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Inducible Expression of Cancer Testis Antigens in MDS Patients Following Treatment with an Oral DNA Methylation Inhibitor (5-azacytidine)

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Inducible Expression of Cancer Testis Antigens in MDS Patients Following Treatment with an Oral DNA Methylation Inhibitor (5-azacytidine) Powered By Docstoc
					                                                                                                                                                                                                   INDUCIBLE EXPRESSION OF CANCER TESTIS ANTIGENS IN MDS PATIENTS FOLLOWING TREATMENT
                                                                                                                                                                                                                  WITH AN ORAL DNA METHYLATION INHIBITOR (5-AZACYTIDINE)
                                                                                                                                                                                                                                                                                1                                                                                                                        1                                                                                2                                            1                                                                                               1                                                         3                                              4
                                                                                                                                                                                                          John Powers, B.S. , Eva Sahakian, Ph.D , Jason A Dubovsky, Ph.D , Tyler Farnum , Emmanuel Berchmans, B.S. , Douglas G McNeel, MD , Barry S. Skikne, MD ,
                                                                                                                                                                                                                                                                                   1                              1
                                                                                                                                                                                                                                                            Rami S. Komrokji, MD and Javier Pinilla-Ibarz, MD, PhD
                                                                                                                                                                                               1 Malignant Hematology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; 2 Ohio State University Medical Center, Columbus, OH; 3 University of Wisconsin, Madison, WI; 4 Celgene Corporation, Overland Park, KS




                                                         ABSTRACT                                                                                                                                                                      FIGURE 1                                                                                                                                                                                                                                                                                        FIGURE 2                                                                                                                                                                                                                       FIGURE 3
Background:The identification of appropriate target antigens is critical to the success of cancer immunotherapies. Cancer-testis antigens        Clinical study patient data and A.                         Patient # Diagnosis
                                                                                                                                                                                                                                      Days on        Cycles
                                                                                                                                                                                                                                                                       HI-E           HI-P          HI-N
                                                                                                                                                                                                                                                                                                                   Marrow                                                     RBC-TI    RBC-TI PLT-TI (56             Hematologic or
                                                                                                                                                                                                                                                                                                                                                                                                                       Transfusion
                                                                                                                                                                                                                                                                                                                                                                                                                                       A.
                                                                                                                                                                                                                                                                                                                                                                                                                                            XAGE-1       FATE1      SSX1     Negative 1   Positive     GAGE-2       PAGE-5   LAGE-1            MAGE-A1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             B.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                -       +
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      A.                                                                                                                    B.
                                                                                                                                                                                                                                       Trial        Completed                                                     Response                                                   (56 Days) (84 Days) Days)
(CTAs) are ectopically expressed immunoprivileged antigens transcriptionally controlled by DNA methylation which have utility as                 treatment schematic
                                                                                                                                                                                                                                                                                                                                                                                                                        Response
                                                                                                                                                                                                                1         AML             54               1             N             N             N               N                                                          N             N              N             N                                                                                                                                                                                                                                                       SSX2
immunotherapeutic targets. 5-azacytidine (AZA), an analogue of cytidine, has been shown to induce expression of CTAs in cancer. We                                                                              2         AML            219               5             N             N             N               N                                                          --            --             --            N                 TPX1       NY-SAR-35 MAGE-A4    Negative 2       Mp17     GAGE-4        LIP1    PAGE-1                                    SSX2                                                                                                                                                                                                                                  SSX2
analyzed matched samples from 37 patients with myeloproliferative disease receiving an oral AZA to determine if an anti-CTA immune                                                                              3         AML            410              14           Y (10)          N             --            mCR (7)                                                     Y (8)         Y (8)           --            Y
                                                                                                                                                                                                                                                                                                                                                                                                                                             NXF2        TSP50     MAGE-B2   MAD-CT-2     h-PAP       MAGE-A3       SPA17    MAGE-E1         NY-ESO-1
                                                        response was generated.                                                                  A) Patient characteristics, of 41                              4
                                                                                                                                                                                                                5
                                                                                                                                                                                                                          AML
                                                                                                                                                                                                                          AML
                                                                                                                                                                                                                                         154
                                                                                                                                                                                                                                         434
                                                                                                                                                                                                                                                           4
                                                                                                                                                                                                                                                          14
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                N
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                              N
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           N
                                                                                                                                                                                                                                                                                                                                                                                                                           N

Methods:Immunoglobulin G (IgG) responses to CTAs were analyzed by High Throughput Immunoblot assay (HTI), 29 CTAs were bound in                  individuals, consisting of Hemato-                             6
                                                                                                                                                                                                                7
                                                                                                                                                                                                                          AML
                                                                                                                                                                                                                          AML
                                                                                                                                                                                                                                         115
                                                                                                                                                                                                                                         240
                                                                                                                                                                                                                                                           3
                                                                                                                                                                                                                                                           6
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                   mCR (4)
                                                                                                                                                                                                                                                                                                                                                                                N
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                              N
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                                             N
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           N
                                                                                                                                                                                                                                                                                                                                                                                                                           Y
                                                                                                                                                                                                                                                                                                                                                                                                                                             SSX4        ADAM2     MAGE-B1   MAD-CT-1         r-PAP      p53       MAGE-A8   SPANXC                      GAGE-7
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                MAD-CT2
a concise pattern to nitrocellulose filters and developed in the following methodology. Patient sera were incubated with the HTI filters and     logic     Lineage      Improvement                             8         AML             72               2             N             N             N               N                                                          --            --             --            N
                                                                                                                                                                                                                                                                                                                                                                                                                                            GAGE-7      SPANXC     MAGE-A8        p53         r-PAP   MAD-CT-1     MAGE-B1   ADAM2                                     SSX4                                                                                                                                                                                                                               MAD-CT-2




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       *
                                                                                                                                                                                                                9       MDS-U           1200              36             N             --            --                                                                         --            --             --            N
any CTA specific antibodies bound to respective antigens and reactive antibodies were detected by secondary anti-human antibodies in a
colorimetric assay. Filters were read by 5 blinded individuals, spots were scored positive or negative against control antigens. These results   (Erythroid, Platlet and Neutro-                               10
                                                                                                                                                                                                               11
                                                                                                                                                                                                                           RA
                                                                                                                                                                                                                           RA
                                                                                                                                                                                                                                         211
                                                                                                                                                                                                                                         154
                                                                                                                                                                                                                                                           7
                                                                                                                                                                                                                                                           4
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                         --
                                                                                                                                                                                                                                                                                       --
                                                                                                                                                                                                                                                                                      Y (1)
                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           N
                                                                                                                                                                                                                                                                                                                                                                                                                           Y                NY-ESO-1    MAGE-E1     SPA17    MAGE-A3      h-PAP       MAD-CT-2     MAGE-B2    TSP50                                    NXF2
were compared to the paired post-therapy sample, a score of 5 was maximal change of pre to post. Those CTAs with strong antibody presence        philic), (WHO score at diagnosis,                             12
                                                                                                                                                                                                               13
                                                                                                                                                                                                                           RA
                                                                                                                                                                                                                           RA
                                                                                                                                                                                                                                         357
                                                                                                                                                                                                                                         594
                                                                                                                                                                                                                                                          10
                                                                                                                                                                                                                                                          18
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                      Y (9)
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           N
                                                                                                                                                                                                                                                                                                                                                                                                                           Y                 SSX2        PAGE-1     LIP1     GAGE-4           Mp17    Negative 2   MAGE-A4 NY-SAR-35                                   TPX1
post-therapy were then confirmed via ELISA resulting in an antibody titer reflected as ug/mL. A standardization of human IgG, as well as a                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      MAD-CT1
                               healthy donor control was used to determine the validity of identified responses.                                 WHO score at therapy induction,                               14
                                                                                                                                                                                                               15
                                                                                                                                                                                                                        RAEB-I
                                                                                                                                                                                                                        RAEB-I
                                                                                                                                                                                                                                         249
                                                                                                                                                                                                                                        1075
                                                                                                                                                                                                                                                           7
                                                                                                                                                                                                                                                          37
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                       Y (4)
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                 mCR (1)
                                                                                                                                                                                                                                                                                                                 mCR (1)
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                               Y (2)
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                             Y (2)
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           Y
                                                                                                                                                                                                                                                                                                                                                                                                                           Y                MAGE-A1      LAGE-1    PAGE-5    GAGE-2       Positive    Negative 1    SSX1      FATE1                      XAGE-1                                                                                                                                                                                                                                           MAD-CT-1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           **
                                                                                                                                                 IPSS score, graded toxicities, and                            16
                                                                                                                                                                                                               17
                                                                                                                                                                                                                        RAEB-I
                                                                                                                                                                                                                        RAEB-I
                                                                                                                                                                                                                                         196
                                                                                                                                                                                                                                         119
                                                                                                                                                                                                                                                           5
                                                                                                                                                                                                                                                           3
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                         --
                                                                                                                                                                                                                                                                                      Y (2)
                                                                                                                                                                                                                                                                                       --
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                 mCR (2)
                                                                                                                                                                                                                                                                                                                 mCR (2)
                                                                                                                                                                                                                                                                                                                                                                                N
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                              N
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           Y
                                                                                                                                                                                                                                                                                                                                                                                                                           Y
Results:When HTI was examined thoroughly several CTAs, MAGEs, SSX2, NY-ESO1 and MAD-CTs were of particular interest. All patients
regardless of pre- or post- therapy exhibited SSX2 expression at high levels this was further confirmed with ELISA exhibiting little to no       prior treatments. The four patients                           18
                                                                                                                                                                                                               19
                                                                                                                                                                                                                        RAEB-I
                                                                                                                                                                                                                        RAEB-I
                                                                                                                                                                                                                                          77
                                                                                                                                                                                                                                         364
                                                                                                                                                                                                                                                           2
                                                                                                                                                                                                                                                           9
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                       --
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                               mCR (2) CR (2)
                                                                                                                                                                                                                                                                                                                                                                                N
                                                                                                                                                                                                                                                                                                                                                                               Y (5)
                                                                                                                                                                                                                                                                                                                                                                                              N
                                                                                                                                                                                                                                                                                                                                                                                             Y (5)
                                                                                                                                                                                                                                                                                                                                                                                                             N
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           N
                                                                                                                                                                                                                                                                                                                                                                                                                           Y
change from pre- to post-therapy. HTI examination of MAD-CT antigens revealed that pre-treatment, MAD-CT-1 and MAD-CT-2 antibody                 with relevant responses (CR, PR or                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             NY-ESO-1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  **
                                                                                                                                                                                                               20       RAEB-I            83               2             N             --            --              N                                                          --            --             --            N
presence, responses were nearly identical, and thirty-four had the exact same response levels. PRAME, a CTA absent from our HTI filter, was
                                                                                                                                                 mCR) are highlighted in yellow. B)
                                                                                                                                                                                                               21       RAEB-I            98               2             N             N             N           mCR (2)                                                        --            --             --            Y
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        +       -




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       ***
evaluated only through ELISA. We determined that eighteen patients presented with an increased anti-PRAME antibody titers at                                                                                   22       RAEB-I           622              19           Y (7)          Y (6)          --        mCR (2) CR (8)                                                   --            --             --            Y                                                                                                                                                                                                                                                                                                                                                                              MAGE-A1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Cancer Testis Antigens
                                                                                                                                                                                                               23       RAEB-I          1150              36             --            N             --              N                                                          --            --             --            N




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 Cancer Testis Antigens
post-treatment when compared to pre-treatment. Of the cancer testis antigens analyzed patients had the highest average changes in antibody       The drug treatment schedule for the                           24       RAEB-I           169               5             N             --            N               N                                                         Y (4)          N              --            Y
                                  titer to PRAME at post-treatment, with a 0.128 ug/mL average increases.                                                                                                      25       RAEB-I           292               7           Y (2)          Y (2)        Y (1)       mCR(1) CR(2)                                                     --            --             --            Y
                                                                                                                                                 Multicenter, sequential design,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                MAGE-A1
                                                                                                                                                                                                                                                                                                                                                                                                                                               Antigens of Interest
                                                                                                                                                                                                               26       RAEB-I           210               5             N             N             N               N                                                          N             N              --            N

Conclusions: Our findings suggest that CTAs could be further pursued as an immunotherapeutic target in myelodysplastic syndrome and that         open-label, phase I dose escalation
                                                                                                                                                                                                               27
                                                                                                                                                                                                               28
                                                                                                                                                                                                                        RAEB-II
                                                                                                                                                                                                                        RAEB-II
                                                                                                                                                                                                                                         209
                                                                                                                                                                                                                                         266
                                                                                                                                                                                                                                                           6
                                                                                                                                                                                                                                                           8
                                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                                         --
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                      Y (1)
                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                     N
                                                                                                                                                                                                                                                                                                                 mCR (2)
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           N
                                                                                                                                                                                                                                                                                                                                                                                                                           Y
                                                                                                                                                                                                                                                                                                                                                                                                                                       C.                                                                                    Screening                                                                              EOS
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             >250 Days                                                                                                                           + NY-ESO-1 response
treatment with AZA could be exploited to modulate antigen expression in combination with immunotherapeutic approaches. Although                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          p= <0.005                                        MAGE-A3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                              -           +
                                                                                                                                                                                                               29       RAEB-II          428              14             N             --          Y (11)        mCR (1)                                                        N             N              --            Y
responses were variable throughout the patients in total, some patients had robust responses at post-treatment. The CTAs, MAGEs, SSX2,           study. All patients received 7 days                           30       RAEB-II          203               6             --            --            N               N                                                          --            --             --            N                                                                                                                                                                                                                                                                                                                 <250 Days                                                                                                                           -NY-ESO-1 response
                                                                                                                                                                                                               31       RAEB-II           94               1             N             N             N               N                                                          --            --             N             N
NY-ESO1 and PRAME, are of particular interest, due to prior and new findings, as well as, on-going clinical trials. Five patients had a robust   of sub-cutaneous Vidaza at 75                                 32       RCMD             738              23             --            N             N               --                                                         --            --             --            N                                                                                                                                                                                                                                                    MAGE-A3
antibody response according to HTI to at least six antigens. Eight patients were of further interest according to ELISA examination. Further                                                                   33       RCMD             358              10             N             N             --              --                                                         N             N              N             N
             data on the relation with clinical response and possible prognostic factors of response to therapy will be presented.               mg/m2 per day x 7 days and the                                34       RCMD             111               3             N             N             N               --                                                         --            --             --            N                                         3

                                                                                                                                                 next cycle was day 28 Oral
                                                                                                                                                                                                               35       RCMD             209               6             N             --            N               --                                                         --            --             --            N




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           ***
                                                                                                                                                                                                               36       RCMD              85               2             N            Y (1)          N               --                                                         --            --             --            Y                                                                                                                                                                                                                                                                                                                                                                              MAGE-A4

                                                                                                                                                 5’-azacytidine was then adminis-                              37
                                                                                                                                                                                                               38
                                                                                                                                                                                                                        RCMD
                                                                                                                                                                                                                         CMML
                                                                                                                                                                                                                                         273
                                                                                                                                                                                                                                        1485
                                                                                                                                                                                                                                                           6
                                                                                                                                                                                                                                                          51
                                                                                                                                                                                                                                                                       Y (6)
                                                                                                                                                                                                                                                                         --
                                                                                                                                                                                                                                                                                       N
                                                                                                                                                                                                                                                                                      Y (2)
                                                                                                                                                                                                                                                                                                     Y
                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                     --
                                                                                                                                                                                                                                                                                                                    SD
                                                                                                                                                                                                                                                                                                                                                                                N
                                                                                                                                                                                                                                                                                                                                                                                --
                                                                                                                                                                                                                                                                                                                                                                                              N
                                                                                                                                                                                                                                                                                                                                                                                              --
                                                                                                                                                                                                                                                                                                                                                                                                             N
                                                                                                                                                                                                                                                                                                                                                                                                             --
                                                                                                                                                                                                                                                                                                                                                                                                                           Y
                                                                                                                                                                                                                                                                                                                                                                                                                           Y
                                                                                                                                                                                                                                                                                                                                                                                                                                                                     4        2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                MAGE-A4
                                                                                                                                                 tered daily for 7 days each 28 days                           39        CMML           1159              42             N             --            Y              SD                                                          Y             Y              --            Y

                                                    BACKGROUND
                                                                                                                                                                                                               40        CMML             62               4             --            N             --           PR-PB                                                         --            --             --            N

                                                                                                                                                 at doses ranging from 120mg to                                41
                                                                                                                                                                                                            Y = Yes
                                                                                                                                                                                                                         CMML
                                                                                                                                                                                                                      N = No
                                                                                                                                                                                                                                         203
                                                                                                                                                                                                                               -- = No data found
                                                                                                                                                                                                                                                           9             Y             Y
                                                                                                                                                                                                                                                    HI-E = Hematologic Improvement (Erythroid)
                                                                                                                                                                                                                                                                                                     --             SD
                                                                                                                                                                                                                                                                                                 HI-N = Hematologic Improvement (Neutrophil)
                                                                                                                                                                                                                                                                                                                                                                                Y             Y              --            Y
                                                                                                                                                                                                                                                                                                                                                                                       HI-P = Hematologic Improvement (Platlet)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                     5        1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          MAGE-E1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     **
                                                                                                                                                 600mg. Maximum tolerated dose                              (#) = Cycle Incident Occurred    CR = Complete Remission        PR = Paritial Remisson      mCR = Marrow Complete Remission
                                                                                                                                                                                                            RBC-TI (#)/ PLT-TI (#) = Red Blood Cell/ Platlet Transfusion Independence for # of Days (Respectively) AML = Acute Myelogenous Leukemia
                                                                                                                                                                                                                                                                                                                                                                                        SD = Stable Disease

The primary downfall of peptide cancer vaccines is the induction and persistence of a biased,                                                                                                               MDS-U = Myelodysplastic Syndrome Unclassafiable RA= Refractory Anemia RAEB-I = RA with excess blasts (5-9%) RAEB-II = RA with Excess Blasts (10-19%)                                                                                                                                                                                                                                                                                                                MAGE-E1
                                                                                                                                                 was 480mg/day.                                             RCMD = Refractory Cytopenia with Multilineage Dysplasia CMML= Chronic Myelomonocytic Leukemia PB= Peripheral Blood                                                                                                                                       6
tolerogenic T-cell population resulting in little to no anti-cancer effect. The ideal peptide candidates
for these vaccines would be cancer restricted and non-self antigens. The large pool of antigens                                                                                                                                                                                                                                                                                                                                                              2                    7                                                                                                                                                                                                                                                                                                                       MAGE-A8
deemed cancer testis antigens (CTA) exhibit cancer restriction and are self antigens excluded from                                                B.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            MAGE-A8
           T-cell anergy making them excellent candidates for peptide vaccination. [1-4]
                                                                                                                                                                      Subcutaneous                                                     Oral                                                                           Oral
It is well known that currently approved anti-cancer drugs, such as the DNA methyltransferase                                                                        Azacitidine (Daily)                                        Azacitidine (Daily)                                                            Azacitidine (Daily)
                                                                                                                                                                                                                                                                                                                                                                                                                                                    1                                                                                                                                                                                                                                                                                                                                                     MAGE-B1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                MAGE-B1
inhibitor 5’-azacytidine, up regulate critical CTAs via demethylation specific enhancement of the                                                                                                                                                                                                                                                                                                                 Continue
                                                                                                                                                                                                                                                                                                                                                                                                                  until end
BORIS (“brother of the regulator of imprinted sites”) transcription factor. Several in-vitro human                                                                                                                                                                                                                                                                                                                of study
models have shown that CTA up regulation is both cancer-specific and durable for months                                                                                                                                          D                    D                                                          D                                                           D
                                                                                                                                                                      D             D                                            a                    a                                                          a                                                           a
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                                       post-treatment. [5-11]                                                                                                         a             a                                                                                                                            y




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                                                                                                                                                                                                                                 y                    y                                                                                                                      y                                                                                                                                                                                                                                                                                                  MAGE-B2                                                                                                                   MAGE-B2
                                                                                                                                                            V         y             y              Drug Holiday                                                            Drug Holiday                                                                                                                                                Patients show with antibody responses at Baseline to several antigens but exhibit augmented levels post drug therapy
                                                                                                                                                            A
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Past studies have identified the potential benefit of utilizing epigenetic modulatory drugs in
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                                                                                                                                                                                                                                                                                                                                                                                                                                       A) The CTA-HTI Filter layout of the 29 Cancer Testis Antigens and control proteins B) An example of an Oral 5-AZA patient’s processed
                                                                                                                                                            A                                                                                                                                                                                                                                                                          HTI Filter C) Antigens of interest including NY-ESO-1 (      ), SSX2 (      ) and MAGEs (1-7 = -A1,-E1, -A4, -B2, -B1, -A8 and –A3




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inducing CTAs in myeloproliferative disorders. These studies showed increased RNA levels of                                                                 T                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Antibody Response Change Pre- to Post- Therapy                                                                              Antibody Response Change Pre- to Post- Therapy
several CTAs in patients’ receiving demethylating agents. Only one recent study showed T-cell                                                               E                                                                                                                                                                                                                                                                          respectfully). MAD-CTs ( -1 and -2 ) were later observed to be of interest and examined
                                                                                                                                                                            Cycle                                                        Cycle                                                                          Cycle
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        NY-ESO-1 antibody activity elucidates other CTAs under methylation control
 sensitivity to MAGE proteins, but humoral reactivity was not examined in these studies. [12-15]                                                                             #1                                                           #2                                                                             #3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        A) Patients on the trial for less than 250 days compared to greater than 250 days show marked difference in antibody response to multiple antigens. Patients on the trial greater
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        than 250 days show significantly increased antibody responses to NY-ESO-1 than those for less time. (B) Patients with NY-ESO-1 antibody reactivity show increased reactivity
                                                        OBJECTIVE                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       to multiple antigens. MAD-CT-1 and MAD-CT-2 expressions are nearly identical (P=<0.05). 2-way ANOVA w/ Bonferroni Post-test was performed using GraphPad Prism
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. (*P=<0.05 **P=<0.01 ***P=<0.001)
      - Elucidate humoral responses to CTAs in responder patients.                                                                                                                                  FIGURE 4                                                                                                                                                                                                                                                                                               FIGURE 5                                                                                                                                                                                                                                                  CONCLUSIONS
                                                                                                                                                                                                                                                                                                                       A.                                                                                                                                                                                                             B.
 - Correlate specific immunological responses to patient outcome.                                                                                                                                                                                                                                                                                                            1.0                                                                                                                                                                                                          0.04
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Responders
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            - Patients undergoing hypomethylating agents show increased humoral responses to several CTAs, particularly NY-ESO-1.
                                                                                                                                                                                                                                                                                                                                                                                                                                                        Responders
                                                                                                                                                                                                                                                                                                                                                                                                                                                        Non-Responders                                                                                                                                              Non-Responders

                - Identify possible target antigens for further study.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   - An elevated antibody response to MAD-CT-1 or MAD-CT-2, at baseline, may indicate patients that are resistant to hypomethylating agents.
                                                                                                                                                     NY-ESO-1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Further studies are needed.
                                    MATERIALS AND METHODS
                                                       Subject Populations                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               - Decreased antibody responses to MAD-CT-1, MAD-CT-2 and SSX2 with increased MAGE-E1 and MAGE-A4, during therapy, may be
Sera were obtained from 41 patients with Myeloproliferative disorders (8 Acute Myelogenous Leukemia,
                                                                                                                                                                                                                                                                                                                                                                             0.5                                                                                                                                                                                                          0.00
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              indicative of patients receptive to further hypomethylating therapies. Further studies are needed.
1 Myelodysplastic Syndrome-Unclassifiable, 4 Refractory Anemia, 13/5 Refractory Anemia with excess
blasts (class I and II respectfully), 6 Refractory Cytopenia with Multilineage Dysplasia and 4 Chronic
                                                                                                                                                                                                                                                                                                                            Antibody Response Change Pre- to Post- Therapy




Myelomonocytic Leukemia). Of these subjects, all patients received hypomethylating agents. All sub-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  REFERENCES




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Antibody Titer Change from Pre- to Post- Therapy
jects gave written institutional review board (IRB)-approved informed consent for their blood products
to be used for immunological research. Blood was collected from multiple sites in the study, shipped to                                                      SSX2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Atanackovic, D., et al. Cancer testis antigen expression and its epigenetic modulation in acute myeloid leukemia. American Journal of Hematology, 2011:86(11) 918-22.
Kansas University Medical Center (Kansas City, KS) for isolation then shipped overnight to H. Lee Mof-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 2. Szmania, S., et al., Immunization with a recombinant MAGE-A3 protein after high dose therapy for myeloma. Journal of Immunotherapy, 2007:30 (8) 847-54.
fitt Cancer Center (Tampa, FL) and stored in aliquots at -80°C until used.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             3. Odunsi, K., et al., Vaccination with an NY-ESO-1 peptide of HLA class I/II specificities induces integrated humoral and T cell responses in ovarian cancer. Proceedings of the
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          National Acadamy of Science U.S.A., 2007:104 (31) 12837-42.
                                                                                                                                                 Antigens




                                     High-Throughput Immunoblot assay (HTI)                                                                                                                     + NY-ESO-1 response
                                                                                                                                                                                                -NY-ESO-1 response
                                                                                                                                                                                                                                                                                                                                                                             0.0                                                                                                                                                                                                          -0.04                                                                        4. Bender, A., et al., LUD 00-009: phase 1 study of intensive course immunization with NY-ESO-1 peptides in HLA-A2 positive patients with NY-ESO-1-expressing cancer. Cancer
We have previously reported the construction of a panel of lambda phage encoding 29 cancer-testis anti-
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Immunity 2007:(7) 16.
gens [16]. Analysis of this panel was conducted similarly to what we have previously reported [11, 16].
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       5. Coral, S., et al., 5-AZA-2'-deoxycytidine in cancer immunotherapy: a mouse to man story. Cancer Research, 2007:67(6) 2900-1; author reply 2901-2.
In brief, XL-1 blue MRF E. coli were grown overnight, collected by centrifugation, resuspended in
10mM MgSO4, and poured in top agarose (LB broth/10mM MgSO4/0.2% maltose/0.7% agarose) over
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       6. Vatolin, S., et al., Conditional expression of the CTCF-paralogous transcriptional factor BORIS in normal cells results in demethylation and derepression of MAGE-A1 and
LB agar in Omniwell plates (Nunc, Rochester, NY). Phage encoding individual (9,000 pfu) CTA’s were                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         reactivation of other cancer-testis genes. Cancer Research, 2005:65(17) 7751-62.
                                                                                                                                                       MAGE-A3
then spotted in replicates onto multiple bacterial agar lawns using a liquid handling robot (Biomek FX,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                7. D'Arcy, V., et al., BORIS, a paralogue of the transcription factor, CTCF, is aberrantly expressed in breast tumours. British Journal of Cancer, 2008:98(3) 571-9.
Beckman, Fullerton, CA). Spotted plates were allowed to sit undisturbed for 15 minutes and then over-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  8. Shichijo, S., et al., Induction of MAGE genes in lymphoid cells by the demethylating agent 5-aza-2'-deoxycytidine. Japanese Journal of Cancer Research, 1996:87(7) 751-6.
laid with nitrocellulose membranes impregnated with 10mM IPTG. Plates were incubated overnight at                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      9. Ghoshal, K., et al., 5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology
37°C. The next day, filters were washed, blocked with TBS (50mM Tris pH 7.2, 100mM NaCl) +                                                                                                                                                                                                                                                                                   -0.5                                                                                                                                                                                                         -0.08                                                                            domain, and nuclear localization signal. Molecular Cell Biology, 2005:25(11) 4727-41.
1%BSA (bovine serum albumin), and then probed overnight with human serum diluted 1:100 in blocking                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     10. Guo, Z.S., et al., De novo induction of a cancer/testis antigen by 5-aza-2'-deoxycytidine augments adoptive immunotherapy in a murine tumor model. Cancer Research,
solution. Subsequently, the membranes were washed, and human IgG was detected with an alkaline                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2006:66(2)1105-13.
phosphatase-conjugated anti-human IgG detection antibody (Sigma, St. Louis, MO). The filters were                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      11. Dubovsky, JA., McNeel DG. Inducible expression of a prostate cancer -testis antigen, SSX-2, following treatment with a DNA methylation inhibitor. Prostate, 2007;67(16)1781-90.
washed again and then developed with 0.3mg/ml nitro-blue tetrazolium chloride (NBT) + 0.15mg/ml                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        12. Garcia-Manero G, Fenaux P. Hypomethylating agents and other novel strategies in myelodysplastic syndromes. Journal of Clinical Oncology, 2011;29(5)516-23.
5-bromo-4-chloro-3’-indolyphosphate p-toluidine salt (BCIP). After development, filters were washed                                                         PRAME                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      13. Goodyear, O., et al., Induction of a CD8+ T-cell response to the MAGE cancer testis antigen by combined treatment with azacitidine and sodium valproate in patients with acute
                                                                                                                                                                                                                                                                            **




with deionized water and immunoreactive plaques were recorded for each filter by visual comparison                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          myeloid leukemia and myelodysplasia. Blood, 2010:116(11)1908-18.
with internal positive (phage encoding human IgG) and negative (empty phage encoding beta-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             14. Karpf, AR., A potential role for epigenetic modulatory drugs in the enhancement of cancer/germ-line antigen vaccine efficacy. Epigenetics, 2006:1(3)116-20.
galactosidase) control plaques.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        15. Sigalotti, L., et al, 5-Aza-2'-deoxycytidine (decitabine) treatment of hematopoietic malignancies: a multimechanism therapeutic approach?. Blood, 2003:101(11)4644-46.
                                                                                                                                                                                                                                                                                                                                                                             -1.0                                                                                                                                                                                                         -0.12
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       16. Dubovsky, JA., Albertini, MR., McNeel, DG. MAD-CT identified as a novel melanoma cancer-testis antigen using phage immunoblot analysis. Journal of Immunotherapy
                              CTA Enzyme-linked Immunosorbent Assay (ELISA)
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           2007;30(7);675-83.
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ELISA was preformed using sera obtained from non-heparinized blood collection tubes. 96 well
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       17. Yan M., Himoudi, et al, Increased PRAME antigen-specific killing of malignant cell lines by low avidity CTL clones, following treatment with 5-Aza-2'-Deoxycytidine. Cancer
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EIA/RIA flat bottom plates were coated O/N with Recombinant Human CTA antigens (PRAME                                                                                                                                                                                                                                                                                                                                                                   Antigens
H00023532-P01, MAGE-A3 H00004102-P01, and SSX2 H00006757-P01 Novus Biologicals) at
                                                                                                                                                                                           Antibody Titer Change from Pre- to Post- Therapy                                                                                                                                                                                                                                                                                                                                                                          Antigens                                              Immunology Immunotherapy 2011:60(9):1243-55.
10ug/mL with 100uL per well. Plates were washed with PBS and blocked. Diluted (1:25) and serially
diluted sera was used in conjunction with a standard of Purified Human IgG antibody (AG711 Millipore)                                            PRAME antibody responses correlate with NY-ESO-1 humoral response.                                                                                            Humoral responses show bias in ELISA and HTI based assays towards Hematologic and Transfusion responses
serially diluted 8 to 0.0625 ug/mL. Incubate O/N at 4C. Sera was removed and the plate was incubated                                             Samples were stratified according to responses seen in HTI. Three antigens (NY-                                                                               A) The antibody response profile of samples run by HTI divided between Responders versus Non-Responders reveal a biased reaction in a number of antigens. (NY-ESO-1,
with a 1:1000 diluted horseradish peroxidase conjugated, anti-human IgG detection antibody (555788                                               ESO-1, SSX2 and MAGE-A3) that were present on the HTI filter were examined                                                                                    MAD-CT-1, MAD-CT-2, SSX2, MAGE-E1, MAGE-A3 and MAGE-A4) B) The antibody response profile of samples run by ELISA divided between Responders versus Non-
BD Biosciences) used in conjunction with SureBlue TMB microwell peroxidase substrate (1-component)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              For High resolution PDF copies scan code
                                                                                                                                                 side by side with PRAME, a known CTA that elicits CTL responses ex-vivo [17].                                                                                 Responders reveal confirmed reaction in a number of antigens. PRAME, although exhibits CTL responses ex-vivo [17], does not show an augmented antibody response between                                                                                                                                                                                                          Free apps available for:                                                                                                                Conflicts of Interest (COI):
(52-00-01 KPL Kirkegaard & Perry Laboratories, Inc.)
                                                                                                                                                 2-way ANOVA w/ Bonferroni Post-test was performed using GraphPad Prism                                                                                        patients.                                                                                                                                                                                                                                                                                                                                                                        iPhone- QRReader                            QR Code
                                                                                                                                                 version 5.00 for Windows, GraphPad Software, San Diego California USA,                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Blackberry- QR Code Scanner Pro- Free                                                                                 Barry S. Skikne, MD: Celgene-Employment, Equity Ownership
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Android- QR Droid
                                                                                                                                                 www.graphpad.com. (**P=<0.01)

				
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