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. 2012 Mar 29;119(13):3142-50.
doi: 10.1182/blood-2011-11-388926. Epub 2012 Jan 20.

Novel myeloma-associated antigens revealed in the context of syngeneic hematopoietic stem cell transplantation

Melinda A Biernacki  1 , Yu-tzu Tai, Guang Lan Zhang, Anselmo Alonso, Wandi Zhang, Rao Prabhala, Li Zhang, Nikhil Munshi, Donna Neuberg, Robert J Soiffer, Jerome Ritz, Edwin P Alyea, Vladimir Brusic, Kenneth C Anderson, Catherine J Wu
Affiliations

Novel myeloma-associated antigens revealed in the context of syngeneic hematopoietic stem cell transplantation

Melinda A Biernacki et al. Blood. .

Abstract

Targets of curative donor-derived graft-versus-myeloma (GVM) responses after allogeneic hematopoietic stem cell transplantation (HSCT) remain poorly defined, partly because immunity against minor histocompatibility Ags (mHAgs) complicates the elucidation of multiple myeloma (MM)-specific targets. We hypothesized that syngeneic HSCT would facilitate the identification of GVM-associated Ags because donor immune responses in this setting should exclusively target unique tumor Ags in the absence of donor-host genetic disparities. Therefore, in the present study, we investigated the development of tumor immunity in an HLA-A0201(+) MM patient who achieved durable remission after myeloablative syngeneic HSCT. Using high-density protein microarrays to screen post-HSCT plasma, we identified 6 Ags that elicited high-titer (1:5000-1:10 000) Abs that correlated with clinical tumor regression. Two Ags (DAPK2 and PIM1) had enriched expression in primary MM tissues. Both elicited Ab responses in other MM patients after chemotherapy or HSCT (11 and 6 of 32 patients for DAPK2 and PIM1, respectively). The index patient also developed specific CD8(+) T-cell responses to HLA-A2-restricted peptides derived from DAPK2 and PIM1. Peptide-specific T cells recognized HLA-A2(+) MM-derived cell lines and primary MM tumor cells. Coordinated T- and B-cell immunity develops against MM-associated Ags after syngeneic HSCT. DAPK1 and PIM1 are promising target Ags for MM-directed immunotherapy.

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Figures

Figure 1
Figure 1
Achievement of durable molecular remission after syngeneic HSCT for treatment of MM in patient A. (A) Levels of serum monoclonal protein (g/dL) become undetectable by 1.5 months after HSCT. Arrows indicate the time points screened by protein microarray. Vertical dashed line indicates time of syngeneic stem cell infusion. (B) Expression of transcripts containing the IgH sequence unique to patient A's tumor is detectable by quantitative real-time PCR in BMMCs (black squares and solid line) and PBMCs (gray squares and line) before syngeneic HSCT, but is undetectable beginning at 24 months after HSCT. Values shown are tumor-specific IgH transcripts normalized to GAPDH transcript expression. “Undetectable” indicates samples with acceptable GAPDH values but absent tumor-specific IgH transcript expression.
Figure 2
Figure 2
Serologic screening identifies high-titer Ab responses against DAPK2, PDGFRB, PIM1, and PRKCB1 developing after syngeneic HSCT. (A) Plasma samples collected from patient A at serial time points before and after HSCT were screened by ProtoArray protein microarray. Subarrays demonstrating DAPK2 reactivity are highlighted in the gray boxes. Spots seen in the bottom right corner of all subarray images are control spots. (B) Arrays were analyzed using 2 methods described previously. Six Ags, DAPK2, PDGFRB, C1orf116, RELA, PIM1, and PRKCB1, elicited significantly greater reactivity after HSCT compared with before HSCT. Significance (Z-scores) scores of Ab reactivity at time points are shown. (C) Ag binding was confirmed by immunoprecipitation of biotinylated target Ags expressed in vitro in rabbit reticulocyte lysate by patient plasma at serial time points. Immunoprecipitated Ags were detected using streptavidin-conjugated secondary Ab. Representative Western blots are shown. The first lane is whole rabbit reticulocyte lysate expressing target Ags that was not subjected to immunoprecipitation. (D) Plasma samples (1:200 dilution) collected from patient A at serial time points before and after HSCT were tested by ELISA (plates coated with 5 μg/mL of recombinant protein). Ab IgG responses against DAPK2 and PIM1 arose and peaked at 3 months after HSCT. (E) Candidate Ags elicit high-titer Ab responses, as indicated by titration studies of serum using ELISA assays. Reactivity remained detectable at dilutions of 1:10 000 (DAPK2) and 1:5000 (PIM1).
Figure 3
Figure 3
Expression of DAPK2 and PIM1 is enriched in primary MM cells. (A) DAPK2 and PIM1 have high transcript expression in primary MM BMMCs (gray triangles), but not in normal PBMCs (black diamonds) by gene-specific quantitative real-time PCR. BM specimens contained > 95% tumor by immunohistochemistry. DAPK2, but not PIM1, was also highly expressed in normal CD19+ B cells (white squares). Bars indicate the median value for transcript expression relative to GAPDH. Statistical significance was determined by 1-sided exact Wilcoxon rank-sum test. (B) Western blotting of cell lysates (20 μg of protein per lane) from normal hematopoietic cells and primary MM BMMCs with Ab against either DAPK2 or PIM1 reveals protein expression in 4 (DAPK2) or 3 (PIM1) of 5 primary MM BMMCs. (C) Western blotting of MM cell line lysates (20 μg of protein per lane) showed expression of DAPK2 and PIM1 in all tested MM cell lines.
Figure 4
Figure 4
MM treatment is associated with the development of Ab responses against DAPK2 and PIM1. (A) Plasma samples from 5 MM patients who demonstrated GVM responses (defined as clinical remission > 2 years) without GVHD were tested by ELISA. Three patients (Pt. 2, black bars; Pt. 3, gray bars; and Pt. 5, white bars) had new or markedly increased Ab reactivity to DAPK2 and/or PIM1 after HSCT. The dashed line indicates a cutoff based on 2 SD above the mean of Ab reactivity of 10 normal donors for each Ag. (B) Ab responses against DAPK2 (black bars) and PIM1 (gray bars) are also seen by ELISA in MM patients treated with standard chemotherapy or autologous HSCT, but are minimal in patients with untreated MM or healthy donors.
Figure 5
Figure 5
DAPK2- and PIM1-derived nonameric peptides elicit T-cell reactivity in post-HSCT PBMCs from patient A. Fresh post-HSCT PBMCs were stimulated ex vivo with 10μM concentrations of individual peptides in the presence of 10 μg/mL of IL-7 for 7 days, then tested by ELISpot with peptide-pulsed autologous matured dendritic cells (PBMC:APC ratio = 1:1). (A) T cells from patient A 2.5 years after HSCT show reactivity to the DAPK2-derived peptide D1, but not to other DAPK2-derived peptides. (B) T cells from patient A 2.75 years after HSCT are strongly reactive to the PIM1-derived peptide P4 and weakly reactive to P1, P2, P5, and P7.
Figure 6
Figure 6
D1- and P4-specific T cells are isolated by tetramer sorting and recognize whole processed and presented Ag. (A) Patient A CD3+ T cells were repeatedly stimulated with either D1 or P4 peptide-pulsed T2 cells before staining with peptide-specific tetramer. Tetramer and CD8 double-positive cells were isolated by FACS (representative FACS plots shown). (B) Sorted D1- or P4-positive T cells were tested by ELISpot against K562 cells stably transfected to express HLA-A2 (K562/A2 cells) that were nucleofected to overexpress either DAPK2 or PIM1. As expected, overexpression of DAPK2 induced apoptosis in nucleofected K562 cells. P4-specific T cells recognized processed and presented PIM1, an effect that was blocked by the class I–blocking Ab w6/32. (C) Sorted D1- or P4-positive T cells were also tested against the A2-positive MM cell lines MC/CAR and IM9 and the A2-negative MM cell lines OPM2 and NCI 4929. Both D1- and P4-specific T cells showed reactivity to HLA-A2–positive cell lines, but not to HLA-A2–negative cell lines. IFN-γ secretion was blocked by co-incubation with w6/32 Ab.
Figure 7
Figure 7
D1- and P4-specific CD8 T cells recognize primary MM tissue from HLA-A2–positive patients. Sorted D1- or P4-positive CD8 T cells were tested by ELISpot against MM BMMCs isolated from fresh BM from HLA-A2–positive donors. D1-specific (A) and P4-specific (B) T cells recognized primary MM BMMCs from HLA-A2–positive donors (n = 5), but not healthy HLA-A2–positive BMMCs (n = 2) or HLA-A2–negative MM BMMCs (n = 4).
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References

    1. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood. 1990;75(3):555–562. - PubMed
    1. Alyea EP, Soiffer RJ, Canning C, et al. Toxicity and efficacy of defined doses of CD4(+) donor lymphocytes for treatment of relapse after allogeneic bone marrow transplant. Blood. 1998;91(10):3671–3680. - PubMed
    1. Lokhorst HM, Schattenberg A, Cornelissen JJ, Thomas LL, Verdonck LF. Donor leukocyte infusions are effective in relapsed multiple myeloma after allogeneic bone marrow transplantation. Blood. 1997;90(10):4206–4211. - PubMed
    1. Crawley C, Lalancette M, Szydlo R, et al. Outcomes for reduced-intensity allogeneic transplantation for multiple myeloma: an analysis of prognostic factors from the Chronic Leukaemia Working Party of the EBMT. Blood. 2005;105(11):4532–4539. - PubMed
    1. Maloney DG, Molina AJ, Sahebi F, et al. Allografting with nonmyeloablative conditioning following cytoreductive autografts for the treatment of patients with multiple myeloma. Blood. 2003;102(9):3447–3454. - PubMed

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