
Cohort B: Extension cohort (10 patients). Progressive disease ON anti‐PD‐1 antibody monotherapy.
Subjects should not have experienced serious and/or life‐threatening toxicity to antibody therapy.
OR
Cohort C: Extension cohort (10 patients). Progressive disease during follow up OFF anti-PD-1 after
clinical benefit (SD/PR/CR) on anti‐PD‐1 antibody therapy. Subjects should not have discontinued
antibody therapy due to serious and/or life‐threatening toxicity
3. At least one measurable parameter according to RECIST 1.1.
4. The patient has an ECOG performance status of 0 or 1
5. The patient is a female of childbearing potential with negative pregnancy test
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6. For women: Agreement to use contraceptive methods with a failure rate of < 1 % per year during the
treatment period and for at least 150 days after the treatment. Safe contraceptive methods for
women are birth control pills, intrauterine device, contraceptive injection, contraceptive implant,
contraceptive patch or contraceptive vaginal ring.
7. For men: Agreement to use contraceptive measures and agreement to refrain from donating sperm
8. 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 or ≥ 5.6 mmol/L
9. Signed declaration of content after oral and written information about the protocol.
5.2
Exclusion criteria
1. The patient has not recovered to grade 0-1 from adverse events due to prior chemotherapy,
radioactive or biological cancer therapy
2. The patient has not recovered from surgery or is less than 4 weeks from major surgery
3. 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
4. The patient is expected to require any other form of systemic antineoplastic therapy while receiving
the treatment
5. The patient has a history of severe clinical autoimmune disease
6. The patient has a history of pneumonitis, organ transplant, human immunodeficiency virus positive,
active hepatitis B or hepatitis C
7. The patient requires systemic steroids for management of immune-related adverse events
experienced on another immunotherapy
8. The patient has active CNS metastases and/or carcinomatous meningitis. However, patients with
subclinical brain metastases < 1 cm can be included (maximum of 4 metastases < 1 cm). (Patients
with previously treated brain metastases may participate provided they are clinically stable. Patients
with untreated brain metastasis will be excluded)
9. The patient has any condition that will interfere with patient compliance or safety (including but not
limited to psychiatric or substance abuse disorders)
10. The patient is pregnant or breastfeeding
11. The patient is unable to voluntarily agree to participate by signed informed consent or assent
12. The patient has an active infection requiring systemic therapy
13. The patient has received a live virus vaccine within 30 days of planned start of therapy
14. Known side effects to Montanide ISA-51
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15. Significant medical disorder according to investigator; e.g. severe asthma or chronic obstructive
lung disease, dysregulated heart disease or dysregulated diabetes mellitus
16. Concurrent treatment with other experimental drugs
17. 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.
18. Severe allergy or anaphylactic reactions earlier in life
5.3
Evaluation before the start of treatment
The following parameters have to be performed within a month of the start of vaccinations (blood tests
within a week):
·
Medical history and clinical examination
·
Performance status according to the ECOG scale
·
Toxicity evaluation
·
Concurrent medical intake
·
Electrocardiogram
·
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 Thyrotropin (TSH), thyroxin (T4), Luteinizing Hormone (LH), Adrenocorticotropic
Hormone (ACTH), Cortisol
o Hepatitis B, hepatitis C (IgG), HIV, HTLV-1(IgG),, Epstein-Barr Virus EBV and
Treponema. .
o Women: Human Chorion Gonadotropin HCG, Follicle stimulating hormone FSH,
Estradiol and Prolactin
o Men: Testosterone
·
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, postmenopausal or have used
adequate and safe prevention for more than 6 months.
·
Diagnostic imaging: PET-CT or CT scan of neck, thorax, abdomen, pelvis and CNS & optionally
extremities.
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Conduct of the study
7.1
Initial patient enrollment and treatment
A minimum of 2 treatments with the peptide vaccine and Nivolumab has to be administered before
inclusion of another patient for each of the first 3 patients included in the study.
1st patient
1st dose 2nd dose
3rd dose
4th dose 5th dose
6th dose
7th dose 9th dose 10th dose
Safety
evaluation
2nd patient
Go/no go
for next
patient
1st dose 2nd dose
3rd dose
4th dose 5th dose 6th dose
Safety
evaluation
3rd patient
Go/no go
for next
patient
1st dose 2nd dose 3rd dose
Safety
evaluation
before
remaining
patients’
inclusion
Dosing of
remaining
patients
can start
in parallel
Go/no go
for
remaining
patients
X
7.2
Treatment plan and treatment schedule
Patients included in the protocol are treated with Nivolumab according to usual guidelines, implying
outpatient IV infusions of 3 mg/kg biweekly until progression. The vaccine is administered on the same day as
the administration start of Nivolumab. The vaccination is administered biweekly for a total of 6 times, then
every fourth week up to week 47, whereupon no additional vaccinations will be given. In total, 15 vaccines
will be administered. A vaccine consists of 100 µg IDO long, 100 µg PD-L1 long1, 25 µl DMSO, 475 µl PBS and
500 µl Montanide. During treatment with Nivolumab a pregnancy test must be done on fertile women every
month.
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.
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Patients who complete all vaccines will continue Nivolumab treatment after standard guidelines.
Health blood samples:
Nivolumab & Peptide vaccine
Females
B-Hæmoglobin
B-Trombocytter
B-Leukocytter
B-Leukocytfraktioner
P-Natrium
P-Kalium
P-Creatinin
P-Carbamid
P-Hydrogencarbonat
P-Albumin
P-Urat
P-ASAT
P-ALAT
P-LDH
P-Basisk Phosphatase
P-Bilirubin(total)
P-Amalyse
P-Glucose
P-TSH
P-Thyroxin (T4)
P-Calcium – ion frit
P-Cortisol
P-LH
P-FSH
Nivolumab & Peptide vaccine
Males
B-Hæmoglobin
B-Trombocytter
B-Leukocytter
B-Leukocytfraktioner
P-Natrium
P-Kalium
P-Creatinin
P-Carbamid
P-Hydrogencarbonat
P-Albumin
P-Urat
P-ASAT
P-ALAT
P-LDH
P-Basisk Phosphatase
P-Bilirubin(total)
P-Amalyse
P-Glucose
P-TSH
P-Thyroxin (T4)
P-Calcium – ion frit
P-Cortisol
P-testosteron
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P-Estradiol
P-Prolactin
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7.4
Follow up
Patients who complete the treatment of vaccines for all 47 weeks will be followed in the specialized unit
at EFEK 3 and 6 months after treatment. Patients who have completed the vaccination treatment will
continue Nivolumab treatment after usual guidelines in parallel with the follow up.
Month
3
6
Day:
1
1
Year: 20__
Toxicity evaluation and performance status
0
0
Clinical examination -
0
0
Health blood samples
0
0
Project blood samples
0
0
PET-CT or CT. scan of neck, thorax,
abdomen, pelvis, and possibly CNS and
0
0
extremities
Duration of treatment:
Treatment with the PD-L1/IDO peptide vaccine is maximum 47 weeks while treatment with Nivolumab is
according to standard treatments, hence as long as there is a clinical effect and the patient tolerates the
treatment.
The duration of treatment for Nivolumab on the basis of median progression free survival (mPFS) is 5.5
months corresponding to each patient receiving an average of 11 treatments. The duration will in practice
last longer as mPFS may underestimate the proportion of patients with treatment benefit due to early
“pseudo” progression prior to response.
Treatment will be stopped if there is a confirmed disease progression according to the immune-related
response criteria or by unacceptable toxicity or need for ongoing prednisolone ≥ 10 mg daily.
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.
7.5
Procedures regarding treatment
Patients will be receiving the peptide vaccine & Nivolumab on the same day. Vaccination will be performed
first, followed by infusion of Nivolumab.
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Peptide vaccine
The vaccine will be administered as a subcutaneous injection of 1 ml on the upper arm altering between
the right and left side.
During administration of the first 3 vaccines, the patient is observed for ½ hour with measurements of pulse
and blood pressure before and after the vaccine is given. The remainder of observations 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 vaccine I protokol MM1636, appendix 18)). When the
vaccine is received, the “receipt of transport” is signed. (SOP: Transport og udlevering af PD-L1/IDO mix og
Montanide, appendix 21).
Nivolumab
Following vaccination and observation, the patient is treated with Nivolumab. Administration of Nivolumab
follows the usual guidelines as if the patient was to be treated with Nivolumab alone. This involves IV
infusions of a 3 mg/kg dose via a filter dropper over ½ an hour following rinse with isotonic sodium
chloride.
Nivolumab is ordered according to usual guidelines with electronic ordering in “Sundhedsplatformen”.
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.
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7.7
Criteria for treatment modifications
Delay of treatment
The treatment will be postponed if the patient experiences non-hematological AE grade ≥ 2,
except fatigue. Otherwise the following criteria are applicable:
Blood samples
Leukocytes
Neutrophilic granulocytes
Thrombocytes
Hemoglobin
Creatinine
ASAT
ALAT
Bilirubin
Other AEs
Skin-related AE
All other AE
Criteria
< 1,5 x 109 (grade 3)
< 1,0 x 109 (grade 3)
< 50 x 109 (grade 3)
< 5 mmol/l (grade 3)
>3 x ULM (grade 3)
>5 x ULN (grade 3)
>5 x ULN (grade 3)
>3 x ULN (grade 3)
≥ grade 3
≥ grade 2
The treatment will be postponed until the severity has been reduced to grade 0 or 1.
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Criteria for end of treatment
1. Unacceptable toxicity
2. Disease progression
3. On patient request
4. Clinical judgement
5. Other treatment
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
Leukocytes
Neutrophilic granulocytes
Thrombocytes
Hemoglobin
Creatinine
ASAT*
ALAT*
Bilirubin**
Criteria
< 1,0 x 109 (≥grade 4)
< 0,5 x 109 (≥grade 4)
< 25 x 109 (≥grade 4)
< 4 mmol/l (≥grade 4)
>4,5 x ULM (≥grade 4)
>8 x ULN (≥grade 4)*
>8 x ULN (≥grade 4)*
>5 x ULN (≥grade 4)**
Other Adverse Events
Bronchospasms or
≥ grad 3
hypersensitivity reactions
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 at predefined evaluation time
points. Pseudo progression where initial tumor growth is seen because of treatment with Nivolumab and
actual disease progression is not seen must be considered.
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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.
8.1
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 3rd vaccine will be replaced with new
participants.
8.2
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
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Manufacture of the peptide vaccine
10.1 Peptides
For detailed description of peptide mixture production see SOP: ”Fremstilling af PD-L1/IDO peptid-mix til
MM1636” (appendix 16)
PD-L1 long1
The peptide sequence:
PD-L1 (9-27)
F-M-T-Y-W-H-L-L-N-A-F-T-V-T-V-P-K-D-L
IDO long
The peptide sequence:
IDO (194-214)
D-T-L-L-K-A-L-L-E-I-A-S-C-L-E-K-A-L-Q-V-F
All amino acid residues are in L-configuration. No disulphide bond is present. The counter ion,
hydrochloride is bound in ionic form.
The peptides are manufactured by solid phase synthesis using the Fmoc (9-fluorenylmethyloxycarbonyl)
approach. The limit on peptide purity is set to no less than 95 %. The peptides are dissolved in dimethyl
sulfoxide (DMSO) and phosphate-buffered saline (PBS). The solution is mixed and filtered through a sterile
0.20 µm filter.
10.2 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.
10.3 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
10.4 Preparing the vaccine
The peptide mixture and Montanide are delivered to the Department of Oncology shortly before use by a
project nurse or laboratory staff from J7. The peptide mixture constitutes 500 µl consisting of 100 µg PD-
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L1 long 1, 100 µg IDO long dissolved in DMSO and PBS in a watery solution. Montanide ISA-51 constitutes
500 µl and right before injection the two solutions are mixed through an I-connector luer lock into a 1 ml
syringe. The syringe is marked with patient name, personal security number, date and time of preparation.
For detailed description of the peptide preparation see SOP “Klargøring ad PD-L1/IDO peptid vaccine til
protokol MM1636”, appendix 17. See also example of labels for the vaccine in appendix 6.
10.5 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.
Ordination of Nivolumab
ATC-kode: L01XC17
Nivolumab is ordered electronically via ”Sundhedsplatformen” from the cytostatic unit at Herlev &
Gentofte Hospital.
See product resume and link: http://ec.europa.eu/health/documents/community-
register/2015/20150717132284/anx_132284_da.pdf.
Nivolumab is labeled in accordance with Annex 13 GMP rules, and a Danish instruction for the staff is
established.
Holder of the Marketing Authorization:
Bristol-Myers Squibb Pharma EEIG
Uxbridge Business Park
Sanderson Road
Uxbridge UB8 1DH
Great Britain
Evaluation
12.1 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.
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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.
12.2 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.
12.3 Statistical considerations
The study is an open phase I/II trial with the primary objective to evaluate safety of the combination
therapy. 50 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.
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12.4 Effect parameters
12.4.1
Primary effect parameter
Primarily, safety and toxicity 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 treatment and in accordance
with the CTCAE criteria. Data collection will take place in accordance with the data source list.
12.4.2
Secondary effect parameter
For the assessment of the immunological response, PD-L1 and IDO 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 using IFN-γ, IL-2 and TNF-α.
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.
12.4.3
Tertiary effect parameter
The clinical effect of the treatment will be assessed by measure of tumor responses by standard imaging
(PET-CT) according to PERCIST and RECIST 1.1 as well as irRC, with the endpoints being objective response
(OR), progression free survival (PFS) and overall survival (OS).17
RECIST
Clinical evaluation will be done in accordance with RECIST 1.1 Guidelines74.
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.
Complete and partial response is to be verified by examination at a minimum of 4 weeks after
documentation of the response at the earliest.
Matched controls will be identified using the DMG Oncology MM database to compare PFS and OS. Since
September 9th 2015 Pembrolizumab has been 1st line treatment in the majority of patients with MM in
Denmark. First, patients treated with Pembrolizumab in the period of 2017-2020 will be identified to collect
patients from the same time period as the patients in the protocol treated with Nivolumab and the PD-
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L1/IDO peptide vaccine. In 2017 and 2018 a total of 319 patients were treated with Pembrolizumab
according to DMG Oncology database.
Controls will be matched according to the following parameters: Age, sex, Performance status (PS), previous
treatment, M-stage, PD-L1 status, BRAF status, LDH level, CRP level.
Propensity scores will be conducted before matching the two groups (control vs treatment). A propensity
score is the probability that a unit with certain characteristics will be assigned to the treatment group (as
opposed to the control group). The scores can be used to reduce or eliminate selection bias by balancing
the characteristics of participants between treated and control groups. When the covariates are balanced, it
becomes much easier to match participants with multiple characteristics (parameters).
We will collect the necessary data approvals in order to extract the register-based control group.
12.5 Immunologic monitoring
12.5.1 Blood samples for immunological monitoring
100 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. Project blood samples will be taken 6 times
the first year and thereafter every third month up to 5 years.
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. (SOP’s: “Isolering af
MNC” and “Nedfrysning af MNC” appendix 19 & 20). 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 PD-L1 and IDO specific CD8 T cells. Blood and
serum samples are processed and stored at Center for Cancer Immune Therapy (CCIT).
12.5.2 Tumor biopsy for tumor immune microenvironment evaluation
Efforts shall be made to take biopsies from the available tumor lesions or involved lymph nodes before the 1st
vaccine and after the 6th 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 the following assays:
·
Immunoscore CR assay will be analyzed performed on the 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 analyzed 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 in positive PD-1 T cells quantification by digital pathology of a single
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FFPE biopsy slide.
·
IHC staining IDO will consist in IDO protein quantification by digital pathology on a single FFPE
biopsy slide.
As well as gene-based assays:
·
Immunosign CR and Pan Cancer Immune profiling on NanoString platform will consist in 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. 15,16
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. 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 limitied number of defined genes will be
performed on the tumor tissue to obtain a ‘immune profile’ to determine which genes- and consequently
which proteins- are expressed in the tumor tissue. Data will be handled according to national laws.
If the patient wishes prior genetic counseling by the chance of discovery of mutations in disease genes
and/or the risk of random finds this can be accommodated by referral to the Clinical Genetics
Department, Rigshospitalet.
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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.
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.).
As described above, we will perform DNA analyzes to clarify the function of the patients’ immune
system. These analyzes will be done by Adaptive Biotechnologies. Data will be stored
pseudonymized and encrypted by Amazon Web Services and Microsoft Azure (both cloud data
centers in the United States). Data will not be deleted from the cloud, but pseudonymization and
encryption protects the patient’s data. Only those responsible for this study have access to these
clouds. The Danish data protection agency has approved Adaptive biotechnologies as a partner in
the trial.
Patients will be informed either by physical attendance or by telephone. They will have time to think and
can always retreat. Patients will sign a new consent and they have the opportunity to ask questions both
before signing and afterwards.
12.5.3 Delayed type hypersensitivity (DTH) & biopsy
Delayed type hypersensitivity will be tested after 6 vaccinations. The test consists of up to a total of 3
intradermal injections of either an aqueous solution of PD-L1/IDO 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 PD-L1/IDO peptides as a sign of
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induction of a functional immune response. The patient can refuse to undergo the DTH-testing without
exclusion of the trial.
12.6 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 up to 5 years after
inclusion. The maximum duration of the research biobank will therefore be 7 years (i.e until 2024)
(i.e. the last person included is still alive after 5 years). 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 12.5.2)
regarding the discovery of known mutations with potential significant impact on patient’s health,
as well as the requirement, that the partner abroad reports back to the primary project managers
in Denmark so that relevant actions can be taken as described in section 12.5.2. 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
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.
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12.7 Risk evaluation
Evaluation of potential risks associated with the peptide vaccines are based on clinical experiences from
other cancer vaccination trials. The risks associated with the immune modulating antibody Nivolumab are
well known. Combination therapy with an immune modulating antibody and a peptide vaccine is also
based on experience from other combination 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
Another theoretical risk is the possibility of inducing autoimmunity when/if the vaccine eliminates the
immunosuppressive PD-L1 and IDO positive cells. PD-L1 specific and IDO specific T cells are present in
unvaccinated healthy individuals as well as cancer patients. Vaccination with PD-L1 and IDO peptides
boosts the activity of these T cells to engage cancer cells and immune cells who express PD-L1 or IDO as
part of the local immunosuppressive microenvironment in cancerous tissues. Normal non-cancerous
tissues express PD-L1 to some degree, and this expression is increased in the case of inflammation. This is
contrasted with cancerous tissues where PD-L1 and IDO is chronically overexpressed. The difference in
expression may provide the therapeutic window allowing PD-L1 specific T cells to discriminate cancer cells
and their tumor-associated immunosuppressive cells from normal cells. When activated, PD-L1 specific T
cells will themselves upregulate PD-1. This means that the activation of the immune response against PD-L1
expressing cells will be self-limiting.
However, in patients with autoimmune diseases or chronic inflammation, PD-L1 and IDO might play a
dampening role, thus these patients are excluded.
Autoimmune reactions are well-known side effects to PD-L1/PD-1 inhibitors with pneumonitis being the
most severe. Due to 3 cases of death related to pneumonitis in the early trials of the PD-1 inhibitors, specific
guidelines for early identification and intervention has been developed, which will be followed in this trial.
These guidelines have successfully reduced the severity of AEs.75
The damage by autoimmune reactions depends on which organ is involved and to which degree. Endocrine
disturbances can occur permanently but mostly transiently in the thyroid gland, hypophysis, adrenal gland
and rarely the pancreas. For these disturbances substitution therapy is available for the duration of the
disturbances.
Grade 1 and 2 autoimmune reactions can in most cases be reversed with anti-histamines or local
corticosteroids without discontinuation of the trial drugs, but higher grade events will lead to
discontinuation and exclusion.
Tolerance
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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.
PET-CT scans are not considered a risk based on the low dose radiation. A full body PET-CT with FDG gives a
radiation dose of around 12 milliSievert (mSv), which is about 5 times the annual background radiation. The
amount of PET-CT scans in this trial is the same as if the patient was to be treated with standard
treatment. 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.
Combination therapy
Combination therapy is becoming more and more common. As mentioned earlier, combining Nivolumab
and Ipilimumab is just approved as first line standard treatment in MM to patients with low expression of
PD-L1. In the studies of monotherapy with Nivolumab around 10-15 % patients experienced grade 3-4 t AE.
When combining PD-1 and CTLA-4 blocking antibodies, grade 3 or 4 toxicity is seen in around 53 % of
patients, thus being quite toxic.
The IDO peptide vaccine in combination with Ipilimumab showed no grade 3-4 AE.12 PD-L1 is like IDO an
immune regulatory molecule with a similar pattern of expression, and combining Nivolumab with the PD-
L1/IDO peptide vaccine is believed to induce toxicity at the same level as if the patient was to be treated
with Nivolumab alone.
This statement is based on other studies where combination with an immune regulatory antibody
(Ipilimumab or Nivolumab) and a peptide-based vaccine did not result in increased toxicity of signifcance
compared to monotherapy of either Ipilimumab or Nivolumab.
Following studies supports the statement. See reference:
·
(Gibney et. Al., 2015) 76
o 30 patients with metastatic melanoma were treated every second week with Nivolumab
and the multi-peptide vaccine (gp100, MART-1, NY-ESO-1 with Montanide ISA 51 VG) for
12 doses followed by maintenance Nivolumab every 12 weeks for 8 doses. Nivolumab in
combination with a multi-peptide vaccine was well tolerated. The common adverse events
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seen in this trial were similar to past studies with Nivolumab alone. The addition of vaccine
led to grade 1 local injection site reactions in a majority of patients.
·
(Bjoern et al., 2016)12
o IDO long peptide has been tested in combination with the anti-CTLA4 check-point
blocking antibody Ipilimumab in 10 patients with MM in a first in man phase I study. 12
There were minimal toxicity and no grade 3-4 toxicities which could be associated with the
IDO vaccine. The majority of patients experienced mild to moderate injection site reactions
at the vaccine site, including itching, swelling and erythema. In most cases symptoms
responded to oral antihistamines and symptoms remitted after completion of vaccine
treatment in all patients. As previous reported to DKMA. One patient developed presumed
ipilimumab-induced colitis, which initially responded to corticosteroids, but later relapsed
while the patient due to unfortunate circumstances was admitted to a local hospital where
the patient died after receiving suboptimal therapy. Colitis is a known severe side effect to
ipilimumab that requires intensive management.
·
(Hodi et al., 2010)25
o In this phase 3 study 676 patients were treated with either Ipilimumab alone (137),
peptide vaccine consisting of the gp100 peptide alone (136) or Ipilimumab plus
gp100 peptide (403). The addition of the gp100 peptide vaccine led to grade 1
local injection site, but the common adverse events where similar with the ones
seen with Ipilimumab alone.
All in all, combination of a peptide vaccine (both consisting of multi peptides or one peptide) and
an immune regulatory antibody (Ipilimumab or Nivolumab) have not been reported to lead to
increas severe toxicity as compared to monotherapy with check point inhibitors.
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13 Monitoring of toxicity
13.1 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:
13.2 Grading of adverse events
The severity of an AE refers to the intensity of the reaction.
Events are graded using CTCAE version 4.0 (Appendix 1). 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).
13.3 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;
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-
results in persistent or significant disability or incapacity;
-
leads to a congenital anomaly or birth defect;
-
is a significant medical event
13.4 Guidelines for adverse events’ possible relation to the treatment
0
No relation– no temporal relation, other etiologies very likely the cause
1
Possible relation – less clear temporal relation, other etiologies likely the cause
2
Probably related – clear temporal relation with recovery at termination of treatment, and not
reasonably explained by the patient’s known clinical condition
3
Related – clear temporal relation with laboratory confirmation or a positive retreatment test
13.5 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)
13.6 Reporting of adverse events and adverse reactions
Investigator reports SAEs, SARs and SUSARs to sponsor within 24 hours. Sponsor reports 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 SAEs
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
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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.
13.7 Known adverse reactions
13.7.1
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, inflammation or erythema. These
reactions are generally classified as mild to moderate and they are typically transient in nature.
Reference is made to attached drug information leaflet. (Appendix 12)
13.7.2
IDO long peptide
The IDO long peptide has already been tested in patients with MM in combination with the immune
modulating antibody Ipilimumab. The vaccine was well tolerated with no grade 3-4 toxicity. Most patients
experienced mild to moderate reactions at the site of injection, i.e. erythema, inflammation and itching. In
most of the cases, symptoms could be treated with oral antihistamine and were typically transient in nature.
12 Reference is made to attached IMPD. (Appendix 5)
13.7.3
PD-L1 long1 peptide
PD-L1 long1 peptide is currently being tested in a phase I first-in-man trial to patients with multiple
myeloma. (EudraCT number 2016-000990-19) Data regarding toxicity does not exist yet.
Firstly, all previous trials with peptide vaccinations have very mild side effects, no matter which proteins
have been targeted, as reviewed by Rahma and colleagues.77 Secondly, vaccination against PD-L1 is in
many ways similar to vaccination against IDO, and there are no apparent reasons why the toxicity should be
higher when vaccinating against PD-L1. Both PD-L1 and IDO are self-proteins with broad expression in
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inflamed or cancerous tissues but low expression in healthy tissues. Both are lymphocyte-suppressive
molecules and immunosuppressive cancer-associated immune cells. The adverse events in our trials with
IDO vaccination have been at the same low levels as that of other peptide vaccines.
In exploratory MOA murine studies, the human sequence as expected does not induce activation of PD-L1
specific T cells in mice, whereas, vaccination with the murine sequence induces strong responses. No
toxicity was found in mice vaccinated with either human PD-L1 long1 & murine PD-L1 long1.
Studies on animals on safety, characterization of the pharmacokinetics, and determination of maximum
tolerated dose (MTD) are not relevant in the case of therapeutic peptide vaccination. (See section 2.12)
Reference is made to attached IMPD. (Appendix 5)
13.7.4 Nivolumab
As described earlier, Nivolumab exerts its effect on T cells by blocking PD-1 whereby the T cells become
more activated and able to kill the tumor cells.
Due to these mechanisms, immune-related AE (irAE) can arise, which in most cases are mild, but can evolve to
be serious and require treatment. Grade 3-4 adverse events are reported in approximately 10-15 %. Half of
the side effects involve the gastrointestinal tract, the endocrine glands, the liver, lungs and the skin. The AE
rarely arise acute, but can be serious/life-threatening if not treated in time. The side effects are generally
manageable and the patients are observed closely by an experienced staff at the Department of Oncology.
Reference is made to attached drug information leaflet for Nivolumab. (Appendix 13)
Handling adverse events
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.
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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 MM. First line standard treatment for patients with malignant melanoma is
both Ipilimumab and Nivolumab in combination or Pembrolizumab/Nivolumab alone depending on PD-L1
expression. Patients treated in this trial are PD-L1 positive patients. Addition of a peptide vaccine to the
existing standard treatment is believed to add no further toxicity. An earlier trial combining Ipilimumab
with an IDO peptide vaccine showed no grade 3-4 AE.
There does not seem to be unacceptable risks or disadvantages regarding the planned treatment based
on the current knowledge on peptide vaccines and combination with immune modulating antibodies.
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.
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. In addition, patients will be followed for PFS and OS for up to 5 years. 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.
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Publications
Cathrine Lund Lorentzen, Julie Westerlin Kjeldsen, 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 2018/19.
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 firm 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.
The project has been initiated by Mads Hald Andersen, Inge Marie Svane, Julie Westerlin Kjeldsen, and
Cathrine Lund Lorentzen.
The PD-L1 and IDO 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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References
1.
Forsea, A. M., Del Marmol, V., De Vries, E., Bailey, E. E. & Geller, A. C. Melanoma
incidence and mortality in Europe: New estimates, persistent disparities. Br. J.
Dermatol. 167, 1124–1130 (2012).
2.
Bhatia, S., Tykodi, S. S. & Thompson, J. a. Treatment of metastatic melanoma: an
overview. Oncology (Williston Park). 23, 488–96 (2009).
3.
Pedoeem, A., Azoulay-Alfaguter, I., Strazza, M., Silverman, G. J. & Mor, A. Programmed
death-1 pathway in cancer and autoimmunity. Clin. Immunol. 153, 145–52 (2014).
4.
Uyttenhove, C. et al. Evidence for a tumoral immune resistance mechanism based on
tryptophan degradation by indoleamine 2,3-dioxygenase. Nat. Med. 9, 1269–1274
(2003).
5.
Søorensen, R. B. et al. The immune system strikes back: Cellular immune responses
against indoleamine 2,3-dioxygenase. PLoS One 4, (2009).
6.
Sorensen RB, Hadrup SR, Svane IM, Hjortso MC, thor Straten P, A. M. Indoleamine 2,3
dioxygenase specific, cytotoxic T cells as immune regulators. Blood 117(7):220, (2001).
7.
Sørensen, R. B. et al. Spontaneous cytotoxic T-cell reactivity against indoleamine 2,3-
dioxygenase-2. Cancer Res. 71, 2038–2044 (2011).
8.
Ahmad, S. M., Borch, T. H., Hansen, M. & Andersen, M. H. PD-L1-specific T cells. Cancer
Immunol. Immunother. 65, 797–804 (2016).
9.
Munir, S. et al. HLA-restricted CTL that are specific for the immune checkpoint ligand
PD-L1 occur with high frequency in cancer patients. Cancer Res. 73, 1764–1776 (2013).
10.
Munir, S. et al. Cutaneous T cell lymphoma cells are targets for immune checkpoint
ligand PD-L1-specific, cytotoxic T cells. Leukemia 27, 2251–2253 (2013).
11.
Munir, S., Andersen, G. H., Svane, I. M. & Andersen, M. H. The immune checkpoint
regulator PD-L1 is a specific target for naturally occurring CD4(+) T cells.
Oncoimmunology 2, e23991 (2013).
12.
Bjoern, J., Iversen, T. Z., Nitschke, N. J., Andersen, M. H. & Svane, I. M. Safety, immune
and clinical responses in metastatic melanoma patients vaccinated with a long peptide
derived from indoleamine 2,3-dioxygenase in combination with ipilimumab.
Cytotherapy 18, 1043–1055 (2016).
13.
Iversen, T. Z. et al. Long-lasting disease stabilization in the absence of toxicity in
metastatic lung cancer patients vaccinated with an epitope derived from indoleamine
2,3 dioxygenase. Clin. Cancer Res. 20, 221–232 (2014).
14.
Pol, J. et al. Trial Watch: Peptide-based anticancer vaccines. Oncoimmunology 4,
e974411 (2015).
15.
Galon, J. et al. Towards the introduction of the ‘Immunoscore’ in the classification of
malignant tumours. J. Pathol. 232, 199–209 (2014).
16.
Chen, P. L. et al. Analysis of immune signatures in longitudinal tumor samples yields
insight into biomarkers of response and mechanisms of resistance to immune
checkpoint blockade. Cancer Discov. 6, 827–837 (2016).
87
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17.
Eisenhauer, E. A. et al. New response evaluation criteria in solid tumours: Revised
RECIST guideline (version 1.1). Eur. J. Cancer 45, 228–247 (2009).
18.
Wahl, R. L., Jacene, H., Kasamon, Y. & Lodge, M. A. From RECIST to PERCIST: Evolving
Considerations for PET response criteria in solid tumors. J. Nucl. Med. 50 Suppl 1,
122S–50S (2009).
19.
Wolchok, J. D. et al. Guidelines for the evaluation of immune therapy activity in solid
tumors: Immune-related response criteria. Clin. Cancer Res. 15, 7412–7420 (2009).
20.
Cancerregisteret. Cancerregisteret 2014 - Tal og analyser, Statens Serum Institut.
(2014).
21.
Mervic, L. Time course and pattern of metastasis of cutaneous melanoma differ
between men and women. PLoS One 7, e32955 (2012).
22.
Balch, C. M. et al. Final version of 2009 AJCC melanoma staging and classification. J.
Clin. Oncol. 27, 6199–206 (2009).
23.
Robert, C. et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N. Engl. J.
Med. 150419053123009 (2015). doi:10.1056/NEJMoa1503093
24.
Roberts, Caroline, et al. Three-year overall survival for patients with advanced
melanoma treated with pembrolizumab in KEYNOTE-001. Abstr. ASCO 2016
25.
Hodi, F. S. et al. Improved survival with ipilimumab in patients with metastatic
melanoma. N. Engl. J. Med. 363, 711–23 (2010).
26.
American Association for Cancer Research. Decade-long survival possible after
ipilimumab. Cancer Discov. 3, OF7 (2013).
27.
Opdivo nivolumab. 1, 1–2 (2016).
28.
Long, G. V. et al. Dabrafenib and trametinib versus dabrafenib and placebo for Val600
BRAF-mutant melanoma: A multicentre, double-blind, phase 3 randomised controlled
trial. Lancet 386, 444–451 (2015).
29.
Chapman, P. B. et al. Improved survival with vemurafenib in melanoma with BRAF
V600E mutation. N. Engl. J. Med. 364, 2507–16 (2011).
30.
Middleton, M. R. et al. Randomized phase III study of temozolomide versus
dacarbazine in the treatment of patients with advanced metastatic malignant
melanoma. J. Clin. Oncol. 18, 158–66 (2000).
31.
Atkins, M. B. et al. High-dose recombinant interleukin 2 therapy for patients with
metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J. Clin.
Oncol. 17, 2105–16 (1999).
32.
Ribas, A. Releasing the Brakes on Cancer Immunotherapy. N. Engl. J. Med. 373, 1490–
1492 (2015).
33.
Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144,
646–674 (2011).
34.
Muller AJ, P. G. Indoleamine 2,3-dioxygenase in immune suppression and cancer. Curr
Cancer Drug Targets 7, 31–40 (2007).
35.
Munn, D. H. et al. GCN2 kinase in T cells mediates proliferative arrest and anergy
88
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induction in response to indoleamine 2,3-dioxygenase. Immunity 22, 633–642 (2005).
36.
Mellor, A. L. Ido expression by dendritic cells: tolerance and tryptophan catabolism.
Nat. Rev. Immunol. 4, 762–774 (2004).
37.
Munn, D. H. et al. Expression of indoleamine 2 , 3-dioxygenase by plasmacytoid
dendritic cells in tumor-draining lymph nodes. J. Clin. Invest. 114, 280–290 (2004).
38.
Munn, D. H. Indoleamine 2, 3-dioxygenase, tumor-induced tolerance and counter-
regulation. Curr. Opin. Immunol. 18, 220–225 (2006).
39.
Uyttenhove, C. Evidence for a tumoral immune resistance mechanism based on
tryptophan degradation by indoleamine 2,3-dioxygenase. Nat. Med. 9, 1269–74 (2003).
40.
Sorensen, R. B. et al. The immune system strikes back: cellular immune responses
against indoleamine 2,3-dioxygenase. PLoS One 4, e6910 (2009).
41.
Sørensen, R. B., Hadrup, S. R., Svane, I. M., Hjortsø, M. C. & Straten, P. Indoleamine 2 ,
3-dioxygenase specific , cytotoxic T cells as immune regulators. 117, 2200–2210
(2011).
42.
Andersen, M. H. The specific targeting of immune regulation: T-cell responses against
Indoleamine 2,3-dioxygenase. Cancer Immunol. Immunother. 61, 1289–1297 (2012).
43.
Ishida, Y., Agata, Y., Shibahara, K. & Honjo, T. Induced expression of PD-1, a novel
member of the immunoglobulin gene superfamily, upon programmed cell death.
EMBO J. 11, 3887–95 (1992).
44.
Wherry, E. J. T cell exhaustion. Nat. Immunol. 131, 492–499 (2011).
45.
Dong, H., Zhu, G., Tamada, K. & Chen, L. B7-H1, a third member of the B7 family, co-
stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 5, 1365–9 (1999).
46.
Tamura, H. et al. B7-H1 costimulation preferentially enhances CD28-independent T-
helper cell function. Blood 97, 1809–16 (2001).
47.
Dong, H. et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential
mechanism of immune evasion. Nat. Med. 8, 793–800 (2002).
48.
Mazanet, M. M. & Hughes, C. C. W. B7-H1 is expressed by human endothelial cells and
suppresses T cell cytokine synthesis. J. Immunol. 169, 3581–8 (2002).
49.
Gong, A.-Y. et al. MicroRNA-513 regulates B7-H1 translation and is involved in IFN-
gamma-induced B7-H1 expression in cholangiocytes. J. Immunol. 182, 1325–33 (2009).
50.
Sugita, S. et al. Human corneal endothelial cells expressing programmed death-ligand
1 (PD-L1) suppress PD-1+ T helper 1 cells by a contact-dependent mechanism.
Investig. Ophthalmol. Vis. Sci. 50, 263–272 (2009).
51.
Eppihimer, M. J. et al. Expression and regulation of the PD-L1 immunoinhibitory
molecule on microvascular endothelial cells. Microcirculation 9, 133–45 (2002).
52.
Nakazawa, A. et al. The Expression and Function of Costimulatory Molecules B7h and
B7-H1 on Colonic Epithelial Cells. Gastroenterology 126, 1347–1357 (2004).
53.
Schoop, R. et al. Suppressed T-cell activation by IFN-gamma-induced expression of
PD-L1 on renal tubular epithelial cells. Nephrol. Dial. Transplant 19, 2713–20 (2004).
54.
Freeman, G. J. et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7
89
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family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192,
1027–34 (2000).
55.
Boussiotis, V. A. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway.
N. Engl. J. Med. 375, 1767–1778 (2016).
56.
Francisco, L. M. et al. PD-L1 regulates the development, maintenance, and function of
induced regulatory T cells. J. Exp. Med. 206, 3015–29 (2009).
57.
Azuma, T. et al. B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells. Blood
111, 3635–43 (2008).
58.
Ribas, A. Adaptive immune resistance: How cancer protects from immune attack.
Cancer Discov. 5, 915–919 (2015).
59.
Keilholz, U. et al. Immunologic monitoring of cancer vaccine therapy: results of a
workshop sponsored by the Society for Biological Therapy. J. Immunother. (1991). 25,
97–138 (2002).
60.
Ahmad, S. M., Larsen, S. K., Svane, I. M. & Andersen, M. H. Harnessing PD-L1-specific
cytotoxic T cells for anti-leukemia immunotherapy to defeat mechanisms of immune
escape mediated by the PD-1 pathway. Leukemia 28, 236–8 (2014).
61.
Ahmad, S. M., Svane, I. M. & Andersen, M. H. The stimulation of PD-L1-specific
cytotoxic T lymphocytes can both directly and indirectly enhance antileukemic
immunity. Blood Cancer J. 4, e230 (2014).
62.
Berke, G. The binding and lysis of target cells by cytotoxic lymphocytes: molecular and
cellular aspects. Annu. Rev. Immunol. 12, 735–73 (1994).
63.
Knuth, A., Wölfel, T. & Meyer zum Büschenfelde, K. H. T cell responses to human
malignant tumours. Cancer Surv. 13, 39–52 (1992).
64.
Melief, C. J. M., Van Hall, T., Arens, R., Ossendorp, F. & Van Der Burg, S. H. Therapeutic
cancer vaccines. J. Clin. Invest. 125, 3401–3412 (2015).
65.
Melief, C. J. M., van Hall, T., Arens, R., Ossendorp, F. & van der Burg, S. H. Therapeutic
cancer vaccines. J. Clin. Invest. 125, 1–12 (2015).
66.
Zhong, X., Bai, C., Gao, W., Strom, T. B. & Rothstein, T. L. Suppression of expression and
function of negative immune regulator PD-1 by certain pattern recognition and
cytokine receptor signals associated with immune system danger. Int. Immunol. 16,
1181–8 (2004).
67.
Bijker, M. S. et al. CD8+ CTL priming by exact peptide epitopes in incomplete Freund’s
adjuvant induces a vanishing CTL response, whereas long peptides induce sustained
CTL reactivity. J Immunol 179, 5033–5040 (2007).
68.
Bijker, M. S. et al. Superior induction of anti-tumor CTL immunity by extended peptide
vaccines involves prolonged, DC-focused antigen presentation. Eur. J. Immunol. 38,
1033–1042 (2008).
69.
Kenter, G. G. et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial
neoplasia. N. Engl. J. Med. 361, 1838–1847 (2009).
70.
Nagata, Y. et al. Clinical significance of HLA class I alleles on postoperative prognosis
of lung cancer patients in Japan. Lung Cancer 65, 91–97 (2009).
90
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4-10-2022
71.
Coukos, G. Defining the critical Hurdals in Cancer Immunotherapy. (2011).
72.
Hoos, A. et al. A clinical development paradigm for cancer vaccines and related
biologics. J. Immunother. 30, 1–15 (2007).
73.
Matsumoto, M. et al. Considerations for non-clinical safety studies of therapeutic
peptide vaccines. Regul. Toxicol. Pharmacol. 70, 254–60 (2014).
74.
Eisenhauer, E. a et al. New response evaluation criteria in solid tumours: revised RECIST
guideline (version 1.1). Eur. J. Cancer 45, 228–47 (2009).
75.
Sunshine, J. & Taube, J. M. PD-1/PD-L1 inhibitors. Curr. Opin. Pharmacol. 23, 32–38
(2015).
76.
Gibney, G. T. et al. Safety, correlative markers, and clinical results of adjuvant
nivolumab in combination with vaccine in resected high-risk metastatic melanoma.
Clin. Cancer Res. 21, 712–720 (2015).
77.
Osama E. Rahma, Emily Gammoh, R. M. Is the ‘3+3’ dose escalation phase 1 clinical
trial design suitable for therapeutic cancer vaccine development? A recommendation
for alternative design. Clin. Cancer Researc 20, 4758–4767 (2014).
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