Haematologica 2003 - Supplements

myeloma cells are able to induce OPN-secretion from host cells.
The fact that myeloma-derived hepatocyte growth factor (HGF)
induced OPN-secretion in a dose-dependent manner from the
osteoblastic cell line Saos-2 supports this finding.
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A novel recombinant bispecific single-chain antibody,
Wue-1xCD3, induces T-cell mediated cytotoxicity
towards human myeloma cells
Dirk Hoenemann1*, Peter Kufer3*, Matthaeus M.
Rimpler1*, Manik Chatterjee1, Freya Riechert1, Kurt
Bommert1, 2, Axel Greiner4, Bernd Dorken1, 2, and Ralf C.
Bargou1
1Humboldt University of Berlin, University Medical Center Charité,
Robert-Roessle-Klinik, Department of Internal Medicine,
Lindenberger Weg 80, Berlin, Germany; 2Max-Delbrueck Center
for Molecular Medicine, Robert-Roessle-Str. 10, Berlin, Germany;
3Institute of Immunology, University of Munich, Munich, Germany;
4Institute of Pathology, University of Wuerzburg, Wuerzburg,
Germany
The development of antibody-based strategies for the treatment of
Multiple Myeloma (MM) has been hampered by the fact that
suitable plasmacell-specific surface antigens have been missing
so far. Although normal and malignant plasmacells express a
number of well characterized surface markers they all have
turned out to be not plasmacell-specific. However, recently a
novel monoclonal antibody, designated Wue-1, has been
generated, which specifically binds to the cell surface of normal
and malignant human plasma cells. Therefore, Wue-1 is an
interesting and promising candidate to develop novel
immunotherapeutic strategies for the treatment of multiple
myeloma. One variant for an antibody-based strategy is the
bispecific antibody approach. In particular, recombinant
bispecific single-chain antibodies are interesting candidates
because they show exceptional biological properties
discriminating these molecules from conventional antibodies. We
have generated a novel MM directed recombinant bispecific
single-chain antibody, Wue-1xCD3 (bscWue-1xCD3) and
analyzed the biological properties of this antibody using the MM
cell line NCI-H929 and primary cells from the bone marrow of
patients with multiple myeloma and autologous or allogeneic
effector T-cells. We were able to show that the bscWue-1xCD3
induces efficient T-cell mediated cell death of NCI-H929 cells
and primary myeloma cells in 10/11 cases analyzed. In contrast to
conventional bispecific antibodies described so far, the
bscWue-1xCD3, is efficacious at low E:T ratios and without T-
cell pre-stimulation. Target cell lysis was specific for Wue-1
antigen positive cells and could be blocked by the Wue-1
monoclonal antibody. Wue-1 antigen negative NALM-6 cells
were not lysed by bscWue-1xCD3 Ab. To our knowledge this is
the first plasma cell-directed bispecific antibody described so far,
showing promising results, and might therefore be a new
potential agent for the treatment of malignant plasma cell
disorders.
074
A recombinant HLA class I-specific single-chain Fv
diabody induces cell death in human myeloma cells
Shuji Ozaki1, Etsuko Sekimoto2, Yoichi Tanaka2, Takashi
Ohshima2, Hironobu Shibata2, Toshihiro Hashimoto2,
Masahiro Abe2, Tetsuya Goto3, Naoki Kimura4, Shin-ichiro
Iida4, Masayuki Tsuchiya4, Toshio Matsumoto2, and
Masaaki Kosaka5
1 Division of Transfusion Medicine, Tokushima University Hospital;
2 Department of Medicine and Bioregulatory Sciences, University
of Tokushima Graduate School of Medicine; 3 Department of
Internal Medicine, Tokushima Red Cross Hospital; 4 Chugai
Pharmaceutical Co., Ltd.; 5 Department of Internal Medicine,
Tokushima Prefecture Hospital of Kaifu, Japan.
Cross-linking of HLA class I molecules has been shown to induce
programmed death of human neoplastic lymphoid cells as well as
activated lymphocytes. We have generated a novel anti-HLA
class I monoclonal antibody (2D7) to examine the possible role of
this molecule expressed on multiple myeloma (MM) cells.
Moreover, a recombinant single-chain Fv diabody was
constructed from the parent mouse IgG antibody. This diabody
(2D7-DB) effectively causes cross-linking of HLA class I
molecules and induces cell death of target cells. In this study, we
compared the levels of HLA class I expression in MM cells and
normal bone marrow cells, and evaluated the in vitro effects of
2D7-DB to induce cytotoxicity and/or apoptosis in MM cells.
Cell surface expression of HLA class I on bone marrow cells was
analyzed by flow cytometry using fluorescein-labeled 2D7 and
phycoerythrin-labeled anti-CD38 antibody for MM cell gating.
Cells were cultured for 48 hours with 2D7-DB or 2D7 in the
presence or absence of F(ab')2 goat anti-mouse IgG (GAM). Cell
viability was determined by trypan blue staining. Induction of
apoptosis was assessed by Annexin V/propidium iodide staining.
HLA class I molecules were more strongly expressed in 13/15 of
patient MM cells than in normal myeloid cells or lymphocytes.
Cross-linking of HLA class I with 2D7 and GAM induced
cytotoxicity of patient MM cells. More importantly, 2D7-DB
alone mediated cytotoxicity in MM cells at 100 ng/mL, but not in
normal myeloid cells or lymphocytes. MM cells treated with
2D7-DB showed cytoplasmic vacuolization and positive staining
for Annexin V. These findings indicate a functional role for HLA
class I molecules in MM cell death and also provide new insights
into the possible therapeutic strategies for targeting this molecule
in MM.
075
Decitabine increases expression of cancer antigens
MAGE-3 and NY-ESO-1 in multiple myeloma cell lines.
Jan Van Droogenbroeck (1), Sushil Gupta (2), Friedel
Nollet (3), Susann Szmania (2), Ramesh Batchu (2), Guilio
Spagnoli (4), Arnold Criel (3), John Shaughnessy (2),
Guido Tricot (2) and Frits van Rhee (2).
(1) AZ Sint Jan, Department of Hematology, Brugge, Belgium.(2)
Myeloma Institute for Research and Therapy, University of
Arkansas for Medical Sciences, Little Rock, AR, USA.(3) AZ Sint
Jan, Laboratory for Hematology, Brugge, Belgium.(4) University of
Basel, Department of Surgery, Switzerland.
NY-ESO-1 and MAGE-3 both belong to the group of Testis
Cancer Antigens (TCA’s). TCA’s are present in testis and
various malignancies, but not in normal tissues. NY-ESO-1 and
MAGE-3 are highly immunogenic, eliciting B-cell and HLAclass
I/class II restricted T-lymphocyte responses. About 20% of
newly diagnosed multiple myeloma (MM) patients express these
antigens, to reach more than double in relapse.
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