The purpose of this phase I study is to investigate the combination of hypomethylating agents
with experimental peptide vaccination against four selected tumor antigens, known to be
upregulated in response to hypomethylating agents, in patients with high risk myelodysplastic
syndrome and acute myeloid leukemia.
Patients with high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), who
cannot be offered curative treatment, have a poor prognosis with a median survival of 11.5
months. There is currently only one registered drug that has been shown to prolong survival
in this patient group; the hypomethylating agent azacitidine (also known as 5-azacytidine or
Vidaza®). The treatment is not curative, but increases the median survival to approximately
two years. The dominant cause of death is progressive disease, and there is a huge need for
new treatment options.
In this study we will combine experimental immunotherapy of peptide vaccination with standard
therapy of azacitidine for treatment of patients with high-risk myelodysplastic syndrome or
acute myeloid leukemia. Our project is expected to expand the knowledge in regard to
combination of immunotherapy and conventional treatment and will be essential for further
development of this treatment modality.
MYELODYSPLASTIC SYNDROME (MDS)
Accumulation of both genetic and epigenetic changes in hematopoietic stem cells is considered
the background for development of MDS. In the early stages of the disease, there is increased
apoptosis, but as the disease progresses to high-risk MDS or AML there is an accumulation of
chromosomal breakage, point mutations and promoter hypermethylation of tumor suppressor
genes, resulting in a more aggressive and proliferative disease. The clinical symptoms are
anemia, repeated infections and bleeding episodes associated with the dysfunctional bone
Currently, the only curative treatment option for MDS is allogeneic bone marrow
transplantation. This is a high-risk treatment, only feasible in younger patients with a
suitable donor. In 2009, a Phase III study (AZA 001) was published, showing that patients
with high-risk MDS and AML with <30% blasts treated with the hypomethylating drug azacitidine
had superior overall survival.
The effect of azacitidine is not immediate in all patients, and it is therefore necessary to
proceed with a minimum 5-6 cycles to determine whether the patient has a response to
treatment. At present, azacitidine is the standard of care for all patients with high-risk
MDS and AML with <30% blasts, who cannot be offered curative treatment. Unfortunately, most
patients who respond to treatment later relapse and die of their disease. The mechanism
behind the clinical efficacy of azacitidine is not fully elucidated and may be
multiparametric. The hypomethylation results in re-expression of tumor suppressor genes that
may serve as a possible mechanism for growth arrest. In addition, an up-regulation of cancer
testis antigens (CTA) has been observed during treatment, which could lead to increased
immune recognition of tumor cells, and hence immune-mediated tumor cell killing.
For participants in this trial, azacitidine is standard therapy and will be administered
according to standard dosing regimens, and participants will be evaluated for treatment
response according to standard criteria.
TUMOR ASSOCIATED ANTIGENS
The principle behind cancer vaccination is based on the immune system's ability to
discriminate between normal and malignant cells. The immune system recognizes an altered
pattern of peptide expression on the surface of the malignant cells. The altered pattern may
involve the expression of qualitatively altered cell proteins, simple overexpression of
normally occurring proteins, or de novo expression of proteins/glycoproteins not normally
expressed in that tissue. This category of immune-recognized proteins is known as tumor
associated antigens. As proteins are degraded in cells, peptides are presented on the surface
of these cells as a complex with tissue type molecules (HLA molecules). T-cells may then
recognize the peptide-HLA complexes, via its T-cell receptor, potentially resulting in
tumor-cell killing, if sufficient priming takes place.
For the present study, the group of cancer testis antigens (CTA's) are of special interest,
since expression of these are known to be regulated by methylation, and therefore affected by
hypo-methylating treatment such as azacitidine. CTA's are known to be immunogenic and are
only expressed at immunoprivileged sites, thus out of reach of immune responses, and on
cancer cells, making them ideal targets for therapeutic cancer vaccination.
We have chosen specifically three CTA's for which abundant re-expression has been shown
following azacitidine treatment, including NY-ESO-1, MAGE-A3 and PRAME. WT-1 is additionally
included as this protein has proven to be an important antigen in hematological malignancies
and is likewise upregulated in response to azacitidine treatment.
The peptides we have chosen have not been used together in this particular combination
before, but separately their use as therapeutic vaccination is well studied. Both smaller
peptide sequences and full length proteins have been utilized.
The use of synthetic long peptides for induction and T-cell responses has shown superior
effects in contrast to minimal peptide sequences in both mice and humans, furthermore
synthetic long peptide has been shown to be efficiently processed and presented to both CD4
and CD8 T-cells of the immune system. Also, long-peptides have the advantage to enable
presentation of several different T-cell epitopes dependent on the patients' HLA type. Thus,
the choice of long peptides will minimize the impact of patients HLA type, and therefore in
the present study allow inclusion of participants without prior selection based on HLA
The peptides we have chosen are between 25 and 29 amino acids and will be mixed as a
suspension with Montanide ISA-51; a commonly used water and oil based vaccine adjuvant,
involved in over 150 registered trials at Clinicaltrail.gov.
Patients with high-risk MDS and AML with <30% blasts are today offered treatment with
azacitidine, 100 mg/m2 for 5 days every 4th week. In the proposed vaccination protocol, we
have chosen to follow this same regimen, but in addition offer vaccination with peptides
derived for cancer testis antigens. Patients will be included after completion of 6 courses
of azacitidine and a treatment evaluation. If the patient shows response to treatment with
azacitidine and the marrow is not hypoplastic, azacitidine treatment will be combined with
peptide vaccination to boost the immune reactivity against selected tumor antigens. Patients
will be vaccinated with a set of 3 vaccinations, with a vaccination on the 1st day of each
azacitidine course, given every 4th week. If treatment is delayed more than 3 weeks the
patient is excluded from the protocol. Patients are evaluated for clinical responses after 6
courses of azacitidine treatment combined with vaccination. Patients that are not
progressing, will continue azacitidine treatment according to standard schedule, and further
vaccinations will be given once every 6th month. Vaccination is continued for up to 2 years
after initiation of vaccination or until disease progression.
PREPARATION OF THE PEPTIDE VACCINE
The peptide vaccine will be prepared under GMP approved laboratory conditions (approved by
the Danish Medicines Agency), which assures the quality of a sterile medicinal product.
Production takes place in room JM702, Herlev Hospital under the approval number 24223.
Below is a brief description of the various procedures. The final vaccine product will hold a
label providing information about the vaccine content and the project number given by the
The vaccination will be targeted against four long peptide sequences from the proteins:
NY-ESO-1 (119-143, 25aa): PGVLLKEFTVSGNILTIRLTAADHR ; PRAME (423-447, 25aa):
LQSLLQHLIGLSNLTHVLYPVPLES ; MAGE-A3 (255-279, 25aa): YLEYRQVPGSDPACYEFLWGPRALV ; WT1
(231-259, 29aa): SQLECMTWNQMNLGATLKGVAAGSSSSVK.
The peptides will be synthesized to >97% purity by an external provider, Pepscan Presto, The
Netherlands. Prior to use, it will be dissolved in DMSO, mixed, and sterile filtered through
a 0.22 micron filter. Endotoxin and bacterial cultivation tests will be carried out on the
preparations. Sterility tests before release of peptides for vaccination treatment is carried
out by hematological laboratory (location 54P4).
Adjuvant: Montanide ISA 51
Montanide ISA 51 is purchased from Seppic Inc., France, in sterile form. We will use 0.5 ml
of Montanide per vaccine.
Preparation of vaccine
The vaccine will be generated as a mix of the peptide-preparation and the adjuvant, on-side,
just prior to injection.
Before use, the peptide mix is subjected to endotoxin tests and microbiological tests
executed by the Department of Microbiology, Herlev Hospital.
This is an open-label Phase I study designed to evaluate the safety of combination therapy of
azacitidine and peptide-vaccination in patients with MDS or AML.
We expect to enroll all participants within 24 months and evaluate the study 12 months
thereafter. Participants will continue with azacitidine until disease progression, and with
vaccination for up to 2 years, or until disease progression. Participants may stop treatment
at any time upon their own request, or will be withdrawn from treatment following disease
progression or severe toxicity during treatment. The inclusion period is expected to begin in
April 2016 and to be finalized in April 2018. Thereafter a follow-up period of maximum 2
years is following. Last participant last protocolled visit date will be at latest, April
Patients with MDS and AML, who receive treatment with azacitidine, will be recruited through
the Department of Hematology at Herlev Hospital. Since all patients are followed in an
outpatient setting, for around six months prior to inclusion while receiving azacitidine
treatment, possible candidates can be identified early and properly informed before a consent
form needs to be signed.
Participation is voluntary and is preceded by written informed consent. Treatment will be
terminated in cases of unacceptable side effects, or upon patient's request at any time. If a
patient is not interested in participation in the trial, treatment will be given according to
the department's usual guidelines. The study is therefore considered ethically sound.
The study follows the guidelines of the Helsinki Agreement and the responsible physician
obtains a permit from the Scientific Ethical Committee, the Danish Health and Medicines
Agency and the Danish Data Protection Agency to conduct the study.
1. Participants must have received 6 courses of azacitidine and been evaluated with
response to treatment.
2. Histologically confirmed high-risk MDS or AML (<30% blasts) and a normo- or
hypercellular marrow after 6 courses of azacitidine.
3. Indication for continued treatment with azacitidine.
4. Age >18 years.
5. Signed consent form after receiving both written and oral information.
6. The patients must be willing to follow the scheduled treatment and sampling.
1. Hypocellular bone marrow after 6 courses of azacitidine.
2. Additional active cancer disease. Participants treated for a second malignancy may be
included if the patient is without evidence of disease at least 2 years after
completion of treatment.
3. Participants with a known hypersensitivity to any of the active substances or to any
of the excipients.
4. Participants with secondary MDS or AML
5. Severe allergies or previous anaphylactic reactions.
6. Active autoimmune disease, for example autoimmune neutropenia/ thrombocytopenia or
hemolytic anemia, systemic lupus erythematosus, Sjögren's syndrome, scleroderma,
myasthenia gravis, Goodpasture's syndrome, Addison's disease, Hashimoto's thyroiditis,
7. Concomitant treatment with systemic immunosuppressive medications (including
prednisone, methotrexate etc.). Participants are allowed to receive up to 10 mg
prednisone at the days of azacitidine injection.
8. Concomitant treatment with other experimental drugs.
9. Concomitant treatment with other systemic anti-cancer therapy.
10. Pregnant or breastfeeding females.