INTRODUCTION

      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
      marrow.

      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.

      AZACITIDINE

      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.

      VACCINE DESIGN

      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
      expression.

      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.

      TRIAL DESIGN

      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.

      STUDY METHOD

      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
      department.

      Peptides

      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.

      Microbiological control

      Before use, the peptide mix is subjected to endotoxin tests and microbiological tests
      executed by the Department of Microbiology, Herlev Hospital.

      EXPERIMENTAL DESIGN

      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
      2020.

      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.

      ETHICAL ASPECTS

      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.