DAPK2 and PIM1 Are Myeloma-Associated Antigens That Elicit Coordinated B and T Cell Immunity After Syngeneic HSCT.

Abstract 2445

Poster Board II-422

Syngeneic hematopoietic stem cell transplantation (HSCT) can produce long-term disease-free survival in patients with hematologic malignancies, including multiple myeloma (MM). Because donor and host are genotypically identical in this setting, allo-immunity is absent and curative responses may result from donor-derived immune responses against tumor-associated antigens. We recently described potent, antigen-specific humoral immune responses arising after myeloablative syngeneic HSCT in an HLA-A2 positive patient with MM who achieved sustained molecular remission lasting over 3 years. Two target antigens of this response, DAPK2 and PIM1, were identified by serologic screening of a recombinant protein array consisting of the expressed products of 8000 open reading frames. Both proteins elicited antigen-specific antibody responses after HSCT that peaked around 3 months post-HSCT and gradually waned thereafter; prior to HSCT these antibody responses were low to undetectable. DAPK2 is a serine-threonine kinase involved in pro-apoptotic signaling, while PIM1 is an oncogene with known associations to B cell malignancies. By Western blot, both proteins demonstrate enriched protein expression in primary myeloma bone marrow compared to normal hematopoietic tissues.

In the current studies, we determined whether antibody responses to DAPK2 and PIM1 were coordinated with T cell immunity against the same proteins following HSCT in this patient. As this patient was HLA-A2 positive, we used established MHC class I peptide binding algorithms to predict candidate peptide epitopes derived from PIM1 and DAPK2. We identified 23 9-mer peptides derived from DAPK2 and PIM1, all with high binding rank scores. All peptides were confirmed to bind to HLA-A2 using T2 binding assays. Ex vivo stimulation of fresh patient PBMC obtained ∼2.5 years post-HSCT with candidate peptides revealed 2 peptides, DAPK2_156–164 (MLLDKNIPI) and PIM1_191-199 (ALLKDTVYT) that elicited strong interferon-γ (IFNγ) secretion consistent with recall responses to these antigens. By IFNγ ELISpot, DAPK2- and PIM1-specific T cells recognized HLA-A2-positive myeloma cell lines MC/CAR and IM9, but not A2-negative OPM1 and NCI4929 lines. T cell recognition was mediated by CD8+ effector cells and was effectively blocked by the MHC class I blocking antibody w6/32. These studies indicate that the predicted peptides are naturally processed and presented by myeloma cells. When tested against CD138+ sorted primary MM marrow cells by IFNγ ELISpot, DAPK2-specific T cells and PIM1-specific T cells recognized 3 of 5 and 1 of 5 A2-positive primary tumor samples, respectively. Neither A2-negative CD138+ tumor samples nor A2-positive normal marrow elicited reactivity from antigen-specific T cells. These data demonstrate that PIM1 and DAPK2 are MM-associated antigens that commonly generate processed epitopes on malignant MM cells. Both proteins elicited coordinated B and T cell immunity in a MM patient who achieved sustained molecular remission after syngeneic HSCT. These data suggest that donor-derived MM-specific adaptive immunity contributes to disease regression following syngeneic HSCT for MM. As PIM1 and DAPK2 represent immunogenic tumor-associated antigens and not allo-antigens, these may be attractive targets for immunotherapy directed at MM outside of the context of allogeneic HSCT.